| 1. |
Arpin C,
Dubois V,
Coulange L,
André C,
Fischer I,
Noury P,
Grobost F,
Brochet JP,
Jullin J,
Dutilh B,
Larribet G,
Lagrange I,
Quentin C,
( 2003 ) Extended-spectrum beta-lactamase-producing Enterobacteriaceae in community and private health care centers. PMID : 14576109 : DOI : 10.1128/aac.47.11.3506-3514.2003 PMC : PMC253776 Abstract >>
In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum beta-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.
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2. |
Partridge SR,
Hall RM,
( 2003 ) The IS1111 family members IS4321 and IS5075 have subterminal inverted repeats and target the terminal inverted repeats of Tn21 family transposons. PMID : 14563872 : DOI : 10.1128/jb.185.21.6371-6384.2003 PMC : PMC219399 Abstract >>
IS5075 and IS4321 are closely related (93.1% identical) members of the IS1111 family that target a specific position in the 38-bp terminal inverted repeats of Tn21 family transposons and that are inserted in only one orientation. They are 1,327 bp long and have identical ends consisting of short inverted repeats of 12 bp with an additional 7 bp (TAATGAG) or 6 bp (AATGAG) to the left of the left inverted repeats and 3 bp (AGA) or 4 bp (AGAT) to the right of the right inverted repeat. Circular forms of IS5075 and IS4321 in which the inverted repeats are separated by abutting terminal sequences (AGATAATGAG) were detected. A similar circular product was found for the related ISPa11. Transposition of IS4321 into the 38-bp target site was detected, but a flanking duplication was not generated. The precisely reconstituted target site was also identified. Over 50 members of the IS1111 family were identified. They encode related transposases, have related inverted repeats, and include related bases that lie outside these inverted repeats. In some, the flanking bases number 5 or 6 on the left and 4 or 3 on the right. Specific target sites were found for several of these insertion sequence (IS) elements. IS1111 family members therefore differ from the majority of IS elements, which are characterized by terminal inverted repeats and a target site duplication, and from members of the related IS110 family, which do not have obvious inverted repeats near their termini.
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3. |
Pang SS,
Duggleby RG,
Schowen RL,
Guddat LW,
( 2004 ) The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate. PMID : 14557277 : DOI : 10.1074/jbc.M304038200 Abstract >>
Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.
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4. |
Woehlke G,
Laussermair E,
Schwarz E,
Oesterhelt D,
Reinke H,
Beyreuther K,
Dimroth P,
( 1992 ) Appendix. Sequence of the beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae: a correction of the C-terminal part. PMID : 1429628 : Abstract >>
N/A
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5. |
Feldmann SD,
Sahm H,
Sprenger GA,
( 1992 ) Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12. PMID : 1324398 : DOI : 10.1007/bf00283840 Abstract >>
The genes xylA and xylB were cloned together with their promoter region from the chromosome of Klebsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xylA encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M(r) of 49,793). The gene xylB encodes the enzyme xylulokinase (XK or XylB) with a calculated M(r) of 51,783 (483 amino acids). The two genes successfully complemented xyl mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylAKp 5' upstream region in high copy number (but lacking an active xylB gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5' upstream regions of xylA on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.
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6. |
Park SY,
Lee SJ,
Oh TK,
Oh JW,
Koo BT,
Yum DY,
Lee JK,
( 2003 ) AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. PMID : 12777494 : DOI : 10.1099/mic.0.26269-0 Abstract >>
Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-L-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-L-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH approximately H approximately D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.
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7. |
Hama H,
Wilson TH,
( 1992 ) Primary structure and characteristics of the melibiose carrier of Klebsiella pneumoniae. PMID : 1339436 : Abstract >>
The melB gene coding for the melibiose carrier of Klebsiella pneumoniae was cloned and sequenced. There were two potential translation initiation sites. It was predicted that the melibiose carrier consists of 471 (or 467) amino acid residues. Seventy-eight percent of the 471 amino acids were identical to the Escherichia coli melibiose carrier. Sugar transport characteristics were studied using an E. coli mel- mutant expressing cloned K. pneumoniae melB gene. Accumulation of melibiose via the K. pneumoniae melibiose carrier was not stimulated by adding NaCl or LiCl which stimulates melibiose accumulation via the E. coli melibiose carrier. Lactose was accumulated only in the presence of LiCl. TMG (methyl-1-thio-beta-D-galactopyranoside) was accumulated in the absence of added NaCl or LiCl. The accumulation was stimulated by LiCl but not by NaCl. Rapid H+ uptake was observed when melibiose or TMG was added to cell suspensions. These results suggest that the preferred cation couplings via K. pneumoniae melibiose carrier are H(+)-melibiose, Li(+)-lactose, and H+/Li(+)-TMG. This coupling spectrum is quite different from that of the E. coli melibiose carrier. It is of special interest that the K. pneumoniae melibiose carrier seems to be lacking the ability to recognize Na+ which is a preferred coupling cation of the E. coli melibiose carrier for all known sugar substrates. Further investigation of these two carriers may give us insight into the Na+ recognition site.
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8. |
Mabilat C,
Lourençao-Vital J,
Goussard S,
Courvalin P,
( 1992 ) A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3. PMID : 1331747 : DOI : 10.1007/bf00286188 Abstract >>
The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.
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9. |
Meulenberg JJ,
Sellink E,
Riegman NH,
Postma PW,
( 1992 ) Nucleotide sequence and structure of the Klebsiella pneumoniae pqq operon. PMID : 1313537 : DOI : 10.1007/bf00280008 Abstract >>
A 6940 bp Klebsiella pneumoniae chromosomal DNA fragment, containing genes involved in pyrroloquinoline quinone (PQQ) biosynthesis, was sequenced. Six open reading frames, pqqA, pqqB, pqqC, pqqD, pqqE and pqqF were identified in the pqq operon, which coded for polypeptides of 2764 (23 amino acids), 33,464, 28,986, 10,436, 42,881 and 83,616 Da, respectively. The transcription startpoint was mapped by primer extension analysis, upstream of pqqA, and promoter boxes could be identified. The gene products of pqqB, pqqC, pqqE and pqqF were detected in maxi-cells and the molecular weights of the proteins corresponded with the molecular weights deduced from the nucleotide sequence. The gene products of pqqA, pqqB, pqqC, pqqD and pqqE show 49%-64% identity in amino acid sequence with those of pqqIV, pqqV, pqqI, pqqII and pqqIII respectively in the cloned pqq cluster of Acinetobacter calcoaceticus. The 84 kDa protein encoded by pqqF, which is not present in the cloned pqq cluster of A. calcoaceticus but which is essential for PQQ biosynthesis in K. pneumoniae and Escherichia coli, seems to belong to a family of proteases.
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10. |
Pai H,
Byeon JH,
Yu S,
Lee BK,
Kim S,
( 2003 ) Salmonella enterica serovar typhi strains isolated in Korea containing a multidrug resistance class 1 integron. PMID : 12760886 : DOI : 10.1128/aac.47.6.2006-2008.2003 PMC : PMC155850 Abstract >>
Six strains of Salmonella enterica serovar Typhi which were resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, tetracycline, and gentamicin were isolated in Korea. This multidrug resistance was transferred by a conjugative plasmid of about 50 kb. The plasmid harbored a class 1 integron, which included six resistance genes, aacA4b, catB8, aadA1, dfrA1, aac(6')-IIa, and the novel blaP2, in that order. All of the isolates showed the same-size plasmids and the same ribotyping patterns, which suggests a clonal spread of these multidrug-resistant isolates.
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11. |
Gerharz T,
Reinelt S,
Kaspar S,
Scapozza L,
Bott M,
( 2003 ) Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA. PMID : 12741850 : DOI : 10.1021/bi0340595 Abstract >>
The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compounds. We recently showed that the separated periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a K(d) of 5.5 microM at pH 7. To identify positively charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titration calorimetry (ITC). In 12 cases, the K(d) for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 microM). In one case (R98A), the K(d) was 6-fold decreased (0.8 microM), whereas in four cases (R66A, H69A, R107A, and K109A) the K(d) was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV circular dichroism (CD) spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the melting point (T(m)) of wild-type CitAP and mutein R98A by 6.2 and 9.5 degrees C, respectively, but had no effect on the T(m) of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.
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12. |
Poirel L,
Decousser JW,
Nordmann P,
( 2003 ) Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene. PMID : 12936998 : DOI : 10.1128/aac.47.9.2938-2945.2003 PMC : PMC182628 Abstract >>
The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum beta-lactamase bla(CTX-M-19) gene in a Klebsiella pneumoniae clinical isolate were determined. This beta-lactamase gene was part of a 4,797-bp transposon inserted inside orf1 of Tn1721. Inside this transposon, bla(CTX-M-19) was bracketed upstream and downstream by insertion sequences ISE cp1B and IS903D, respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy ISEcp1B element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of ISE cp1B and a second sequence farther away, resembling its second inverted repeat. Additionally, ISEcp1B provided -35 and -10 promoter sequences, contributing to the high-level expression of the bla(CTX-M-19) gene. Southern blot analysis failed to identify a reservoir of ISEcp1-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of ISEcp1-like elements to mobilize and to promote the expression of beta-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.
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13. |
Chen FJ,
Lauderdale TL,
Ho M,
Lo HJ,
( 2003 ) The roles of mutations in gyrA, parC, and ompK35 in fluoroquinolone resistance in Klebsiella pneumoniae. PMID : 12959405 : DOI : 10.1089/107662903322286472 Abstract >>
In a survey of 541 Klebsiella pneumoniae isolates from 44 hospitals in Taiwan, three distinct populations were identified by the disk diffusion method according to the disribution of zone diameters of ciprofloxacin. Isolates with resistant, reduced-susceptible, and susceptible to fluoroquinolone were defined as CIP zone diameters of < or = 15 mm, 16-26 mm, and > or = 27 mm, respectively. Thus, in addition to 38 (7%) resistant isolates, there were 30 (5.5%) reduced-susceptible isolates and 473 (87.5%) susceptible isolates. A total of 34 isolates consisting of nine resistant, 13 reduced-susceptible, and 12 susceptible isolates were assessed for point mutations in gyrA and parC and the outer membrane profiles. The susceptibility to fluoroquinolone of 13 reduced-susceptible isolates was not altered in the presence of carbonyl cyanide m-chlorophenylhydrazone, an efflux inhibitor, showing that efflux is not a major contributor to reduced susceptibility. In addition to single mutation in gyrA, OmpK35 porin loss can also be the first step for developing fluoroquinolone resistance. No strain possesses a parC mutation without the simultaneous presence of a gyrA mutation, suggesting that mutations in parC play a complementary role for higher-level of fluoroquinolone resistance and fluoroquinolone resistance is a multistep process.
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14. |
Sun J,
van den Heuvel J,
Soucaille P,
Qu Y,
Zeng AP,
( N/A ) Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria. PMID : 12675558 : DOI : 10.1021/bp025739m Abstract >>
The dihydroxyacetone (dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol. In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions. Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniae MGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes. The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridium butyricum. Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C. perfringens are located in two separate organization units on the chromosome. Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K. pneumoniae and C. freundii have high similarities (80-95%) but lower similarities to those of the Clostridium species (30-80%). Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K. pneumoniae or C. freundii, suggesting two different types of dha regulon in the Clostridium species studied. The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied. In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K. pneumoniae in addition to the known ATP-dependent dha kinase. This finding gives new insights into the regulation of glycerol metabolism in K. pneumoniae and explains some hitherto not well understood experimental observations.
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15. |
Correia M,
Boavida F,
Grosso F,
Salgado MJ,
Lito LM,
Cristino JM,
Mendo S,
Duarte A,
( 2003 ) Molecular characterization of a new class 3 integron in Klebsiella pneumoniae. PMID : 12936982 : DOI : 10.1128/aac.47.9.2838-2843.2003 PMC : PMC182612 Abstract >>
Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum beta-lactamase was encoded by bla(GES-1), previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located on a new class 3 integron. The integron was 2863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The bla(GES-1) gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between bla(OXA-10)-type and aac(6')-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the bla(GES-1) gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.
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16. |
Destoumieux-Garzón D,
Thomas X,
Santamaria M,
Goulard C,
Barthélémy M,
Boscher B,
Bessin Y,
Molle G,
Pons AM,
Letellier L,
Peduzzi J,
Rebuffat S,
( 2003 ) Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli. PMID : 12890026 : DOI : 10.1046/j.1365-2958.2003.03610.x Abstract >>
The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 microM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB- and energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron-siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB- and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.
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17. |
Morris D,
O'Hare C,
Glennon M,
Maher M,
Corbett-Feeney G,
Cormican M,
( 2003 ) Extended-spectrum beta-lactamases in Ireland, including a novel enzyme, TEM-102. PMID : 12878521 : DOI : 10.1128/aac.47.8.2572-2578.2003 PMC : PMC166109 Abstract >>
Organisms producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Ireland. A total of 925 isolates of ampicillin-resistant members of the Enterobacteriaceae were received from six hospitals in Ireland over a 3-year period from September 1996 to September 1999. Isolates were screened for ESBL production by the double-disk diffusion (DDD) method. DDD-positive isolates that were (i) confirmed as ESBL producers by National Committee for Clinical Laboratory Standards (NCCLS) confirmatory testing and (ii) susceptible to cefoxitin by disk diffusion were considered ESBL producers. By these criteria, 27 (3%) of the ampicillin-resistant members of the Enterobacteriaceae studied were categorized as ESBL producers. Molecular typing suggested that some intra- and interhospital spread of ESBL-producing isolates had occurred. DNA sequencing of amplified bla(TEM) and bla(SHV) genes resulted in the detection of a novel bla(TEM) ESBL gene, bla(TEM-102) in two isolates (Klebsiella pneumoniae and Enterobacter cloacae) received from the same hospital but isolated from different patients. The study suggests dissemination of ESBL-producing bacteria within the health care system in Ireland and emphasizes the need for measures to control such spread.
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18. |
Seputiene V,
Motiejūnas D,
Suziedelis K,
Tomenius H,
Normark S,
Melefors O,
Suziedeliene E,
( 2003 ) Molecular characterization of the acid-inducible asr gene of Escherichia coli and its role in acid stress response. PMID : 12670971 : DOI : 10.1128/jb.185.8.2475-2484.2003 PMC : PMC152617 Abstract >>
Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.
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19. |
Galimand M,
Courvalin P,
Lambert T,
( 2003 ) Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. PMID : 12878520 : DOI : 10.1128/aac.47.8.2565-2571.2003 PMC : PMC166065 Abstract >>
A self-transferable plasmid of ca. 80 kb, pIP1204, conferred multiple-antibiotic resistance to Klebsiella pneumoniae BM4536, which was isolated from a urinary tract infection. Resistance to beta-lactams was due to the bla(TEM1) and bla(CTX-M) genes, resistance to trimethroprim was due to the dhfrXII gene, resistance to sulfonamides was due to the sul1 gene, resistance to streptomycin-spectinomycin was due to the ant3"9 gene, and resistance to nearly all remaining aminoglycosides was due to the aac3-II gene and a new gene designated armA (aminoglycoside resistance methylase). The cloning of armA into a plasmid in Escherichia coli conferred to the new host high-level resistance to 4,6-disubstituted deoxystreptamines and fortimicin. The deduced sequence of ArmA displayed from 37 to 47% similarity to those of 16S rRNA m(7)G methyltransferases from various actinomycetes, which confer resistance to aminoglycoside-producing strains. However, the low guanine-plus-cytosine content of armA (30%) does not favor an actinomycete origin for the gene. It therefore appears that posttranscriptional modification of 16S rRNA can confer high-level broad-range resistance to aminoglycosides in gram-negative human pathogens.
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20. |
Reinelt S,
Hofmann E,
Gerharz T,
Bott M,
Madden DR,
( 2003 ) The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain. PMID : 12867417 : DOI : 10.1074/jbc.M305864200 Abstract >>
The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central beta-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.
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21. |
Orriss GL,
Erni B,
Schirmer T,
( 2003 ) Crystal structure of the IIB(Sor) domain of the sorbose permease from Klebsiella pneumoniae solved to 1.75A resolution. PMID : 12662934 : DOI : 10.1016/s0022-2836(03)00215-8 Abstract >>
The phosphoenolpyruvate transferase system (PTS) is the major pathway by which bacteria import hexose sugars across the plasma membrane. The PTS transfers a phosphoryl group sequentially via several components from the glycolytic intermediate phosphoenolpyruvate (PEP) to the translocated sugar. It is comprised of the two general proteins enzyme I and HPr, and a sugar-specific enzyme II complex. Sugar translocation is through the membrane domain of the enzyme II complex. The enzyme II complex can belong to one of six families based upon sequence similarity, with the sorbose transporter from Klebsiella pneumoniae a member of the mannose family.The structure of the IIB(Sor) domain was solved to 1.75A resolution by molecular replacement. It has a central core of seven parallel beta-strands surrounded by a total of six alpha-helices. Three helices cover the front face, one the back face with the remaining two capping the central beta-sheet at the top and bottom. The catalytic His15 residue is situated on the surface-exposed loop between strand 1 and helix 1. In addition to the features previously observed in the homologous IIB(Lev) domain from Bacillus subtilis we see new features in the IIB(Sor) structure. First, the catalytic His15 side-chain is fixed in a specific conformation by forming a short hydrogen bond with Asp10, which in turn makes a salt-bridge with Arg8. Second, as observed in other phosphoproteins, an arginine residue (Arg12) is well poised to stabilize a phosphoryl group on His15. Third, we see an Asp/His pair reminiscent of that observed in the IIA(Man) domain from Escherichia coli. Finally, docking of IIA(Man) to IIB(Sor) shows that Arg12 in its current conformation is well positioned to assist the subsequent transfer of the phosphoryl group onto the sugar in line with previous mutagenesis studies.
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22. |
Essa AM,
Julian DJ,
Kidd SP,
Brown NL,
Hobman JL,
( 2003 ) Mercury resistance determinants related to Tn21, Tn1696, and Tn5053 in enterobacteria from the preantibiotic era. PMID : 12604550 : DOI : 10.1128/aac.47.3.1115-1119.2003 PMC : PMC149298 Abstract >>
Three mer transposons from the Murray collection of preantibiotic enterobacteria show >99% sequence identity to current isolates. Tn5073 is most closely related to Tn5036 and Tn1696, and Tn5074 is most closely related to Tn5053. Tn5075 is most closely related to Tn21 but lacks integron In2 and is flanked by insertion elements.
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23. |
Horinouchi N,
Ogawa J,
Sakai T,
Kawano T,
Matsumoto S,
Sasaki M,
Mikami Y,
Shimizu S,
( 2003 ) Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production. PMID : 12839746 : DOI : 10.1128/aem.69.7.3791-3797.2003 PMC : PMC165126 Abstract >>
The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2'-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.
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24. |
Lai YC,
Lin GT,
Yang SL,
Chang HY,
Peng HL,
( 2003 ) Identification and characterization of KvgAS, a two-component system in Klebsiella pneumoniae CG43. PMID : 12583907 : DOI : 10.1111/j.1574-6968.2003.tb11507.x Abstract >>
A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.
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25. |
Smith Moland E,
Hanson ND,
Herrera VL,
Black JA,
Lockhart TJ,
Hossain A,
Johnson JA,
Goering RV,
Thomson KS,
( 2003 ) Plasmid-mediated, carbapenem-hydrolysing beta-lactamase, KPC-2, in Klebsiella pneumoniae isolates. PMID : 12615876 : DOI : 10.1093/jac/dkg124 Abstract >>
Four isolates of Klebsiella pneumoniae obtained from patients at a Maryland medical centre exhibited reduced susceptibility to carbapenems and were found to produce the novel, class A, plasmid-mediated, carbapenem-hydrolysing enzyme, KPC-2. This enzyme has 99% identity with the plasmid-mediated, carbapenem-hydrolysing enzyme KPC-1, reported previously in a North Carolina K. pneumoniae isolate. The KPC-2-producing isolates were either susceptible or intermediate to imipenem and meropenem, unlike the KPC-1-producing isolate, which was resistant to these agents. Detection of KPC-2 may be a problem for clinical laboratories because in this study it was associated with positive extended-spectrum beta-lactamase (ESBL) confirmation tests (clavulanate-potentiated activities of ceftriaxone, ceftazidime, cefepime and aztreonam). Therefore, a failure to recognize the significance of reduced carbapenem susceptibility in the isolates that remained susceptible to imipenem or meropenem could have resulted in the isolates being incorrectly identified as ESBL producers.
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26. |
Izquierdo L,
Merino S,
Regué M,
Rodriguez F,
Tomás JM,
( 2003 ) Synthesis of a Klebsiella pneumoniae O-antigen heteropolysaccharide (O12) requires an ABC 2 transporter. PMID : 12591881 : DOI : 10.1128/jb.185.5.1634-1641.2003 PMC : PMC148082 Abstract >>
A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K. pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5alpha. A total of eight open reading frames (ORFs) (wb(O12) gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb(O12) cluster revealed an interesting coincidence with the wb(O4) cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background.
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27. |
Lobo C,
Sanchez M,
Garbi C,
Ferrer E,
Martinez-Iñigo MJ,
Allende JL,
Martín C,
Casasús L,
Alonso R,
Gibello A,
Martin M,
( 2002 ) Immobilized native bacteria as a tool for bioremediation of soils and waters: implementation and modeling. PMID : 12805921 : DOI : 10.1100/tsw.2002.211 PMC : PMC6009742 Abstract >>
Based on 3,4-dihydroxyphenylacetate (3,4-DHPA) dioxygenase amino acid sequence and DNA sequence data for homologous genes, two different oligonucleotides were designed. These were assayed to detect 3,4-DHPA related aromatic compound-degrading bacteria in soil samples by using the FISH method. Also, amplification by PCR using a set of ERIC primers was assayed for the detection of Pseudomonas GCH1 strain, which used in the soil bioremediation process. A model was developed to understand and predict the behavior of bacteria and pollutants in a bioremediation system, taking into account fluid dynamics, molecular/cellular scale processes, and biofilm formation.
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28. |
Nurizzo D,
Shewry SC,
Perlin MH,
Brown SA,
Dholakia JN,
Fuchs RL,
Deva T,
Baker EN,
Smith CA,
( 2003 ) The crystal structure of aminoglycoside-3'-phosphotransferase-IIa, an enzyme responsible for antibiotic resistance. PMID : 12628253 : DOI : 10.1016/s0022-2836(03)00121-9 Abstract >>
A major factor in the emergence of antibiotic resistance is the existence of enzymes that chemically modify common antibiotics. The genes for these enzymes are commonly carried on mobile genetic elements, facilitating their spread. One such class of enzymes is the aminoglycoside phosphotransferase (APH) family, which uses ATP-mediated phosphate transfer to chemically modify and inactivate aminoglycoside antibiotics such as streptomycin and kanamycin. As part of a program to define the molecular basis for aminoglycoside recognition and inactivation by such enzymes, we have determined the high resolution (2.1A) crystal structure of aminoglycoside-3'-phosphotransferase-IIa (APH(3')-IIa) in complex with kanamycin. The structure was solved by molecular replacement using multiple models derived from the related aminoglycoside-3'-phosphotransferase-III enzyme (APH(3')-III), and refined to an R factor of 0.206 (R(free) 0.238). The bound kanamycin molecule is very well defined and occupies a highly negatively charged cleft formed by the C-terminal domain of the enzyme. Adjacent to this is the binding site for ATP, which can be modeled on the basis of nucleotide complexes of APH(3')-III; only one change is apparent with a loop, residues 28-34, in a position where it could fold over an incoming nucleotide. The three rings of the kanamycin occupy distinct sub-pockets in which a highly acidic loop, residues 151-166, and the C-terminal residues 260-264 play important parts in recognition. The A ring, the site of phosphoryl transfer, is adjacent to the catalytic base Asp190. These results give new information on the basis of aminoglycoside recognition, and on the relationship between this phosphotransferase family and the protein kinases.
|
29. |
Partridge SR,
Hall RM,
( 2003 ) In34, a complex In5 family class 1 integron containing orf513 and dfrA10. PMID : 12499211 : DOI : 10.1128/aac.47.1.342-349.2003 PMC : PMC149023 Abstract >>
A complex class 1 integron, In34, found in a conjugative plasmid from a multidrug-resistant Klebsiella pneumoniae strain isolated in 1997 at a hospital in Sydney, Australia, was shown to have a backbone related to that of In2, which belongs to the In5 family. In In34, the aadB gene cassette replaces the aadA1a cassette in In2, and two additional resistance genes, dfrA10 and aphA1, that are not part of a gene cassette are present. The aphA1 gene is in a Tn4352-like transposon that is located in the tniA gene. The dfrA10 gene lies adjacent to a 2,154-bp DNA segment, known as the common region, that contains an open reading frame predicting a product of 513 amino acids (Orf513). Orf513 is 66 and 55% identical to the products of two further open reading frames that, like the common region, are found adjacent to antibiotic resistance genes. A 27-bp conserved sequence was found at one end of each type of common region. The loss of dfrA10 due to homologous recombination between flanking direct repeats and incorporation of the excised circle by homologous recombination were demonstrated. Part of In34 is identical to the sequenced portion of In7, which is from a multidrug-resistant Escherichia coli strain that had been isolated 19 years earlier in the same hospital. In34 and In7 are in plasmids that contain the same six resistance genes conferring resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, neomycin, tobramycin, trimethoprim, and sulfonamides, but the plasmid backbones appear to be unrelated, suggesting that translocation of a multiple-drug-resistance-determining region as well as horizontal transfer may have occurred.
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30. |
Liao DI,
Reiss L,
Turner I,
Dotson G,
( 2003 ) Structure of glycerol dehydratase reactivase: a new type of molecular chaperone. PMID : 12517345 : Abstract >>
The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B(12) from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 A resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa alpha subunits and two globular 14 kDa beta subunits. The alpha subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the beta subunit resembles that of the beta subunit of glycerol dehydratase, except that it lacks some coenzyme B(12) binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.
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31. |
Harada H,
Ishikawa H,
( 1997 ) Phylogenetical relationship based on groE genes among phenotypically related Enterobacter, Pantoea, Klebsiella, Serratia and Erwinia species. PMID : 12501307 : Abstract >>
In an attempt to define the phylogenetical relationship among 17 phenotypically related species of genera Enterobacter, Pantoea, Serratia, Klebsiella and Erwinia, we determined almost all of their groE operon sequences using the polymerase chain reaction direct sequencing method. The number of nucleotide substitutions per site was 0.12+/-0.030. The value was 3.6-fold higher than that of 16S rDNA. As a result, we were successful in constructing molecular phylogenetic trees which had a finer resolution than that based on the 16S rDNA sequences. The phylogenetic trees based on the nucleotide sequences and deduced amino acid sequences of groE operons indicated that the members of genera Enterobacter, Pantoea and Klebsiella were closely related to each other, while Serratia and Erwinia species except Erwinia carotovora, made distinct clades. The close relationship between Enterobacter aerogenes and Klebsiella pneumoniae, that had been suggested by biochemical tests and DNA hybridization, was also supported by our molecular phylogenetic trees.
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32. |
Liao DI,
Dotson G,
Turner I,
Reiss L,
Emptage M,
( 2003 ) Crystal structure of substrate free form of glycerol dehydratase. PMID : 12538056 : Abstract >>
Glycerol dehydratase (GDH) and diol dehydratase (DDH) are highly homologous isofunctional enzymes that catalyze the elimination of water from glycerol and 1,2-propanediol (1,2-PD) to the corresponding aldehyde via a coenzyme B(12)-dependent radical mechanism. The crystal structure of substrate free form of GDH in complex with cobalamin and K(+) has been determined at 2.5 A resolution. Its overall fold and the subunit assembly closely resemble those of DDH. Comparison of this structure and the DDH structure, available only in substrate bound form, shows the expected change of the coordination of the essential K(+) from hexacoordinate to heptacoordinate with the displacement of a single coordinated water by the substrate diol. In addition, there appears to be an increase in the rigidity of the K(+) coordination (as measured by lower B values) upon the binding of the substrate. Structural analysis of the locations of conserved residues among various GDH and DDH sequences has aided in identification of residues potentially important for substrate preference or specificity of protein-protein interactions.
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33. |
Brenwald NP,
Jevons G,
Andrews JM,
Xiong JH,
Hawkey PM,
Wise R,
( 2003 ) An outbreak of a CTX-M-type beta-lactamase-producing Klebsiella pneumoniae: the importance of using cefpodoxime to detect extended-spectrum beta-lactamases. PMID : 12493817 : DOI : 10.1093/jac/dkg051 Abstract >>
N/A
|
34. |
Wang H,
Kelkar S,
Wu W,
Chen M,
Quinn JP,
( 2003 ) Clinical isolates of Enterobacteriaceae producing extended-spectrum beta-lactamases: prevalence of CTX-M-3 at a hospital in China. PMID : 12543694 : DOI : 10.1128/aac.47.2.790-793.2003 PMC : PMC151729 Abstract >>
The prevalence of extended-spectrum beta-lactamase-producing strains was demonstrated in 5 of 44 (11.4%) Escherichia coli, 17 of 43 (39.5%) Klebsiella pneumoniae, 3 of 50 (6.0%) Enterobacter cloacae, and 2 of 25 (8.0%) Citrobacter freundii strains at a teaching hospital in China. Nineteen of these 27 strains expressed CTX-M-3 beta-lactamase (pI 8.6). A subset of the clinical isolates expressing the CTX-M-3 enzyme, tested by pulsed-field gel electrophoresis, revealed multiple clones. Five isolates expressed a novel enzyme, SHV-43 (pI 8.0), which had two substitutions (Leu113Phe and Thr149Ser) compared with SHV-1.
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35. |
Poirel L,
Héritier C,
Podglajen I,
Sougakoff W,
Gutmann L,
Nordmann P,
( 2003 ) Emergence in Klebsiella pneumoniae of a chromosome-encoded SHV beta-lactamase that compromises the efficacy of imipenem. PMID : 12543688 : DOI : 10.1128/aac.47.2.755-758.2003 PMC : PMC151740 Abstract >>
A Klebsiella pneumoniae isolate was identified that had reduced susceptibility to several expanded-spectrum cephalosporins and imipenem. That isolate produced a chromosome-encoded SHV-type beta-lactamase, SHV-38, that had an alanine to valine substitution in position Ambler 146 compared to beta-lactamase SHV-1. The kinetic parameters for purified beta-lactamases SHV-38 and SHV-1 showed that the hydrolytic spectrum of SHV-38 included only ceftazidime and imipenem. This report is the first example of an SHV-type beta-lactamase capable of hydrolyzing imipenem.
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36. |
Decré D,
Verdet C,
Raskine L,
Blanchard H,
Burghoffer B,
Philippon A,
Sanson-Le-Pors MJ,
Petit JC,
Arlet G,
( 2002 ) Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area. PMID : 12407124 : DOI : 10.1093/jac/dkf193 Abstract >>
We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.
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37. |
Marquordt C,
Fang Q,
Will E,
Peng J,
von Figura K,
Dierks T,
( 2003 ) Posttranslational modification of serine to formylglycine in bacterial sulfatases. Recognition of the modification motif by the iron-sulfur protein AtsB. PMID : 12419807 : DOI : 10.1074/jbc.M209435200 Abstract >>
Calpha-formylglycine is the catalytic residue of sulfatases. Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. The modifying enzymes are unknown. AtsB, an iron-sulfur protein, is strictly required for modification of Ser(72) in the periplasmic sulfatase AtsA of Klebsiella pneumoniae. Here we show (i) that AtsB is a cytosolic protein acting on newly synthesized serine-type sulfatases, (ii) that AtsB-mediated FGly formation is dependent on AtsA's signal peptide, and (iii) that the cytosolic cysteine-type sulfatase of Pseudomonas aeruginosa can be converted into a substrate of AtsB if the cysteine is substituted by serine and a signal peptide is added. Thus, formylglycine formation in serine-type sulfatases depends both on AtsB and on the presence of a signal peptide, and AtsB can act on sulfatases of other species. AtsB physically interacts with AtsA in a Ser(72)-dependent manner, as shown in yeast two-hybrid and GST pull-down experiments. This strongly suggests that AtsB is the serine-modifying enzyme and that AtsB relies on a cytosolic function of the sulfatase's signal peptide.
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38. |
Leverstein-van Hall MA,
Fluit AC,
Paauw A,
Box AT,
Brisse S,
Verhoef J,
( 2002 ) Evaluation of the Etest ESBL and the BD Phoenix, VITEK 1, and VITEK 2 automated instruments for detection of extended-spectrum beta-lactamases in multiresistant Escherichia coli and Klebsiella spp. PMID : 12354869 : DOI : 10.1128/jcm.40.10.3703-3711.2002 PMC : PMC130880 Abstract >>
Seventy-four isolates of multiresistant Escherichia coli and Klebsiella spp. recovered during a 3-year period and 17 control strains with genotypically identified beta-lactamases were tested for the production of extended-spectrum beta-lactamases (ESBLs) by using the Etest and the VITEK 1, VITEK 2, and Phoenix automated instruments. The use of the Etest was evaluated by investigating its accuracy in detecting the ESBLs of the control strains and by comparing interpretation results of laboratory technicians and experts. The accuracy of the Etest was 94%. With the Etest as the reference for the clinical strains and the genotype as the reference for the control strains, the automated instruments detected the ESBLs with accuracies of 78% (VITEK 2), 83% (VITEK 1), and 89% (Phoenix). No significant difference between the systems with regard to the control strains was detected. The VITEK 2 did, however, perform less well than the Phoenix (P = 0.03) on the collection of clinical isolates, mainly because of its high percentage of indeterminate test results (11%). No significant difference between the performances of the VITEK 1 and either the VITEK 2 or the Phoenix was found. However, because of its associated BDXpert system the Phoenix showed the best performance. The Etest was found to be an accurate test but was limited by its indeterminate results (4%), its inability to differentiate between K1 hyperproduction and ESBLs, questionable guidelines concerning mutants inside the inhibition zones, and the inability of the technicians to recognize subtle zone deformations.
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39. |
Huston WM,
Jennings MP,
McEwan AG,
( 2002 ) The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition. PMID : 12354238 : DOI : 10.1046/j.1365-2958.2002.03132.x Abstract >>
Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.
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40. |
Crowley B,
Benedí VJ,
Doménech-Sánchez A,
( 2002 ) Expression of SHV-2 beta-lactamase and of reduced amounts of OmpK36 porin in Klebsiella pneumoniae results in increased resistance to cephalosporins and carbapenems. PMID : 12384391 : DOI : 10.1128/aac.46.11.3679-3682.2002 PMC : PMC128712 Abstract >>
A Klebsiella pneumoniae clinical isolate was resistant to cefoxitin, cefotaxime, ceftazidime, ceftazidime-clavulanate, piperacillin-tazobactam (MICs, >256 micro g/ml in all cases), and meropenem (MIC, 16 micro g/ml) and was intermediate to imipenem (MIC, 8 micro g/ml). Decreased expression of the OmpK36 porin and expression of an SHV-2 beta-lactamase contributed to the observed resistance to these beta-lactam-containing agents.
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41. |
Yamanishi M,
Yunoki M,
Tobimatsu T,
Sato H,
Matsui J,
Dokiya A,
Iuchi Y,
Oe K,
Suto K,
Shibata N,
Morimoto Y,
Yasuoka N,
Toraya T,
( 2002 ) The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol. PMID : 12230560 : DOI : 10.1046/j.1432-1033.2002.03151.x Abstract >>
Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.
|
42. |
Sarno R,
McGillivary G,
Sherratt DJ,
Actis LA,
Tolmasky ME,
( 2002 ) Complete nucleotide sequence of Klebsiella pneumoniae multiresistance plasmid pJHCMW1. PMID : 12384346 : DOI : 10.1128/aac.46.11.3422-3427.2002 PMC : PMC128720 Abstract >>
The multiresistance plasmid pJHCMW1, harbored by a clinical Klebsiella pneumoniae strain isolated from a neonate with meningitis, was sequenced. A circular sequence of 11,354 bp was generated, of which 7,993 bp make up Tn1331, a transposon including the antibiotic resistance genes aac(6')-Ib, aadA1, bla(OXA-9), and bla(TEM-1). The gene aac(6')-Ib is included in a gene cassette, and both aadA1 and bla(OXA-9) are included in a single-gene cassette that may have arisen as a consequence of a recombination event involving two integrons. The pJHCMW1 plasmid replicates through a ColE1-like RNA-regulated mechanism, includes a functional oriT, and two loci with similarity to XerCD site-specific recombination target sites involved in plasmid stabilization by the resolution of multimers. One of these two loci, mwr, is active and has been the subject of previous studies, and the other, dxs, is not functional but binds the recombinase XerD with low affinity. Two additional open reading frames were identified, one with low similarity to two hypothetical membrane proteins from Mycobacterium tuberculosis and Mycobacterium leprae and the other with low similarity to psiB, a gene encoding a function that facilitates the establishment of the transferring plasmid in the recipient bacterial cell during the process of conjugation.
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43. |
Mayer SM,
Gormal CA,
Smith BE,
Lawson DM,
( 2002 ) Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco). PMID : 12133839 : DOI : 10.1074/jbc.M205888200 Abstract >>
The x-ray crystal structure of NifV(-) Klebsiella pneumoniae nitrogenase MoFe protein (NifV(-) Kp1) has been determined and refined to a resolution of 1.9 A. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.
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44. |
Yin-Ching C,
Jer-Horng S,
Ching-Nan L,
Ming-Chung C,
( 2002 ) Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe. PMID : 12127794 : Abstract >>
A gene, designated khe, that encodes a haemolysin of Klebsiella pneumoniae CMC-1 has been cloned and sequenced. When expressed in Escherichia coli, a unique peptide of approximately 20kDa was identified. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 486bp encoding a 162 amino acid polypeptide with an estimated pI of 6.77. No extensive sequence homology could be identified between khe and any reported sequence at either the nucleotide or amino acid level. Furthermore, DNA hybridizations under high stringency conditions failed to show any cross hybridizations to several bacteria including K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. These data indicate that we have cloned a unique gene, which is highly conserved among tested K. pneumoniae isolates.
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45. |
Thompson J,
Lichtenthaler FW,
Peters S,
Pikis A,
( 2002 ) Beta-glucoside kinase (BglK) from Klebsiella pneumoniae. Purification, properties, and preparative synthesis of 6-phospho-beta-D-glucosides. PMID : 12110692 : DOI : 10.1074/jbc.M206397200 Abstract >>
ATP-dependent beta-glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC) exists as a homotetramer composed of non-covalently linked subunits of M(r) approximately 33,000. Determination of the first 28 residues from the N terminus of the protein allowed the identification and cloning of bglK from genomic DNA of K. pneumoniae. The open reading frame (ORF) of bglK encodes a 297-residue polypeptide of calculated M(r) 32,697. A motif of 7 amino acids (AFD(7)IG(9)GT) near the N terminus may comprise the ATP-binding site, and residue changes D7G and G9A yielded catalytically inactive proteins. BglK was progressively inactivated (t(12) approximately 19 min) by N-ethylmaleimide, but ATP afforded considerable protection against the inhibitor. By the presence of a centrally located signature sequence, BglK can be assigned to the ROK (Repressor, ORF, Kinase) family of proteins. Preparation of (His6)BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200-500 mg) of ten 6-phospho-beta-d-glucosides, including cellobiose-6'-P, gentiobiose-6'-P, cellobiitol-6-P, salicin-6-P, and arbutin-6-P. These (and other) derivatives are substrates for phospho-beta-glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho-beta-d-glucosides have been determined by fast atom bombardment-negative ion spectrometry, thin-layer chromatography, and (1)H and (13)C NMR spectroscopy. The recently sequenced genomes of two Listeria species, L. monocytogenes EGD-e and L. innocua CLIP 11262, contain homologous genes (lmo2764 and lin2907, respectively) that encode a 294-residue polypeptide (M(r) approximately 32,200) that exhibits approximately 58% amino acid identity with BglK. The protein encoded by the two genes exhibits beta-glucoside kinase activity and cross-reacts with polyclonal antibody to (His6)BglK from K. pneumoniae. The location of lmo2764 and lin2907 within a beta-glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog in Listeria species.
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46. |
Fukushima M,
Kakinuma K,
Kawaguchi R,
( 2002 ) Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence. PMID : 12149329 : DOI : 10.1128/jcm.40.8.2779-2785.2002 PMC : PMC120687 Abstract >>
Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.
|
47. |
Kim J,
Shin HS,
Seol SY,
Cho DT,
( 2002 ) Relationship between blaSHV-12 and blaSHV-2a in Korea. PMID : 11815566 : DOI : 10.1093/jac/49.2.261 Abstract >>
In contrast to the USA and Europe, where SHV-2, SHV-4 and SHV-5 are the prevalent extended-spectrum SHV enzymes, in Korea SHV-2a and SHV-12 are the most frequently identified extended-spectrum SHV enzymes. A 6.6 kb BamHI fragment containing the bla(SHV-12) gene of strain K7746 isolated from one university hospital in Korea was cloned into the pCRScriptCAM vector. Sequencing of the constructed recombinant plasmid pK7746-C1 revealed that the immediate upstream sequence of the bla(SHV-12) gene showed little similarity to the part of the prototype bla(SHV-1) gene due to the insertion of an IS26 element next to the -10 region. Instead, the upstream sequences of bla(SHV-12) retained 100% DNA identity with the part of plasmid pMPA2a from Klebsiella pneumoniae KPZU-3 carrying bla(SHV-2a). The restriction map of the inserted 6.6 kb DNA fragment of plasmid pK7746-C1 was also homologous to that of plasmid pMPA2a, suggesting a common lineage of bla(SHV-12) and bla(SHV-2a). We also studied, using PCR, the upstream non-coding region of several SHV beta-lactamase genes for the presence of IS26 sequence. The flanking IS26 sequence in the immediate upstream region of the bla(SHV) gene was not detected in five standard strains producing SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. However, IS26 was detected in all 69 clinical strains producing SHV-2a or SHV-12 isolated from three university hospitals in Korea during 1993-1999. The above findings suggest a direct evolution of SHV-12 from SHV-2a, not from SHV-2 to -5, and it is considered to be one of the reasons for the absolute predominance of SHV-2a and SHV-12 in Korea.
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48. |
Grabbe R,
Kuhn A,
Schmitz RA,
( 2001 ) Cloning, sequencing and characterization of Fnr from Klebsiella pneumoniae. PMID : 11816975 : DOI : 10.1023/a:1012060730647 Abstract >>
The transcription factor Fnr (fumarate nitrate reductase regulator) globally regulates gene expression in response to oxygen deprivation in Escherichia coli. We report here the cloning and sequencing of the fnr gene from the facultative anaerobic bacterium Klebsiella pneumoniae M5al, another member of the enteric bacteria. The deduced amino acid sequence of K. pneumoniae fnr showed very high similarity (98% amino acid identity) to the Fnr protein from E. coli and contained the four essential cysteine residues which are presumed to build the oxygen-sensing [4Fe4S]+2 center. Transfer of the K. pneumoniae gene to a fnr mutant of E. coli complemented the mutation and permitted synthesis of nitrate reductase and fumarate reductase during anaerobic growth. A gene fusion between K. pneumoniae fnr and glutathione S-transferase was constructed and expressed in E. coli under anaerobic conditions in order to make the protein available in preparative amounts. The overproduced protein was purified by glutathione-Sepharose 4B affinity chromatography in the absence of oxygen, and biochemically characterized.
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49. |
Fang CT,
Chen HC,
Chuang YP,
Chang SC,
Wang JT,
( 2002 ) Cloning of a cation efflux pump gene associated with chlorhexidine resistance in Klebsiella pneumoniae. PMID : 12019132 : DOI : 10.1128/aac.46.6.2024-2028.2002 PMC : PMC127239 Abstract >>
Expression libraries of a chlorhexidine-resistant Klebsiella pneumoniae strain were constructed and transformed into Escherichia coli XLOLR. Twenty chlorhexidine-resistant transformants were obtained after selection. All clones contained a novel 903-nucleotide locus. Its sequences were compatible with a cation efflux pump, and the locus was thus designated as cepA. Retransformation using cepA-containing plasmids conferred chlorhexidine resistance to both XLOLR and a chlorhexidine-sensitive K. pneumoniae strain. Therefore, CepA is associated with chlorhexidine resistance and may act as a cation efflux pump.
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50. |
Rasheed JK,
Anderson GJ,
Queenan AM,
Biddle JW,
Oliver A,
Jacoby GA,
Bush K,
Tenover FC,
( 2002 ) TEM-71, a novel plasmid-encoded, extended-spectrum beta-lactamase produced by a clinical isolate of Klebsiella pneumoniae. PMID : 12019125 : DOI : 10.1128/aac.46.6.2000-2003.2002 PMC : PMC127224 Abstract >>
TEM-71, a novel extended-spectrum beta-lactamase from a Klebsiella pneumoniae clinical isolate, had an isoelectric point of 6.0 and a substrate profile showing preferential hydrolysis of cefotaxime over ceftazidime. It differed from TEM-1 by two substitutions, Gly238Ser and Glu240Lys, and was under the control of the strong P4 promoter.
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51. |
Pons AM,
Zorn N,
Vignon D,
Delalande F,
Van Dorsselaer A,
Cottenceau G,
( 2002 ) Microcin E492 is an unmodified peptide related in structure to colicin V. PMID : 11751140 : DOI : 10.1128/aac.46.1.229-230.2002 PMC : PMC126999 Abstract >>
The pore-forming microcin E492 was purified by solid-phase extraction and reversed-phase high-pressure liquid chromatography. Its molecular mass was 7,886 Da. The entire 84-amino-acid sequence was determined. There is no postranslational modification in the secreted microcin, and the sequence has homologies with the sequence of the microcin colicin V.
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52. |
Vinogradov E,
Frirdich E,
MacLean LL,
Perry MB,
Petersen BO,
Duus J?,
Whitfield C,
( 2002 ) Structures of lipopolysaccharides from Klebsiella pneumoniae. Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains. PMID : 11986326 : DOI : 10.1074/jbc.M202683200 Abstract >>
Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.
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53. |
Dauga C,
( 2002 ) Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. PMID : 11931166 : DOI : 10.1099/00207713-52-2-531 Abstract >>
Phylogenetic trees showing the evolutionary relatedness of Enterobacteriaceae based upon gyrB and 16S rRNA genes were compared. Congruence among trees of these molecules indicates that the genomes of these species are not completely mosaic and that molecular systematic studies can be carried out. Phylogenetic trees based on gyrB sequences appeared to be more reliable at determining relationships among Serratia species than trees based on 16S rRNA gene sequences. gyrB sequences from Serratia species formed a monophyletic group validated by significant bootstrap values. Serratia fonticola had the most deeply branching gyrB sequence in the Serratia monophyletic group, which was consistent with its atypical phenotypic characteristics. Klebsiella and Enterobacter genera seemed to be polyphyletic, but the branching patterns of gyrB and 16S rRNA gene trees were not congruent. Enterobacter aerogenes was grouped with Klebsiella pneumoniae on the gyrB phylogenetic tree, which supports that this species could be transferred to the Klebsiella genus. Unfortunately, 16S rRNA and gyrB phylogenetic trees gave conflicting evolutionary relationships for Citrobacter freundii because of its unusual gyrB evolutionary process. gyrB lateral gene transfer was suspected for Hafnia alvei. Saturation of gyrB genes was observed by the pairwise comparison of Proteus spp., Providencia alcalifaciens and Morganella morganii sequences. Depending on their level of variability, 16S rRNA gene sequences were useful for describing phylogenetic relationships between distantly related Enterobacteriaceae, whereas gyrB sequence comparison was useful for inferring intra- and some intergeneric relationships.
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54. |
Cao V,
Lambert T,
Courvalin P,
( 2002 ) ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum beta-lactamase CTX-M-17. PMID : 11959547 : DOI : 10.1128/aac.46.5.1212-1217.2002 PMC : PMC127148 Abstract >>
The resistance of Klebsiella pneumoniae BM4493, isolated in Ho Chi Minh City, Vietnam, to cefotaxime and aztreonam was due to production of a novel beta-lactamase, CTX-M-17. The bla(CTX-M-17) gene was borne by 7,086-bp plasmid pIP843, which was entirely sequenced and which was found to belong to the ColE1 family. The 876-bp bla(CTX-M-17) gene differed from bla(CTX-M-14) by 2 nucleotides, which led to the single amino acid substitution Glu289-->Lys. bla(CTX-M-17) was flanked upstream by an ISEcp1-like element and downstream by an insertion sequence (IS) IS903 variant designated IS903-C. The transcriptional start site of bla(CTX-M-17) was located 109 nucleotides upstream from the initiation codon in the ISEcp1-like element, which also provided the promoter sequences. Plasmid pIP843, which was non-self-transferable and nonmobilizable, contained five open reading frames transcribed in the same orientation. Regions homologous to sequences coding for putative RNA II and RNA I transcripts, a rom gene, which is involved in initiation of replication, and a cer-like gene, which is responsible for the stability of ColE1-like plasmids, were identified. Consensus sequences for putative replication (oriV) and transfer (oriT) origins were present. Results of primer extension experiments indicated that ISEcp1 provides the promoter for expression of bla(CTX-M-17) and may contribute to dissemination of this gene.
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55. |
Pai H,
Lee HJ,
Choi EH,
Kim J,
Jacoby GA,
( 2001 ) Evolution of TEM-related extended-spectrum beta-lactamases in Korea. PMID : 11709362 : DOI : 10.1128/AAC.45.12.3651-3653.2001 PMC : PMC90891 Abstract >>
TEM-52, differing from TEM-1 by having the substitutions Glu-104-->Lys, Met-182-->Thr, and Gly-238-->Ser, has previously been described as the most prevalent extended-spectrum beta-lactamase (ESBL) in Korea. In a further survey, we discovered the ESBLs TEM-15, which is like TEM-52 but lacks the substitution at residue 182, and TEM-88, which is like TEM-52 with an additional Gly-196-->Asp substitution. TEM-88 retained the activity of TEM-52 against moxalactam. Otherwise, the kinetic properties of the three ESBLs failed to show an advantage to this evolution.
|
56. |
Schneider K,
Kästner CN,
Meyer M,
Wessel M,
Dimroth P,
Bott M,
( 2002 ) Identification of a gene cluster in Klebsiella pneumoniae which includes citX, a gene required for biosynthesis of the citrate lyase prosthetic group. PMID : 11948157 : DOI : 10.1128/jb.184.9.2439-2446.2002 PMC : PMC134981 Abstract >>
The biosynthesis of the 2'-(5"-phosphoribosyl)-3'-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >or=40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.
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57. |
Poirel L,
Naas T,
Le Thomas I,
Karim A,
Bingen E,
Nordmann P,
( 2001 ) CTX-M-type extended-spectrum beta-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. PMID : 11709308 : DOI : 10.1128/AAC.45.12.3355-3361.2001 PMC : PMC90837 Abstract >>
Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniae ILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 beta-lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 beta-lactamase. The amino acid sequence of the CTX-M-18 beta-lactamase differed from that of the CTX-M-9 beta-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. The bla(CTX-M-18) and bla(CTX-M-19) genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.
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58. |
Shmara A,
Weinsetel N,
Dery KJ,
Chavideh R,
Tolmasky ME,
( 2001 ) Systematic analysis of a conserved region of the aminoglycoside 6'-N-acetyltransferase type Ib. PMID : 11709299 : DOI : 10.1128/AAC.45.12.3287-3292.2001 PMC : PMC90828 Abstract >>
Alanine-scanning mutagenesis was applied to the aminoglycoside 6'-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42 degrees C (D. Panaite and M. Tolmasky, Plasmid 39:123-133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.
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59. |
Tran JH,
Jacoby GA,
( 2002 ) Mechanism of plasmid-mediated quinolone resistance. PMID : 11943863 : DOI : 10.1073/pnas.082092899 PMC : PMC122823 Abstract >>
Quinolones are potent antibacterial agents that specifically target bacterial DNA gyrase and topoisomerase IV. Widespread use of these agents has contributed to the rise of bacterial quinolone resistance. Previous studies have shown that quinolone resistance arises by mutations in chromosomal genes. Recently, a multiresistance plasmid was discovered that encodes transferable resistance to quinolones. We have cloned the plasmid-quinolone resistance gene, termed qnr, and found it in an integron-like environment upstream from qacE Delta 1 and sulI. The gene product Qnr was a 218-aa protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. Qnr had pentapeptide repeat domains of 11 and 28 tandem copies, separated by a single glycine with a consensus sequence of A/C D/N L/F X X. Because the primary target of quinolones is DNA gyrase in Gram-negative strains, we tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Purified Qnr-His(6) protected Escherichia coli DNA gyrase from inhibition by ciprofloxacin. Gyrase protection was proportional to the concentration of Qnr-His(6) and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His(6) was lost by boiling the protein and involved neither quinolone inactivation nor independent gyrase activity. Protection of topoisomerase IV, a secondary target of quinolone action in E. coli, was not evident. How Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.
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60. |
Lagos R,
Baeza M,
Corsini G,
Hetz C,
Strahsburger E,
Castillo JA,
Vergara C,
Monasterio O,
( 2001 ) Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin. PMID : 11679081 : DOI : 10.1046/j.1365-2958.2001.02630.x Abstract >>
Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.
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61. |
Allen AE,
Booth MG,
Frischer ME,
Verity PG,
Zehr JP,
Zani S,
( 2001 ) Diversity and detection of nitrate assimilation genes in marine bacteria. PMID : 11679368 : DOI : 10.1128/AEM.67.11.5343-5348.2001 PMC : PMC93313 Abstract >>
A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.
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62. |
Lai YC,
Peng HL,
Chang HY,
( 2001 ) Identification of genes induced in vivo during Klebsiella pneumoniae CG43 infection. PMID : 11598090 : DOI : 10.1128/IAI.69.11.7140-7145.2001 PMC : PMC100105 Abstract >>
A novel in vivo expression technology (IVET) was performed to identify Klebsiella pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a functional galU gene as the reporter, an IVE promoter could render the galU mutant virulent while maintaining the white nonmucoid colony phenotype. A total of 20 distinct sequences were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn on under iron deprivation, whereas the expression of another five genes was found to be activated in the presence of paraquat, a superoxide generator.
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63. |
Wardwell SA,
Yang YT,
Chang HY,
San KY,
Rudolph FB,
Bennett GN,
( 2001 ) Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824. PMID : 11687934 : DOI : 10.1038/sj/jim/7000179 Abstract >>
Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added.
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64. |
Arlet G,
Nadjar D,
Herrmann JL,
Donay JL,
Lagrange PH,
Philippon A,
( 2001 ) Plasmid-mediated rifampin resistance encoded by an arr-2-like gene cassette in Klebsiella pneumoniae producing an ACC-1 class C beta-lactamase. PMID : 11583008 : DOI : 10.1128/aac.45.10.2971-2972.2001 PMC : PMC90768 Abstract >>
N/A
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65. |
Peters ED,
Leverstein-van Hall MA,
Box AT,
Verhoef J,
Fluit AC,
( 2001 ) Novel gene cassettes and integrons. PMID : 11557503 : DOI : 10.1128/AAC.45.10.2961-2964.2001 PMC : PMC90765 Abstract >>
An increase in multiresistant Enterobacteriaceae was observed at one of the departments of the University Medical Center Utrecht. Nine different integrons and 17 gene cassettes were found, including the new gene cassette aadA8. This cassette was highly related to aadA3 and aadA2. In addition, an unknown promoter sequence was found for two integrons.
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66. |
Chaves J,
Ladona MG,
Segura C,
Coira A,
Reig R,
Ampurdanés C,
( 2001 ) SHV-1 beta-lactamase is mainly a chromosomally encoded species-specific enzyme in Klebsiella pneumoniae. PMID : 11557480 : DOI : 10.1128/AAC.45.10.2856-2861.2001 PMC : PMC90742 Abstract >>
The nature of the SHV-1 beta-lactamase gene was analyzed in 97 epidemiologically unrelated Klebsiella pneumoniae strains isolated from clinical samples. beta-Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of beta-lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples with K. pneumoniae TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with EcoRI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 beta-lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 beta-lactamase originated from the K. pneumoniae chromosome.
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67. |
Reisbig MD,
Hanson ND,
( 2002 ) The ACT-1 plasmid-encoded AmpC beta-lactamase is inducible: detection in a complex beta-lactamase background. PMID : 11864960 : DOI : 10.1093/jac/49.3.557 Abstract >>
The purpose of this study was to identify the genetic organization and inducibility of bla(ACT-1) in a clinical isolate of Klebsiella pneumoniae possessing at least five different beta-lactamases. The genetic organization of the bla(ACT-1)/ampR region is identical to those of inducible chromosomal ampC genes. RNA analysis using primer extension demonstrated a five-fold increase in bla(ACT-1) transcript production on exposure to cefoxitin. These findings are significant because induction was detected in a complicated beta-lactamase background. In addition, this report is the first to describe an inducible plasmid-encoded AmpC beta-lactamase of Enterobacter cloacae origin.
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68. |
Chang FY,
Siu LK,
Fung CP,
Huang MH,
Ho M,
( 2001 ) Diversity of SHV and TEM beta-lactamases in Klebsiella pneumoniae: gene evolution in Northern Taiwan and two novel beta-lactamases, SHV-25 and SHV-26. PMID : 11502506 : DOI : 10.1128/aac.45.9.2407-2413.2001 PMC : PMC90669 Abstract >>
A total of 113 blood culture isolates of Klebsiella pneumoniae from 10 hospitals in northern Taiwan were studied for SHV and TEM beta-lactamase production. bla(SHV) was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of bla(SHV-1) were found at positions T18A (ThrACC-->AlaGCC), L35Q (LeuCTA-->GluCAA), and M129V (MetATG-->ValGTG) for SHV-25 and at position A187T (AlaGCC-->ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillin-clavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV beta-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum beta-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the beta-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV beta-lactamases in Taiwan.
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69. |
Silva J,
Gatica R,
Aguilar C,
Becerra Z,
Garza-Ramos U,
Velázquez M,
Miranda G,
Leaños B,
Solórzano F,
Echániz G,
( 2001 ) Outbreak of infection with extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Mexican hospital. PMID : 11526149 : DOI : 10.1128/jcm.39.9.3193-3196.2001 PMC : PMC88317 Abstract >>
Thirty-one strains of Klebsiella pneumoniae (including 10 duplicates) from 21 septicemic pediatric patients (age, <2 months) were studied during a 4-month period (June to October 1996) in which the fatality rate was 62% (13 of 21). These isolates identified by the API 20E system yielded the same biotype. Pulsed-field gel electrophoresis experiments revealed the same clone in 31 strains. The isolates were multidrug-resistant but were still susceptible to ciprofloxacin, imipenem, and cefoxitin. A 135-kb plasmid was harbored in all of the isolates. No transconjugants were obtained that were resistant to ampicillin, cefotaxime, tetracycline, or gentamicin. Isoelectric focusing for beta-lactamases was performed on all strains, and three bands with pIs of 5.4, 7.6, and 8.2 were obtained. Of these, the pI 8.2 beta-lactamase had an extended-spectrum beta-lactamase phenotype. PCR amplification of both TEM- and SHV-type genes was obtained. The sequence analysis of the SHV PCR product indicated a mutation corresponding to the SHV-5 beta-lactamase.
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70. |
Thompson J,
Robrish SA,
Immel S,
Lichtenthaler FW,
Hall BG,
Pikis A,
( 2001 ) Metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. Participation and properties of sucrose-6-phosphate hydrolase and phospho-alpha-glucosidase. PMID : 11473129 : DOI : 10.1074/jbc.M106504200 Abstract >>
Klebsiella pneumoniae is presently unique among bacterial species in its ability to metabolize not only sucrose but also its five linkage-isomeric alpha-d-glucosyl-d-fructoses: trehalulose, turanose, maltulose, leucrose, and palatinose. Growth on the isomeric compounds induced a protein of molecular mass approximately 50 kDa that was not present in sucrose-grown cells and which we have identified as an NAD(+) and metal ion-dependent 6-phospho-alpha-glucosidase (AglB). The aglB gene has been cloned and sequenced, and AglB (M(r) = 49,256) has been purified from a high expression system using the chromogenic p-nitrophenyl alpha-glucopyranoside 6-phosphate as substrate. Phospho-alpha-glucosidase catalyzed the hydrolysis of a wide variety of 6-phospho-alpha-glucosides including maltose-6'-phosphate, maltitol-6-phosphate, isomaltose-6'-phosphate, and all five 6'-phosphorylated isomers of sucrose (K(m) approximately 1-5 mm) yet did not hydrolyze sucrose-6-phosphate. By contrast, purified sucrose-6-phosphate hydrolase (M(r) approximately 53,000) hydrolyzed only sucrose-6-phosphate (K(m) approximately 80 microm). Differences in molecular shape and lipophilicity potential between sucrose and its isomers may be important determinants for substrate discrimination by the two phosphoglucosyl hydrolases. Phospho-alpha-glucosidase and sucrose-6-phosphate hydrolase exhibit no significant homology, and by sequence-based alignment, the two enzymes are assigned to Families 4 and 32, respectively, of the glycosyl hydrolase superfamily. The phospho-alpha-glucosidase gene (aglB) lies adjacent to a second gene (aglA), which encodes an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system. We suggest that the products of the two genes facilitate the phosphorylative translocation and subsequent hydrolysis of the five alpha-d-glucosyl-d-fructoses by K. pneumoniae.
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71. |
Noah C,
Brabetz W,
Gronow S,
Brade H,
( 2001 ) Cloning, sequencing, and functional analysis of three glycosyltransferases involved in the biosynthesis of the inner core region of Klebsiella pneumoniae lipopolysaccharide. PMID : 11521078 : Abstract >>
The genes encoding the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (waaA) and heptosyltransferases I (waaC) and II (waaF) in Klebsiella pneumoniae were cloned from a DNA library by functional complementation of corresponding Escherichia coli and Salmonella enterica mutants. Sequence analyses revealed extensive homologies of the deduced proteins to their counterparts in other Enterobacteriaceae. However, differences were evident with regard to the chromosomal organization of the genes. To perform in vitro studies, the waaA, waaC and waaF genes were subcloned and expressed in the Gram-positive host Corynebacterium glutamicum. WaaA was characterized as a bifunctional enzyme capable of transferring two Kdo residues to a synthetic bisphosphorylated tetraacyl-lipid A precursor of E. coli (compound 406). In contrast, waaC and waaF were shown to encode specific glycosyltransferases catalyzing the consecutive transfer of two L-glycero-D-manno-heptose residues to Kdo(2)-406.
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72. |
Arpin C,
Labia R,
Andre C,
Frigo C,
El Harrif Z,
Quentin C,
( 2001 ) SHV-16, a beta-lactamase with a pentapeptide duplication in the omega loop. PMID : 11502518 : DOI : 10.1128/aac.45.9.2480-2485.2001 PMC : PMC90681 Abstract >>
A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 microg/ml), ticarcillin (MIC of 512 microg/ml), and ceftazidime (MIC of 128 microg/ml) and susceptible to all other beta-lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum beta-lactamase (ESBL). Transconjugants in Escherichia coli were obtained at low levels (10(-7) per donor cell) and exhibited a similar beta-lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 microg/ml). The ESBL, pI 7.6, was encoded by a large plasmid (>100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR using bla(SHV)-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla(SHV-1) or bla(SHV-16) in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.
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73. |
Wilde C,
Bachellier S,
Hofnung M,
Clément JM,
( 2001 ) Transposition of IS1397 in the family Enterobacteriaceae and first characterization of ISKpn1, a new insertion sequence associated with Klebsiella pneumoniae palindromic units. PMID : 11443073 : DOI : 10.1128/JB.183.15.4395-4404.2001 PMC : PMC95333 Abstract >>
IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.
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74. |
Cloeckaert A,
Baucheron S,
Chaslus-Dancla E,
( 2001 ) Nonenzymatic chloramphenicol resistance mediated by IncC plasmid R55 is encoded by a floR gene variant. PMID : 11451703 : DOI : 10.1128/AAC.45.8.2381-2382.2001 PMC : PMC90660 Abstract >>
The IncC plasmid R55, initially described in the 1970s and isolated from Klebsiella pneumoniae, confers nonenzymatic chloramphenicol resistance. The gene coding for this resistance was cloned and sequenced and shows 95 to 97% nucleotide identity with the recently reported floR gene from Salmonella enterica serovar Typhimurium DT104 and from Escherichia coli animal isolates, respectively, conferring cross-resistance to florfenicol.
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75. |
Yan JJ,
Ko WC,
Wu JJ,
( 2001 ) Identification of a plasmid encoding SHV-12, TEM-1, and a variant of IMP-2 metallo-beta-lactamase, IMP-8, from a clinical isolate of Klebsiella pneumoniae. PMID : 11451699 : DOI : 10.1128/AAC.45.8.2368-2371.2001 PMC : PMC90656 Abstract >>
A multidrug-resistant plasmid encoding TEM-1, SHV-12, and a variant of IMP-2 metallo-beta-lactamase, designated IMP-8, was identified from a clinical isolate of Klebsiella pneumoniae. There are four nucleotide differences between bla(IMP-2) and bla(IMP-8), resulting in two amino acid differences. bla(IMP-8) was also found to be carried by an integron-borne gene cassette similar to the bla(IMP-2) cassette.
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76. |
Regué M,
Climent N,
Abitiu N,
Coderch N,
Merino S,
Izquierdo L,
Altarriba M,
Tomás JM,
( 2001 ) Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis. PMID : 11371519 : DOI : 10.1128/JB.183.12.3564-3573.2001 PMC : PMC95232 Abstract >>
A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.
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77. |
Kariuki S,
Corkill JE,
Revathi G,
Musoke R,
Hart CA,
( 2001 ) Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya. PMID : 11408239 : DOI : 10.1128/AAC.45.7.2141-2143.2001 PMC : PMC90616 Abstract >>
Nine Klebsiella pneumoniae isolates, six from blood and three from cerebrospinal fluid of newborn babies at Kenyatta National Hospital, Nairobi, Kenya, were analyzed for the mechanism of cephalosporin resistance. By using pulsed-field gel electrophoresis of XbaI-digested chromosomal DNA, all the nine isolates were found to be clonal. PCR and direct sequencing revealed a novel extended-spectrum beta-lactamase, which we designated CTX-M-12. It has a more potent hydrolytic activity against cefotaxime than against ceftazidime and a pI of 9.0 and is encoded on a large self-transferable ca. 160-kbp plasmid.
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78. |
Riley MA,
Pinou T,
Wertz JE,
Tan Y,
Valletta CM,
( 2001 ) Molecular characterization of the klebicin B plasmid of Klebsiella pneumoniae. PMID : 11407916 : DOI : 10.1006/plas.2001.1519 Abstract >>
The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.
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79. |
Brisse S,
Verhoef J,
( 2001 ) Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. PMID : 11411715 : DOI : 10.1099/00207713-51-3-915 DOI : 10.1099/00207713-51-3-915 Abstract >>
The infra-specific phylogenetic diversity and genetic structure of both Klebsiella pneumoniae and Klebsiella oxytoca was investigated using a combination of randomly amplified polymorphic DNA (RAPD) analysis, sequencing of gyrA and parC genes, and automated ribotyping. After RAPD analysis with four independent primers of 120 clinical isolates collected from 22 European hospitals in 13 countries, K. pneumoniae isolates fell into three clusters and K. oxytoca isolates fell into two clusters, while Klebsiella planticola isolates formed a sixth cluster. Each cluster was geographically widespread. K. pneumoniae cluster I (KpI) accounted for 80% of the isolates of this species and included reference strains of the three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. Clusters KpII and KpIII were equally represented, as were the two K. oxytoca clusters. Individualization of each cluster was fully confirmed by phylogenetic analysis of gyrA and parC gene sequences. In addition, sequence data supported the evolutionary separation of K. pneumoniae from a phylogenetic group including K. oxytoca, Klebsiella terrigena, K. planticola and Klebsiella ornithinolytica. Automated ribotyping using Mlu I appeared suitable for identification of each Klebsiella cluster. The adonitol fermentation test was found to be useful for cluster identification in K. pneumoniae, since it was negative in all strains of clusters KpIII and in some KpII strains, but always positive in cluster KpI. The usefulness of gyrA and parC sequence data for population genetics and cluster identification in bacteria was demonstrated, even for the phylogenetic positioning of quinolone-resistant isolates.
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80. |
Yigit H,
Queenan AM,
Anderson GJ,
Domenech-Sanchez A,
Biddle JW,
Steward CD,
Alberti S,
Bush K,
Tenover FC,
( 2001 ) Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. PMID : 11257029 : DOI : 10.1128/AAC.45.4.1151-1161.2001 PMC : PMC90438 Abstract >>
A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three beta-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla(SHV) and bla(TEM) genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, bla(KPC-1), was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing beta-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing beta-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase, KPC-1, although alterations in porin expression may also play a role.
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81. |
Thompson J,
Robrish SA,
Pikis A,
Brust A,
Lichtenthaler FW,
( 2001 ) Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. PMID : 11322729 : DOI : 10.1016/s0008-6215(01)00028-3 Abstract >>
Not only sucrose but the five isomeric alpha-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at 0-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6'-O-phosphorylated alpha-D-glucosyl-fructoses are hydrolyzed by an inducible (approximately 49-50 Kda) phospho-alpha-glucosidase from Family 4 of the glycosylhydrolase superfamily.
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82. |
Corkill JE,
Cuevas LE,
Gurgel RQ,
Greensill J,
Hart CA,
( 2001 ) SHV-27, a novel cefotaxime-hydrolysing beta-lactamase, identified in Klebsiella pneumoniae isolates from a Brazilian hospital. PMID : 11266422 : DOI : 10.1093/jac/47.4.463 Abstract >>
From a collection of cefotaxime-resistant Klebsiella pneumoniae isolated from neonatal blood culture specimens in a maternity hospital in Aracaju, Brazil, two isolates (strains KPBRZ-842 and -843, indistinguishable by pulsed-field gel electrophoresis) were found to produce beta-lactamases with isoelectric points (pI) of 5.4 and 8.2, respectively. Using a gel overlay method, cefotaxime hydrolysis was shown to be associated with the pI 8.2 protein. Nucleotide sequencing of the gene encoding the pI 8.2 beta-lactamase revealed a bla(SHV-ESBL)-type gene differing from the gene encoding SHV-1 by three silent point mutations, and a fourth that resulted in an amino acid substitution, aspartate for glycine, at position 156. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-27.
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83. |
Otte S,
Lengeler JW,
( 2001 ) The mtl genes and the mannitol-1-phosphate dehydrogenase from Klebsiella pneumoniae KAY2026. PMID : 11164312 : DOI : 10.1111/j.1574-6968.2001.tb09473.x Abstract >>
The mtl operon of Klebsiella pneumoniae KAY2026 (formerly Aerobacter aerogenes 1033-5P14) was shown to contain as the promoter-proximal gene mtlA, encoding a D-mannitol-specific enzyme II transporter (IICBA(Mtl)). This gene is followed by mtlD, coding for a mannitol-1-phosphate dehydrogenase (MtlD, 382 amino acid residues), and mtlR (MtlR, 195 amino acid residues) coding for a putative repressor, gene mtlR overlaps the termination codon of mtlD. The DNA and protein sequences are highly similar to the corresponding genes (81% identical bp) and proteins (79-85% identical amino acids) of Escherichia coli K-12. A truncated form of MtlD lacking the 162 C-terminal amino acid residues still shows 10% dehydrogenase activity which may explain the controversy in the literature concerning the properties of mannitol-phosphate and other medium-length dehydrogenases.
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84. |
Orencia MC,
Yoon JS,
Ness JE,
Stemmer WP,
Stevens RC,
( 2001 ) Predicting the emergence of antibiotic resistance by directed evolution and structural analysis. PMID : 11224569 : DOI : 10.1038/84981 Abstract >>
Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations (approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.
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85. |
Otagiri M,
Kurisu G,
Ui S,
Takusagawa Y,
Ohkuma M,
Kudo T,
Kusunoki M,
( 2001 ) Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms. PMID : 11173520 : DOI : 10.1093/oxfordjournals.jbchem.a002845 Abstract >>
The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.
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86. |
Fortineau N,
Poirel L,
Nordmann P,
( 2001 ) Plasmid-mediated and inducible cephalosporinase DHA-2 from Klebsiella pneumoniae. PMID : 11157909 : DOI : 10.1093/jac/47.2.207 Abstract >>
A Klebsiella pneumoniae strain resistant to cefoxitin and oxyimino-cephalosporins was cultured from a child hospitalized in Paris, France, in 1992. This isolate harboured a beta-lactamase gene located on an approximately 200 kb non-self-transferable plasmid. The beta-lactamase identified, DHA-2, shared 99% amino acid identity with the AmpC enzyme of Morganella morganii. DHA-2 was a point-mutant derivative of DHA-1 identified previously in a Salmonella enteritidis isolate. DHA-2 expression was inducible due to an ampR regulatory gene. This is the first report of an inducible and plasmid-located cephalosporinase from K. pneumoniae.
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87. |
Yuan M,
Hall LM,
Hoogkamp-Korstanje J,
Livermore DM,
( 2001 ) SHV-14, a novel beta-lactamase variant in Klebsiella pneumoniae isolates from Nijmegen, The Netherlands. PMID : 11120985 : DOI : 10.1128/AAC.45.1.309-311.2001 PMC : PMC90280 Abstract >>
Four ceftazidime-resistant isolates of a Klebsiella pneumoniae strain were collected from intensive care unit patients in Nijmegen, The Netherlands. These isolates had TEM-29 and SHV-14 beta-lactamases. SHV-14 is a novel variant, with two substitutions compared with the sequence of SHV-1: Ile8Phe and Arg43Ser. Its gene also had a silent C-->T mutation at nucleotide 481. The SHV-14 enzyme had slightly higher V(max) rates than SHV-1 for oxyimino-aminothiazolyl cephalosporins, but this activity was insufficient for the enzyme to count as an extended-spectrum beta-lactamase.
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88. |
Essack SY,
Hall LM,
Pillay DG,
McFadyen ML,
Livermore DM,
( 2001 ) Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum beta-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa. PMID : 11120950 : DOI : 10.1128/AAC.45.1.88-95.2001 PMC : PMC90245 Abstract >>
beta-Lactamase production was investigated in cultures of 25 Klebsiella pneumoniae isolates isolated at a hospital in Durban, South Africa, in 1994 and 1996. Twenty of these isolates gave ceftazidime MIC/ceftazidime plus clavulanate MIC ratios of >/=8, implying production of extended-spectrum beta-lactamases (ESBLs), and DNA sequencing identified an ESBL gene (bla(TEM-53)) in a further two isolates. Pulsed-field gel electrophoresis (PFGE) defined 4 distinct strains among the 12 isolates collected in 1994 and 9 distinct strains among the 13 isolates collected in 1996. In three cases, multiple isolates from single patients varied in their PFGE profiles and antibiograms, implying mixed colonization or infection. Isoelectric focusing and DNA hybridization found both TEM and SHV enzymes and their genes in all 25 isolates. Many isolates had multiple identical or different beta-lactamase gene variants, with at least 84 bla(SHV) and bla(TEM) gene copies among the 25 organisms. Sequencing identified the genes for the SHV-1, -2, and -5 enzymes and for four new SHV types (SHV-19, -20, -21, and -22). These new SHV variants had novel mutations remote from sites known to affect catalytic activity. Sequencing also found the genes for TEM-1, TEM-53, and one novel type, TEM-63. All the isolates had multiple and diverse plasmids. These complex and diverse patterns of ESBL production and strain epidemiology are far removed from the concept of an ESBL outbreak and suggest a situation in which ESBL production has become endemic and in which evolution is generating a wide range of enzyme combinations. This complexity and diversity complicates patient management and the design of antibiotic use policies.
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89. |
Grabbe R,
Klopprogge K,
Schmitz RA,
( 2001 ) Fnr Is required for NifL-dependent oxygen control of nif gene expression in Klebsiella pneumoniae. PMID : 11157952 : DOI : 10.1128/JB.183.4.1385-1393.2001 PMC : PMC95013 Abstract >>
In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by NifL in response to molecular oxygen and combined nitrogen. We recently showed that K. pneumoniae NifL is a flavoprotein, which apparently senses oxygen through a redox-sensitive, conformational change. We have now studied the oxygen regulation of NifL activity in Escherichia coli and K. pneumoniae strains by monitoring its inhibition of NifA-mediated expression of K. pneumoniae phi (nifH'-'lacZ) fusions in different genetic backgrounds. Strains of both organisms carrying fnr null mutations failed to release NifL inhibition of NifA transcriptional activity under oxygen limitation: nif induction was similar to the induction under aerobic conditions. When the transcriptional regulator Fnr was synthesized from a plasmid, it was able to complement, i.e., to relieve NifL inhibition in the fnr mutant backgrounds. Hence, Fnr appears to be involved, directly or indirectly, in NifL-dependent oxygen regulation of nif gene expression in K. pneumoniae. The data indicate that in the absence of Fnr, NifL apparently does not receive the signal for anaerobiosis. We therefore hypothesize that in the absence of oxygen, Fnr, as the primary oxygen sensor, activates transcription of a gene or genes whose product or products function to relieve NifL inhibition by reducing the flavin adenine dinucleotide cofactor under oxygen-limiting conditions.
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90. |
Lai YC,
Yang SL,
Peng HL,
Chang HY,
( 2000 ) Identification of genes present specifically in a virulent strain of Klebsiella pneumoniae. PMID : 11083844 : DOI : 10.1128/iai.68.12.7149-7151.2000 PMC : PMC97829 Abstract >>
Klebsiella pneumoniae is a common cause of septicemia and urinary tract infections. The PCR-supported genomic subtractive hybridization was employed to identify genes specifically present in a virulent strain of K. pneumoniae. Analysis of 25 subtracted DNA clones has revealed 19 distinct nucleotide sequences. Two of the sequences were found to be the genes encoding the transposase of Tn3926 and a capsule polysaccharide exporting enzyme. Three sequences displayed moderate homology with bvgAS, which encodes a two-component signal transduction system in Bordetella pertussis. The rest of the sequences did not exhibit homology with any known genes. The distribution of these novel sequences varied greatly in K. pneumoniae clinical isolates, reflecting the heterogeneous nature of the K. pneumoniae population.
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91. |
Naas T,
Benaoudia F,
Massuard S,
Nordmann P,
( 2000 ) Integron-located VEB-1 extended-spectrum beta-lactamase gene in a Proteus mirabilis clinical isolate from Vietnam. PMID : 11062188 : DOI : 10.1093/jac/46.5.703 Abstract >>
A clinical isolate of Proteus mirabilis Lil-1 was obtained from a Vietnamese patient hospitalized in Paris, France. This isolate was resistant to cephalosporins, and there was marked synergy between cephalosporins and clavulanic acid together with unusual synergy between cefoxitin and cefuroxime. PCR analysis revealed the presence of blaVEB-1, an integron-located gene coding for an extended-spectrum beta-lactamase (ESBL) identified previously in an Escherichia coli isolate MG-1 from Vietnam. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1-containing integron along with flanking sequences were amplified from P. mirabilis Lil-1 whole-cell DNA. A novel class 1 integron, In55, was identified that contained, in addition to intI1, qacEDelta1, sul1 and Orf5 genes, an 8 kb variable region. This region was comparable in size to that found previously in E. coli MG-1, but different from those previously identified in two Pseudomonas aeruginosa isolates from Thailand. In55 was located on a 190 kb self-transferable plasmid, which was different in size and structure from that found in E. coli MG-1. The finding of blaVEB-1 on different plasmids and integrons in enterobacterial isolates underlines the interspecies spread of this novel ESBL gene.
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92. |
Hoenke S,
Wild MR,
Dimroth P,
( 2000 ) Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase. PMID : 11052675 : DOI : 10.1021/bi0011532 Abstract >>
Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.
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93. |
Hoenke S,
Schmid M,
Dimroth P,
( 2000 ) Identification of the active site of phosphoribosyl-dephospho-coenzyme A transferase and relationship of the enzyme to an ancient class of nucleotidyltransferases. PMID : 11052676 : DOI : 10.1021/bi001154u Abstract >>
Malonate decarboxylase from Klebsiella pneumoniae contains an acyl carrier protein (MdcC) to which a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group is attached via phosphodiester linkage to serine 25. We have shown in the preceding paper in this issue that the formation of this phosphodiester bond is catalyzed by a phosphoribosyl-dephospho-coenzyme A transferase MdcG with the substrate 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA that is synthesized from ATP and dephospho-coenzyme A by the triphosphoribosyl transferase MdcB. The reaction catalyzed by MdcG is related to nucleotidyltransfer reactions, and the enzyme indeed catalyzes unphysiological nucleotidyltransfer, e.g., adenylyltransfer from ATP to apo acyl carrier protein (ACP). These unspecific side reactions are favored at high Mg(2+) concentrations. A sequence motif including D134 and D136 of MdcG is a signature of all nucleotidyltransferases. It is known from the well-characterized mammalian DNA polymerase beta that this motif is at the active site of the enzyme. Site-directed mutagenesis of D134 and/or D136 of MdcG to alanine abolished the transfer of the prosthetic group to apo ACP, but the binding of triphosphoribosyl-dephospho-CoA to MdcG was not affected. Evidence is presented that similar to MdcG, MadK encoded by the malonate decarboxylase operon of Malonomonas rubra and CitX from the operon encoding citrate lyase in Escherichia coli are phosphoribosyl-dephospho-CoA transferases catalyzing the attachment of the phosphoribosyl-dephospho-CoA prosthetic group to their specific apo ACPs.
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94. |
Lagrange PH,
Philippon A,
Herrmann J,
Donay L,
Verdet C,
Rouveau M,
Nadjar D,
( 2000 ) Outbreak of Klebsiella pneumoniae producing transferable AmpC-type beta-lactamase (ACC-1) originating from Hafnia alvei. PMID : 10828397 : DOI : 10.1111/j.1574-6968.2000.tb09133.x Abstract >>
Fifty-two strains of Klebsiella pneumoniae producing an AmpC-type plasmid-mediated beta-lactamase were isolated from 13 patients in the same intensive care unit between March 1998 and February 1999. These strains were resistant to ceftazidime, cefotaxime and ceftriaxone, but susceptible to cefoxitin, cefepime and aztreonam. Plasmid content and genomic DNA restriction pattern analysis suggested dissemination of a single clone. Two beta-lactamases were identified, TEM-1 and ACC-1. We used internal bla(ACC-1) primers, to sequence PCR products obtained from two unrelated strains of Hafnia alvei. Our results show that the ACC-1 beta-lactamase was derived from the chromosome-encoded AmpC-type enzyme of H. alvei.
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95. |
Osborn AM,
da Silva Tatley FM,
Steyn LM,
Pickup RW,
Saunders JR,
( 2000 ) Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons. PMID : 10974114 : DOI : 10.1099/00221287-146-9-2267 Abstract >>
The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.
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96. |
Rasheed JK,
Anderson GJ,
Yigit H,
Queenan AM,
Doménech-Sánchez A,
Swenson JM,
Biddle JW,
Ferraro MJ,
Jacoby GA,
Tenover FC,
( 2000 ) Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18. PMID : 10952583 : DOI : 10.1128/aac.44.9.2382-2388.2000 PMC : PMC90073 Abstract >>
Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum beta-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single beta-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla(SHV) gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla(SHV)-specific probe indicated that a bla(SHV) gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla(SHV)-related gene revealed that it differs from bla(SHV-1) at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.
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97. |
Wu SM,
Tsai SH,
Yan JJ,
( 2000 ) Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan. PMID : 10817689 : DOI : 10.1128/aac.44.6.1438-1442.2000 PMC : PMC89893 Abstract >>
Twenty (8.5%) of 234 nonrepetitive clinical isolates of Klebsiella pneumoniae from southern Taiwan were found to produce extended-spectrum beta-lactamases (ESBLs): 10 strains produced SHV-12, 4 produced SHV-5, 2 produced a non-TEM non-SHV ESBL with a pI of 8.3, 3 produced a novel AmpC beta-lactamase designated CMY-8 with a pI of 8.25, and 1 produced SHV-12 and an unidentified AmpC enzyme with a pI of 8.2. The CMY-8 enzyme confers a resistance phenotype similar to CMY-1 and MOX-1, and sequence comparisons showed high homologies (>95%) of nucleotide and amino acid sequences among these three enzymes. Plasmid and pulse-field gel electrophoresis analyses revealed that all isolates harboring an SHV-derived ESBL were genetically unrelated, indicating that dissemination of resistance plasmids is responsible for the spread of SHV ESBLs among K. pneumoniae in this area. All three isolates carrying CMY-8 had identical genotypic patterns, suggesting the presence of an epidemic strain.
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98. |
Przondo-Mordarska H,
Stankiewicz M,
Miaczy?ska B,
Pa?ucha A,
Fiett J,
( 2000 ) A novel complex mutant beta-lactamase, TEM-68, identified in a Klebsiella pneumoniae isolate from an outbreak of extended-spectrum beta-lactamase-producing Klebsiellae. PMID : 10817699 : DOI : 10.1128/aac.44.6.1499-1505.2000 PMC : PMC89903 Abstract >>
Twenty-two Klebsiella pneumoniae and two K. oxytoca extended-spectrum beta-lactamase (ESBL)-producing isolates were collected in 1996 from patients in two pediatric wards of the University Hospital in Wroc?aw, Poland. Molecular typing has revealed that the K. pneumoniae isolates represented four different epidemic strains. Three kinds of enzymes with ESBL activity (pI values of 5.7, 6.0, and 8.2) were identified. The pI 6.0 beta-lactamases belonged to the TEM family, and sequencing of the bla(TEM) genes amplified from representative isolates revealed that these enzymes were TEM-47, previously identified in K. pneumoniae isolates from pediatric hospitals in L?dz and Warsaw. One of the TEM-47-producing strains from Wroc?aw was very closely related to the isolates from the other cities, and this indicated countrywide spread of the epidemic strain. The pI 5.7 beta-lactamase was produced by a single K. pneumoniae isolate for which, apart from oxyimino-beta-lactams, the MICs of beta-lactam-inhibitor combinations were also remarkably high. Sequencing revealed that this was a novel TEM beta-lactamase variant, TEM-68, specified by the following combination of mutations: Gly238Ser, Glu240Lys, Thr265Met, and Arg275Leu. The new enzyme has most probably evolved from TEM-47 by acquiring the single substitution of Arg275, which before was identified only twice in enzymes with inhibitor resistance (IR) activity. TEM-68 was shown to be a novel complex mutant TEM beta-lactamase (CMT-2) which combines strong ESBL activity with relatively weak IR activity and, when expressed in K. pneumoniae, is able to confer high-level resistance to a wide variety of beta-lactams, including inhibitor combinations. This data confirms the role of the Arg275Leu mutation in determining IR activity and documents the first isolation of K. pneumoniae producing the complex mutant enzyme.
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99. |
Kido N,
( 2000 ) A single amino acid substitution in a mannosyltransferase, WbdA, converts the Escherichia coli O9 polysaccharide into O9a: generation of a new O-serotype group. PMID : 10762260 : DOI : 10.1128/jb.182.9.2567-2573.2000 PMC : PMC111322 Abstract >>
wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-alphaMan-1,2-alphaMan-1,3-alphaMan-1, 3-alphaMan-1. The equivalent structural O polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-alphaMan-1,2-alphaMan-1, 2-alphaMan-1,3-alphaMan-1,3-alphaMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdA genes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.
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100. |
Colloms S,
Blakely G,
Tolmasky ME,
( 2000 ) Stability by multimer resolution of pJHCMW1 is due to the Tn1331 resolvase and not to the Escherichia coli Xer system. PMID : 10746761 : DOI : 10.1099/00221287-146-3-581 Abstract >>
The plasmid pJHCMW1 encodes resistance to several aminoglycosides and beta-lactams and consists of a copy of the transposon Tn1331, a region including the replication functions, and a sequence with homology to ColE1 cer, designated mwr. In this work, the role of this cer-like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied. The Escherichia coli Xer site-specific recombination system acts at sites such as ColE1 cer to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance. Despite its high similarity to ColE1 cer, the pJHCMW1 mwr was a poor substrate for Xer recombination in E. coli and did not contribute significantly to plasmid stability. Instead, the Tn1331 co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1. Although Xer recombination at pJHCMW1 mwr was inefficient in E. coli, the recombination that did occur was dependent on ArgR, PepA, XerC and XerD. A supercoiled circular DNA molecule containing two pJHCMW1 mwr sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD in vitro, confirming that pJHCMW1 mwr is a functional recombination site. However, unlike cer, some Holliday-junction-containing product could be detected for mwr in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions. Binding experiments demonstrated that XerD bound to pJHCMW1 mwr core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD.
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101. |
Momma M,
( 2000 ) Cloning and sequencing of the maltohexaose-producing amylase gene of Klebsiella pneumoniae. PMID : 10737206 : DOI : 10.1271/bbb.64.428 Abstract >>
The molecular characterization of the maltohexaose-producing amylase gene of Klebsiella pneumoniae revealed an open reading frame in which 2,031 base pairs encode a protein of 677 amino acids with a calculated molecular weight of 75,921. The amylase gene had high similarities of 73.6% in DNA sequence and 79.3% in deduced amino acid sequence with the periplasmic alpha-amylase MalS gene of Escherichia coli.
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102. |
Izquierdo L,
Nogueras MM,
Altarriba M,
Merino S,
( 2000 ) Cloning and sequencing of the Klebsiella pneumoniae O5 wb gene cluster and its role in pathogenesis. PMID : 10768928 : DOI : 10.1128/iai.68.5.2435-2440.2000 PMC : PMC97443 Abstract >>
One representative recombinant clone encoding Klebsiella pneumoniae O5-antigen lipopolysaccharide (LPS) was found upon screening for serum resistance in a cosmid-based genomic library of K. pneumoniae KT769 (O5:K57) introduced into Escherichia coli DH5alpha. A total of eight open reading frames (wb(O5) gene cluster) were necessary to produce K. pneumoniae O5-antigen LPS in E. coli K-12. The enzymatic activities proposed for the wb(O5) gene cluster are in agreement with the activities proposed for the biosynthesis of K. pneumoniae O5-antigen LPS. Using the complete DNA sequence of the K. pneumoniae wb(O5) gene cluster, we obtained (by single or double recombination) genetically well-characterized mutants devoid only of this O5-antigen LPS. Finally, using these O5(-) mutants and the corresponding wild-type strains or complemented mutants with the wb(O5) gene cluster (O5(+) strains), we found that the presence of K. pneumoniae O5-antigen LPS is essential for some pathogenic features like serum resistance, adhesion to uroepithelial cells, and colonization (experimental infections) of the urinary tract in rats.
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103. |
Hall LM,
Savelkoul PH,
Yuan M,
( 2000 ) SHV-13, a novel extended-spectrum beta-lactamase, in Klebsiella pneumoniae isolates from patients in an intensive care unit in Amsterdam. PMID : 10722518 : DOI : 10.1128/aac.44.4.1081-1084.2000 PMC : PMC89819 Abstract >>
Eleven clonally related Klebsiella pneumoniae isolates were examined. These had been isolated at an intensive care unit in Amsterdam in 1994. Their resistance was associated with a conjugative 170-kb plasmid which encoded a novel SHV beta-lactamase designated SHV-13. The SHV-13 enzyme had two substitutions compared with SHV-1: Leu35Gln and Gly238Ala. It hydrolyzed cefotaxime much more rapidly than ceftazidime or aztreonam.
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104. |
Le Thomas I,
Naas T,
Poirel L,
( 2000 ) Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. PMID : 10681329 : DOI : 10.1128/aac.44.3.622-632.2000 PMC : PMC89737 Abstract >>
Klebsiella pneumoniae ORI-1 was isolated in 1998 in France from a rectal swab of a 1-month-old girl who was previously hospitalized in Cayenne Hospital, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferable plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistance profile antagonized by the addition of clavulanic acid, tazobactam, or imipenem. The gene for GES-1 (Guiana extended-spectrum beta-lactamase) was cloned, and its protein was expressed in Escherichia coli DH10B, where this pI-5. 8 beta-lactamase of a ca. 31-kDa molecular mass conferred resistance to oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum beta-lactamases (ESBLs). The highest percentage of amino acid identity was obtained with the carbenicillinase GN79 from Proteus mirabilis; with YENT, a chromosome-borne penicillinase from Yersinia enterocolitica; and with L-2, a chromosome-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% amino acid identity each). However, a dendrogram analysis showed that GES-1 clustered within a class A ESBL subgroup together with ESBLs VEB-1 and PER-1. Sequencing of a 7,098-bp DNA fragment from plasmid pTK1 revealed that the GES-1 gene was located on a novel class 1 integron named In52 that was characterized by (i) a 5' conserved segment containing an intI1 gene possessing two putative promoters, P(1) and P(2), for coordinated expression of the downstream antibiotic resistance genes and an attI1 recombination site; (ii) five antibiotic gene cassettes, bla(GES-1), aac(6')Ib' (gentamicin resistance and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel chloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomycin resistance); and (iii) a 3' conserved segment consisting of qacEDelta1 and sulI. The bla(GES-1) and aadA2 gene cassettes were peculiar, since they lacked a typical 59-base element. This work identified the second class A ESBL gene of a non-TEM, non-SHV series which was located in the plasmid and integron, thus providing it additional means for its spread and its expression.
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105. |
Hujer AM,
Bonafede M,
Hutton R,
Carias LL,
Rice LB,
( 2000 ) High-level expression of chromosomally encoded SHV-1 beta-lactamase and an outer membrane protein change confer resistance to ceftazidime and piperacillin-tazobactam in a clinical isolate of Klebsiella pneumoniae. PMID : 10639363 : DOI : 10.1128/aac.44.2.362-367.2000 PMC : PMC89684 Abstract >>
We describe Klebsiella pneumoniae 15571, a clinical isolate resistant to ceftazidime MIC = 32 microg/ml) and piperacillin-tazobactam (MICs = 1,024 and 128 microg/ml). K. pneumoniae 15571 expresses a single beta-lactamase with a pI of 7.6. However, when cloned in a high-copy-number vector in Escherichia coli, this bla(SHV-1) gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described bla(SHV-1) genes. Outer membrane protein (OMP) analysis of K. pneumoniae 15571 revealed a decrease in the quantity of a minor 45-kDa OMP in comparison to that in K. pneumoniae 44NR, a low-level ampicillin-resistant strain that also expresses a chromosomally determined bla(SHV-1). Crude beta-lactamase enzyme extracts from K. pneumoniae 15571 produced roughly 200-fold more beta-lactamase activity than K. pneumoniae 44NR. Northern hybridization analysis revealed that this difference was explainable by quantifiable differences in transcription of the bla(SHV-1) gene in the two strains. Primer extension analysis of bla(SHV-1) mRNA from K. pneumoniae 15571 and 44NR indicated that the transcriptional start sites were identical in the two strains. DNA sequencing of the promoter regions upstream of the of bla(SHV-1) open reading frames in the two K. pneumoniae strains revealed an A-->C change in the second position of the -10 region in K. pneumoniae 44NR compared to that in 15571. Site-directed mutagenesis of the cloned K. pneumoniae 15571 bla(SHV-1), in which the A in the second position of the 15571 -10 region was changed to a C, resulted in a substantial lowering of the MIC of ampicillin. When the levels of beta-lactamase enzyme expression in E. coli were compared, the bla(SHV-1) downstream of the altered -10 region produced 17-fold less beta-lactamase enzyme. These results indicate that elevated levels of ceftazidime resistance can result from a combination of increased enzyme production and minor OMP changes and that levels of chromosomally encoded SHV-1 beta-lactamase production can vary substantially with a single-base-pair change in promoter sequence.
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106. |
Chai W,
Wu SQ,
( 1999 ) General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al. PMID : 10572131 : PMC : PMC103690 Abstract >>
Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon. Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of nasR gene expression. We constructed strains bearing single-copy Phi(nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus. The expression of DeltarhaBS::[Phi(nasR-lacZ)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma(N) (sigma(54)) proteins. Sequence inspection of the nasR control region reveals an apparent sigma(N)-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma(N)-dependent nasR promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta-galactosidase expression indistinguishable from Phi(nasR-lacZ) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription. Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107-117, 1988).
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107. |
Jurenaite S,
Lubys A,
( 1999 ) Structural organization and regulation of the plasmid-borne type II restriction-modification system Kpn2I from Klebsiella pneumoniae RFL2. PMID : 10518615 : DOI : 10.1093/nar/27.21.4228 PMC : PMC148698 Abstract >>
Kpn 2I enzymes of a type II restriction-modification (R-M) system from the bacterium Klebsiella pneumoniae strain RFL2 recognize the sequence 5'-TCCGGA-3'. The Kpn 2I R-M genes have been cloned and expressed in Escherichia coli. DNA sequence analysis revealed the presence of two convergently transcribed open reading frames (ORFs) coding for a restriction endonuclease (Enase) of 301 amino acids (34. 8 kDa) and methyltransferase (Mtase) of 375 amino acids (42.1 kDa). The 3'-terminal ends of these genes (kpn2IR and kpn2IM, respectively) overlap by 11 bp. In addition, a small ORF (gene kpn2IC) capable of coding for a protein of 96 amino acids in length (10.6 kDa) was found upstream of kpn2IM. The direction of kpn2IC transcription is opposite to that of kpn2IM. The predicted amino acid sequence of this ORF includes a probable helix-turn-helix motif. We show that the product of kpn2IC represses expression of the Kpn 2I Mtase but has no influence on expression of the Enase gene. Such a mode of regulation is unique among R-M systems analyzed so far. The Kpn 2I R-M is located on the K.pneumoniae RFL2 plasmid pKp4.3, which is able to replicate in E.coli cells.
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108. |
Lawson DM,
Gormal CA,
Mayer SM,
( 1999 ) New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein. PMID : 10525412 : DOI : 10.1006/jmbi.1999.3107 Abstract >>
The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.
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109. |
Ullmann U,
Schneider I,
Jungwirth R,
Sahly H,
( 1999 ) A novel type of AmpC beta-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. PMID : 10428914 : PMC : PMC89392 Abstract >>
A Klebsiella pneumoniae strain resistant to oxyimino cephalosporins was cultured from respiratory secretions of a patient suffering from nosocomial pneumonia in Kiel, Germany, in 1997. The isolate harbors a bla resistance gene located on a transmissible plasmid. An Escherichia coli transconjugant produces a beta-lactamase with an isoelectric point of 7.7 and a resistance phenotype characteristic of an AmpC (class 1) beta-lactamase except for low MICs of cephamycins. The bla gene was cloned and sequenced. It encodes a protein of 386 amino acids with the active site serine of the S-X-X-K motif at position 64, as is characteristic for class C beta-lactamases. Multiple alignment of the deduced amino acid sequence with 21 other AmpC beta-lactamases demonstrates only very distant homology, reaching at maximum 52.3% identity for the chromosomal AmpC beta-lactamase of Serratia marcescens SR50. The beta-lactamase of K. pneumoniae KUS represents a new type of AmpC-class enzyme, for which we propose the designation ACC-1 (Ambler class C-1).
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110. |
Bott M,
Scapozza L,
Pfister K,
Meyer M,
Perozzo R,
Reinelt S,
( 1999 ) The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor. PMID : 10447894 : DOI : 10.1046/j.1365-2958.1999.01536.x Abstract >>
The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327-547) showed constitutive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB-D56N formed a stable complex with MalE-CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45-176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 46.3 kJ mol-1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2- is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.
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111. |
Poirel L,
Karim A,
Nordmann P,
( 1999 ) Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum beta-lactamase VEB-1 in Pseudomonas aeruginosa. PMID : 10427724 : DOI : 10.1111/j.1574-6968.1999.tb13691.x Abstract >>
A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum beta-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacE delta 1 and sull genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum beta-lactamase gene.
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112. |
Dierks T,
Miech C,
Balleininger M,
Schmidt B,
von Figura K,
( 1999 ) The iron sulfur protein AtsB is required for posttranslational formation of formylglycine in the Klebsiella sulfatase. PMID : 10336424 : DOI : 10.1074/jbc.274.22.15375 Abstract >>
The catalytic residue of eukaryotic and prokaryotic sulfatases is a alpha-formylglycine. In the sulfatase of Klebsiella pneumoniae the formylglycine is generated by posttranslational oxidation of serine 72. We cloned the atsBA operon of K. pneumoniae and found that the sulfatase was expressed in inactive form in Escherichia coli transformed with the structural gene (atsA). Coexpression of the atsB gene, however, led to production of high sulfatase activity, indicating that the atsB gene product plays a posttranslational role that is essential for the sulfatase to gain its catalytic activity. This was verified after purification of the sulfatase from the periplasm of the cells. Peptide analysis of the protein expressed in the presence of AtsB revealed that half of the polypeptides carried the formylglycine at position 72, while the remaining polypeptides carried the encoded serine. The inactive sulfatase expressed in the absence of AtsB carried exclusively serine 72, demonstrating that the atsB gene is required for formylglycine modification. This gene encodes a 395-amino acid residue iron sulfur protein that has a cytosolic localization and is supposed to directly or indirectly catalyze the oxidation of the serine to formylglycine.
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113. |
Schmitz RA,
( 1999 ) NifL of Klebsiella pneumoniae: redox characterization in relation to the nitrogen source. PMID : 10350621 : DOI : 10.1016/s0167-4838(99)00075-8 Abstract >>
In Klebsiella pneumoniae, NifL modulates the activity of the transcriptional activator NifA in response to combined nitrogen or external molecular oxygen. We recently showed that K. pneumoniae NifL is a flavoprotein which apparently senses oxygen through a redox-sensitive, conformational change. In order to study whether the nitrogen signal might be transmitted to NifA through a stable modification of NifL we characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL carried FAD as cofactor independent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was -277+/-5 mV at pH 8.0 and 25 degrees C, as determined by reduction with dithionite or with enzymatic reduction by xanthine oxidase in the presence of methyl viologen as redox mediator. When synthesized under nitrogen-limiting conditions, NifL showed a redox potential of -274+/-6 mV at pH 8.0 and 25 degrees C. Fully reduced NifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that for NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, however, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium.
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114. |
Hernández-Allés S,
Martínez-Martínez L,
Albertí S,
( 1999 ) Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance. PMID : 10217760 : PMC : PMC93711 Abstract >>
Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.
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115. |
Knox JR,
Bonomo RA,
Nukaga M,
( 1999 ) Structure of the SHV-1 beta-lactamase. PMID : 10231522 : DOI : 10.1021/bi990136d Abstract >>
The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established. The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A. The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution. Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively. Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A. Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet. All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1. However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1. A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.
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116. |
Rosenbusch J,
Benedí V,
Schirmer T,
Phale P,
( 1999 ) Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae. PMID : 10196126 : Abstract >>
Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure. The crystal structure of OmpK36 has been determined to a resolution of 3.2 A by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel beta barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF. The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength.
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117. |
Rahn A,
Drummelsmith J,
( 1999 ) Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae. PMID : 10094716 : PMC : PMC93651 Abstract >>
Group 1 capsules of Escherichia coli are similar to the capsules produced by strains of Klebsiella spp. in terms of structure, genetics, and patterns of expression. The striking similarities between the capsules of these organisms prompted a more detailed investigation of the cps loci encoding group 1 capsule synthesis. Six strains of K. pneumoniae and 12 strains of E. coli were examined. PCR analysis showed that the clusters in these strains are conserved in their chromosomal locations. A highly conserved block of four genes, orfX-wza-wzb-wzc, was identified in all of the strains. The wza and wzc genes are required for translocation and surface assembly of E. coli K30 antigen. The conservation of these genes points to a common pathway for capsule translocation. A characteristic JUMPstart sequence was identified upstream of each cluster which may function in conjunction with RfaH to inhibit transcriptional termination at a stem-loop structure found immediately downstream of the "translocation-surface assembly" region of the cluster. Interestingly, the sequence upstream of the cps clusters in five E. coli strains and one Klebsiella strain indicated the presence of IS elements. We propose that the IS elements were responsible for the transfer of the cps locus between organisms and that they may continue to mediate recombination between strains.
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118. |
Arlet G,
Philippon A,
( 1999 ) Sequences of the genes for the TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases. PMID : 10103213 : PMC : PMC89239 Abstract >>
The sequences of the blaTEM genes encoding TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases were determined. Analysis of the deduced amino acid sequences indicated that TEM-20 and TEM-29 were derived from TEM-1 and that TEM-21 and TEM-22 were derived from TEM-2. The substitutions involved were Ser-238 and Thr-182 for TEM-20; His-164 for TEM-29; Lys-104, Arg-153, and Ser-238 for TEM-21; and Lys-104, Gly-237, and Ser-238 for TEM-22. The promoter region of the blaTEM-22 gene was identical to that of blaTEM-3. High-level production of TEM-20 could result from a 135-bp deletion which combined the -35 region of the Pa promoter with the -10 region of the P3 promoter and a G-->T transition in the latter motif.
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119. |
Wilson J,
Berger BJ,
( 1999 ) Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae. PMID : 10074065 : PMC : PMC93571 Abstract >>
An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.
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120. |
Vo AT,
van Duijkeren E,
Fluit AC,
Gaastra W,
( 2007 ) Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses. PMID : 17521833 : DOI : 10.1016/j.vetmic.2007.04.027 Abstract >>
The aim of the present study was to contribute to the knowledge on extended-spectrum beta-lactamases (ESBL's), AmpC beta-lactamases and integrons in Enterobacteriaceae isolated from horses, which is still limited. The susceptibility of 1581 clinical isolates from animals to ceftiofur was tested. Most of these isolates (n=1347) originated from horses. Seven ceftiofur-resistant equine isolates (four Escherichia coli and three Klebsiella pneumoniae) were identified and all seven were multidrug-resistant. These isolates were further studied for the presence of ESBL's, AmpC beta-lactamases and class 1 integrons. The potential for the horizontal transfer of resistance genes among these clinical isolates was also studied. ESBL-type resistance genes were found in five isolates, AmpC-type genes in one isolates and integrons in six isolates. Nucleotide sequence analysis revealed that the isolates carried the bla(CTX-M-1), bla(CMY-2), bla(TEM-1) and/or bla(SHV-1) genes. This is the first report describing the in vitro conjugal transfer of the bla(CTX-M-1) genes from a clinical E. coli isolate to Salmonella isolates. Gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2 and aadA5), and trimethoprim (dfrA1, drfA12 and dfrA17) were found on the integrons present in the isolates. The cassette arrays of the dfrA17-aadA5 and dfrA1-aadA1 genes in the two integrons of a single E. coli isolate have not yet been described before. To our knowledge this is the first report on ESBL's and AmpC beta-lactamases in equine E. coli and Klebsiella isolates.
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121. |
Chong Y,
Lee K,
( 1998 ) Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 years after the discovery? PMID : 10097678 : DOI : 10.3349/ymj.1998.39.6.520 Abstract >>
The dogma that ampC genes are located exclusively on the chromosome was dominant until about 10 years ago. Since 1989 over 15 different plasmid-encoded AmpC beta-lactamases have been reported from several countries. Most of these enzymes evolved in two clusters. The major cluster includes several enzymes with a high similarity to CMY-2, which is the closest related chromosomal AmpC enzyme of Citrobacter freundii. A second cluster centers around CMY-1. It is less homogeneous and not closely related chromosomal AmpC enzymes. Molecular diversification by amino acid substitutions does not usually translate into a change in the resistance phenotype. At this time, CMY-2 appears to be the most prevalent and widely distributed. Further global increase of prevalence and diversity of plasmidic AmpC beta-lactamases have to be anticipated in the next millenium.
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122. |
Ambrozic Avgustin J,
Keber R,
Zerjavic K,
Orazem T,
Grabnar M,
( 2007 ) Emergence of the quinolone resistance-mediating gene aac(6')-Ib-cr in extended-spectrum-beta-lactamase-producing Klebsiella isolates collected in Slovenia between 2000 and 2005. PMID : 17846136 : DOI : 10.1128/AAC.01480-06 PMC : PMC2151430 Abstract >>
Seventy-four nonrepetitive uropathogenic fluoroquinolone-resistant or -intermediate extended-spectrum-beta-lactamase-producing Klebsiella isolates from Slovenia were screened for the presence of plasmid-mediated quinolone resistance genes. None of the known qnr genes were detected. The aac(6')-Ib-cr allele was detected on plasmids from 25 transconjugants for which the ciprofloxacin MIC was higher than for the recipient Escherichia coli strain.
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123. |
Brasme L,
Nordmann P,
Fidel F,
Lartigue MF,
Bajolet O,
Poirel L,
Forte D,
Vernet-Garnier V,
Madoux J,
Reveil JC,
Alba-Sauviat C,
Baudinat I,
Bineau P,
Bouquigny-Saison C,
Eloy C,
Lafaurie C,
Siméon D,
Verquin JP,
Noël F,
Strady C,
De Champs C,
( 2007 ) Incidence of class A extended-spectrum beta-lactamases in Champagne-Ardenne (France): a 1 year prospective study. PMID : 17804424 : DOI : 10.1093/jac/dkm319 Abstract >>
To assess the frequency and diversity of extended spectrum beta-lactamases (ESBLs) in the Champagne-Ardenne region France, and to identify genetic elements associated with the bla(CTX-M) genes. During 2004, all the non-duplicate isolates of Pseudomonas aeruginosa and Acinetobacter baumannii resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime, screening samples excluded, were collected in 10 public hospitals and 3 private clinics. bla genes were sequenced and bla(CTX-M) environment characterized by PCR mapping. In Enterobacteriaceae (138/21 861; 0.6%), ESBLs were predominantly TEM-24 (n = 52; 37.7%) and CTX-M-15 (n = 37; 26.8%). Three new enzymes were identified, CTX-M-61 (CTX-M-1 group), TEM- and SHV-type. A. baumannii (n = 5) produced VEB-1 and P. aeruginosa (n = 2) SHV-2a. ISEcp1 was detected in 22/27 strains, disrupted in 7 of them. The IS903-like element was downstream of bla(CTX-M-14) and bla(CTX-M-16). ISCR1 was found upstream of bla(CTX-M-2) and bla(CTX-M-9), and ISCR1 and bla(CTX-M-2) were located on a sul1-type class 1 integron. In comparison with 2001-02, ESBL distribution among Enterobacteriaceae showed an increase in CTX-M-type (44.9% vs 3.7% P < 10(-7)) due to Escherichia coli CTX-M-15 and to the almost total disappearance of TEM-3 (0.9% vs 51.2%). E. coli was the most frequent species (50.0% vs 5.1% in 1998) despite a similar prevalence to that in 1998 (0.5% vs 0.2%). A careful detection of bla(CTX-M)-type spread to other species would help to anticipate clonal endemics such as those observed in Enterobacter aerogenes TEM-24.
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124. |
Chen YT,
Lauderdale TL,
Liao TL,
Shiau YR,
Shu HY,
Wu KM,
Yan JJ,
Su IJ,
Tsai SF,
( 2007 ) Sequencing and comparative genomic analysis of pK29, a 269-kilobase conjugative plasmid encoding CMY-8 and CTX-M-3 beta-lactamases in Klebsiella pneumoniae. PMID : 17526756 : DOI : 10.1128/AAC.00167-07 PMC : PMC1932545 Abstract >>
A 269-kilobase conjugative plasmid, pK29, from a Klebsiella pneumoniae strain was sequenced. The plasmid harbors multiple antimicrobial resistance genes, including those encoding CMY-8 AmpC-type and CTX-M-3 extended-spectrum beta-lactamases in the common backbone of IncHI2 plasmids. Mechanisms for dissemination of the resistance genes are highlighted in comparative genomic analyses.
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125. |
Smith CA,
Caccamo M,
Kantardjieff KA,
Vakulenko S,
( 2007 ) Structure of GES-1 at atomic resolution: insights into the evolution of carbapenamase activity in the class A extended-spectrum beta-lactamases. PMID : 17704567 : DOI : 10.1107/S0907444907036955 Abstract >>
The structure of the class A extended-spectrum beta-lactamase GES-1 from Klebsiella pneumoniae has been determined to 1.1 A resolution. GES-1 has the characteristic active-site disulfide bond of the carbapenemase family of beta-lactamases and has a structure that is very similar to those of other known carbapenemases, including NMC-A, SME-1 and KPC-2. Most residues implicated in the catalytic mechanism of this class of enzyme are present in the GES-1 active site, including Ser70, which forms a covalent bond with the carbonyl C atom of the beta-lactam ring of the substrate during the formation of an acyl-enzyme intermediate, Glu166, which is implicated as both the acylation and deacylation base, and Lys73, which is also implicated as the acylation base. A water molecule crucial to catalysis is observed in an identical location as in other class A beta-lactamases, interacting with the side chains of Ser70 and Glu166. One important residue, Asn170, also normally a ligand for the hydrolytic water, is missing from the GES-1 active site. This residue is a glycine in GES-1 and the enzyme is unable to hydrolyze imipenem. This points to this residue as being critically important in the hydrolysis of this class of beta-lactam substrate. This is further supported by flexible-docking studies of imipenem with in silico-generated Gly170Asn and Gly170Ser mutant GES-1 enzymes designed to mimic the active sites of imipenem-hydrolyzing point mutants GES-2 and GES-5.
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126. |
Vogler AP,
Lengeler JW,
( 1991 ) Comparison of the sequences of the nagE operons from Klebsiella pneumoniae and Escherichia coli K12: enhanced variability of the enzyme IIN-acetylglucosamine in regions connecting functional domains. PMID : 1745234 : DOI : 10.1007/bf00290677 Abstract >>
The nagE operon, encoding the enzyme II specific for N-acetylglucosamine (EIINag), and adjacent DNA from the chromosome of Klebsiella pneumoniae were sequenced and compared with the corresponding sequence from Escherichia coli K12. The deduced EIINag sequences differ in 72 out of 651 amino acids, the K. pneumoniae sequence being three residues longer. The amino acid differences were distributed unevenly, and were most frequent in regions connecting the three functional domains of the protein. In the nagE-nagB intergenic region, two promoter, two operator, and one CAP consensus sequence with regulatory functions were highly conserved. The nag structural genes from both species were very similar (83% DNA similarity; 89% amino acid similarity) except for frequent AT to GC exchanges in the wobble base of codons in K. pneumoniae DNA relative to the E. coli DNA.
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127. |
Young JM,
Park DC,
( 2007 ) Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences. PMID : 17451899 : DOI : 10.1016/j.syapm.2007.03.002 Abstract >>
Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.
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128. |
Schneider I,
Markovska R,
Keuleyan E,
Sredkova M,
Rachkova K,
Mitov I,
Bauernfeind A,
( 2007 ) Dissemination and persistence of a plasmid-mediated TEM-3-like beta-lactamase, TEM-139, among Enterobacteriaceae in Bulgaria. PMID : 17382521 : DOI : 10.1016/j.ijantimicag.2006.12.014 Abstract >>
During a survey of extended-spectrum beta-lactamases (ESBLs) in Bulgaria from 1996 to 2003, a TEM-3-like ESBL was detected in strains of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii and Klebsiella oxytoca from three centres in three different towns. The nucleotide sequence of the cloned gene was identical to that of TEM-3, except for one substitution (C347A) causing an amino acid exchange at position 49 from leucine to methionine. This TEM-3 variant with both a unique nucleotide and amino acid sequence was designated TEM-139. Transformants producing TEM-3 or TEM-139 expressed identical beta-lactam resistance phenotypes. TEM-139 was the only TEM-type ESBL detected in the surveyed hospitals (seven centres in three towns). TEM-139 is a natural variant of TEM-3 with an amino acid exchange without informational content, detectable only by molecular procedures, e.g. a nucleotide-specific polymerase chain reaction.
|
129. |
Remeli GA,
Pacca CC,
Silva GC,
Almeida MT,
Rubio FG,
Nogueira ML,
Nogueira MC,
( 2007 ) OKP-B-14, a new OKP-B variant isolated from Klebsiella pneumoniae in Brazil. PMID : 17442542 : DOI : 10.1016/j.ijantimicag.2007.01.011 Abstract >>
N/A
|
130. |
Yu WL,
Fung CP,
Ko WC,
Cheng KC,
Lee CC,
Chuang YC,
( 2007 ) Polymerase chain reaction analysis for detecting capsule serotypes K1 and K2 of Klebsiella pneumoniae causing abscesses of the liver and other sites. PMID : 17357063 : DOI : 10.1086/512686 Abstract >>
N/A
|
131. |
Lau HY,
Clegg S,
Moore TA,
( 2007 ) Identification of Klebsiella pneumoniae genes uniquely expressed in a strain virulent using a murine model of bacterial pneumonia. PMID : 17369011 : DOI : 10.1016/j.micpath.2007.01.001 PMC : PMC1892313 Abstract >>
Klebsiella pneumoniae is a gram-negative bacterium of significant clinical importance. This study examines the differential pulmonary host anti-bacterial responses towards two clinical isolates of K. pneumoniae. Intratracheal inoculation with 7 x 10(4)CFU of strain 43816 induced 100% mortality in C57BL/6J mice within 5 days post infection, whereas infection with 5 x 10(5)CFU of strain IA565 resulted in 100% survival. Infection with strain 43816 resulted in significant pulmonary and peripheral blood bacterial burden and induction of the chemokines MIP-2, KC and MCP-1 by 24h post infection. In contrast, IA565-infected mice displayed basal chemokine levels and no detectable bacteria by 24h post inoculation were isolated from lungs or peripheral blood. These data indicate an apparent lack of pathogenicity of strain IA565. Since little is known about Klebsiella-specific virulence genes, we have utilized PCR-based genomic DNA and cDNA suppressive subtractive hybridization and identified nine DNA sequences unique to the pathogenic strain of K. pneumoniae 43816. These sequences were highly homologous to enteric bacterial genes regulating iron uptake, fimbrial-mediated adhesion, energy production and conversion, transcriptional regulation, signal transduction, restriction endonuclease activity, and membrane transport.
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132. |
Palasubramaniam S,
Karunakaran R,
Gin GG,
Muniandy S,
Parasakthi N,
( 2007 ) Imipenem-resistance in Klebsiella pneumoniae in Malaysia due to loss of OmpK36 outer membrane protein coupled with AmpC hyperproduction. PMID : 17337225 : DOI : 10.1016/j.ijid.2007.01.005 Abstract >>
N/A
|
133. |
Rodríguez-Martínez JM,
Velasco C,
García I,
Cano ME,
Martínez-Martínez L,
Pascual A,
( 2007 ) Characterisation of integrons containing the plasmid-mediated quinolone resistance gene qnrA1 in Klebsiella pneumoniae. PMID : 17368003 : DOI : 10.1016/j.ijantimicag.2007.02.003 Abstract >>
The aim of this study was to determine the structural relationships of qnrA1 and other resistance genes in four integrons contained in four clinical isolates of Klebsiella pneumoniae. In the four integrons, the sequences surrounding qnrA1 were similar to those described for pMG252 (accession no. AY070235). The four integrons carried a class 1 integrase gene belonging to the complex class 1 integron. Three of the strains contained an identical integron coding for resistance to beta-lactams, aminoglycosides, chloramphenicol and trimethoprim. The fourth strain contained a different integron coding for resistance to beta-lactams, aminoglycosides and chloramphenicol. Downstream of the last integron, copies of IS6100 and IS26 were present. We describe two new and different integrons containing qnrA1. These integrons code for resistance to different groups of antimicrobial agents from K. pneumoniae clinical strains isolated in the USA.
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134. |
Possot O,
d'Enfert C,
Reyss I,
Pugsley AP,
( 1992 ) Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein. PMID : 1738317 : DOI : 10.1111/j.1365-2958.1992.tb00841.x Abstract >>
The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide-binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytoplasmic protein. A representative PulE-beta-galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF-lacZ gene fusions that encoded hybrid proteins with relatively high levels of beta-galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K-12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide-binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.
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135. |
Bogaerts P,
Galimand M,
Bauraing C,
Deplano A,
Vanhoof R,
De Mendonca R,
Rodriguez-Villalobos H,
Struelens M,
Glupczynski Y,
( 2007 ) Emergence of ArmA and RmtB aminoglycoside resistance 16S rRNA methylases in Belgium. PMID : 17224412 : DOI : 10.1093/jac/dkl527 Abstract >>
16S rRNA methylase-mediated high-level resistance to aminoglycosides has been reported recently in clinical isolates of Gram-negative bacilli only from a limited number of countries. This study was conducted to investigate the occurrence of this type of resistance in clinical isolates of Enterobacteriaceae from two Belgian hospitals and the characteristics of the strains. We screened for high-level gentamicin, tobramycin and amikacin resistance in clinical isolates of Enterobacteriaceae consecutively collected between 2000 and 2005 at two laboratories by PCR for the armA, rmtA and rmtB 16S rRNA methylase genes. The beta-lactamase presence in the strains was also determined by phenotypic and genotypic methods. Overall armA genes were detected in 18 Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae and Citrobacter amalonaticus whereas rmtB was detected in a single E. coli isolate. The rmtA gene was not found. All 16S rRNA methylase-bearing strains produced extended-spectrum beta-lactamases (ESBLs), predominantly type CTX-M-3, as well as various types of beta-lactamases. In the majority of the strains, the armA gene was carried by conjugative plasmids of the IncL/M incompatibility group whereas rmtB was borne by an IncFI plasmid. This is the first report of the emergence of 16S rRNA methylases in Enterobacteriaceae in Belgium. The rapid spread of multidrug-resistant isolates producing both ESBLs and 16S rRNA methylases raises clinical concern and may become a major therapeutic threat in the future.
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136. |
Bae IK,
Lee YN,
Jeong SH,
Lee K,
Lee H,
Kwak HS,
Woo GJ,
( 2007 ) High prevalence of SHV-12 and the emergence of CTX-M-12 in clinical isolates of Klebsiella pneumoniae from Korea. PMID : 17227703 : DOI : 10.1016/j.ijantimicag.2006.10.009 Abstract >>
N/A
|
137. |
da Fonseca EL,
Vieira VV,
Cipriano R,
Vicente AC,
( 2007 ) Emergence of blaGES-5 in clinical colistin-only-sensitive (COS) Pseudomonas aeruginosa strain in Brazil. PMID : 17284538 : DOI : 10.1093/jac/dkl517 Abstract >>
N/A
|
138. |
Fu Y,
Zhang F,
Zhang W,
Chen X,
Zhao Y,
Ma J,
Bao L,
Song W,
Ohsugi T,
Urano T,
Liu S,
( 2007 ) Differential expression of bla(SHV) related to susceptibility to ampicillin in Klebsiella pneumoniae. PMID : 17276039 : DOI : 10.1016/j.ijantimicag.2006.10.015 Abstract >>
Klebsiella pneumoniae usually shows intrinsic resistance to ampicillin and other beta-lactams. bla(SHV) is thought to be a key beta-lactamase gene responsible for this intrinsic resistance to ampicillin. Nevertheless, surveys of clinical strains reveal that some isolates of K. pneumoniae that carry bla(SHV) remain susceptible to ampicillin. To explore susceptibility to ampicillin in relation to bla(SHV) in K. pneumoniae, we analysed the existence and transcription of bla(SHV) and determined beta-lactamase activity as well as the susceptibilities to clinically relevant beta-lactams, including ampicillin in 160K. pneumoniae isolates from China. In total, 141 isolates (88.1%) were detected as bla(SHV)-positive, 20 (14.2%) of which were found to be broadly susceptible to all beta-lactams tested, including ampicillin. Among the 20 broadly susceptible isolates, sequencing of bla(SHV) revealed synonymous point mutations in 19 isolates and a premature stop codon in 1 isolate. Reverse transcription-polymerase chain reaction failed to detect bla(SHV) mRNA in five isolates (25%). The results demonstrate that differential expression of bla(SHV) in clinical isolates of K. pneumoniae can affect susceptibility to ampicillin.
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139. |
Mazzariol A,
Roelofsen E,
Koncan R,
Voss A,
Cornaglia G,
( 2007 ) Detection of a new SHV-type extended-spectrum beta-lactamase, SHV-31, in a Klebsiella pneumoniae strain causing a large nosocomial outbreak in The Netherlands. PMID : 17178800 : DOI : 10.1128/AAC.00909-06 PMC : PMC1803151 Abstract >>
A Klebsiella pneumoniae strain resistant to third-generation cephalosporins was isolated in the eastern Netherlands. The strain was found to carry a novel extended-spectrum beta-lactamase, namely, SHV-31. The combination of the two mutations by which SHV-31 differs from SHV-1, namely, L35Q and E240K, had previously only been described in association with one or more additional mutations.
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140. |
Wei ZQ,
Du XX,
Yu YS,
Shen P,
Chen YG,
Li LJ,
( 2007 ) Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. PMID : 17145797 : DOI : 10.1128/AAC.01053-06 PMC : PMC1797727 Abstract >>
A carbapenem-resistant isolate of Klebsiella pneumoniae producing class A carbapenemase KPC-2 was identified in Zhejiang, China. The KPC-2 gene was located on an approximately 60-kb plasmid in a genetic environment partially different from that of blaKPC-2 in the isolates from the United States and Colombia.
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141. |
Vignoli R,
Cordeiro N,
Seija V,
Schelotto F,
Radice M,
Ayala J,
Power P,
Gutkind G,
( N/A ) [Genetic environment of CTX-M-2 in Klebsiella pneumoniae isolates from hospitalized patients in Uruguay]. PMID : 17037256 : Abstract >>
We studied two CTX-M-2-producing Klebsiella pneumoniae clinical strains, K96005 and K13, isolated from hospitalized patients in Uruguay, during 1996 and 2003, respectively. The genomic surroundings of bla(CTX-M-2) were characterized by PCR-mapping and DNA sequencing. Our results show that blaCTX-M-2 is included in a complex class-1 integron (InK13), associated with an orf513 in both isolates. The genetic array of the integron, aac(6')-lb, bla(OxA,2), orfD (gene cassette region), associated with an orf513-bla(CTX-M-2), seems to be widely disseminated over the Rio de la Plata region.
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142. |
Doloy A,
Verdet C,
Gautier V,
Decré D,
Ronco E,
Hammami A,
Philippon A,
Arlet G,
( 2006 ) Genetic environment of acquired bla(ACC-1) beta-lactamase gene in Enterobacteriaceae isolates. PMID : 16982793 : DOI : 10.1128/AAC.00619-06 PMC : PMC1693989 Abstract >>
We studied the genetic organization of bla(ACC-1) in 14 isolates of Enterobacteriaceae from France, Tunisia, and Germany. In a common ancestor, ISEcp1 was likely involved in the mobilization of this gene from the Hafnia alvei chromosome to a plasmid. Other genetic events involving insertion sequences (particularly IS26), transposons (particularly Tn1696), or sulI-type integrons have occurred, leading to complex genetic environments.
|
143. |
Ktari S,
Arlet G,
Mnif B,
Gautier V,
Mahjoubi F,
Ben Jmeaa M,
Bouaziz M,
Hammami A,
( 2006 ) Emergence of multidrug-resistant Klebsiella pneumoniae isolates producing VIM-4 metallo-beta-lactamase, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC beta-lactamase in a Tunisian university hospital. PMID : 17015633 : DOI : 10.1128/AAC.00663-06 PMC : PMC1694011 Abstract >>
Klebsiella pneumoniae clinical isolates resistant to carbapenems were recovered from 11 patients in the hospital of Sfax, Tunisia. The isolates were closely related as shown by pulsed-field gel electrophoresis, and they produced VIM-4 metallo-enzyme, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC enzyme. The bla(VIM-4) gene is part of a class 1 integron.
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144. |
Bae IK,
Lee BH,
Hwang HY,
Jeong SH,
Hong SG,
Chang CL,
Kwak HS,
Kim HJ,
Youn H,
( 2006 ) A novel ceftazidime-hydrolysing extended-spectrum beta-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. PMID : 16785225 : DOI : 10.1093/jac/dkl252 Abstract >>
To characterize a novel ceftazidime-hydrolysing CTX-M mutant, designated CTX-M-54, produced by Klebsiella pneumoniae clinical isolate BDK0419 and to investigate its genetic environment. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the genetic organization of the blaCTX-M-54 gene was investigated by PCR and sequencing of the regions surrounding this gene. Kinetic parameters were determined from purified CTX-M-54. The strain BDK0419 contained a transferable plasmid with a molecular size of approximately 21 kbp that carries both blaSHV-2a and blaCTX-M-54 beta-lactamase genes, along with two other plasmids. The blaCTX-M-54 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903-like element. CTX-M-54 had a P167Q substitution within the omega loop region of class A beta-lactamases compared with the sequence of CTX-M-3. The MIC of ceftazidime for K. pneumoniae BDK0419 was 16-fold higher than that of cefotaxime; however, the kinetic parameter of CTX-M-54 against ceftazidime revealed a low catalytic efficiency. This work shows once again that novel CTX-M enzymes with an expanded activity towards ceftazidime through a single amino acid substitution can be identified from clinical isolates. Thus, detection of CTX-M enzymes can no longer be based solely on the resistance phenotypes of clinical isolates towards ceftazidime and cefotaxime.
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145. |
Melano RG,
Davidson RJ,
Musgrave HL,
Forward KR,
( 2006 ) Cephalosporin resistance in Klebsiella pneumoniae from Nova Scotia, Canada. PMID : 16769193 : DOI : 10.1016/j.diagmicrobio.2006.04.016 Abstract >>
From 2116 Klebsiella pneumoniae strains isolated between January 2001 and December 2002 in Nova Scotia, Canada, 25 (1.18%) showed a reduced susceptibility to cefoxitin or extended-spectrum cephalosporins. Narrow-spectrum beta-lactamase genes (bla(SHV-11), bla(SHV-1), bla(SHV-26), bla(SHV-32), bla(SHV-36), and bla(SHV-40)) were the most prevalent. Four new variants were identified (bla(LEN-17), bla(OKP-B-13), bla(OKP-B-14), and bla(OKP-A-11)), representing the 1st description of bla(OKP) in the Americas. Among the extended-spectrum beta-lactamase (ESBL) genes, bla(SHV-2), bla(SHV2a), bla(SHV-12), and bla(CTX-M-15) were detected (ESBL prevalence of 0.14%). Nineteen strains were resistant to cefoxitin (MIC, 32 to >256 microg/mL). Nevertheless, an AmpC-like activity was detected in only 1 strain, which expressed CMY-2. The combined effects of narrow-spectrum beta-lactamase production and decreased or nonexpression of OmpK35/36 porins did not account for the cefoxitin resistance observed in some of these strains.
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146. |
Chang BJ,
Huang YJ,
Chan CH,
Hsu L,
Peng HL,
Chang HY,
Yew TR,
Liu CH,
Chi S,
( 2006 ) Measurement of the adhesive force between a single Klebsiella pneumoniae type 3 fimbria and collagen IV using optical tweezers. PMID : 16997275 : DOI : 10.1016/j.bbrc.2006.08.190 Abstract >>
Type 3 fimbriae are important adhesive filaments that assist Klebsiella pneumoniae to establish an infection. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins, although the difference has not been determined physically. For this reason, the adhesive force between type 3 fimbriae and collagen IV were measured using optical tweezers. The measured force data displayed a periodic histogram thus Fourier analysis was applied to group it to extract the adhesive force of a single molecular pair. Specifically, we showed that grouping should begin with an offset at the first half of the period. Finally, we first present the adhesive force between each mrkD(V2)-, mrkD(V3)-, and mrkD(V4)-expressed fimbriae and collagen IV is 2.03, 3.79, and 2.87 pN, respectively. This result can be referred to further research on mrkD allelic effect on bacteria infection.
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147. |
Zheng P,
Sun J,
van den Heuvel J,
Zeng AP,
( 2006 ) Discovery and investigation of a new, second triose phosphate isomerase in Klebsiella pneumoniae. PMID : 16697481 : DOI : 10.1016/j.jbiotec.2006.03.034 Abstract >>
In this study, a tpi1 gene encoding for the enzyme triose phosphate isomerase in Klebsiella pneumoniae DSM2026 was knocked out in an effort to metabolically engineer this strain as a model system for the production of 1,3-propanediol. Investigations of the tpi1 knockout mutant led to the discovery of a second tpi gene (tpi2) in this organism. The new tpi2 gene was cloned and sequenced. The coding region of the tpi2 gene contains 795bp (base pairs) and the deduced protein consists of 265 amino acids. Sequence comparison of TPI2 proteins in different organisms revealed the presence of a highly conserved signature A-Y-E-P-V-W-A-I-G-[EDVS]-[GKNASH], which is nearly the same as the reported TPI consensus signature. The tpi1 gene of K. pneumoniae DSM2026 shows a high sequence similarity to that of E. coli, whereas, the tpi2 gene resembles more its relatives in the alpha-proteobacteria, suggesting that they evolve from different ancestors. The overexpression of the tpi2 gene restores the growth deficiency of tpi1 knockout mutant on the minimal medium containing glucose or glycerol. Furthermore, the catalytic activity of this new triose phosphate isomerase was confirmed in both tpi1 knockout mutant and tpi2 over-expressing strain by enzyme assays. For the first time, the co-existence of two tpi genes in an enteric bacterium is experimentally confirmed.
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148. |
Totir MA,
Padayatti PS,
Helfand MS,
Carey MP,
Bonomo RA,
Carey PR,
van den Akker F,
( 2006 ) Effect of the inhibitor-resistant M69V substitution on the structures and populations of trans-enamine beta-lactamase intermediates. PMID : 17002290 : DOI : 10.1021/bi060990m PMC : PMC2596060 DOI : 10.1021/bi060990m PMC : PMC2596060 Abstract >>
The objective of this study was to determine the molecular factors that lead to beta-lactamase inhibitor resistance for the M69V variant in SHV-1 beta-lactamase. With mechanism-based inhibitors, the beta-lactamase forms an acyl-enzyme intermediate that consists of a trans-enamine derivative in the active site. This study focuses on these intermediates by introducing the E166A mutation that greatly retards deacylation. Thus, by comparing the properties of the E166A and M69V/E166A forms, we can explore the consequences of the resistance mutation at the level of the enamine acyl-enzyme forms. The reactions between the beta-lactamase and the inhibitors tazobactam, sulbactam, and clavulanic acid are followed in single crystals of the enzymes by using a Raman microscope. The resulting Raman difference spectroscopic data provide detailed information about conformational events involving the enamine species as well as an estimate of their populations. The Raman difference spectra for each of the inhibitors in the E166A and M69V/E166A variants are very similar. In particular, detailed analysis of the main enamine Raman vibration near 1595 cm(-1) reveals that the structure and flexibility of the enamine fragments are essentially identical for each of the three inhibitors in E166A and in the M69V/E166A double mutant. This finding is in accord with the X-ray-derived structures, presented herein at 1.6-1.75 A resolution, of the trans-enamine intermediates formed by the three inhibitors in M69V/E166A. However, a comparison of Raman results for M69V/E166A and E166A shows that the M69V mutation results in a 40%, 25%, and negligible reductions in the enamine population when the beta-lactamase crystals are soaked in 5 mM tazobactam, clavulanic acid, and sulbactam solutions, respectively. The levels of enamine from tazobactam and clavulanic acid can be increased by increasing the concentrations of inhibitor in the mother liquor. Thus, the sensitivity of population levels to the inhibitor concentration in the mother liquor focuses attention on the properties of the encounter complex preceding acylation. It is proposed that for small ligands, such as tazobactam, sulbactam, and clavulanic acid, the positioning of the lactam ring in the active site in the correct orientation for acylation is only one of a number of poorly defined conformations. For tazobactam and clavulanic acid, the correctly oriented encounter complex is even less likely in the M69V variant, leading to a reduction in the level of inhibition of the enzyme via formation of the acyl-enzyme intermediate and the onset of resistance. Analysis of the X-ray structures of the three intermediates in M69V/E166A demonstrates that, compared to the structures for the E166A form, the oxyanion hole becomes smaller, providing one explanation for why acylation may be less efficient following the M69V substitution.
|
149. |
Allen BL,
Gerlach GF,
Clegg S,
( 1991 ) Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae. PMID : 1670938 : DOI : 10.1128/jb.173.2.916-920.1991 PMC : PMC207091 Abstract >>
The nucleotide sequence of six genes involved in the expression of type 3 fimbriae of Klebsiella pneumoniae was determined. In addition to the genes that encode the fimbrial subunit (mrkA) and adhesion (mrkD), the mrkB, mrkC, and mrkE genes appear to be involved in assembly of the fimbrial filament and regulation of type 3 fimbrial expression. The mrkF gene product is required to maintain the stability of the fimbrial filament on the cell surface.
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150. |
Cambau E,
Lascols C,
Sougakoff W,
Bébéar C,
Bonnet R,
Cavallo JD,
Gutmann L,
Ploy MC,
Jarlier V,
Soussy CJ,
Robert J,
( 2006 ) Occurrence of qnrA-positive clinical isolates in French teaching hospitals during 2002-2005. PMID : 16961639 : DOI : 10.1111/j.1469-0691.2006.01529.x Abstract >>
Bacteria harbouring the novel qnrA plasmid-mediated mechanism of quinolone resistance have been described in different countries, but the frequency of their occurrence has not been investigated. In total, 1,468 clinical isolates of Enterobacteriaceae with quinolone resistance or extended-spectrum beta-lactamase (ESBL) phenotypes were collected from eight teaching hospitals in France during 2002-2005 and screened for qnrA. Overall, 28 isolates (22 Enterobacter cloacae, three Klebsiella pneumoniae, one Citrobacter freundii, one Klebsiella oxytoca and one Proteus mirabilis) were positive for qnrA, representing 1.9% of all isolates, 3.3% of ESBL-producing isolates (22% of the E. cloacae isolates) and 0% of non-ESBL-producing isolates. The prevalence of qnrA among consecutive ESBL-producing isolates in 2004 from the eight hospitals was 2.8% (18/639). Of the qnrA-positive isolates, 100% were intermediately-resistant or resistant to nalidixic acid, and 75% to ciprofloxacin. Twenty-one of the 22 qnrA-positive E. cloacae isolates were obtained from two hospitals in the Paris area, and molecular typing and plasmid content analysis showed clonal relationships for five, three and two isolates, respectively. The qnrA genetic environment was similar to that of the In36 integron. The remaining two isolates had qnrA variants (30 and 29 nucleotide differences, respectively, compared with the original sequence) and an unknown genetic environment. The ESBL gene associated with qnrA was bla(SHV-12) in most of the isolates, but bla(PER-1) and bla(SHV-2a) were found in two isolates. In France, it appears that qnrA-positive isolates are predominantly E. cloacae isolates producing SHV-12, and may be associated with the dissemination of an In36-like integron.
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151. |
Chen YT,
Shu HY,
Li LH,
Liao TL,
Wu KM,
Shiau YR,
Yan JJ,
Su IJ,
Tsai SF,
Lauderdale TL,
( 2006 ) Complete nucleotide sequence of pK245, a 98-kilobase plasmid conferring quinolone resistance and extended-spectrum-beta-lactamase activity in a clinical Klebsiella pneumoniae isolate. PMID : 16940067 : DOI : 10.1128/AAC.00456-06 PMC : PMC1635178 Abstract >>
A plasmid containing the qnrS quinolone resistance determinant and the gene encoding the SHV-2 beta-lactamase has been discovered from a clinical Klebsiella pneumoniae strain isolated in Taiwan. The complete 98-kb sequence of this plasmid, designated pK245, was determined by using a whole-genome shotgun approach. Transfer of pK245 conferred low-level resistance to fluoroquinolones in electroporant Escherichia coli epi300. The sequence of the immediate region surrounding qnrS in pK245 is nearly identical (>99% identity) to those of pAH0376 from Shigella flexneri and pINF5 from Salmonella enterica serovar Infantis, the two other qnrS-carrying plasmids reported to date, indicating a potential common origin. Other genes conferring resistance to aminoglycosides (aacC2, strA, and strB), chloramphenicol (catA2), sulfonamides (sul2), tetracycline (tetD), and trimethoprim (dfrA14) were also detected in pK245. The dfrA14 gene is carried on a class I integron. Several features of this plasmid, including three separate regions containing putative replicons, a partitioning-control system, and a type II restriction modification system, suggest that it may be able to replicate and adapt in a variety of hosts. Although no critical conjugative genes were detected, multiple insertion sequence elements were found scattered throughout pK245, and these may facilitate the dissemination of the antimicrobial resistance determinants. We conclude that pK245 is a chimera which acquired its multiple antimicrobial resistance determinants horizontally from different sources. The identification of pK245 plasmid expands the repertoire of the coexistence of quinolone and extended-spectrum-beta-lactam resistance determinants in plasmids carried by various species of the family Enterobacteriaceae in different countries.
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152. |
Muratani T,
Kobayashi T,
Matsumoto T,
( 2006 ) Emergence and prevalence of beta-lactamase-producing Klebsiella pneumoniae resistant to cephems in Japan. PMID : 16701983 : DOI : 10.1016/j.ijantimicag.2006.03.007 Abstract >>
Forty-six cephem-resistant Klebsiella pneumoniae strains with minimum inhibitory concentrations>8 microg/mL for cefpodoxime and cefmetazole were selected from clinical isolates obtained between 2000 and 2002 from eight hospitals on Northern Kyushu Island, Japan. We investigated the mechanisms of resistance to cephems in these 46 K. pneumoniae isolates. The results of isoelectric focusing of beta-lactamases produced by these isolates, polymerase chain reaction for detection of various Class A, Class B and Class C beta-lactamases, and determination of the sequence of the beta-lactamase structural gene showed that most of these isolates had various types of broad-spectrum beta-lactamases. Of the 46 isolates, 2 were CMY-2 beta-lactamase producers and 41 were DHA-1 beta-lactamase producers. Forty of the 41 DHA-1 beta-lactamase producers simultaneously produced SHV-12 extended-spectrum beta-lactamase (ESBL), and the remaining isolate simultaneously produced SHV-27. Furthermore, one DHA-1 and SHV-12 beta-lactamase producer also produced IMP-1 beta-lactamase. The only broad-spectrum beta-lactamase with another isolate was IMP-1. Chromosomal DNA restriction fragment analysis using XbaI suggested that nosocomial infection due to DHA-1 and SHV-12 beta-lactamase producers had occurred at two centres. This is the first report of nosocomial infection due to DHA-1 beta-lactamase-producing K. pneumoniae including other plasmid-encoded AmpC beta-lactamases in Japan. The mechanisms of resistance of 44 of the 46 isolates to cephalosporins and cephamycins were ESBL production and/or plasmid-encoded AmpC beta-lactamase and/or IMP-1 beta-lactamase production. For two isolates, the mechanism of resistance to could not be identified. These results show that it is necessary to minimise the prevalence of these resistant strains as it will be a very serious problem if organisms producing these broad-spectrum beta-lactamases increase in clinical situations. It is important to detect these strains sooner and to perform rigorous infection control earlier.
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153. |
Yeh KM,
Chang FY,
Fung CP,
Lin JC,
Siu LK,
( 2006 ) magA is not a specific virulence gene for Klebsiella pneumoniae strains causing liver abscess but is part of the capsular polysaccharide gene cluster of K. pneumoniae serotype K1. PMID : 16687604 : DOI : 10.1099/jmm.0.46368-0 Abstract >>
N/A
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154. |
Mendonça N,
Ferreira E,
Caniça M,
( 2006 ) Occurrence of a novel SHV-type enzyme (SHV-55) among isolates of Klebsiella pneumoniae from Portuguese origin in a comparison study for extended-spectrum beta-lactamase-producing evaluation. PMID : 16938422 : DOI : 10.1016/j.diagmicrobio.2006.06.023 Abstract >>
Fifty-five isolates of Klebsiella pneumoniae were evaluated for extended-spectrum beta-lactamase (ESBL) detection and confirmation, using MIC testing by agar dilution, broth microdilution, and the ESBL E-Test (AB Biodisk, Solna, Sweden), according to reference laboratory criteria (RLC) and Clinical and Laboratory Standards Institute (CLSI) guidelines. The RLC classify as ESBL producers those strains for which any MIC of cephalosporins is 3-fold lower in the presence of 2 mug/mL of clavulanate. The E-Test was the only to show 100% sensitivity and specificity to detect ESBL-producer strains with either set of guidelines. MIC determination by agar dilution or broth microdilution, using NCCLS guidelines, showed sensitivity of 92.9%. Nucleotide sequencing allowed the identification of a new ESBL (SHV-55). Overall, this gold standard method confirmed the production of 18 ESBL producers, 36 non-ESBL producers, from which 9 were false ESBL producers (suggesting hyperproduction) and 1 presumptive ESBL TEM-derived. New guidelines for ESBL detection and reliable methods of ESBL identification are required.
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155. |
Aubert D,
Naas T,
Héritier C,
Poirel L,
Nordmann P,
( 2006 ) Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of beta-lactam resistance genes. PMID : 16952941 : DOI : 10.1128/JB.00375-06 PMC : PMC1595497 Abstract >>
IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla(VEB-1) and bla(OXA-48). 5' Rapid amplification of cDNA ends experiments revealed that expression of these beta-lactamase genes was driven by the outward-directed promoter, P(out), located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.
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156. |
Lee KY,
Hopkins JD,
Syvanen M,
( 1991 ) Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae. PMID : 1662755 : DOI : 10.1111/j.1365-2958.1991.tb00826.x Abstract >>
A new aminoglycoside resistance gene (aphA1-IAB) confers high-level resistance to neomycin. The sequence of aphA1-IAB is closely related to aphA1 found in the transposons Tn4352, Tn903 and Tn602. For example, aphA1-IAB differs from aphA1-903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA1-IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA1-903 enzyme. A parsimonious phylogenetic tree suggests that aphA1-IAB evolved from an ancestral form that is closely related or identical to the aphA1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA1-IAB, as well as its convergence toward aphA3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA1-IAB evolved under the selective pressure of neomycin use in relatively recent times.
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157. |
Yong D,
Choi YS,
Roh KH,
Kim CK,
Park YH,
Yum JH,
Lee K,
Chong Y,
( 2006 ) Increasing prevalence and diversity of metallo-beta-lactamases in Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae from Korea. PMID : 16641469 : DOI : 10.1128/AAC.50.5.1884-1886.2006 PMC : PMC1472216 Abstract >>
Among imipenem-nonsusceptible isolates, acquired metallo-beta-lactamase genes were detected in 36 of 581 (6.2%) Pseudomonas aeruginosa isolates, 42 of 44 (95.4%) other Pseudomonas species, and 136 of 513 (26.5%) Acinetobacter species from 2003 to 2004 at a Korean hospital. Overall, bla(VIM-2)-like genes were the most prevalent and were also detected in Enterobacteriaceae, including Klebsiella pneumoniae.
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158. |
Seiflein TA,
Lawrence JG,
( 2006 ) Two transsulfurylation pathways in Klebsiella pneumoniae. PMID : 16885444 : DOI : 10.1128/JB.00347-06 PMC : PMC1540059 Abstract >>
In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-gamma-synthase and cystathionine-beta-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-beta-synthase and cystathionine-gamma-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.
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159. |
Alves MS,
Dias RC,
de Castro AC,
Riley LW,
Moreira BM,
( 2006 ) Identification of clinical isolates of indole-positive and indole-negative Klebsiella spp. PMID : 16928968 : DOI : 10.1128/JCM.00940-06 PMC : PMC1594763 Abstract >>
Biochemical methods employed to classify bacterial species have limitations and may have contributed to the taxonomic complexity recently reported for the genus Klebsiella. The objective of the present study was to apply a simple biochemical test panel to classify a collection of human Klebsiella isolates. We found that with only three additional tests, it is possible to place most isolates in a defined species. Analysis of a 512-bp sequence of the rpoB gene was used as the reference. A total of 16 conventional and 4 supplementary tests were used to evaluate 122 recent isolates identified as Klebsiella from 120 patients, isolated at the clinical laboratory of a university hospital in Minas Gerais, Brazil. Of these, 102 (84%) isolates were identified as Klebsiella pneumoniae or Klebsiella variicola, 19 (15%) as Klebsiella oxytoca, and 1 (1%) as Raoultella planticola. Enterobacterial repetitive intergenic consensus-PCR typing revealed a diversity of genotypes. rpoB gene sequencing confirmed the phenotypic identification and detected five K. variicola isolates among the K. pneumoniae/K. variicola group. Three additional tests that include growth at 10 degrees C and histamine and d-melezitose assimilation should be considered essential tests for the typing of Klebsiella isolates.
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160. |
Jacoby GA,
Walsh KE,
Mills DM,
Walker VJ,
Oh H,
Robicsek A,
Hooper DC,
( 2006 ) qnrB, another plasmid-mediated gene for quinolone resistance. PMID : 16569827 : DOI : 10.1128/AAC.50.4.1178-1182.2006 PMC : PMC1426915 Abstract >>
A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 beta-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to >or=95% homogeneity. As little as 5 pM of QnrB-His6 protected purified DNA gyrase against inhibition by 2 microg/ml (6 microM) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 beta-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.
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161. |
Yu WL,
Chen SC,
Hung SW,
Chuang YC,
Chung JG,
Chen IC,
Wu LT,
( 2006 ) Genetic association of blaSHV-5 with transposable elements IS26 and IS5 in Klebsiella pneumoniae from Taiwan. PMID : 16842580 : DOI : 10.1111/j.1469-0691.2006.01488.x Abstract >>
A cloned 5,248-bp EcoRI fragment from the Klebsiella pneumoniae transferable plasmid pKP53 (> 70 kb) containing bla(SHV-5) was sequenced. Insertion sequences IS26 and IS5 were found downstream from bla(SHV-5). The DNA sequences of the genetic environment surrounding bla(SHV-5) were homologous to plasmid p1658/97 from Escherichia coli, containing a truncated recF gene and a truncated deoR gene upstream and downstream from bla(SHV-5), respectively. RecF may be involved in bla(SHV-5) translocation to the plasmid by RecF-dependent recombination. This novel genetic environment may be associated with the successful proliferation and/or expression of SHV-5 in K. pneumoniae strains from Taiwan.
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162. |
Boddicker JD,
Anderson RA,
Jagnow J,
Clegg S,
( 2006 ) Signature-tagged mutagenesis of Klebsiella pneumoniae to identify genes that influence biofilm formation on extracellular matrix material. PMID : 16861646 : DOI : 10.1128/IAI.00129-06 PMC : PMC1539622 Abstract >>
Klebsiella pneumoniae causes urinary tract infections, respiratory tract infections, and septicemia in susceptible individuals. Strains of Klebsiella frequently produce extended-spectrum beta-lactamases, and infections with these strains can lead to relatively high mortality rates (approximately 15%). Other virulence factors include production of an antiphagocytic capsule and outer membrane lipopolysaccharide (LPS), which mediates serum resistance, as well as fimbriae on the surface of the bacteria. Type 1 fimbriae mediate adherence to many types of epithelial cells and may facilitate adherence of the bacteria to the bladder epithelium. Type 3 fimbriae can bind in vitro to the extracellular matrix of urinary and respiratory tissues, suggesting that they mediate binding to damaged epithelial surfaces. In addition, type 3 fimbriae are required for biofilm formation by Klebsiella pneumoniae on plastics and human extracellular matrix; thus, they may facilitate the formation of treatment-resistant biofilm on indwelling plastic devices, such as catheters and endotracheal tubing. The presence of these devices may cause tissue damage, allowing Klebsiella to grow as a biofilm on exposed tissue basement membrane components. Though in vivo biofilm growth may be an important step in the infection process, little is known about the genetic factors required for biofilm formation by Klebsiella pneumoniae. Thus, we performed signature-tagged mutagenesis to identify factors produced by K. pneumoniae strain 43816 that are required for biofilm formation. We identified mutations in the cps capsule gene cluster, previously unidentified transcriptional regulators, fimbrial, and sugar phosphotransferase homologues, as well as genetic loci of unknown function, that affect biofilm formation.
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163. |
Loli A,
Tzouvelekis LS,
Tzelepi E,
Carattoli A,
Vatopoulos AC,
Tassios PT,
Miriagou V,
( 2006 ) Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1. PMID : 16870645 : DOI : 10.1093/jac/dkl302 Abstract >>
To elucidate the mechanisms responsible for the diversity of beta-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals. Five VPKP clinical isolates were studied. MICs of beta-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS-PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. beta-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with bla(VIM)- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing. Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred beta-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla(VIM)- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of bla(VIM-1). Cloning and sequencing showed In-e541-like integrons truncated at the 5'CS by insertion of IS26 elements at two different positions. A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the bla(VIM-1)-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.
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164. |
Villegas MV,
Lolans K,
Correa A,
Suarez CJ,
Lopez JA,
Vallejo M,
Quinn JP,
N/A N/A,
( 2006 ) First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America. PMID : 16870793 : DOI : 10.1128/AAC.00186-06 PMC : PMC1538657 Abstract >>
The plasmid-mediated class A carbapenemase KPC-2 was isolated from unrelated Klebsiella pneumoniae isolates in Medellin, Colombia. These KPC enzymes are the first from South America and the second isolation outside of the United States. The expanding geographic spread of KPC carbapenemases underscores the importance of clinical recognition of these enzymes.
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165. |
Yu Y,
Ji S,
Chen Y,
Zhou W,
Wei Z,
Li L,
Ma Y,
( 2007 ) Resistance of strains producing extended-spectrum beta-lactamases and genotype distribution in China. PMID : 16533535 : DOI : 10.1016/j.jinf.2006.01.014 Abstract >>
To investigate the resistance of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases (ESBLs) and the genotyping of ESBLs in China. MICs of 12 antibiotics against 50 strains (by random selection) of ESBLs-producing E. coli and K. pneumoniae were determined by E-test. The genotypes of ESBLs were analyzed by PCR, DNA sequencing and isoelectric focusing. The susceptibility rate of 50 isolates was 100% in imipenem, 60%-80% in cefoperazone/sulbactam, ceftazidime and piperacillin/tazobactam, and lower in other antimicrobial agents tested. Only 6.0% of the isolates were sensitive to cefotaxime. Four hundred and forty-seven of 509 isolates had been confirmed the genotype of ESBLs. Four hundred and sixteen strains produced only one type of ESBLs, including CTX-M-14 (271 strains), CTX-M-3 (70 strains), CTX-M-24 (35 strains), CTX-M-22 (8 strains), CTX-M-15 (4 strains), CTX-M-9 (4 strains), CTX-M-28 (3 strains), CTX-M-12 (1 strain), CTX-M-13 (1 strain), CTX-M-27 (1 strain), CTX-M-29 (1 strain), SHV-12 (10 strains), SHV-5 (4 strains), SHV-2 (2 strains), and SHV-9 (1 strain). Thirty isolates carried two or three types of ESBLs, and producing CTX-M-14 and CTX-M-3 together were the most common type. The resistance of E. coli and K. pneumonia producing ESBLs in China was a serious issue and CTX-M type ESBLs were the most common genotype. CTX-M-14 was the predominant genotype. Some isolates produced two or three ESBLs.
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166. |
Bagattini M,
Crivaro V,
Di Popolo A,
Gentile F,
Scarcella A,
Triassi M,
Villari P,
Zarrilli R,
( 2006 ) Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit. PMID : 16531430 : DOI : 10.1093/jac/dkl077 Abstract >>
To investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy. Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating. Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified beta-lactamase genes identified a novel bla(TEM) ESBL (bla(TEM-136)) along with bla(SHV-1) in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and bla(TEM-1) and bla(SHV-12) in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an approximately 80 kb plasmid containing bla(SHV-12) and bla(TEM-1), was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 x 10(-6) and 1 x 10(-6) cfu/recipient cell, respectively. The selection of ESBL-producing clones and the transfer of the bla(SHV-12) ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.
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167. |
Chuang YP,
Fang CT,
Lai SY,
Chang SC,
Wang JT,
( 2006 ) Genetic determinants of capsular serotype K1 of Klebsiella pneumoniae causing primary pyogenic liver abscess. PMID : 16453259 : DOI : 10.1086/499968 Abstract >>
Primary pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae is an emerging infectious disease. Capsular serotype K1 and the magA gene have been reported to be associated with this disease. The prevalence of magA was determined by polymerase chain reaction (PCR). The sequences of the magA flanking region were completed by inverse PCR and direct sequencing. Serotyping was performed by double immunodiffusion. Insertion mutations and trans-complementation were used to define the K1 genetic determination region. Thirty-five of 42 strains from patients with PLA were magA positive, whereas only 1 of 32 non-PLA strains was magA positive. All 36 magA-positive strains were serotype K1, and the 38 magA-negative strains were not (36/36 vs. 0/38; P<.0001). Sequencing of the magA flanking region revealed a putative capsular polysaccharide synthesis (cps) region; this region was 25 kb in length and contained 20 open reading frames (ORFs); of these ORFs, 9 were cotranscribed as part of an operon and differed from both MGH78578 and the Chedid strain. Mutation of 4 genes in this region turned the mutant strains anti-K1 negative. Trans-complementation restored the K1 phenotype. The operon containing magA is responsible for capsular serotype K1 of K. pneumoniae. Several loci in the operon are unique determinants of K1 strains.
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168. |
Schmid K,
Ebner R,
Jahreis K,
Lengeler JW,
Titgemeyer F,
( 1991 ) A sugar-specific porin, ScrY, is involved in sucrose uptake in enteric bacteria. PMID : 1649946 : DOI : 10.1111/j.1365-2958.1991.tb00769.x Abstract >>
During the molecular analysis of a plasmid-coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al., 1988). Loss of this protein (Mr 58 kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km for sucrose transport in whole cells from 10 microM in wild-type cells to 300 microM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild-type cells, which remained unchanged at about 10 microM, and reflects the activity of the sucrose-specific Enzymell of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid-coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins.
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169. |
Lupa B,
Lyon D,
Gibbs MD,
Reeves RA,
Wiegel J,
( 2005 ) Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases. PMID : 15979273 : DOI : 10.1016/j.ygeno.2005.05.002 Abstract >>
Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous (approximately 15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5alpha (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity.
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170. |
Wachino J,
Kurokawa H,
Suzuki S,
Yamane K,
Shibata N,
Kimura K,
Ike Y,
Arakawa Y,
( 2006 ) Horizontal transfer of blaCMY-bearing plasmids among clinical Escherichia coli and Klebsiella pneumoniae isolates and emergence of cefepime-hydrolyzing CMY-19. PMID : 16436707 : DOI : 10.1128/AAC.50.2.534-541.2006 PMC : PMC1366887 Abstract >>
Nine Escherichia coli and 5 Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital between 1995 and 1997. All nine E. coli isolates and one K. pneumoniae isolate carried bla(CMY-9), while the other four K. pneumoniae isolates harbored a variant of bla(CMY-9), namely, bla(CMY-19). The pulsed-field gel electrophoresis patterns of the nine CMY-9-producing E. coli isolates were almost identical, suggesting their clonal relatedness, while those of the five K. pneumoniae isolates were divergent. Plasmid profiles, Southern hybridization, and conjugation assays revealed that the genes for the CMY-9 and the CMY-19 beta-lactamases were located on very similar conjugative plasmids in E. coli and K. pneumoniae. The genetic environment of bla(CMY-19) was identical to that of bla(CMY-9). A single amino acid substitution, I292S, adjacent to the H-10 helix region was observed between CMY-9 and CMY-19. This substitution was suggested to be responsible for the expansion of the hydrolyzing activity against several broad-spectrum cephalosporins, and this finding was consistent with the kinetic parameters determined with purified enzymes. These findings suggest that the bla(CMY-19) genes found in the four K. pneumoniae isolates might have originated from bla(CMY-9) gene following a point mutation and dispersed among genetically different K. pneumoniae isolates via a large transferable plasmid.
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171. |
Peng HL,
Wang PY,
Wu CM,
Hwang DC,
Chang HY,
( 1992 ) Cloning, sequencing and heterologous expression of a Klebsiella pneumoniae gene encoding an FAD-independent acetolactate synthase. PMID : 1644303 : DOI : 10.1016/0378-1119(92)90500-o Abstract >>
The gene encoding the valine-resistant and FAD-independent acetolactate synthase of Klebsiella pneumoniae was isolated and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and it exhibited an open reading frame of 1680 bp in length. In vivo expression of the acetolactate synthase-encoding gene in E. coli revealed a single 60-kDa protein which is consistent with the molecular weight calculated from the deduced amino acid sequence of the gene product. The gene product shares about 20-30% homology with the acetolactate synthases of E. coli, yeast and higher plants.
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172. |
Siebor E,
Péchinot A,
Duez JM,
Neuwirth C,
( 2005 ) One new LEN enzyme and two new OKP enzymes in Klebsiella pneumoniae clinical isolates and proposed nomenclature for chromosomal beta-lactamases of this species. PMID : 15980410 : DOI : 10.1128/AAC.49.7.3097-3098.2005 PMC : PMC1168668 Abstract >>
N/A
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173. |
Zarnayová M,
Siebor E,
Péchinot A,
Duez JM,
Bujdáková H,
Labia R,
Neuwirth C,
( 2005 ) Survey of Enterobacteriaceae producing extended-spectrum beta-lactamases in a Slovak hospital: dominance of SHV-2a and characterization of TEM-132. PMID : 15980402 : DOI : 10.1128/AAC.49.7.3066-3069.2005 PMC : PMC1168689 Abstract >>
Eighty-five extended-spectrum beta-lactamase-producing Enterobacteriaceae from a Slovak hospital have been studied. SHV-2a was predominant, but other variants have been detected, namely, SHV-5, SHV-12, TEM-12, TEM-15, and TEM-132, which differed from TEM-1 by amino acid substitutions R164H, E240K, and I173V and had kinetic properties similar to those of TEM-28.
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174. |
Pourreza A,
Witherspoon M,
Fox J,
Newmark J,
Bui D,
Tolmasky ME,
( 2005 ) Mutagenesis analysis of a conserved region involved in acetyl coenzyme A binding in the aminoglycoside 6'-N-acetyltransferase type Ib encoded by plasmid pJHCMW1. PMID : 15980378 : DOI : 10.1128/AAC.49.7.2979-2982.2005 PMC : PMC1168681 Abstract >>
Alanine scanning of motif A in the pJHCMW1-encoded aminoglycoside 6'-N-acetyltransferase type Ib identified amino acids important for the ability of the enzyme to confer wild-type levels of resistance to kanamycin and amikacin. The replacement of two amino acids, D117 or L120, with alanine residues resulted in complete loss of the resistance phenotype.
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175. |
Verdet C,
Benzerara Y,
Gautier V,
Adam O,
Ould-Hocine Z,
Arlet G,
( 2006 ) Emergence of DHA-1-producing Klebsiella spp. in the Parisian region: genetic organization of the ampC and ampR genes originating from Morganella morganii. PMID : 16436717 : DOI : 10.1128/AAC.50.2.607-617.2006 PMC : PMC1366880 Abstract >>
Eleven Klebsiella pneumoniae clinical isolates and one Klebsiella oxytoca clinical isolate showing various pulsed-field gel electrophoresis types and producing an inducible DHA-1 class C beta-lactamase were isolated in the Parisian region between 1998 and 2003. The aim of this study was to compare the genetic organization of the bla(DHA-1) genes in this collection of clinical isolates. In four isolates, the Morganella morganii-derived genomic region containing bla(DHA-1) was inserted in an entire complex sul1-type integron, including a region common to In6-In7 (CR1), as previously described in a bla(DHA-1)-producing Salmonella enterica serovar Enteritidis KF92 isolate from Saudi Arabia in 1992. Different gene cassette arrays were characterized in each of these integrons. In two of them, an additional 10-kb fragment was inserted between the CR1 and the M. morganii-derived region and was similar to the sap (ABC transporter family) and psp (phage shock protein) operons originated from Salmonella enterica serovar Typhimurium. The length of the M. morganii region was variable, suggesting that several independent recombination events have occurred and that open reading frame orf513 encodes a recombinase involved in the mobilization of the resistance genes. The genetic organization of bla(DHA-1) was identical in the eight other isolates. This structure is likely derived from a complex integron following the insertion of IS26, leading to the deletion of the first part of integron. The horizontal transfer of one plasmid carrying that truncated integron was shown for seven of these isolates.
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176. |
Yong D,
Lim Y,
Song W,
Choi YS,
Park DY,
Lee H,
Yum JH,
Lee K,
Kim JM,
Chong Y,
( 2005 ) Plasmid-mediated, inducible AmpC beta-lactamase (DHA-1)-producing Enterobacteriaceae at a Korean hospital: wide dissemination in Klebsiella pneumoniae and Klebsiella oxytoca and emergence in Proteus mirabilis. PMID : 15936167 : DOI : 10.1016/j.diagmicrobio.2005.03.008 Abstract >>
The aim of the study was to investigate the phenotypic and genetic characteristics of recently emerging cefoxitin-resistant and induction-positive isolates of Escherichia coli, Klebsiella species, and Proteus mirabilis. Strains of Enterobacteriaceae were isolated at a Korean tertiary care hospital between June and December 2002. Induction was tested using cefoxitin and aztreonam disks, the blaDHA allele was detected by PCR, and pulsed-field gel electrophoresis (PFGE) patterns were also analyzed. Among the cefoxitin-resistant isolates, 2.7% of E. coli, 21.1% of Klebsiella pneumoniae, 32.0% of Klebsiella oxytoca, and 8.3% of P. mirabilis isolates showed induction, and were blaDHA-1 allele positive. To the best of our knowledge, this is the first report of blaDHA-1 in P. mirabilis. The MICs of ceftazidime, cefotaxime, and aztreonam increased significantly by higher inoculum, suggesting that their clinical usefulness is limited. Presence of multiple PFGE patterns and identical patterns in some isolates suggest that the widely disseminated blaDHA-1 in Klebsiella species was because of both horizontal and clonal spread.
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177. |
Chavan M,
Rafi H,
Wertz J,
Goldstone C,
Riley MA,
( 2005 ) Phage associated bacteriocins reveal a novel mechanism for bacteriocin diversification in Klebsiella. PMID : 15883889 : DOI : 10.1007/s00239-004-0263-9 Abstract >>
Ninety-six isolates of Klebsiella pneumoniae and K. oxytoca were recovered from wild mammals in Australia. 14.6% of these bacteria produce killing phenotypes that suggest the production of bacteriocin toxins. Cloning and sequencing of the gene clusters encoding two of these killing phenotypes revealed two instances of a bacteriocin associated with a bacteriophage gene, the first such genetic organization described. The newly identified klebicin C gene cluster was discovered in both K. pneumoniae and K. oxytoca. The newly identified klebicin D gene cluster was detected in K. oxytoca. Protein sequence comparisons and phylogenetic inference suggest that klebicin C is most closely related to the rRNase group of colicins (such as colicin E4), while klebicin D is most closely related to the tRNase group of colicins (such as colicin D). The klebicin C and D gene clusters have similar genetic and regulatory organizations. In both cases, an operon structure is inferred consisting of a phage-associated open reading frame and klebicin activity and associated immunity genes. This novel bacteriophage/bacteriocin organization may provide a novel mechanism for the generation of bacteriocin diversity in Klebsiella.
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178. |
Hernández JR,
Martínez-Martínez L,
Cantón R,
Coque TM,
Pascual A,
N/A N/A,
( 2005 ) Nationwide study of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases in Spain. PMID : 15855544 : DOI : 10.1128/AAC.49.5.2122-2125.2005 PMC : PMC1087612 Abstract >>
Clonal dissemination of extended-spectrum beta-lactamases (ESBL) in 170 Escherichia coli isolates and 70 Klebsiella pneumoniae isolates from a nationwide study of 40 Spanish centers in 2000 was not observed in most centers. The most prevalent ESBL were CTX-M-9 (27.3%), SHV-12 (23.9%), and CTX-M-14 (20.5%) for E. coli and TEM-3 (16.7%) and TEM-4 (25%) for K. pneumoniae. A new ESBL, TEM-133, with mutations L21F, E104K, and R164S, was identified.
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179. |
Regué M,
Izquierdo L,
Fresno S,
Piqué N,
Corsaro MM,
Naldi T,
De Castro C,
Waidelich D,
Merino S,
Tomás JM,
( 2005 ) A second outer-core region in Klebsiella pneumoniae lipopolysaccharide. PMID : 15937181 : DOI : 10.1128/JB.187.12.4198-4206.2005 PMC : PMC1151721 Abstract >>
Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide beta-Glcp(1-6)-alpha-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide alpha-Hep(1-4)-alpha-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-D-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model.
|
180. |
Pournaras S,
Ikonomidis A,
Tzouvelekis LS,
Tokatlidou D,
Spanakis N,
Maniatis AN,
Legakis NJ,
Tsakris A,
( 2005 ) VIM-12, a novel plasmid-mediated metallo-beta-lactamase from Klebsiella pneumoniae that resembles a VIM-1/VIM-2 hybrid. PMID : 16304191 : DOI : 10.1128/AAC.49.12.5153-5156.2005 PMC : PMC1315972 Abstract >>
A transferable plasmid from Klebsiella pneumoniae carried a class 1 integron containing bla(VIM-12), a novel bla(VIM)-type gene, flanked by two copies of aacA7. bla(VIM-12) was clustered between bla(VIM-1) and bla(VIM-2) and differed from bla(VIM-1) by 18 nucleotides that were all located at the 3' end and matched the corresponding nucleotides in bla(VIM-2). The bla(VIM-12)-associated 59-base element was identical to that described in bla(VIM-2) alleles.
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181. |
Fevre C,
Passet V,
Weill FX,
Grimont PA,
Brisse S,
( 2005 ) Variants of the Klebsiella pneumoniae OKP chromosomal beta-lactamase are divided into two main groups, OKP-A and OKP-B. PMID : 16304190 : DOI : 10.1128/AAC.49.12.5149-5152.2005 PMC : PMC1315974 Abstract >>
Two bla(OKP) subgroups were found, diverging by 4.2%. Subgroups bla(OKP-A) (10 enzyme variants, pIs from 7.1 to 8.3) and bla(OKP-B) (11 variants, pI 7.1) showed similar antibiotic susceptibilities. Sequencing of rpoB, gyrA, and mdh demonstrated a concordant subdivision of Klebsiella pneumoniae phylogenetic group KpII into two subgroups, KpII-A and KpII-B.
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182. |
Kassis-Chikhani N,
Decré D,
Gautier V,
Burghoffer B,
Saliba F,
Mathieu D,
Samuel D,
Castaing D,
Petit JC,
Dussaix E,
Arlet G,
( 2006 ) First outbreak of multidrug-resistant Klebsiella pneumoniae carrying blaVIM-1 and blaSHV-5 in a French university hospital. PMID : 16284103 : DOI : 10.1093/jac/dki389 Abstract >>
We studied eight imipenem-resistant isolates of Klebsiella pneumoniae involved in an outbreak in a French teaching hospital. The eight isolates were recovered from clinical specimens or rectal swabs. Antibiotic susceptibilities were determined using standard agar diffusion and dilution methods including synergy tests. PFGE was used to study the relatedness of isolates. Genes encoding beta-lactamases were characterized by transfer assays, specific amplification and cloning. The eight isolates were closely related by PFGE analysis and highly related to a K. pneumoniae strain from Greece. They were highly resistant to beta-lactams, including aztreonam and imipenem (MIC > or =32 mg/L), and were positive by the imipenem-EDTA disc synergy test. Isolates were also resistant to aminoglycosides, newer quinolones and sulfamethoxazole, and showed an intermediate level of resistance to tetracycline. VIM-1 and SHV-5 beta-lactamases were revealed in all isolates by PCR. The analysis of plasmid contents of Escherichia coli DH10B electroporants expressing the VIM-1 beta-lactamase or the SHV-5 beta-lactamase confirmed that the two enzymes were coded by two different plasmids. The bla(VIM-1) gene was part of a class 1 integron that also included aac6, dhfrI and aadA genes and was similar to those reported from strains isolated in Greece. This study confirms the potential risk of spread of multiresistant bacteria with international transfer of patients.
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183. |
Beis K,
Srikannathasan V,
Liu H,
Fullerton SW,
Bamford VA,
Sanders DA,
Whitfield C,
McNeil MR,
Naismith JH,
( 2005 ) Crystal structures of Mycobacteria tuberculosis and Klebsiella pneumoniae UDP-galactopyranose mutase in the oxidised state and Klebsiella pneumoniae UDP-galactopyranose mutase in the (active) reduced state. PMID : 15843027 : DOI : 10.1016/j.jmb.2005.02.057 PMC : PMC3326527 Abstract >>
Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the d-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH- co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25 A. The structures of Klebsiella pneumoniae mutase with FAD and with FADH- bound have been determined to 2.2 A and 2.35 A resolution, respectively. This is the first report of the FADH(-)-containing structure. Two flavin-dependent mechanisms for the enzyme have been proposed, one, which involves a covalent adduct being formed at the flavin and the other based on electron transfer. Using our structural data, we have examined the two mechanisms. The electron transfer mechanism is consistent with the structural data, not surprisingly, since it makes fewer demands on the precise positioning of atoms. A model based on a covalent adduct FAD requires repositioning of the enzyme active site and would appear to require the isoalloxazine ring of FADH- to buckle in a particular way. However, the FADH- structure reveals that the isoalloxazine ring buckles in the opposite sense, this apparently requires the covalent adduct to trigger profound conformational changes in the protein or to buckle the FADH- opposite to that seen in the apo structure.
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184. |
Soge OO,
Queenan AM,
Ojo KK,
Adeniyi BA,
Roberts MC,
( 2006 ) CTX-M-15 extended-spectrum (beta)-lactamase from Nigerian Klebsiella pneumoniae. PMID : 16319181 : DOI : 10.1093/jac/dki429 Abstract >>
In this study, extended-spectrum beta-lactamases (ESBLs) were characterized from 30 selected multidrug-resistant Klebsiella pneumoniae strains isolated from patients with community-acquired urinary tract infections from Southwest Nigeria. The beta-lactamases were phenotypically characterized using isoelectric focusing, genotypically characterized using PCR assays and hybridization of the PCR products. Two of the bla(CTX-M) genes were completely sequenced. The location of the CTX-M-type genes was determined using transformation, DNA-DNA hybridization, PCR assays and hybridization of the PCR products from the Escherichia coli transformants. All 30 isolates produced at least one beta-lactamase. Seventeen of the isolates were resistant to cefotaxime, and had > or =100-fold reduction in susceptibility with cefotaxime plus clavulanic acid (4 mg/L), indicating the presence of an ESBL. The 17 isolates were shown to have bla(CTX-M) genes that were associated with large plasmids (> or =58 kb), which also carried a tetracycline resistance gene, tet(A), and various aminoglycoside resistance genes. Two CTX-M-type genes were sequenced and had amino acid sequences indistinguishable from previously sequenced CTX-M-15 beta-lactamases. The ISEcp1 element was located upstream of bla(CTX-M-15) in the same position as previously described. In addition, 23 of the isolates produced TEM beta-lactamases, 27 produced SHV beta-lactamases and four produced AmpC beta-lactamases. Thirty K. pneumoniae produced multiple beta-lactamases, with 57% producing CTX-M enzymes. This is the first characterization of CTX-M-15-positive K. pneumoniae in Western Africa.
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185. |
Eckert C,
Gautier V,
Arlet G,
( 2006 ) DNA sequence analysis of the genetic environment of various blaCTX-M genes. PMID : 16291869 : DOI : 10.1093/jac/dki398 Abstract >>
Over a 3 year period (2000-2003) 21 Escherichia coli, 5 Klebsiella pneumoniae, 1 Serratia marcescens and 1 Proteus mirabilis producing CTX-M-type beta-lactamase were collected from five different hospitals in Paris, France. This study was conducted to analyse the genetic environment of these 28 bla(CTX-M) genes. Antimicrobial susceptibility testing was performed by the disc diffusion method and MICs of various beta-lactams were determined by an agar dilution method. PCR was used to detect and sequence alleles encoding CTX-M, TEM, SHV and CMY enzymes. The genetic environment was analysed by amplification and direct sequencing using various set of PCR primers or cloning in pBK-CMV. Sequence analysis revealed that these isolates contained seven different bla(CTX-M) genes: bla(CTX-M-1) (4 strains), bla(CTX-M-2) (2 strains), bla(CTX-M-3) (4 strains), bla(CTX-M-9) (1 strain), bla(CTX-M-14) (5 strains), bla(CTX-M-15) (11 strains), bla(CTX-M-24) (1 strain). TEM-1 was associated with CTX-M-type enzymes in 15 isolates. Two strains produced both CTX-M-15 and SHV-2 or CTX-M-14 and CMY-2. In 25 strains the insertion sequence ISEcp1 was located upstream of the 5' end of the bla(CTX-M) gene. Among these strains, in five isolates, ISEcp1 was disrupted by insertion sequences such as IS26 (in three of them) or IS1 or IS10. Insertion sequence IS903 was found downstream of bla(CTX-M-14) or bla(CTX-M-24). Examination of the other three bla(CTX-M) genes (two bla(CTX-M-2) and one bla(CTX-M-9)) by cloning, sequencing and PCR analysis revealed the presence of complex Class 1 integrons, In35, an integron similar to In60 and a novel integron. This work further confirmed the predominant role of ISEcp1 in the mobilization of bla(CTX-M) genes of the CTX-M-1 cluster and the presence of In35, of an integron similar to In60 and a novel complex Class 1 integron.
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186. |
Baraniak A,
Fiett J,
Mrówka A,
Walory J,
Hryniewicz W,
Gniadkowski M,
( 2005 ) Evolution of TEM-type extended-spectrum beta-lactamases in clinical Enterobacteriaceae strains in Poland. PMID : 15855509 : DOI : 10.1128/AAC.49.5.1872-1880.2005 PMC : PMC1087658 Abstract >>
Seventeen extended-spectrum beta-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these beta-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different bla(TEM-1) gene alleles were precursors of the ESBL genes: bla(TEM-1A), which was the ancestor of bla(TEM-93), and bla(TEM-1F), from which all the remaining genes originated. The evolution of the bla(TEM-1F)-related genes most probably consisted of three major separate lineages, one of which, including bla(TEM-4), bla(TEM-47), bla(TEM-48), bla(TEM-49), bla(TEM-68), and bla(TEM-94), was highly structured itself and could have been initiated by the bla(TEM-25) gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related bla(TEM) genes, which suggested either the intense diversification or transposition of bla(TEM) genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.
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187. |
Liu DQ,
Liu H,
Gao XL,
Leak DJ,
Zhou NY,
( 2005 ) Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1. PMID : 15782938 : DOI : 10.1016/j.micres.2004.09.003 Abstract >>
3-Hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 is an enzyme that utilizes 3-hydroxybenzoate (3-HBA) as substrate yielding gentisate. Site-directed mutagenesis was carried out to define which residues may be involved in catalytic reaction. Substitution of arginine to glutamate at position 169 of the enzyme resulted in the complete loss of catalytic activity. This indicated Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis.
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188. |
Naas T,
Nordmann P,
Vedel G,
Poyart C,
( 2005 ) Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France. PMID : 16189140 : DOI : 10.1128/AAC.49.10.4423-4424.2005 PMC : PMC1251528 Abstract >>
N/A
|
189. |
Oliver A,
Coque TM,
Alonso D,
Valverde A,
Baquero F,
Cantón R,
( 2005 ) CTX-M-10 linked to a phage-related element is widely disseminated among Enterobacteriaceae in a Spanish hospital. PMID : 15793141 : DOI : 10.1128/AAC.49.4.1567-1571.2005 PMC : PMC1068625 Abstract >>
CTX-M-10 has been widely disseminated among multiple clones of several species of Enterobacteriaceae, harboring seemingly different plasmids, for over a decade in Ram?n y Cajal University Hospital, Madrid, Spain. Cloning and sequencing of a 12.2-kb DNA fragment from plasmid pRYCE21 from Klebsiella pneumoniae strain KP4aC revealed a novel phage-related element immediately upstream of bla(CTX-M-10) conserved among different CTX-M-10-producing strains. This is the first report showing an extended-spectrum-beta-lactamase gene linked to a phage-related element.
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190. |
Wu LT,
Hung SW,
Chuang YC,
Chen HE,
Jones RN,
Yu WL,
( 2005 ) Identification of a novel cephalosporinase (DHA-3) in Klebsiella pneumoniae isolated in Taiwan. PMID : 16216104 : DOI : 10.1111/j.1469-0691.2005.01252.x Abstract >>
A strain of Klebsiella pneumoniae resistant to cefoxitin and oxyimino-cephalosporins, but susceptible to cefepime, was isolated from an adult patient hospitalised in Taichung, Taiwan. Isoelectric focusing revealed three beta-lactamases with isoelectric points of 5.4, 8.2 and 7.9, respectively. Following PCR with plasmid DNA templates and gene sequencing, these enzymes were shown to correspond to TEM-1, SHV-5 and a novel DHA-1-like enzyme (designated DHA-3). The bla genes for TEM-1 and SHV-5 were transferable, but the bla(DHA-3) gene was non-self-transferable in conjugation experiments. All three bla genes were successfully introduced by electrotransformation into an Escherichia coli recipient (DH5alpha), resulting in a similar resistance profile to that observed in the original donor strain. Other K. pneumoniae strains producing DHA-1-like enzymes have been identified previously in Taiwan, and this report suggests that DHA-type beta-lactamases are continuing to emerge in this country.
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191. |
van der Rest ME,
Siewe RM,
Abee T,
Schwarz E,
Oesterhelt D,
Konings WN,
( 1992 ) Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae. PMID : 1577734 : Abstract >>
The gene of the sodium-dependent citrate transport system from Klebsiella pneumoniae (citS) is located on plasmid pES3 (Schwarz, E., and Oesterhelt, D. (1985) EMBO J. 4, 1599-1603) and encodes a 446-amino acid protein. Transport of citrate via this citrate transport protein (CitS) is dependent on the presence of sodium ions and is inhibited by magnesium ions. The delta pH (pH gradient across the membrane) is the major driving force for uptake. It is postulated that, in analogy with the proton-dependent citrate carrier (CitH) of K. pneumoniae (van der Rest, M. E., Abee, T., Molenaar, D., and Konings, W. N. (1990) Eur. J. Biochem. 195, 71-77), only one of the protonated species of citrate is recognized by CitS and that citrate is translocated across the membrane in symport with protons and sodium ions. The hydrophobicity profile of CitS suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments. These segments are not centered around a hydrophilic core as has been suggested for other transport proteins, but the protein is asymmetrical with seven transmembrane segments in front of a large hydrophilic loop and five after this loop. The amino acid sequence is highly similar to a citrate transport system of Lactococcus lactis subsp. lactis var. diacetylactis (CitP) (David, S., van der Rest, M. E., Driessen, A. J. M., Simons, G., and de Vos, W. M. (1990) J. Bacteriol. 172, 5789-5794) and less similar to CitH of K. pneumoniae. We conclude that the citS gene of K. pneumoniae encodes a sodium-dependent citrate transport system that belongs to a novel subclass of transport proteins.
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192. |
Brisse S,
Duijkeren Ev,
( 2005 ) Identification and antimicrobial susceptibility of 100 Klebsiella animal clinical isolates. PMID : 15708829 : DOI : 10.1016/j.vetmic.2004.11.010 Abstract >>
The objectives of this study were to determine the distribution of Klebsiella species and phylogenetic groups in animal clinical samples and to determine the levels of antimicrobial resistance of animal Klebsiella clinical isolates. One hundred Klebsiella veterinary clinical isolates were identified using gyrA PCR-RFLP and rpoB gene sequencing as a confirmatory method. Klebsiella pneumoniae phylogenetic group KpI was dominant (78 isolates), but KpII, KpIII (K. variicola), K. oxytoca, K. planticola and K. terrigena were also represented. The relative frequencies in animal infections of Klebsiella species and phylogenetic groups were similar to those observed in human nosocomial infections, suggesting that similar ecological and molecular factors cause Klebsiella infections in both situations. Resistance was common against ampicillin (99%) and cephalexin (43%) but not against ceftazidime, ceftiofur, tetracycline, enrofloxacin, gentamicin and trimethoprim-sulfamethoxazole. Thirteen isolates resistant to three or more antimicrobials or combinations thereof were found, but acquired antimicrobial resistance remains lower among animal isolates than among human nosocomial isolates.
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193. |
Duncan MJ,
Mann EL,
Cohen MS,
Ofek I,
Sharon N,
Abraham SN,
( 2005 ) The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts. PMID : 16118220 : DOI : 10.1074/jbc.M501249200 Abstract >>
Type 1 fimbriae of enterobacteria are heteropolymeric organelles of adhesion composed of FimH, a mannose-binding lectin, and a shaft composed primarily of FimA. We compared the binding activities of recombinant clones expressing type 1 fimbriae from Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium for gut and uroepithelial cells and for various soluble mannosylated proteins. Each fimbria was characterized by its capacity to bind particular epithelial cells and to aggregate mannoproteins. However, when each respective FimH subunit was cloned and expressed in the absence of its shaft as a fusion protein with MalE, each FimH bound a wide range of mannose-containing compounds. In addition, we found that expression of FimH on a heterologous fimbrial shaft, e.g. K. pneumoniae FimH on the E. coli fimbrial shaft or vice versa, altered the binding specificity of FimH such that it closely resembled that of the native heterologous type 1 fimbriae. Furthermore, attachment to and invasion of bladder epithelial cells, which were mediated much better by native E. coli type 1 fimbriae compared with native K. pneumoniae type 1 fimbriae, were found to be dependent on the background of the fimbrial shaft (E. coli versus K. pneumoniae) rather than the background of the FimH expressed. Thus, the distinct binding specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of their respective FimH subunits, but are also modulated by the fimbrial shaft on which each FimH subunit is presented, possibly through conformational constraints imposed on FimH by the fimbrial shaft. The capacity of type 1 fimbrial shafts to modulate the tissue tropism of different enterobacterial species represents a novel function for these highly organized structures.
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194. |
Diancourt L,
Passet V,
Verhoef J,
Grimont PA,
Brisse S,
( 2005 ) Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. PMID : 16081970 : DOI : 10.1128/JCM.43.8.4178-4182.2005 PMC : PMC1233940 Abstract >>
A multilocus sequence typing (MLST) scheme was developed for Klebsiella pneumoniae. Sequences of seven housekeeping genes were obtained for 67 K. pneumoniae strains, including 19 ceftazidime- and ciprofloxacin-resistant isolates. Forty distinct allelic profiles were identified. MLST data were validated against ribotyping and showed high (96%) discriminatory power. The MLST approach provides unambiguous data useful for the epidemiology of K. pneumoniae isolates.
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195. |
Wei ZQ,
Chen YG,
Yu YS,
Lu WX,
Li LJ,
( 2005 ) Nosocomial spread of multi-resistant Klebsiella pneumoniae containing a plasmid encoding multiple beta-lactamases. PMID : 16091442 : DOI : 10.1099/jmm.0.46151-0 Abstract >>
Six Klebsiella pneumoniae isolates that exhibited resistance to a wide spectrum of antibiotics were recovered from the intensive care units in the First Affiliated Hospital, Zhejiang University, Hangzhou, China. All isolates contained two plasmids of approximately 95 kb and 200 kb. The 95 kb plasmid was shown to be transferable by conjugation experiments. Isoelectric focusing patterns of the beta-lactamases extracted from the six transconjugants were identical, displaying five pI bands: 5.4, 7.75, 8.0, 8.2 and 8.4. The band corresponding to a pI of 7.75 could be inhibited by cloxacillin but not clavulanic acid, while the other bands could be inhibited by clavulanic acid but not cloxacillin. The 95 kb plasmid was digested with HindIII and a recombinant plasmid pT948 was obtained. The insert was found to contain blaDHA-1, regulatory gene ampR and an insertion element (IS26), which was downstream of blaDHA-1. PCR and DNA sequencing results confirmed that the 95 kb plasmid encoded at least four beta-lactamase genes: blaTEM-1, blaSHV-12), blaCTX-M-3 and blaDHA-1. Epidemiological typing by PFGE of the six clinical isolates of K. pneumoniae demonstrated identical genotypic patterns. In conclusion, all results indicated that the six multi-drug resistant clinical isolates of K. pneumoniae most probably originated from one clone and caused a localized epidemic in the intensive care units.
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196. |
Tórtola MT,
Lavilla S,
Miró E,
González JJ,
Larrosa N,
Sabaté M,
Navarro F,
Prats G,
( 2005 ) First detection of a carbapenem-hydrolyzing metalloenzyme in two enterobacteriaceae isolates in Spain. PMID : 16048967 : DOI : 10.1128/AAC.49.8.3492-3494.2005 PMC : PMC1196258 Abstract >>
Two strains of Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, bla(VIM-1) was found to be carried on a gene cassette inserted into a class 1 integron. The bla(VIM-1)-containing integron was located on a transferable plasmid.
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197. |
Ben-David I,
Price SE,
Bortz DM,
Greineder CF,
Cohen SE,
Bauer AL,
Jackson TL,
Younger JG,
( 2005 ) Dynamics of intrapulmonary bacterial growth in a murine model of repeated microaspiration. PMID : 16014897 : DOI : 10.1165/rcmb.2005-0053OC PMC : PMC2715355 Abstract >>
To study the change in intrapulmonary bacterial growth rate over time during Gram-negative pneumonia, a two-hit model of recurrent bacterial aspiration was developed in mice. A mutant of Klebsiella pneumoniae was isolated that could be distinguished from the wild type when cultured on appropriate media. These strains were intranasally administered, 4 h apart, to mice whose lungs were quantitatively cultured 24 h later. The relative burden of each aspirated inoculum was determined, and, using the administered dose and the number of bacteria from each inoculum present at the end of the experiment, first-order growth constants for each inoculum were calculated. Results indicate that after an initial aspiration of this organism, subsequently aspirated bacteria proliferate more slowly. When two aspirations occurred 4 h apart, the bacteria aspirated first represented 96% of total lung burden at 24 h. The growth constant of the second inoculum was related to the magnitude of the first inoculum in an inverse, nonlinear fashion. When parallel experiments were performed in complement C3-deficient mice, no suppression of the second inoculum was noted, suggesting that early upregulation of antibacterial activity in the lung is a C3-mediated event.
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198. |
Thomas L,
Espedido B,
Watson S,
Iredell J,
N/A N/A,
( 2005 ) Forewarned is forearmed: antibiotic resistance gene surveillance in critical care. PMID : 16021691 : DOI : 10.1016/j.jhin.2004.11.016 Abstract >>
N/A
|
199. |
Partridge SR,
Hall RM,
( 2004 ) Complex multiple antibiotic and mercury resistance region derived from the r-det of NR1 (R100). PMID : 15504849 : DOI : 10.1128/AAC.48.11.4250-4255.2004 PMC : PMC525457 Abstract >>
The sequence of the 45.2-kb multidrug and mercury resistance region of pRMH760, a large plasmid from a clinical isolate of Klebsiella pneumoniae collected in 1997 in Australia, was completed. Most of the modules found in the resistance determinant (r-det), or Tn2670, region of NR1 (also known as R100), isolated from a Shigella flexneri strain in Japan in the late 1950s, were present in pRMH760 but in a different configuration. The location was also different, with the Tn2670-derived region flanked by the transposition module of Tn1696 and a mercury resistance module almost identical to one found in the plasmid pDU1358. This arrangement is consistent with a three-step process. First, the r-det was circularized via homologous recombination between the IS1 elements and reincorporated at a new location, possibly in a different plasmid, via homologous recombination between the 5'-conserved (5'-CS) or 3'-CS of the In34 integron in the r-det and the same region of a second class 1 integron in a Tn1696 relative. Subsequently, resolvase-mediated recombination between the res sites in the r-det and a second mercury resistance transposon removed one end of the Tn1696-like transposon and part of the second transposon. Other events occurring within the r-det-derived portion have also contributed to the formation of the pRMH760 resistance region. Tn2 or a close relative that includes the bla(TEM-1b) gene had moved into the Tn21 mercury resistance module with subsequent deletion of the adjacent sequence, and all four 38-bp inverted repeats corresponding to Tn21 family transposon termini have been interrupted by an IS4321-like element.
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200. |
Dubois V,
Poirel L,
Arpin C,
Coulange L,
Bebear C,
Nordmann P,
Quentin C,
( 2004 ) SHV-49, a novel inhibitor-resistant beta-lactamase in a clinical isolate of Klebsiella pneumoniae. PMID : 15504885 : DOI : 10.1128/AAC.48.11.4466-4469.2004 PMC : PMC525401 Abstract >>
A clinical strain of Klebsiella pneumoniae carried the bla(SHV-49) gene, encoding a novel inhibitor-resistant beta-lactamase of pI 7.6, derived from SHV-1 by the single substitution M69I. It also harbored a gene differing from bla(SHV-11) by four silent mutations and coding for a penicillinase. Both genes were chromosome located and might represent either a species-specific gene or an acquired resistance gene.
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201. |
Munday CJ,
Boyd DA,
Brenwald N,
Miller M,
Andrews JM,
Wise R,
Mulvey MR,
Hawkey PM,
( 2004 ) Molecular and kinetic comparison of the novel extended-spectrum beta-lactamases CTX-M-25 and CTX-M-26. PMID : 15561863 : DOI : 10.1128/AAC.48.12.4829-4834.2004 PMC : PMC529179 Abstract >>
CTX-M-25 is a novel extended-spectrum beta-lactamase isolated from a single Canadian Escherichia coli isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-25) gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The bla(CTX-M-26) gene was detected on a 100-kb plasmid isolated from a Klebsiella pneumoniae strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the bla(CTX-M-25)-harboring plasmid. Both CTX-M genes were located downstream of ISEcp1, although the copy upstream of bla(CTX-M-25) was disrupted by IS50-A. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (kcat values, 33 and 0.005 s(-1) for CTX-M-25 and CTX-M-26, respectively).
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202. |
Woodford N,
Tierno PM,
Young K,
Tysall L,
Palepou MF,
Ward E,
Painter RE,
Suber DF,
Shungu D,
Silver LL,
Inglima K,
Kornblum J,
Livermore DM,
( 2004 ) Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center. PMID : 15561858 : DOI : 10.1128/AAC.48.12.4793-4799.2004 PMC : PMC529220 Abstract >>
From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant Klebsiella pneumoniae. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a blaKPC allele encoding a novel variant, KPC-3, with a His(272)-->Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing beta-lactamase variant to have been reported in members of the Enterobacteriaceae, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.
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203. |
Austin S,
Dixon R,
( 1992 ) The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. PMID : 1534752 : PMC : PMC556689 Abstract >>
The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites.
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204. |
Chin V,
Valinluck V,
Magaki S,
Ryu J,
( 2004 ) KpnBI is the prototype of a new family (IE) of bacterial type I restriction-modification system. PMID : 15475385 : DOI : 10.1093/nar/gnh134 PMC : PMC524312 Abstract >>
KpnBI is a restriction-modification (R-M) system recognized in the GM236 strain of Klebsiella pneumoniae. Here, the KpnBI modification genes were cloned into a plasmid using a modification expression screening method. The modification genes that consist of both hsdM (2631 bp) and hsdS (1344 bp) genes were identified on an 8.2 kb EcoRI chromosomal fragment. These two genes overlap by one base and share the same promoter located upstream of the hsdM gene. Using recently developed plasmid R-M tests and a computer program RM Search, the DNA recognition sequence for the KpnBI enzymes was identified as a new 8 nt sequence containing one degenerate base with a 6 nt spacer, CAAANNNNNNRTCA. From Dam methylation and HindIII sensitivity tests, the methylation loci were predicted to be the italicized third adenine in the 5' specific region and the adenine opposite the italicized thymine in the 3' specific region. Combined with previous sequence data for hsdR, we concluded that the KpnBI system is a typical type I R-M system. The deduced amino acid sequences of the three subunits of the KpnBI system show only limited homologies (25 to 33% identity) at best, to the four previously categorized type I families (IA, IB, IC, and ID). Furthermore, their identity scores to other uncharacterized putative genome type I sequences were 53% at maximum. Therefore, we propose that KpnBI is the prototype of a new 'type IE' family.
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205. |
Lee JH,
Jung HI,
Jung JH,
Park JS,
Ahn JB,
Jeong SH,
Jeong BC,
Lee JH,
Lee SH,
( 2004 ) Dissemination of transferable AmpC-type beta-lactamase (CMY-10) in a Korean hospital. PMID : 15383166 : DOI : 10.1089/mdr.2004.10.224 Abstract >>
To determine dissemination and genotype of AmpC beta-lactamases and an extended-spectrum beta-lactamase among clinical isolates of Enterobacteriaceae, we performed antibiotic susceptibility testing, pI determination, induction test, plasmid profiles, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. Among the 51 clinical isolates collected from a university hospital in Korea, six isolates were resistant to cephamycins. All six isolates produced a plasmid-encoded AmpC-type beta-lactamase, CMY-10. Five strains also produced one or more other beta-lactamases: SHV-12, an extended-spectrum beta-lactamase (five isolates); TEM-1, a class A beta-lactamase (two isolates); and a chromosomal AmpC beta-lactamase (one isolate, a strain of Enterobacter aerogenes, which produced all four of the beta-lactamases that were identified). One of six isolates produced only CMY-10. ERIC-PCR analysis revealed that dissemination of CMY-10 and SHV-12 was due to a clonal outbreak of a resistant strain and to the interspecies spread of resistance to cephamycins and broad-spectrum beta-lactams in Korea. CMY-10 beta-lactamase genes that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized from six clinical isolates. A sequence identical to the common regions in In6, In7, and a novel integron from pSAL-1 was found upstream from blaCMY-10 gene at nucleotides 1-71. A total of 15 nucleotides (I-15) or 18 nucleotides (I-18) between position 71 and 72 were inserted into the blaCMY-10 gene. The blaCMY-10 gene might be inserted into a sul1-type complex integron by I-15 or I-18.
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206. |
Werts C,
Charbit A,
Bachellier S,
Hofnung M,
( 1992 ) DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin. PMID : 1535683 : DOI : 10.1007/bf00265433 Abstract >>
We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145-167, 173-187, 197-226, 237-300, 311-329, and 367-387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding.(ABSTRACT TRUNCATED AT 250 WORDS)
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207. |
Wachino J,
Doi Y,
Yamane K,
Shibata N,
Yagi T,
Kubota T,
Ito H,
Arakawa Y,
( 2004 ) Nosocomial spread of ceftazidime-resistant Klebsiella pneumoniae strains producing a novel class a beta-lactamase, GES-3, in a neonatal intensive care unit in Japan. PMID : 15155185 : DOI : 10.1128/AAC.48.6.1960-1967.2004 PMC : PMC415581 Abstract >>
Klebsiella pneumoniae strain KG525, which showed high-level resistance to broad-spectrum cephalosporins, was isolated from the neonatal intensive care unit (NICU) of a Japanese hospital in March 2002. The ceftazidime resistance of strain KG525 was transferable to Escherichia coli CSH-2 by conjugation. Cloning and sequence analysis revealed that production of a novel extended-spectrum class A beta-lactamase (pI 7.0), designated GES-3, which had two amino acid substitutions of M62T and E104K on the basis of the sequence of GES-1, was responsible for resistance in strain KG525 and its transconjugant. The bla(GES-3) gene was located as the first gene cassette in a class 1 integron that also contained an aacA1-orfG fused gene cassette and one unique cassette that has not been described in other class 1 integrons and ended with a truncated 3' conserved segment by insertion of IS26. Another five ceftazidime-resistant K. pneumoniae strains, strains KG914, KG1116, KG545, KG502, and KG827, which were isolated from different neonates during a 1-year period in the same NICU where strain KG525 had been isolated, were also positive for GES-type beta-lactamase genes by PCR. Pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR analyses displayed genetic relatedness among the six K. pneumoniae strains. Southern hybridization analysis with a GES-type beta-lactamase gene-specific probe showed that the locations of bla(GES) were multiple and diverse among the six strains. These findings suggest that within the NICU setting genetically related K. pneumoniae strains carrying the bla(GES) gene were ambushed with genetic rearrangements that caused the multiplication and translocation of the bla(GES) gene.
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208. |
Chen YT,
Chang HY,
Lai YC,
Pan CC,
Tsai SF,
Peng HL,
( 2004 ) Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. PMID : 15276215 : DOI : 10.1016/j.gene.2004.05.008 Abstract >>
We have determined the entire DNA sequence of pLVPK, which is a 219-kb virulence plasmid harbored in a bacteremic isolate of Klebsiella pneumoniae. A total of 251 open reading frames (ORFs) were annotated, of which 37% have homologous genes of known function, 31% match the hypothetical genes in the GenBank database, and the remaining 32% are novel sequences. The obvious virulence-associated genes carried by the plasmid are the capsular polysaccharide synthesis regulator rmpA and its homolog rmpA2, and multiple iron-acquisition systems, including iucABCDiutA and iroBCDN siderophore gene clusters, Mesorhizobium loti fepBC ABC-type transporter, and Escherichia coli fecIRA, which encodes a Fur-dependent regulatory system for iron uptake. In addition, several gene clusters homologous with copper, silver, lead, and tellurite resistance genes of other bacteria were also identified. Identification of a replication origin consisting of a repA gene lying in between two sets of iterons suggests that the replication of pLVPK is iteron-controlled and the iterons are the binding sites for the repA to initiate replication and maintain copy number of the plasmid. Genes homologous with E. coli sopA/sopB and parA/parB with nearby direct DNA repeats were also identified indicating the presence of an F plasmid-like partitioning system. Finally, the presence of 13 insertion sequences located mostly at the boundaries of the aforementioned gene clusters suggests that pLVPK was derived from a sequential assembly of various horizontally acquired DNA fragments.
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209. |
Wachino J,
Doi Y,
Yamane K,
Shibata N,
Yagi T,
Kubota T,
Arakawa Y,
( 2004 ) Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A beta-lactamase, GES-4, possessing a single G170S substitution in the omega-loop. PMID : 15273099 : DOI : 10.1128/AAC.48.8.2905-2910.2004 PMC : PMC478515 Abstract >>
The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type beta-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new beta-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 microg/ml) and beta-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 +/- 1.7 microM). The GES-4 enzyme had a single G170S substitution in the Omega-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of beta-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla(GES-4) genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla(GES-4) genes found in strain KG502 were almost identical to that of bla(GES-3) in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla(GES-4) gene found in strain KG502 might well emerge from a point mutation in the bla(GES-3) gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.
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210. |
Chen H,
Ponniah G,
Salonen N,
Blum P,
( 2004 ) Culture-independent analysis of fecal enterobacteria in environmental samples by single-cell mRNA profiling. PMID : 15294770 : DOI : 10.1128/AEM.70.8.4432-4439.2004 PMC : PMC492453 Abstract >>
A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.
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211. |
Vourli S,
Giakkoupi P,
Miriagou V,
Tzelepi E,
Vatopoulos AC,
Tzouvelekis LS,
( 2004 ) Novel GES/IBC extended-spectrum beta-lactamase variants with carbapenemase activity in clinical enterobacteria. PMID : 15135524 : DOI : 10.1016/j.femsle.2004.03.028 Abstract >>
Two clinical isolates, an Escherichia coli and a Klebsiella pneumoniae, with decreased susceptibility to carbapenems were studied. This phenotype was associated with production of novel GES/IBC variant beta-lactamases, designated GES-3 (from E. coli) and GES-4 (from K. pneumoniae), exhibiting carbapenemase activity. Both enzymes possessed Ser at Ambler's position 170 instead of Gly found in the beta-lactamases GES-1 and IBC-1 that lack carbapenemase activity. Additionally, position 104 in GES-4 was occupied by a Lys as in IBC-1. bla(GES-3) and bla(GES-4) occurred as gene cassettes in the variable regions of class 1 integrons carried by plasmids. The structure of the GES-4-encoding integron was similar to that of previously described IBC-1 integrons. The GES-3-encoding integron was, most likely, truncated at the 3' conserved segment.
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212. |
Soltero-Higgin M,
Carlson EE,
Gruber TD,
Kiessling LL,
( 2004 ) A unique catalytic mechanism for UDP-galactopyranose mutase. PMID : 15133501 : DOI : 10.1038/nsmb772 Abstract >>
The flavoenzyme uridine 5'-diphosphate (UDP)-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). The latter is an essential precursor to the cell wall arabinogalactan of Mycobacterium tuberculosis. The catalytic mechanism for this enzyme had not been elucidated. Here, we provide evidence for a mechanism in which the flavin cofactor assumes a new role. Specifically, the N5 of the reduced anionic flavin cofactor captures the anomeric position of the galactose residue with release of UDP. Interconversion of the isomers occurs via a flavin-derived iminium ion. To trap this putative intermediate, we treated UGM with radiolabeled UDP-Galp and sodium cyanoborohydride; a radiolabeled flavin-galactose adduct was obtained. Ultraviolet-visible spectroscopy and mass spectrometry indicate that this product is an N5-alkyl flavin. We anticipate that the clarification of the catalytic mechanism for UGM will facilitate the development of anti-mycobacterial agents.
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213. |
Magnusson OT,
Toyama H,
Saeki M,
Rojas A,
Reed JC,
Liddington RC,
Klinman JP,
Schwarzenbacher R,
( 2004 ) Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone. PMID : 15148379 : DOI : 10.1073/pnas.0402640101 PMC : PMC419531 Abstract >>
The biosynthesis of pyrroloquinoline quinone (PQQ), a vitamin and redox cofactor of quinoprotein dehydrogenases, is facilitated by an unknown pathway that requires the expression of six genes, pqqA to -F. PqqC, the protein encoded by pqqC, catalyzes the final step in the pathway in a reaction that involves ring cyclization and eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic-acid to PQQ. Herein, we describe the crystal structures of PqqC and its complex with PQQ and determine the stoichiometry of H2O2 formation and O2 uptake during the reaction. The PqqC structure(s) reveals a compact seven-helix bundle that provides the scaffold for a positively charged active site cavity. Product binding induces a large conformational change, which results in the active site recruitment of amino acid side chains proposed to play key roles in the catalytic mechanism. PqqC is unusual in that it transfers redox equivalents to molecular oxygen without the assistance of a redox active metal or cofactor. The structure of the enzyme-product complex shows additional electron density next to R179 and C5 of PQQ, which can be modeled as O2 or H2O2, indicating a site for oxygen binding. We propose a reaction sequence that involves base-catalyzed cyclization and a series of quinone-quinol tautomerizations that are followed by cycles of O2/H2O2-mediated oxidations.
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214. |
Koh TH,
Wang GC,
Sng LH,
Koh TY,
( 2004 ) CTX-M and plasmid-mediated AmpC-producing Enterobacteriaceae Singapore. PMID : 15224678 : DOI : 10.3201/eid1006.030726 PMC : PMC3323164 Abstract >>
N/A
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215. |
Thomas X,
Destoumieux-Garzón D,
Peduzzi J,
Afonso C,
Blond A,
Birlirakis N,
Goulard C,
Dubost L,
Thai R,
Tabet JC,
Rebuffat S,
( 2004 ) Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity. PMID : 15102848 : DOI : 10.1074/jbc.M400228200 Abstract >>
Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74-84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-l-serine linked via a C-glycosidic linkage to a beta-d-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74-84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl(3). By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides.
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216. |
Haeggman S,
Löfdahl S,
Paauw A,
Verhoef J,
Brisse S,
( 2004 ) Diversity and evolution of the class A chromosomal beta-lactamase gene in Klebsiella pneumoniae. PMID : 15215087 : DOI : 10.1128/AAC.48.7.2400-2408.2004 PMC : PMC434173 Abstract >>
We investigated the diversity of the chromosomal class A beta-lactamase gene in Klebsiella pneumoniae in order to study the evolution of the gene. A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified in K. pneumoniae and of different chromosomal beta-lactamase variants previously identified. Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for other K. pneumoniae beta-lactamase). Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups. One new SHV variant, seven new LEN variants, and four OKP variants were identified. The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0. The nucleotide sequences of the housekeeping genes gyrA, coding for subunit A of gyrase, and mdh, coding for malate dehydrogenase, were also determined. Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively. This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families. We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome of K. pneumoniae.
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217. |
Chou HC,
Lee CZ,
Ma LC,
Fang CT,
Chang SC,
Wang JT,
( 2004 ) Isolation of a chromosomal region of Klebsiella pneumoniae associated with allantoin metabolism and liver infection. PMID : 15213119 : DOI : 10.1128/IAI.72.7.3783-3792.2004 PMC : PMC427404 Abstract >>
Klebsiella pneumoniae liver abscess with metastatic complications is an emerging infectious disease in Taiwan. To identify genes associated with liver infection, we used a DNA microarray to compare the transcriptional profiles of three strains causing liver abscess and three strains not associated with liver infection. There were 13 clones that showed higher RNA expression levels in the three liver infection strains, and 3 of these 13 clones contained a region that was absent in MGH 78578. Sequencing of the clones revealed the replacement of 149 bp of MGH 78578 with a 21,745-bp fragment in a liver infection strain, NTUH-K2044. This 21,745-bp fragment contained 19 open reading frames, 14 of which were proven to be associated with allantoin metabolism. The K2044 (DeltaallS) mutant showed a significant decrease of virulence in intragastric inoculation of BALB/c mice, and the prevalence of this chromosomal region was significantly higher in strains associated with liver abscess than in those that were not (19 or 32 versus 2 of 94; P = 0.0001 [chi(2) test]). Therefore, the 22-kb region may play a role in K. pneumoniae liver infection and serve as a marker for rapid identification.
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218. |
Mulvey MR,
Bryce E,
Boyd D,
Ofner-Agostini M,
Christianson S,
Simor AE,
Paton S,
N/A N/A,
( 2004 ) Ambler class A extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals. PMID : 15047521 : DOI : 10.1128/aac.48.4.1204-1214.2004 PMC : PMC375296 Abstract >>
This report describes a study carried out to gain baseline information on the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. in Canada. A total of 29,323 E. coli and 5,156 Klebsiella sp. isolates were screened at 12 participating sites. Of these, 505 clinically significant, nonrepeat isolates displaying reduced susceptibility to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. A total of 116 isolates were confirmed to be ESBL producers. PCR and sequence analysis revealed the presence of TEM-11 (n = 1), TEM-12 (n = 1), TEM-29 (n = 1), TEM-52 (n = 4), CTX-M-13 (n = 1), CTX-M-14 (n = 15), CTX-M-15 (n = 11), SHV-2 (n = 2), SHV-2a (n = 12), SHV-5 (n = 6), SHV-12 (n = 45), and SHV-30 (n = 2). Five novel beta-lactamases were identified and designated TEM-115 (n = 2), TEM-120 (n = 1), SHV-40 (n = 2), SHV-41 (n = 4), and SHV-42 (n = 1). In addition, no molecular mechanism was identified for five isolates displaying an ESBL phenotype. Macrorestriction analysis of all ESBL isolates was conducted, as was restriction fragment length polymorphism analysis of plasmids harboring ESBLs. Although a "clonal" distribution of isolates was observed at some individual sites, there was very little evidence suggesting intrahospital spread. In addition, examples of identical or closely related plasmids that were identified at geographically distinct sites across Canada are given. However, there was considerable diversity with respect to plasmid types observed.
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219. |
Rosenblueth M,
Martínez L,
Silva J,
Martínez-Romero E,
( 2004 ) Klebsiella variicola, a novel species with clinical and plant-associated isolates. PMID : 15053318 : DOI : 10.1078/0723-2020-00261 Abstract >>
A new Klebsiella species, K. variicola, is proposed on the basis of total DNA-DNA hybridization, on the monophyly observed in the phylogenetic analysis derived from the sequences of rpoB, gyrA, mdh, infB, phoE and nifH genes and on distinct phenotypic traits. The bacteria from this new species seem to be genetically isolated from K. pneumoniae strains, do not ferment adonitol and were obtained from plants (such as banana, rice, sugar cane and maize) and hospitals. The type strain is F2R9T (= ATCC BAA-830T = CFNE 2004T).
|
220. |
Yuanyuan Z,
Yang C,
Baishan F,
( 2004 ) Cloning and sequence analysis of the dhaT gene of the 1,3-propanediol regulon from Klebsiella pneumoniae. PMID : 15049372 : Abstract >>
1,3-Propanediol oxidoreductase encoded by dhaT gene, a gene of 1,3-propanediol regulon, is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. DhaT gene was amplified from the genome of K. pneumoniae, sequenced and its amino acid sequence deduced. A predicted secondary structure and 3D-structural model was constructed by homology modelling. Based on these results, we infer that 1,3-propanediol oxidoreductase belongs to NAD(P)-dependent alcohol dehydrogenase group III of iron-activated dehydrogenases.
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221. |
Fang Q,
Peng J,
Dierks T,
( 2004 ) Post-translational formylglycine modification of bacterial sulfatases by the radical S-adenosylmethionine protein AtsB. PMID : 14749327 : DOI : 10.1074/jbc.M313855200 Abstract >>
C(alpha)-Formylglycine (FGly) is the catalytic residue of sulfatases. FGly is generated by post-translational modification of a cysteine (prokaryotes and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. AtsB of Klebsiella pneumoniae is directly involved in FGly generation from serine. AtsB is predicted to belong to the newly discovered radical S-adenosylmethionine (SAM) superfamily. By in vivo and in vitro studies we show that SAM is the critical co-factor for formation of a functional AtsB.SAM.sulfatase complex and for FGly formation by AtsB. The SAM-binding site of AtsB involves (83)GGE(85) and possibly also a juxtaposed FeS center coordinated by Cys(39) and Cys(42), as indicated by alanine scanning mutagenesis. Mutation of these and other conserved cysteines as well as treatment with metal chelators fully impaired FGly formation, indicating that all three predicted FeS centers are crucial for AtsB function. It is concluded that AtsB oxidizes serine to FGly by a radical mechanism that is initiated through reductive cleavage of SAM, thereby generating the highly oxidizing deoxyadenosyl radical, which abstracts a hydrogen from the serine-C(beta)H(2)-OH side chain.
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222. |
Peng HL,
Fu TF,
Liu SF,
Chang HY,
( 1992 ) Cloning and expression of the Klebsiella pneumoniae galactose operon. PMID : 1478918 : DOI : 10.1093/oxfordjournals.jbchem.a123947 Abstract >>
The entire galactose (gal) operon of Klebsiella pneumoniae was isolated and functionally analyzed in Escherichia coli. The genes encoding galactokinase (galK), galactose-1-phosphate uridyltransferase (galT), and UDP-galactose-4-epimerase (galE) were mapped by complementation analysis. The gene order E-T-K was found to be identical to that of Salmonella spp. and E. coli. Analysis of the nucleotide sequence in the control region revealed significant homology with that of E. coli. Two major sites for transcriptional initiation, both mapped to a cytosyl residue, were identified by primer extension. When the operon is expressed in E. coli, the K. pneumoniae gal gene products make up about 30% of the total cellular proteins. The presence of a powerful promoter responsible for high level synthesis of the gal proteins was also demonstrated using beta-galactosidase as reporter.
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223. |
Poirel L,
Héritier C,
Tolün V,
Nordmann P,
( 2004 ) Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. PMID : 14693513 : DOI : 10.1128/aac.48.1.15-22.2004 PMC : PMC310167 Abstract >>
Klebsiella pneumoniae strain 11978 was isolated in Turkey in 2001 and was found to be resistant to all beta-lactams, including carbapenems. Cloning and expression in Escherichia coli identified five beta-lactamases, including two novel oxacillinases. The beta-lactamase OXA-48 hydrolyzed imipenem at a high level and was remotely related (less than 46% amino acid identity) to the other oxacillinases. It hydrolyzed penicillins and imipenem but not expanded-spectrum cephalosporins. The bla(OXA-48) gene was plasmid encoded and not associated with an integron, in contrast to most of the oxacillinase genes. An insertion sequence, IS1999, was found immediately upstream of bla(OXA-48). Another plasmid that encoded a second oxacillinase gene, bla(OXA-47), located inside a class 1 integron was identified in K. pneumoniae 11978. OXA-47 had a narrow spectrum of hydrolysis activity and did not hydrolyze ceftazidime or imipenem, as is found for the beta-lactamase (OXA-1) to which it is related. In addition, beta-lactamases TEM-1 and SHV-2a were expressed from the same K. pneumoniae isolate. Analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of ca. 36 kDa. Thus, the high-level resistance to beta-lactams of this clinical isolate resulted from peculiar beta-lactamases and modification of outer membrane proteins.
|
224. |
Sajidan A,
Farouk A,
Greiner R,
Jungblut P,
Müller EC,
Borriss R,
( 2004 ) Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1. PMID : 14727093 : DOI : 10.1007/s00253-003-1530-1 Abstract >>
Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.
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225. |
Miranda G,
Castro N,
Leaños B,
Valenzuela A,
Garza-Ramos U,
Rojas T,
Solórzano F,
Chihu L,
Silva J,
( 2004 ) Clonal and horizontal dissemination of Klebsiella pneumoniae expressing SHV-5 extended-spectrum beta-lactamase in a Mexican pediatric hospital. PMID : 14715728 : DOI : 10.1128/jcm.42.1.30-35.2004 PMC : PMC321705 Abstract >>
One hundred eighty-four clinical isolates of Klebsiella pneumoniae were recovered from August 1996 to October 1997 at the Pediatric Hospital of the Instituto Mexicano del Seguro Social in Mexico City, Mexico. Most of the isolates were collected from the neonatal intensive care unit and infant wards, which are located on the same floor of the hospital. Isolates were genotypically compared by pulsed-field gel electrophoresis with XbaI restriction of chromosomal DNA. Of 184 clinical isolates, 91 belonged to cluster A and comprised three subtypes (A1, A2, and A3), while 93 isolates, comprising two minor clones, B (10 isolates) and C (7 isolates), and 76 unique patterns, were considered unrelated isolates (URI). Susceptibility patterns were indistinguishable in both groups. Fifty extended-spectrum beta-lactamase-producing isolates, including 34 from clone A and 16 from URI, were examined for further studies. Molecular and genetic analysis showed that 47 of 50 clinical isolates expressed the SHV-5 beta-lactamase. This enzyme, in combination with TEM-1, was encoded in a >or=170-kb conjugative plasmid. Results indicate that dissemination of this resistance was due to clonal and horizontal spread.
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226. |
Nelson EC,
Segal H,
Elisha BG,
( 2003 ) Outer membrane protein alterations and blaTEM-1 variants: their role in beta-lactam resistance in Klebsiella pneumoniae. PMID : 14613957 : DOI : 10.1093/jac/dkg486 Abstract >>
The aim of the study was to characterize the genetic basis of resistance to selected beta-lactam antibiotics in two clinical isolates of Klebsiella pneumoniae. K. pneumoniae strains were isolated from two hospitalized patients. One of the strains was resistant to amoxicillin, co-amoxiclav, cefuroxime, piperacillin and cefoxitin but susceptible to all the other cephalosporins tested. The second strain displayed a similar phenotype except that it was resistant to piperacillin/tazobactam and susceptible to cefoxitin. PCR assays and DNA sequencing showed that the cefoxitin-susceptible strain contained a novel blaTEM-1 variant downstream of the strong Pa/Pb promoter. SDS-PAGE analysis of the outer membrane proteins (OMPs) did not identify OmpK35 and suggested reduced expression of OmpK36 in this strain. Following passage in non-selective media, expression of OmpK36 was restored with a concomitant increase in cefuroxime susceptibility. A similar experimental approach identified blaTEM-1C in the cefoxitin-resistant K. pneumoniae strain. This strain was deficient in OmpK35 and OmpK36; absence of the latter protein was due to the presence of IS1 in the ompK36 regulatory region. Resistance to selected beta-lactams in two clinical isolates of K. pneumoniae was due to interplay between the expression of OMPs and TEM-1.
|
227. |
Cobbe N,
Heck MM,
( 2004 ) The evolution of SMC proteins: phylogenetic analysis and structural implications. PMID : 14660695 : DOI : 10.1093/molbev/msh023 Abstract >>
The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.
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228. |
Wertz JE,
Goldstone C,
Gordon DM,
Riley MA,
( 2003 ) A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. PMID : 14640415 : Abstract >>
A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.
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229. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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230. |
Drieux L,
Bourgeois-Nicolaos N,
Cremniter J,
Lawrence C,
Jarlier V,
Doucet-Populaire F,
Sougakoff W,
( 2011 ) Accumulation of carbapenemase-producing Gram-negative bacteria in a single patient linked to the acquisition of multiple carbapenemase producers and to the in vivo transfer of a plasmid encoding VIM-1. PMID : 21570257 : DOI : 10.1016/j.ijantimicag.2011.03.017 Abstract >>
N/A
|
231. |
Dimri GP,
Das HK,
( 1990 ) Cloning and sequence analysis of gyrA gene of Klebsiella pneumoniae. PMID : 2155395 : DOI : 10.1093/nar/18.1.151 PMC : PMC330215 Abstract >>
The gene gyrA encoding the DNA gyrase A subunit of Klebsiella pneumoniae has been cloned in the plasmid pBR322. Bases of about 3.5 Kb DNA have been sequenced to locate the gyrA gene. An open reading frame of 2628 nucleotides coding for a 97 KD protein has been identified. Homology to the extent of about 85% was detected at the nucleotide level and about 90% at the amino acid level, when the sequences were compared with that of Escherichia coli gyrA. Some very interesting differences have, however, been found in the promoter region.
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232. |
Kornacker MG,
Pugsley AP,
( 1990 ) Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsiella pneumoniae UNF5023. PMID : 2181242 : DOI : 10.1111/j.1365-2958.1990.tb02016.x Abstract >>
The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.
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233. |
Samuelsen ?,
Toleman MA,
Hasseltvedt V,
Fuursted K,
Leegaard TM,
Walsh TR,
Sundsfjord A,
Giske CG,
( 2011 ) Molecular characterization of VIM-producing Klebsiella pneumoniae from Scandinavia reveals genetic relatedness with international clonal complexes encoding transferable multidrug resistance. PMID : 21595797 : DOI : 10.1111/j.1469-0691.2011.03532.x Abstract >>
VIM-producing Klebsiella pneumoniae (VPKP) has been identified as a source of hospital outbreaks and is prevalent particularly in the Mediterranean region. In this study we have characterized eight VPKP isolates identified in Scandinavia during 2005-2008. With the exception of one isolate, all were from patients with recent history of hospitalization abroad (Greece, n = 6; Turkey, n = 1). Multilocus sequence typing (MLST) resulted in five sequence types (STs), ST36 (n = 1), ST147 (n = 4), ST272 (n = 1), ST273 (n = 1) and ST383 (n = 1), which except for ST272 were part of putative international clonal complexes. All were multidrug resistant due to the presence of other resistance determinants, including extended-spectrum �]-lactamases (CTX-M-3, SHV-5 and SHV-12), 16S rRNA methylases (ArmA) and plasmid-mediated quinolone resistance determinants (QnrS). One isolate harboured a novel VIM-variant (VIM-26) while VIM-1 and VIM-19 were detected in six and one isolate, respectively. Two different genetic structures surrounding the bla(VIM) gene were identified in four isolates. In two isolates bla(VIM-1) and bla(VIM-26) were located in an integron similar to In-e541 (intI1;bla(VIM-1/-26);aacA7; dhfrI;aadA1;3'CS) while in the other two isolates bla(VIM-1) was located in an integron lacking 3'CS but with an IS26 element in the 3'end (intI1;bla(VIM-1);aac(6')-Ib;IS26), as identified in the IncN plasmid pKOX105. The bla(VIM) -genes were located on transferable plasmids ranging from ?40 to ?240 kb and associated with Tn21 in four isolates. PCR-based replicon typing indicated association of bla(VIM) with IncN (n = 3) and A/C (n = 1) broad-host-range plasmids but also with unknown replicons (n = 4). In conclusion, Scandinavian VPKP is associated with importation and genetically related to international clones encoding transferable plasmid-mediated multidrug resistance.
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234. |
Titgemeyer F,
Eisermann R,
Hengstenberg W,
Lengeler JW,
( 1990 ) The nucleotide sequence of ptsH gene from Klebsiella pneumoniae. PMID : 2186369 : DOI : 10.1093/nar/18.7.1898 PMC : PMC330621 Abstract >>
N/A
|
235. |
van der Rest ME,
Schwarz E,
Oesterhelt D,
Konings WN,
( 1990 ) DNA sequence of a citrate carrier of Klebsiella pneumoniae. PMID : 2186908 : DOI : 10.1111/j.1432-1033.1990.tb15502.x Abstract >>
The citrate transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz E. and D. Oesterhelt (1985) EM BO J. 4, 1599-1603] has been subcloned in Escherichia coli DH1. The DNA sequence of a 1723-base fragment that codes for the citrate carrier has been determined and the gene product has been characterized with the T7 promoter system. The DNA fragment contains an open reading frame of 1332 base pairs and codes for a protein of 444 amino acids. The hydropathy profile suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments centered around a hydrophilic core. The gene for the citrate carrier has 66% similarity with a citrate carrier determinant from a naturally occurring plasmid responsible for secondary transport of citrate across the cytoplasmic membrane of E. coli.
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236. |
Magesh H,
Kamatchi C,
Vaidyanathan R,
Sumathi G,
( N/A ) Identification of plasmid-mediated quinolone resistance genes qnrA1, qnrB1 and aac(6')-1b-cr in a multiple drug-resistant isolate of Klebsiella pneumoniae from Chennai. PMID : 21860107 : DOI : 10.4103/0255-0857.83910 Abstract >>
Resistance to fluoroquinolones, a commonly prescribed antimicrobial for Gram-negative and Gram-positive microorganisms, is of importance in therapy. The purpose of this study was to screen for the presence of Plasmid-Mediated Quinolone Resistance (PMQR) determinants in clinical isolates of Klebsiella pneumoniae. Extended-Spectrum Beta-Lactamase (ESBL) isolates of K. pneumoniae collected during October 2009 were screened by the antimicrobial susceptibility test. The plasmids from these isolates were analysed by specific Polymerase chain Reaction (PCR) for qnrA, qnrB and aac(6')-1b. The amplified products were sequenced to confirm the allele. Our analysis showed that 61% out of the 23 ESBL K. pneumoniae isolates were resistant to ciprofloxacin and 56% to levofloxacin. The PMQR was demonstrated by transforming the plasmids from two isolates P12 and P13 into E. coli JM109. The PMQR gene qnrA was found in 16 isolates and qnrB in 11 isolates. The plasmid pKNMGR13 which conferred an minimum inhibitory concentration (MIC) of more than 240 �gg/ml in sensitive E. coli was found to harbour the qnrA1 and qnrB1 allele. Furthermore, the gene aac(6')-1b-cr encoding a variant aminoglycoside 6'-N Acetyl transferase which confers resistance to fluoroquinolones was found in the same plasmid. Our report shows the prevalence of PMQR mediated by qnrA and qnrB in multidrug-resistant K. pneumoniae isolates from Chennai. A multidrug-resistant plasmid conferring high resistance to ciprofloxacin was found to harbour another PMQR gene, aac(6')-1b-cr mutant gene. This is the first report screening for PMQR in K. pneumoniae isolates from India.
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237. |
Nematzadeh S,
Shoeib N,
Shahcheraghi F,
Fereshteh S,
Feizabadi MM,
Mehdi FM,
Nikbin VS,
Sadat NV,
Nasehi L,
Leila N,
( N/A ) Molecular characterization of CTX-M�]-lactamases among Klebsiella pneumoniae isolated from patients at Tehran hospitals. PMID : 21860105 : DOI : 10.4103/0255-0857.83908 Abstract >>
Plasmid-encoded CTX-M-group of extended-spectrum �]-lactamases (ESBLs) represent a significant and rapidly emerging problem in most part of the world. The aim of the present study was to describe the prevalence of CTX-M producing Klebsiella pneumoniae at Tehran hospitals. Clinical isolates of K. pneumoniae (n=250) were collected from 10 hospitals of Tehran. Susceptibility to antimicrobial agents, MIC of cefotaxime and ESBLs production of collected isolates were detected. All ESBL-producing isolates were screened for bla CTX-M genes using PCR and DNA sequencing. Molecular typing of bla(CTX-M) harboring isolates was performed by Pulsed-field gel electrophoresis assay. Of 250 K. pneumoniae clinical isolates, 102 isolates revealed ESBLs - phenotype. PCR assay and sequencing detected bla(CTX-M) genes in 71.5% (n= 73) of ESBL-producing isolates. The prevalence of CTX-M -I and CTX-M-III clusters among these isolates was 35.61% (n=26) and 21.9 % (n=16) respectively. Coexistence of CTX-M -I and CTX-M-III clusters was found among 42.5% (n= 31) of isolates. Of 102 isolates that were positive in the phenotypic confirmatory test (PCT), 29 isolates (28.4%) did not produce any amplicons in PCR for bla(CTX-M) gene. The results of PCR for CTX-M -II and CTX-M-IV clusters were also negative. Analysis of the 31 CTX-M producing K. pneumoniae isolates by PFGE typing showed 26 distinct patterns. The bla CTX-M genes are widespread among Iranian isolates of K. pneumoniae. PFGE demonstrated the high diversity of K. pneumoniae harboring bla(CTX-M) in our study.
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238. |
Bogaerts P,
de Castro RR,
Deplano A,
Bouchahrouf W,
Tsobo C,
Denis O,
Glupczynski Y,
( 2011 ) Detection of a VIM-27-producing Klebsiella pneumoniae isolate in a patient following surgical tourism in Greece. PMID : 21746946 : DOI : 10.1128/AAC.00688-11 PMC : PMC3165348 Abstract >>
N/A
|
239. |
Bialek-Davenet S,
Marcon E,
Leflon-Guibout V,
Lavigne JP,
Bert F,
Moreau R,
Nicolas-Chanoine MH,
( 2011 ) In vitro selection of ramR and soxR mutants overexpressing efflux systems by fluoroquinolones as well as cefoxitin in Klebsiella pneumoniae. PMID : 21464248 : DOI : 10.1128/AAC.00156-11 PMC : PMC3101381 Abstract >>
The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression.
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240. |
Potron A,
Nordmann P,
Lafeuille E,
Al Maskari Z,
Al Rashdi F,
Poirel L,
( 2011 ) Characterization of OXA-181, a carbapenem-hydrolyzing class D beta-lactamase from Klebsiella pneumoniae. PMID : 21768505 : DOI : 10.1128/AAC.00481-11 PMC : PMC3186949 Abstract >>
Klebsiella pneumoniae KP3 was isolated from a patient transferred from India to the Sultanate of Oman. K. pneumoniae KP3 was resistant to all �]-lactams, including carbapenems, and expressed the carbapenem-hydrolyzing �]-lactamase OXA-181, which differs from OXA-48 by four amino acid substitutions. Compared to OXA-48, OXA-181 possessed a very similar hydrolytic profile. The bla(OXA-181) gene was located on a 7.6-kb ColE-type plasmid and was linked to the insertion sequence ISEcp1. The ISEcp1-mediated one-ended transposition of bla(OXA-181) was also demonstrated.
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241. |
Roy Chowdhury P,
Ingold A,
Vanegas N,
Martínez E,
Merlino J,
Merkier AK,
Castro M,
González Rocha G,
Borthagaray G,
Centrón D,
Bello Toledo H,
Márquez CM,
Stokes HW,
( 2011 ) Dissemination of multiple drug resistance genes by class 1 integrons in Klebsiella pneumoniae isolates from four countries: a comparative study. PMID : 21518841 : DOI : 10.1128/AAC.01529-10 PMC : PMC3122386 Abstract >>
A comparative genetic analysis of 42 clinical Klebsiella pneumoniae isolates, resistant to two or more antibiotics belonging to the broad-spectrum �]-lactam group, sourced from Sydney, Australia, and three South American countries is presented. The study focuses on the genetic contexts of class 1 integrons, mobilizable genetic elements best known for their role in the rapid evolution of antibiotic resistance among Gram-negative pathogens. It was found that the class 1 integrons in this cohort were located in a number of different genetic contexts with clear regional differences. In Sydney, IS26-associated Tn21-like transposons on IncL/M plasmids contribute greatly to the dispersal of integron-associated multiple-drug-resistant (MDR) loci. In contrast, in the South American countries, Tn1696-like transposons on an IncA/C plasmid(s) appeared to be disseminating a characteristic MDR region. A range of mobile genetic elements is clearly being recruited by clinically important mobile class 1 integrons, and these elements appear to be becoming more common with time. This in turn is driving the evolution of complex and laterally mobile MDR units and may further complicate antibiotic therapy.
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242. |
Papagiannitsis CC,
Kotsakis SD,
Petinaki E,
Vatopoulos AC,
Tzelepi E,
Miriagou V,
Tzouvelekis LS,
( 2011 ) Characterization of metallo-beta-lactamase VIM-27, an A57S mutant of VIM-1 associated with Klebsiella pneumoniae ST147. PMID : 21518835 : DOI : 10.1128/AAC.00238-11 PMC : PMC3122466 Abstract >>
VIM-27 metallo-�]-lactamase, an Ala(57) �� Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla(VIM-27) was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most �]-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.
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243. |
Diene SM,
Bruder N,
Raoult D,
Rolain JM,
( 2011 ) Real-time PCR assay allows detection of the New Delhi metallo-�]-lactamase (NDM-1)-encoding gene in France. PMID : 21497063 : DOI : 10.1016/j.ijantimicag.2011.02.006 Abstract >>
In this study, we report the development of a rapid real-time polymerase chain reaction (PCR) assay with TaqMan probe to detect the New Delhi metallo-�]-lactamase (NDM-1)-encoding gene directly from bacterial isolates. The specificity of the assay was verified in silico as well as with a large panel of 84 clinically relevant bacteria, including the Klebsiella pneumoniae NCTC 13443 NDM-1-positive reference strain. Using this assay retrospectively on a local series of 44 K. pneumoniae isolates from Marseille Hospitals (France), it was possible to detect and identify an NDM-1-producing K. pneumoniae strain isolated from bronchoalveolar lavage in April 2010 from a French patient repatriated from India after a motorbike accident. Standard PCR amplification and sequencing of the entire NDM-1 gene from this isolate was also performed and the amino acid sequence showed 100% homology with the NDM-1 protein from the K. pneumoniae reference strain. We believe that this real-time PCR assay would be a powerful tool that could be added to other molecular detection assays such as detection of KPC- or OXA-encoding genes for rapid screening and/or identification of carbapenem-resistant bacterial isolates from patients returning from the Asian continent that could be implemented in a point-of-care strategy.
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244. |
Gomez SA,
Pasteran FG,
Faccone D,
Tijet N,
Rapoport M,
Lucero C,
Lastovetska O,
Albornoz E,
Galas M,
N/A N/A,
Melano RG,
Corso A,
Petroni A,
( 2011 ) Clonal dissemination of Klebsiella pneumoniae ST258 harbouring KPC-2 in Argentina. PMID : 21851480 : DOI : 10.1111/j.1469-0691.2011.03600.x Abstract >>
The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla(KPC-2) was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla(KPC-2) in an unusual genetic environment flanked by ISKpn8-�Gbla(TEM-1) and ISKpn6-like. bla(KPC-2) was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla(KPC-2) in Tn4401a.
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245. |
Wang D,
Wang H,
Qi Y,
Liang Y,
Zhang J,
Yu L,
( 2011 ) Novel variants of the qnrB gene, qnrB31 and qnrB32, in Klebsiella pneumoniae. PMID : 21816942 : DOI : 10.1099/jmm.0.034272-0 Abstract >>
Quinolone resistance in the family Enterobacteriaceae is mostly attributed to the accumulation of mutations in the bacterial enzymes targeted by fluoroquinolones: DNA gyrase and DNA topoisomerase IV. Here we isolated the Klebsiella pneumoniae strains KP3606 and KP4707 from different specimens from 2008 to 2010 in Taizhou Municipal Hospital of China, and discovered a new subtype qnrB31, for which the GenBank accession number is HQ418999, and another new subtype qnrB32, for which the GenBank accession number is HQ704413. Susceptibility testing showed that KP3606 had a reduced susceptibility (MIC ?0.5 ?g ml(-1)) to quinolones, while KP4707 was resistant to quinolones. Of all qnrB alleles, the novel variants the qnrB32 gene and qnrB31 gene have the highest amino acid identity. The results suggested that of all the various genes involved in resistance to quinolones, the qnrB gene is the most likely to be mutated, and plasmids might play a role in the dissemination and evolution of qnrB genes.
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246. |
Mataseje LF,
Boyd DA,
Willey BM,
Prayitno N,
Kreiswirth N,
Gelosia A,
Poutanen SM,
Low DE,
Jenkins SG,
Katz K,
Mulvey MR,
( 2011 ) Plasmid comparison and molecular analysis of Klebsiella pneumoniae harbouring bla(KPC) from New York City and Toronto. PMID : 21406433 : DOI : 10.1093/jac/dkr092 Abstract >>
This study examined Klebsiella pneumoniae clinical isolates and their bla(KPC) plasmids to determine potential relatedness of the isolates and their plasmids harbouring carbapenem resistance mechanisms. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae from New York City (NYC) (n = 19) and Toronto (n = 2) were typed by PFGE and multilocus sequence typing (MLST). bla(KPC)-harbouring plasmids were transformed into Escherichia coli DH10B(TM), restricted using EcoRI and analysed for bla content and replicon (rep) type. Susceptibility profiles for clinical and transformed strains were determined by automated microbroth dilution using CLSI breakpoints. Outer membrane protein (OMP) genes were analysed by sequencing of ompk35 and ompk36. PFGE analysis identified 17 related strains (? 80% similarity; 11 KPC-2, 6 KPC-3) where ST258 was the dominant clonal type. All clinical isolates contained both bla(SHV) and bla(TEM-1) and, with the exception of one isolate, were multidrug resistant (MDR). Transformed KPC plasmids (n = 21) carried TEM-1 (n = 18) and were MDR (n = 5). Three plasmid clusters, repFIIA (n = 10), repR (n = 3) and an unknown type (n = 3), were observed. repFllA plasmids were observed from both NYC and Toronto strains. OMP gene analysis revealed premature stop codons in ompk35 and numerous deletions and insertions in ompk36. The dissemination of bla(KPC) is due both to carriage of similar KPC-harbouring plasmids within genetically distinct K. pneumoniae and to clonal spread of K. pneumoniae with unrelated KPC plasmids.
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247. |
Wang P,
Hu F,
Xiong Z,
Ye X,
Zhu D,
Wang YF,
Wang M,
( 2011 ) Susceptibility of extended-spectrum-beta-lactamase-producing Enterobacteriaceae according to the new CLSI breakpoints. PMID : 21752977 : DOI : 10.1128/JCM.00222-11 PMC : PMC3165602 Abstract >>
In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum �]-lactamases (ESBLs) and confirmatory tests. The aim of this study was to determine how many ESBL-producing strains of three common species of Enterobacteriaceae test susceptible using the new breakpoints. As determined with the CLSI screening and confirmatory tests, 382 consecutive ESBL-producing strains were collected at Huashan Hospital between 2007 and 2008, including 158 strains of Escherichia coli, 164 of Klebsiella pneumoniae, and 60 of Proteus mirabilis. Susceptibility was determined by the CLSI agar dilution method. CTX-M-, TEM-, and SHV-specific genes were determined by PCR amplification and sequencing. bla(CTX-M) genes alone or in combination with bla(SHV) were present in 92.7% (354/382) of these ESBL-producing strains. Forty-two (25.6%) strains of K. pneumoniae harbored SHV-type ESBLs alone or in combination. No TEM ESBLs were found. Utilizing the new breakpoints, all 382 strains were resistant to cefazolin, cefotaxime, and ceftriaxone, while 85.0 to 96.7% of P. mirabilis strains tested susceptible to ceftazidime, cefepime, and aztreonam, 41.8 to 45.6% of E. coli strains appeared to be susceptible to ceftazidime and cefepime, and 20.1% of K. pneumoniae were susceptible to cefepime. In conclusion, all ESBL-producing strains of Enterobacteriaceae would be reported to be resistant to cefazolin, cefotaxime, and ceftriaxone by using the new CLSI breakpoints, but a substantial number of ESBL-containing P. mirabilis and E. coli strains would be reported to be susceptible to ceftazidime, cefepime, and aztreonam, which is likely due to the high prevalence of CTX-M type ESBLs.
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248. |
Park YJ,
Yu JK,
Park KG,
Park YG,
Lee S,
Kim SY,
Jeong SH,
( 2011 ) Prevalence and contributing factors of nonsusceptibility to imipenem or meropenem in extended-spectrum �]-lactamase-producing Klebsiella pneumoniae and Escherichia coli. PMID : 21397426 : DOI : 10.1016/j.diagmicrobio.2010.12.012 Abstract >>
Among the extended-spectrum �]-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli, 3.9% of K. pneumoniae showed nonsusceptibility to imipenem or meropenem; and their mechanism was the combination of ESBL and/or plasmid-mediated AmpC �]-lactamase production and porin loss. The presence of bla(CTX-M-14) and loss of OmpK36 were associated with higher carbapenem MICs.
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249. |
Partridge SR,
Thomas LC,
Ginn AN,
Wiklendt AM,
Kyme P,
Iredell JR,
( 2011 ) A novel gene cassette, aacA43, in a plasmid-borne class 1 integron. PMID : 21422220 : DOI : 10.1128/AAC.01582-10 PMC : PMC3101451 Abstract >>
A novel gene cassette, aacA43, was identified in the aadB-aacA43-oxa10-smr2 cassette array in a class 1 integron. Like related aminoglycoside-(6')-acetyltransferases, AacA43 confers clinically relevant resistance to kanamycin, tobramycin, and some less-used aminoglycosides but not to gentamicin. Although transferable on an IncL/M plasmid, aacA43 was identified in only two different Klebsiella pneumoniae strains (14 isolates), one Escherichia coli strain (2 isolates), and one Enterobacter cloacae strain in a survey of patients in a Sydney intensive care unit in 2004-2005.
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250. |
Veras DL,
Alves LC,
Brayner FA,
Guedes DR,
Maciel MA,
Rocha CR,
de Souza Lopes AC,
( 2011 ) Prevalence of the bla (SHV) gene in Klebsiella pneumoniae isolates obtained from hospital and community infections and from the microbiota of healthy individuals in Recife, Brazil. PMID : 21359845 : DOI : 10.1007/s00284-011-9899-z Abstract >>
The aim of this study was to determine the prevalence of the bla (SHV) gene in Klebsiella pneumoniae isolates from hospital and community infections and from the normal microbiota of healthy individuals in Recife, PE, Brazil. Fifty-two K. pneumoniae isolates were analyzed regarding the presence of the bla (SHV) gene, using PCR, and eight isolates were analyzed by DNA sequencing. This gene was detected in 16 isolates from hospital infections, four from community infections, and nine from the normal microbiota. This was the first study to find the bla (SHV) gene in K. pneumoniae isolates from the normal microbiota. Through DNA sequencing of eight K. pneumoniae isolates from hospital and community infections, with a resistance phenotype indicative of extended-spectrum �]-lactamase production, a new SHV variant named SHV-122 was found. We also detected the presence of bla (SHV-1), bla (SHV-11), bla (SHV-28), and bla (SHV-108). The results show that in Recife, Brazil, K. pneumoniae isolates that presented resistance to oxyimino-�]-lactams had high prevalence and diversity of the bla (SHV) gene. We also conclude that there was a high presence of the bla (SHV) gene among isolates from the normal microbiota of healthy individuals.
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251. |
Santos C,
Caetano T,
Ferreira S,
Ramalheira E,
Mendo S,
( 2011 ) A novel complex class 1 integron found in a Klebsiella pneumoniae isolate from Portugal. PMID : 21722254 : DOI : 10.1111/j.1469-0691.2010.03416.x Abstract >>
Klebsiella pneumoniae Kp1 carrying a novel complex class 1 integron was isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro, central Portugal. The integron consists of two variable regions (VRs); VR1 was previously described in Escherichia coli and Vibrio cholerae, and VR2 contains an In37-like structure and is located downstream of an ISCR1 element. The integron was found on a plasmid of 225 kb. The qnrB10 gene, although present, is not associated with the complex class 1 integron.
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252. |
Tsai YK,
Fung CP,
Lin JC,
Chen JH,
Chang FY,
Chen TL,
Siu LK,
( 2011 ) Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence. PMID : 21282452 : DOI : 10.1128/AAC.01275-10 PMC : PMC3067157 Abstract >>
OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (�GompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (�GompK35) had no significant effect. A double deletion of ompK35 and ompK36 (�GompK35/36) further increased these MICs to high-level resistance and led to 8- and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the �GompK35 mutation resulted in a significant (? 4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a �GompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the �GompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both �GompK36 and �GompK35/36 mutants. In a mouse peritonitis model, the �GompK35 mutant showed no change in virulence, and the �GompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the �GompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.
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253. |
Wu JJ,
Wang LR,
Liu YF,
Chen HM,
Yan JJ,
( 2011 ) Prevalence and characteristics of ertapenem-resistant Klebsiella pneumoniae isolates in a Taiwanese university hospital. PMID : 21352075 : DOI : 10.1089/mdr.2010.0115 Abstract >>
This study was conducted to investigate the prevalence and characteristics of ertapenem-resistant (ETP-R) Klebsiella pneumoniae isolates at a Taiwanese hospital. The disk-diffusion tests revealed that the rate of ertapenem resistance among all isolates collected in 2008 was 13.5%, and the resistance rate among bloodstream isolates increased from 0% to 13.6% between 2001 and 2008. Eighty-two nonduplicate ETP-R isolates collected in 2008 were examined. Seventy-four (90.2%) isolates of them had extended-spectrum �]-lactamases (CTX-M- and SHV-type), AmpC enzymes (DHA-1 and CMY-2), and IMP-8 metallo-�]-lactamase alone or in combination, and an extremely high prevalence of fluoroquinolone resistance (95.1%) and plasmid-mediated quinolone resistance determinants (90.2%) were also observed. Eighteen ETP-R but imipenem-susceptible isolates were selected and compared with 18 imipenem-nonsusceptible isolates collected before 2008. Sequence analyses revealed genetic disruptions of OmpK36 in 11 imipenem-nonsusceptible and 6 imipenem-susceptible isolates, respectively, and OmpK35 disruptions in 10 isolates for both groups. For the isolates with intact ompK36, sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests decreased expression of OmpK36 in 5 of 7 imipenem-nonsusceptible isolates and 3 of 12 imipenem-susceptible isolates. In conclusion, the increasing prevalence of ertapenem resistance that was predominantly attributed to noncarbapenemase-mediated resistance mechanisms in K. pneumoniae is becoming a serious treat to patients in Taiwan.
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254. |
Tijet N,
Alexander DC,
Richardson D,
Lastovetska O,
Low DE,
Patel SN,
Melano RG,
( 2011 ) New Delhi metallo-beta-lactamase, Ontario, Canada. PMID : 21291614 : DOI : 10.3201/eid1702.101561 PMC : PMC3204783 Abstract >>
N/A
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255. |
Huang ZM,
Mi JR,
Sheng YQ,
Zou YX,
Chu QJ,
Ge LW,
Yang HY,
( 2010 ) [Study on pan-resistant Klebsiella pneumoniae harboring blaKPC-2 type carbapenemase gene from a hospital outbreak in Huzhou, Zhejiang]. PMID : 21163037 : Abstract >>
To investigate the status of genotype of the KPC (Klebsiella pneumoniae carbapenemase)-encoding genes in Pan-resistant K. Pneumoniae, isolated from the 98th Hospital of People's Liberation Army, Huzhou district, Zhejiang province, China. 19 strains of Pan-resistant K. pneumoniae were isolated from the inpatients between November, 2008 and July, 2009. Phenotypic confirmatory test for suspected carbapenemases production were carried out by Modified Hodge test. Carbapenemase gene of bla(KPC) was analyzed by PCR and verified by DNA sequencing. In 19 strains of K. pneumoniae, the positive rates of Modified Hodge test and gene of bla(KPC) were both 100.0%. These genes all belonged to bla(KPC-2) subtype confirmed by nucleotide sequence analysis. Among them, the bla(KPC-2) gene sequence of the HZ001 strain (its original serial number was HZ9871) had been registered in GenBank (GenBank Accession Number: GU086225). All of the Pan-resistant K. pneumoniae isolated from the inpatients harbored bla(KPC-2) type carbapenemases gene and causing an outbreak in a hospital. Carbapenemases that producing type KPC-2 might be the major reason which causing the resistance to Carbapenems antibiotics.
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256. |
Liu Y,
Wang H,
Sun X,
Yang H,
Wang Y,
Song W,
( 2011 ) Study on mechanisms of colonization of nitrogen-fixing PGPB, Klebsiella pneumoniae NG14 on the root surface of rice and the formation of biofilm. PMID : 21132569 : DOI : 10.1007/s00284-010-9835-7 Abstract >>
Plant growth-promoting bacteria (PGPB) refer to the bacteria beneficial to plants, and they may affect the growth and development of plants directly or indirectly. This article studied the activities of nitrogen fixation and colonization of a strain of PGPB, Klebsiella pneumoniae NG14, which was isolated from the rice root surface. The results showed that NG14 harbouring the nifH gene had nitrogenase activity, (15)N(2)-fixing activity, and was able to colonize on the root surface and within the cavity of root vascular tissues of rice. Using proteomics technology to study the differences and changes of membrane proteins (MP) of NG14 bacterial biofilm in non-biological surface, 28 proteins showing significant differences before and after the formation of bacterial biofilm have been identified, in which the precursors of membrane pore protein OmpC relevant to osmotic stress resistance was up-regulated. This study would have positive significance on further understanding of the direct and indirect promotion effects of PGPB and related mechanisms.
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257. |
Montealegre MC,
Correa A,
Briceño DF,
Rosas NC,
De La Cadena E,
Ruiz SJ,
Mojica MF,
Camargo RD,
Zuluaga I,
Marin A,
Quinn JP,
Villegas MV,
N/A N/A,
( 2011 ) Novel VIM metallo-beta-lactamase variant, VIM-24, from a Klebsiella pneumoniae isolate from Colombia. PMID : 21282438 : DOI : 10.1128/AAC.01208-10 PMC : PMC3088182 Abstract >>
We report the emergence of a novel VIM variant (VIM-24) in a Klebsiella pneumoniae isolate in Colombia. The isolate displays MICs for carbapenems below the resistance breakpoints, posing a real challenge for its detection. The blaVIM-24 gene was located within a class 1 integron carried on a large plasmid. Further studies are needed to clarify its epidemiological and clinical impact.
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258. |
Yue L,
Chen X,
Li S,
Liao X,
Zhuang N,
Zhang Y,
Liu YH,
( 2011 ) First report of plasmid-mediated quinolone resistance qnrA1 gene in Klebsiella pneumoniae isolate of animal origin. PMID : 21235404 : DOI : 10.1089/fpd.2010.0737 Abstract >>
One QnrA1-producing Klebsiella pneumoniae isolate GDKA1 from chicken was detected. The qnrA1 gene on plasmid pGDKA1 was located in a genetic environment similar to that in In36 on plasmid pHSH1 and could be cotransferred to Escherichia coli J53 Az(R) with other resistances by a conjugation experiment. Upstream of the qnrA1 gene, there was a class I integron with the dfrA27 and aadA2 cassettes. Similar genetic environments of qnrA1 in Enterobacteriaceae isolates from both human and animal origin might, to some extent, demonstrate similar mechanisms of qnrA distribution. The presence of qnrA1 in health animal commensal bacteria should be worthy of note. This is the first report of qnrA1 in K. pneumoniae and dfrA27 in an Enterobacteriaceae isolate of animal origin.
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259. |
Reyss I,
Pugsley AP,
( 1990 ) Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. PMID : 2129543 : DOI : 10.1007/bf00633815 Abstract >>
DNA sequence analysis, Tnpho and Tntac-1, mutagenesis, deletion analysis, expression under bacteriophage T7 gene 10 promoter control, subcellular fractionation and complementation tests were used to study the function of DNA located in the centre of the pulC-O operon from Klebsiella oxytoca strain UNF5023. The characterized region of the operon includes five genes (pulG, pulH, pulI, pulJ and pulK) coding for apparently integral inner membrane proteins which are required for pullulanase secretion. The results presented here and previously show that the pulC-O operon contains at least 11 pullulanase secretion genes.
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260. |
Zhu YL,
Zhang XN,
Gao F,
Cheng J,
Hu LF,
Ma T,
Yin J,
Ye Y,
Li JB,
( 2011 ) ACT-6, a novel plasmid-encoded class C �]-lactamase in a Klebsiella pneumoniae isolate from China. PMID : 21304534 : DOI : 10.1038/ja.2011.1 Abstract >>
The purpose of this study was to investigate the phenotypic and molecular characterization of a novel plasmid-mediated AmpC-type �]-lactamase in Klebsiella pneumoniae E701 isolated from Anhui province in China. In comparison with the ACT-1, sequence analysis revealed that there were 43 point mutations in the coding gene, and 10 of which led to amino-acid substitution. Resistance could be transferred by conjugation or transformation with plasmid DNA into E. coli JM109, which was due to the production of a �]-lactamase with an isoelectric point of 8.4 named ACT-6. Cloning, expression, purification and kinetics were carried out to study the characterization of the novel AmpC-type �]-lactamase. The results of MIC determinations and substrate profiles showed there was no significant difference in the activities of the novel enzyme and ACT-1. Moreover, the class 1 integron and the whole open reading frame of the novel AmpC-type �]-lactamase from K.pneumoniae E701 were detectable in the same size plasmid. This is the first report on the emergence of the novel ACT-6 type �]-lactamases in K. pneumoniae.
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261. |
Dashti AA,
Jadaon MM,
Amyes SG,
( 2010 ) Retrospective study of an outbreak in a Kuwaiti hospital of multidrug-resistant Klebsiella pneumoniae possessing the new SHV-112 extended-spectrum beta-lactamase. PMID : 21123157 : DOI : 10.1179/joc.2010.22.5.335 Abstract >>
Patients infected with bacteria producing extendedspectrum beta-lactamases (ESBL) are at higher risk of mortality and morbidity. Several mutations in genes encoding SHV, tem and CTX-M beta-lactamases have been associated with ESBL activity. This paper describes a new SHV mutation in ESBL-producing strains of Klebsiella pneumoniae isolated in Kuwait. The study included 13 K. penumoniae strains isolated from patients admitted to the Amiri hospital of Kuwait. The production of ESBL in all strains was confirmed by Vitek system and E-test. All the ESBL genes were amplified by PCR and examined by DNA sequencing. All these ESBL-positive isolates were resistant to ceftazidime and cefotaxime. DNA sequencing revealed an A815G point mutation in the bla (SHV)gene causing an asparagine (AAT) to aspartic acid (GAT) mutation at position 253 of the enzyme. This new mutation was assigned the unique number SHV-112, and the Genebank accession number EU477409. This study reports a new mutation in the SHV gene in K. pneumoniae with ESBL capability. There could be other mutations still to be found in ESBL genes of K. pneumoniae in Kuwait and probably in other middle eastern countries, and researchers in the region should make use of molecular techniques to look for more novel mutations in ESBL-producing strains of K. pneumoniae.
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262. |
Zhao J,
Dang H,
( 2011 ) Identification of a globally distributed clinical streptomycin-resistance plasmid and other resistance determinants in a coastal bay of China. PMID : 21054449 : DOI : 10.1111/j.1472-765X.2010.02958.x Abstract >>
To study streptomycin-resistant bacteria isolated from Jiaozhou Bay and their molecular determinants of resistance. Twenty-seven tetracycline-resistant and 49 chloramphenicol-resistant bacterial isolates from surface seawater of Jiaozhou Bay were selected for investigation. More than 88% of these isolates were resistant to streptomycin. Half of the streptomycin-resistant bacteria harboured the strA-strB gene pair, and six isolates carried Tn5393-like transposons by PCR detection. The p9123-related plasmids containing the sul2-strA-strB gene cluster were characterized in two environmental Escherichia coli isolates. Transposon Tn5393 was first identified on a Klebsiella pneumoniae plasmid, which also carried Tn1721, estP and umu genes responsible for antimicrobial and insecticide resistance. Coresistance to streptomycin and tetracycline or chloramphenicol was found with high frequency. p9123-related plasmid and Tn5393 transposon may contribute to the wide distribution and spread of the strA-strB gene pair in Jiaozhou Bay. The detection of streptomycin-resistance plasmid pQ1-1 from Jiaozhou Bay seawater bacteria and human bacterial pathogens from USA indicates its global dissemination and transmission, across different components of the microbiota on earth. Streptomycin resistance can be recognized as an important bioindicator of environmental quality, owing to its association with anthropogenic pollution and the multidrug-resistant microbiota.
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263. |
Koh TH,
Khoo CT,
Wijaya L,
Leong HN,
Lo YL,
Lim LC,
Koh TY,
( 2010 ) Global spread of New Delhi metallo-�]-lactamase 1. PMID : 21109168 : DOI : 10.1016/S1473-3099(10)70274-7 Abstract >>
N/A
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264. |
Zou LK,
Wang HN,
Zeng B,
Zhang AY,
Li JN,
Li XT,
Tian GB,
Wei K,
Zhou YS,
Xu CW,
Yang ZR,
( 2011 ) Phenotypic and genotypic characterization of �]-lactam resistance in Klebsiella pneumoniae isolated from swine. PMID : 21035968 : DOI : 10.1016/j.vetmic.2010.09.030 Abstract >>
Little is known about the antimicrobial resistance mechanisms in Klebsiella pneumoniae from swine in China. Thus, this paper aims to demonstrate the �]-lactam resistance phenotypes and genotypes of K. pneumoniae isolates from swine in southwestern China, detect possible new �]-lactamase variants, and determine whether or not the variants differ in their antibiotic resistance. Isolates from 58 unrelated diseased swine were collected from 61 pig farms in southwestern China from 2007 to 2009. Among the 58 isolates, 75.8-100% were resistant to �]-lactam, 62.0-68.97% to fluoroquinolone, 44.8-46.55% to aminoglycoside, and 8.62-17.24% to �]-lactam inhibitors. PCR amplification and DNA sequencing showed that bla(TEM-1) was detected in 100% (n=58) of the isolates, bla(SHV) in 82.76% (n=48), bla(CTX-M) in 39.66% (n=23), and bla(OKP) in 17.24% (n=10). The bla(SHV) types included bla(SHV-1), bla(SHV-11), bla(SHV-12), and bla(SHV-27). None of the isolates harbored bla(KPC), bla(LEN), or bla(GES) gene. Four novel variants (bla(OKP-A-13), bla(OKP-A-14), bla(OKP-A-15), and bla(OKP-A-16)) were identified among the 10 OKP �]-lactamase-producing K. pneumoniae isolates resistant to ampicillin, amoxicillin, oxacillin, cefalexin, and cefadroxil. Plasmid analysis and PCR amplification indicated that bla(TEM-1) genes were detected in the total plasmid. Molecular typing by pulsed-field gel electrophoresis revealed the presence of 10 distinct pulsotypes of OKP producer isolates. Plasmid DNA digested with XbaI yielded two to six bands of ca. 0.15-30 kb. Transformants of the 10 OKP producer isolates showed no differences in their antibiotic susceptibility, except for the pulsotype B transformant, which carried bla(CTX-M). In China, �]-lactam resistance appeared to be common among K. pneumoniae isolates from swine, suggesting that K. pneumoniae may be a reservoir for the dissemination of �]-lactam resistance among Chinese pig farms.
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265. |
Bojer MS,
Struve C,
Ingmer H,
Hansen DS,
Krogfelt KA,
( 2010 ) Heat resistance mediated by a new plasmid encoded Clp ATPase, ClpK, as a possible novel mechanism for nosocomial persistence of Klebsiella pneumoniae. PMID : 21085699 : DOI : 10.1371/journal.pone.0015467 PMC : PMC2976762 Abstract >>
Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermotolerant providing a conceivable explanation of its persistence in the hospital environment. This marked phenotype is mediated by a novel type of Clp ATPase, designated ClpK. The clpK gene is encoded by a conjugative plasmid and we find that the clpK gene alone renders an otherwise sensitive E. coli strain resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections.
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266. |
Jiang Y,
Yu D,
Wei Z,
Shen P,
Zhou Z,
Yu Y,
( 2010 ) Complete nucleotide sequence of Klebsiella pneumoniae multidrug resistance plasmid pKP048, carrying blaKPC-2, blaDHA-1, qnrB4, and armA. PMID : 20547789 : DOI : 10.1128/AAC.00137-10 PMC : PMC2934982 Abstract >>
The Klebsiella pneumoniae multidrug resistance plasmid pKP048 was completely sequenced. This plasmid carries several important resistance determinants, such as bla(KPC-2), bla(DHA-1), qnrB4, and armA, which confer resistance to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, respectively. Analysis of the finished 151,188-bp sequence data revealed 163 putative genes, 108 of which were assigned functions such as replication, stable inheritance, antibiotic resistance, a mobile element, conjugal transfer, and a restriction-modification system, showing the strong phylogenetic mosaicism and plasticity of the plasmid.
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267. |
N/A N/A,
( 2010 ) Detection of Enterobacteriaceae isolates carrying metallo-beta-lactamase - United States, 2010. PMID : 20577157 : Abstract >>
During January-June 2010, three Enterobacteriaceae isolates carrying a newly described resistance mechanism, the New Delhi metallo-beta-lactamase (NDM-1), were identified from three U.S. states at the CDC antimicrobial susceptibility laboratory. This is the first report of NDM-1 in the United States, and the first report of metallo-beta-lactamase carriage among Enterobacteriaceae in the United States. These isolates, which include an Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, carry blaNDM-1, which confers resistance to all beta-lactam agents except aztreonam (a monobactam antimicrobial); all three isolates were aztreonam resistant, presumably by a different mechanism. In the United Kingdom, where these organisms are increasingly common, carriage of Enterobacteriaceae containing blaNDM-1 has been closely linked to receipt of medical care in India and Pakistan. All three U.S. isolates were from patients who received recent medical care in India.
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268. |
Ong CL,
Beatson SA,
Totsika M,
Forestier C,
McEwan AG,
Schembri MA,
( 2010 ) Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species. PMID : 20576143 : DOI : 10.1186/1471-2180-10-183 PMC : PMC2900259 Abstract >>
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.
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269. |
De Araujo C,
Balestrino D,
Roth L,
Charbonnel N,
Forestier C,
( 2010 ) Quorum sensing affects biofilm formation through lipopolysaccharide synthesis in Klebsiella pneumoniae. PMID : 20600864 : DOI : 10.1016/j.resmic.2010.05.014 Abstract >>
Biofilm formation by Klebsiella pneumoniae is modulated by quorum sensing through the synthesis of interspecies AI-2 autoinducers. We characterized in K. pneumoniae the genes homologous to those described in Escherichia coli involved in AI-2 transport, and created two isogenic mutants deleted of lsrCD and tqsA. The levels of extracellular AI-2 with lsrCD and tqsA knockout mutants showed increased and lowered concentrations of AI-2, respectively. The level of transcripts of luxS, the gene responsible for AI-2 synthesis, was increased in sessile cells of the tqsA mutant. In contrast, the expression of the AI-2 import regulator genes lsrR and lsrK was decreased. In addition, the two mutants lsrCD and tqsA formed biofilms with greater biomass but impaired architecture. Since exopolysaccharides play a main role in K. pneumoniae biofilm formation, we investigated their relationship with AI-2 synthesis. None of the mutations in luxS and the AI-2 transport systems affected the expression of three capsule polysaccharide-related genes (wzi, wza and wzx), but all induced an increase in the expression of two lipopolysaccharide (LPS)-synthesis-related genes, wbbM and wzm. AI-2 therefore seems to act as a regulator of biofilm formation and LPS synthesis in sessile K. pneumoniae cells.
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270. |
Ruiz E,
Rojo-Bezares B,
Sáenz Y,
Olarte I,
Esteban I,
Rocha-Gracia R,
Zarazaga M,
Torres C,
( 2010 ) Outbreak caused by a multi-resistant Klebsiella pneumoniae strain of new sequence type ST341 carrying new genetic environments of aac(6')-Ib-cr and qnrS1 genes in a neonatal intensive care unit in Spain. PMID : 20547103 : DOI : 10.1016/j.ijmm.2010.04.014 Abstract >>
An outbreak due to a Klebsiella pneumoniae clone occurred in a neonatal intensive care unit of a Spanish Hospital in which three newborns were infected (all with gestational age ?29 weeks; two of them died) and seven were colonized (gestational age >32 weeks; none died). One K. pneumoniae strain per patient was further characterized. The 10 strains showed an indistinguishable pulsed-field-gel-electrophoresis pattern, were typed in the phylogenetic group KpI and were ascribed into a new sequence type registered as ST341. All 10 strains presented the same multiple-antibiotic-resistant phenotype, showed extended-spectrum-beta-lactamase production, and harbored the bla(CTX-M-15), bla(SHV-11), bla(OXA-1,)aac(6')-Ib-cr, qnrS1, aac(3)-II, aph(3')-Ia and aadA5 resistance genes. No class 1 or class 2 integrons were detected. The bla(CTX-M-15) gene presented the following genetic environment: ISEcp1-bla(CTX-M-15)-orf477. These strains contained two copies of the aac(6')-Ib-cr gene included in the following new genetic environments: aac(3)-II-IS26-aac(6')-Ib-cr-bla(OXA-1) and aac(3)-II-IS26-�GcatB3-bla(OXA-1)-aac(6')-Ib-cr (registered at GenBank with accession numbers GQ438247 and GQ438248, respectively). The genetic environment of the qnrS1 gene (IS26-�GISEcl2-qnrS1) (GenBank accession number GQ438249) was also not described previously. The aac(6')-Ib-cr, qnrS1, bla(CTX-M-15), aac(3)-II, and bla(OXA-1) genes, located in a plasmid of 33.5 kb, could be transferred to Escherichia coli by transformation.
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271. |
Austin S,
Kundrot C,
Dixon R,
( 1991 ) Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function. PMID : 2041769 : DOI : 10.1093/nar/19.9.2281 PMC : PMC329431 Abstract >>
A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.
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272. |
Billot-Klein D,
Gutmann L,
Collatz E,
( 1990 ) Nucleotide sequence of the SHV-5 beta-lactamase gene of a Klebsiella pneumoniae plasmid. PMID : 2088203 : DOI : 10.1128/aac.34.12.2439 PMC : PMC172080 Abstract >>
The nucleotide sequence of the SHV-5 beta-lactamase gene, subcloned from a plasmid of Klebsiella pneumoniae, was determined. The amino acid changes thought to be responsible for the extended substrate profile of SHV-5 are Gly----Ser234 and Glu----Lys235. SHV-5 is identical to SHV-4, except for Leu----Arg201, which accounts for the difference in apparent pI of the two enzymes.
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273. |
Prosser GA,
Patterson AV,
Ackerley DF,
( 2010 ) uvrB gene deletion enhances SOS chromotest sensitivity for nitroreductases that preferentially generate the 4-hydroxylamine metabolite of the anti-cancer prodrug CB1954. PMID : 20727918 : DOI : 10.1016/j.jbiotec.2010.08.007 Abstract >>
CB1954 is an anti-cancer prodrug that can be reduced at either of two nitro groups to form cytotoxic metabolites. We describe here two efficient and previously uncharacterized nitroreductases, YfkO from Bacillus subtilis which reduces CB1954 exclusively at the 4-NO(2) position, and NfsA from Klebsiella pneumoniae which preferentially reduces the 2-NO(2) group. Utilizing these novel enzymes, together with three previously characterized nitroreductases, we demonstrate that the Escherichia coli SOS-chromotest assay can differentially detect the 4-nitro versus 2-nitro reduction products of CB1954 following deletion of the nucleotide excision repair gene uvrB, but not mismatch repair (mutS) or methyltransferase (ada/ogt) genes. These findings may hold significance for identification and selection of nitroreductases for CB1954-mediated gene therapy, particularly when targeting tumors that are deficient in nucleotide excision repair. Moreover, we demonstrate that comparative SOS chromotest analysis in wild type and uvrB mutant strains can be used to determine whether or not nucleotide excision repair plays a significant role in processing DNA damage resulting from activation of different nitroaromatic prodrugs.
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274. |
Pournaras S,
Poulou A,
Voulgari E,
Vrioni G,
Kristo I,
Tsakris A,
( 2010 ) Detection of the new metallo-beta-lactamase VIM-19 along with KPC-2, CMY-2 and CTX-M-15 in Klebsiella pneumoniae. PMID : 20522444 : DOI : 10.1093/jac/dkq190 Abstract >>
To report the identification of the metallo-beta-lactamase (MBL) variant VIM-19 in a Klebsiella pneumoniae clinical strain co-producing KPC-2 carbapenemase, CMY-2 cephalosporinase and CTX-M-15 extended-spectrum beta-lactamase. MICs were determined by agar dilution. Phenotypic tests were performed to detect carbapenemase production. PCR and nucleotide sequencing were used for the identification of bla gene types and mapping of the integron carrying the MBL gene. The location of the MBL and KPC alleles was investigated by mating experiments, plasmid analysis and PCR assays. Imipenem, meropenem and ertapenem MICs for the study strain were 32, 16 and 64 mg/L, respectively. The strain carried bla(TEM-1), bla(CMY-2), bla(KPC-2) and bla(CTX-M-15) genes along with the gene bla(VIM-19), which was located in a class 1 integron as the first gene cassette, followed by aacA6, dfrA1 and aadA1 cassettes. Mating experiments, plasmid analysis and PCR assays revealed that bla(VIM-19) and bla(CMY-2) were carried on an approximately 150 kb self-transferable plasmid, while bla(KPC-2) and bla(TEM-1) were on an approximately 70 kb self-transferable plasmid; bla(CTX-M-15) was non-transferable. The detection of the new MBL, VIM-19, which has enhanced carbapenemase activity, along with KPC-2, CMY-2 and CTX-M-15 is of concern. Further spread of the respective strains or plasmids may have serious consequences for antimicrobial chemotherapy.
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275. |
Perez F,
Endimiani A,
Ray AJ,
Decker BK,
Wallace CJ,
Hujer KM,
Ecker DJ,
Adams MD,
Toltzis P,
Dul MJ,
Windau A,
Bajaksouzian S,
Jacobs MR,
Salata RA,
Bonomo RA,
( 2010 ) Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination. PMID : 20513702 : DOI : 10.1093/jac/dkq191 PMC : PMC2904665 Abstract >>
Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained bla(OXA-23), and four isolates possessed bla(OXA-24/40). Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing bla(KPC-2) or bla(KPC-3) were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.
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276. |
Miriagou V,
Papagiannitsis CC,
Kotsakis SD,
Loli A,
Tzelepi E,
Legakis NJ,
Tzouvelekis LS,
( 2010 ) Sequence of pNL194, a 79.3-kilobase IncN plasmid carrying the blaVIM-1 metallo-beta-lactamase gene in Klebsiella pneumoniae. PMID : 20660690 : DOI : 10.1128/AAC.00665-10 PMC : PMC2944605 Abstract >>
The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla(VIM-1), aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.
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277. |
García-Fernández A,
Miriagou V,
Papagiannitsis CC,
Giordano A,
Venditti M,
Mancini C,
Carattoli A,
( 2010 ) An ertapenem-resistant extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae clone carries a novel OmpK36 porin variant. PMID : 20660683 : DOI : 10.1128/AAC.01301-09 PMC : PMC2944588 Abstract >>
Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and �]-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem- and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem.
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278. |
Locatelli C,
Scaccabarozzi L,
Pisoni G,
Moroni P,
( 2010 ) CTX-M1 ESBL-producing Klebsiella pneumoniae subsp. pneumoniae isolated from cases of bovine mastitis. PMID : 20720020 : DOI : 10.1128/JCM.00941-10 PMC : PMC2953115 Abstract >>
N/A
|
279. |
Wecksler SR,
Stoll S,
Iavarone AT,
Imsand EM,
Tran H,
Britt RD,
Klinman JP,
( 2010 ) Interaction of PqqE and PqqD in the pyrroloquinoline quinone (PQQ) biosynthetic pathway links PqqD to the radical SAM superfamily. PMID : 20737074 : DOI : 10.1039/c0cc00968g Abstract >>
pqqD is one of six genes required for PQQ production in Klebsiella pneumoniae. Herein, we demonstrate that PqqD interacts specifically with the radical SAM enzyme PqqE, causing a perturbation in the electronic environment around the [4Fe-4S](+) clusters. This interaction redirects the role for PqqD in PQQ biosynthesis.
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280. |
Kristóf K,
Tóth A,
Damjanova I,
Jánvári L,
Konkoly-Thege M,
Kocsis B,
Koncan R,
Cornaglia G,
Szego E,
Nagy K,
Szabó D,
( 2010 ) Identification of a blaVIM-4 gene in the internationally successful Klebsiella pneumoniae ST11 clone and in a Klebsiella oxytoca strain in Hungary. PMID : 20410063 : DOI : 10.1093/jac/dkq133 Abstract >>
N/A
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281. |
Landman D,
Babu E,
Shah N,
Kelly P,
Bäcker M,
Bratu S,
Quale J,
( 2010 ) Activity of a novel aminoglycoside, ACHN-490, against clinical isolates of Escherichia coli and Klebsiella pneumoniae from New York City. PMID : 20667885 : DOI : 10.1093/jac/dkq278 Abstract >>
Reports of Enterobacteriaceae resistant to all commonly used antimicrobial agents, including �]-lactams, fluoroquinolones and aminoglycosides, are increasing in hospitals worldwide. The activity of ACHN-490, a next-generation aminoglycoside, was examined against clinical isolates of Escherichia coli and Klebsiella pneumoniae from hospitals in New York City, an area where multidrug-resistant organisms are endemic. Unique patient isolates of E. coli and K. pneumoniae were gathered from 16 hospitals located in New York City in 2009 and underwent susceptibility testing to aminoglycosides and ACHN-490. Subsets of isolates were characterized by PCR for the presence of genes encoding aminoglycoside-modifying enzymes, ribosomal methylases and KPC-type carbapenemases. Although most isolates of E. coli were susceptible to the aminoglycosides, the MIC(90) values of gentamicin, tobramycin and amikacin were 32, 8 and 4 mg/L, respectively. The MIC(90) of ACHN-490 was 1 mg/L. Multidrug resistance, including resistance to aminoglycosides and the presence of bla(KPC), was much more common in isolates of K. pneumoniae. However, the MIC(90) of ACHN-490 for K. pneumoniae was also 1 mg/L. The MICs of ACHN-490 did not correlate with the presence of commonly recovered aminoglycoside-modifying enzymes. Bactericidal activity was evident in most isolates at concentrations 4�� the MIC. The novel aminoglycoside ACHN-490 retains activity against most isolates of E. coli and K. pneumoniae, including multidrug-resistant strains. Additional studies examining the roles of efflux systems and outer membrane permeability alterations are recommended in isolates with reduced susceptibility to this agent.
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282. |
Zong Z,
Partridge SR,
Iredell JR,
( 2010 ) ISEcp1-mediated transposition and homologous recombination can explain the context of bla(CTX-M-62) linked to qnrB2. PMID : 20421399 : DOI : 10.1128/AAC.00041-10 PMC : PMC2897309 Abstract >>
bla(CTX-M-62), a C508T variant of bla(CTX-M-3b), was transferred from Klebsiella pneumoniae JIE137 on a conjugative plasmid together with a class 1 integron containing the dfrA12-gcuF-aadA2 cassette array, ISCR1, and qnrB2. bla(CTX-M-62) lies between intact and rearranged copies of ISEcp1 in a configuration that can be explained by a combination of transposition and homologous recombination and which also illustrates the ability of ISEcp1 to mobilize an adjacent gene as part of transposition units of different sizes.
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283. |
Leavitt A,
Chmelnitsky I,
Carmeli Y,
Navon-Venezia S,
( 2010 ) Complete nucleotide sequence of KPC-3-encoding plasmid pKpQIL in the epidemic Klebsiella pneumoniae sequence type 258. PMID : 20696875 : DOI : 10.1128/AAC.00175-10 PMC : PMC2944570 Abstract >>
We have determined the entire DNA sequence of plasmid pKpQIL, the bla(KPC-3)-carrying plasmid harbored by the carbapenem-resistant Klebsiella pneumoniae clone sequence type 258 (ST 258) in Israel. pKpQIL is a 113,637-bp, self-transmissible plasmid that belongs to the incompatibility group IncFII. It consists of a large backbone of a pKPN4-like plasmid and carries the bla(KPC-3)-containing Tn4401a transposon of a pNYC-like plasmid.
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284. |
Kumarasamy KK,
Toleman MA,
Walsh TR,
Bagaria J,
Butt F,
Balakrishnan R,
Chaudhary U,
Doumith M,
Giske CG,
Irfan S,
Krishnan P,
Kumar AV,
Maharjan S,
Mushtaq S,
Noorie T,
Paterson DL,
Pearson A,
Perry C,
Pike R,
Rao B,
Ray U,
Sarma JB,
Sharma M,
Sheridan E,
Thirunarayan MA,
Turton J,
Upadhyay S,
Warner M,
Welfare W,
Livermore DM,
Woodford N,
( 2010 ) Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. PMID : 20705517 : DOI : 10.1016/S1473-3099(10)70143-2 PMC : PMC2933358 Abstract >>
Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-beta-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK. Enterobacteriaceae isolates were studied from two major centres in India--Chennai (south India), Haryana (north India)--and those referred to the UK's national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene bla(NDM-1) was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan. We identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries. The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.
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285. |
Turton JF,
Perry C,
Elgohari S,
Hampton CV,
( 2010 ) PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. PMID : 20110386 : DOI : 10.1099/jmm.0.015198-0 Abstract >>
A multiplex PCR is described which detects capsular types K1, K2, K5, K54 and K57, which are those most associated with invasive disease or pathogenicity, a further capsular type (K20), two putative virulence factors (rmpA and wcaG) and the 16S-23S internal transcribed spacer unit of Klebsiella pneumoniae, facilitating identification of this organism. wcaG encodes capsular fucose production and was associated with capsular types K1 and K54, but was also found in strains of other capsular types; 18 of the 543 isolates screened were PCR-positive for this gene. An eight-locus variable number tandem repeat (VNTR) scheme was designed, which provided discrimination at a level similar to that afforded by PFGE among a panel of 36 isolates representing 29 PFGE types. All isolates tested of the virulent K1 clone of CC23, associated with pyogenic liver abscesses, shared the same VNTR profile, which may be helpful in identifying this clone; such isolates were also PCR-positive for allS. These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.
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286. |
Tolmasky ME,
( 1990 ) Sequencing and expression of aadA, bla, and tnpR from the multiresistance transposon Tn1331. PMID : 1963948 : Abstract >>
A fragment of Tn1331 including tnpR, aac, aadA, and a bla gene which encodes lower levels of resistance to ampicillin and carbenicillin as compared to those mediated by the TEM beta-lactamase was sequenced. The polypeptide encoded by the bla gene has homology with the OXA-1, PSE-2, and OXA-2 proteins. Genes aac and bla are upstream and downstream respectively of aadA, and are both flanked by recombinational hot spots. Tn1331 has 520-bp direct repeats which include parts of the tnpR and TEM bla genes. Evolutionary models for the genesis of Tn1331 are proposed.
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287. |
Feizabadi MM,
Delfani S,
Raji N,
Majnooni A,
Aligholi M,
Shahcheraghi F,
Parvin M,
Yadegarinia D,
( 2010 ) Distribution of bla(TEM), bla(SHV), bla(CTX-M) genes among clinical isolates of Klebsiella pneumoniae at Labbafinejad Hospital, Tehran, Iran. PMID : 19961397 : DOI : 10.1089/mdr.2009.0096 Abstract >>
Extended-spectrum beta-lactamase (ESBL)-producing isolates of Klebsiella pneumoniae have been increasingly recognized in the hospital settings in Iran as well as throughout the world. The aim of this study was to detect and determine the genes encoding the ESBLs including bla(TEM), bla(SHV), and bla(CTX-M) groups among the K. pneumoniae isolates at Labbafinejad Hospital by polymerase chain reaction (PCR) and characterize them by direct sequencing of PCR products. Eighty-nine isolates were isolated from patients at different wards during March 2008-March 2009. They were identified as K. pneumoniae using biochemical tests. Susceptibility of isolates to 17 different antimicrobial agents was determined using agar disk diffusion method. The phenotypic confirmatory test was used to screen the isolates for production of ESBLs. To amplify the bla(SHV) the template DNA was extracted by boiling method. Plasmid DNA was extracted using minipreparation kit and used as template in PCR for detection of bla(TEM) and bla(CTX-M). The selected PCR products were sequenced and analyzed. All 89 strains were susceptible to imipenem. The rates of resistance to different antibiotics were in the following order: aztronam (79.7%), cefexime (67.4%), cefpodoxime (66.2%), cefotaxime (65.1%), ceftazidime (61.7%). The phenotypic confirmatory test detected 62 isolates (69.7%) as ESBL-producing K. pneumoniae. The prevalence of genes encoding ESBLs were as follows: bla(TEM) 54% (n = 48), bla(SHV) 67.4% (n = 60), bla(CTX-M-I) 46.51% (n = 40), and bla(CTX-M-III) 29% (n = 25). The bla(CTX-M-II) and bla(CTX-M-IV) were not detected. All bla(TEM) types were characterized as bla(TEM-1) and all bla(CTX-M-I) were identified as bla(CTX-M-15). The SHV types were characterized as SHV-5, SHV-11, and SHV-12. The rate of ESBL at Labbafinejad Hospital was 25% increase in a 4-year study that ended in March 2009. It appears that bla(TEM-1), bla(SHV-5), bla(SHV-11), bla(SHV-12), and bla(CTX-M-15) are the dominant ESBLs among the resistant strains of K. pneumoniae in Iran.
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288. |
Yi H,
Xi Y,
Liu J,
Wang J,
Wu J,
Xu T,
Chen W,
Chen B,
Lin M,
Wang H,
Zhou M,
Li J,
Xu Z,
Jin S,
Bao Q,
( PloS one ) PMID : 20066042 : DOI : 10.1371/journal.pone.0008601 PMC : PMC2797631 Abstract >>
Klebsiella pneumoniae is a clinically significant species of bacterium which causes a variety of diseases. Clinical treatment of this bacterial infection is greatly hindered by the emergence of multidrug-resistant strains. The resistance is largely due to the acquisition of plasmids carrying drug-resistant as well as pathogenic genes, and its conjugal transfer facilitates the spread of resistant phenotypes. The 70,057 bp plasmid pKF3-70, commonly found in Klebsiella pneumoniae, is composed of five main functional modules, including regions involved in replication, partition, conjugation, transfer leading, and variable regions. This plasmid is more similar to several Escherichia coli plasmids than any previously reported K. pneumoniae plasmids and pKF3-70 like plasmids share a common and conserved backbone sequence. The replication system of the pKF3-70 is 100% identical to that of RepFII plasmid R100 from E. coli. A beta-lactamase gene ctx-m-14 with its surrounding insertion elements (ISEcp1, truncated IS903 and a 20 bp inverted repeat sequence) may compose an active transposon which is directly bordered by two putative target repeats ATTAC."
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289. |
Tato M,
Coque TM,
Baquero F,
Cantón R,
( 2010 ) Dispersal of carbapenemase blaVIM-1 gene associated with different Tn402 variants, mercury transposons, and conjugative plasmids in Enterobacteriaceae and Pseudomonas aeruginosa. PMID : 19901094 : DOI : 10.1128/AAC.00783-09 PMC : PMC2798558 Abstract >>
The emergence of bla(VIM-1) within four different genetic platforms from distinct Enterobacteriaceae and Pseudomonas aeruginosa isolates in an area with a low prevalence of metallo-beta-lactamase producers is reported. Forty-three VIM-1-producing isolates (including 19 Enterobacter cloacae, 2 Escherichia coli, and 2 P. aeruginosa isolates, 18 Klebsiella pneumoniae isolate, and 2 Klebsiella oxytoca isolate) recovered from 2005 to 2007 and corresponding to 15 pulsed-field gel electrophoresis types were studied. The Enterobacteriaceae isolates corresponded to a hospital outbreak, and the P. aeruginosa isolates were sporadically recovered. The genetic context of the integrons carrying bla(VIM-1) (arbitrarily designated types A, B, C, and D) was characterized by PCR mapping based on known Tn402 and mercury transposons and further sequencing. Among Enterobacteriaceae isolates, bla(VIM-1) was part of integrons located either in an In2-Tn402 element linked to Tn21 (type A; In110-bla(VIM-1)-aacA4-aadA1) or in a Tn402 transposon lacking the whole tni module [type B; In113-bla(VIM-1)-aacA4-dhfrII (also called dfrB1)-aadA1-catB2] and the transposon was associated with an IncHI2 or IncI1 plasmid, respectively. Among P. aeruginosa isolates, bla(VIM-1) was part of a new gene cassette array located in a defective Tn402 transposon carrying either tniBDelta3 and tniA (type C; bla(VIM-1)-aadA1) or tniC and DeltatniQ (type D; bla(VIM-1)-aadB), and both Tn402 variants were associated with conjugative plasmids of 30 kb. The dissemination of bla(VIM-1) was associated with different genetic structures and bacterial hosts, depicting a complex emergence and evolutionary network scenario in our facility, Ram?n y Cajal University Hospital, Madrid, Spain. Knowledge of the complex epidemiology of bla(VIM-1) is necessary to control this emerging threat.
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290. |
Verdet C,
Gautier V,
Chachaty E,
Ronco E,
Hidri N,
Decré D,
Arlet G,
( 2009 ) Genetic context of plasmid-carried blaCMY-2-like genes in Enterobacteriaceae. PMID : 19596889 : DOI : 10.1128/AAC.00753-08 PMC : PMC2737857 Abstract >>
Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of blaCMY-2-like genes reflected the replicon type, usually IncA/C or IncI1. These blaCMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.
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291. |
Papanicolaou GA,
Medeiros AA,
Jacoby GA,
( 1990 ) Novel plasmid-mediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae. PMID : 1963529 : DOI : 10.1128/aac.34.11.2200 PMC : PMC172023 Abstract >>
Klebsiella pneumoniae isolates from 11 patients at the Miriam Hospital were identified as resistant to cefoxitin and ceftibuten as well as to aztreonam, cefotaxime, and ceftazidime. Resistance could be transferred by conjugation or transformation with plasmid DNA into Escherichia coli and was due to the production of a beta-lactamase with an isoelectric point of 8.4 named MIR-1. In E. coli, MIR-1 conferred resistance to aztreonam, cefotaxime, ceftazidime, ceftibuten, ceftriaxone, and such alpha-methoxy beta-lactams as cefmetazole, cefotetan, cefoxitin, and moxalactam. In vitro, MIR-1 hydrolyzed cephalothin and cephaloridine much more rapidly than it did penicillin G, ampicillin, or carbenicillin. Cefotaxime was hydrolyzed at 10% the rate of cephaloridine. Cefoxitin inactivation could only be detected by a microbiological test. The inhibition profile of MIR-1 was similar to that of chromosomally mediated class I beta-lactamases. Potassium clavulanate had little effect on cefoxitin or cefibuten resistance and was a poor inhibitor of MIR-1 activity. Cefoxitin or imipenem did not induce MIR-1. The gene determining MIR-1 was cloned on a 1.4-kb AccI-PstI fragment. Under stringent conditions, probes for TEM-1 and SHV-1 genes and the E. coli ampC gene failed to hybridize with the MIR-1 gene. However, a provisional sequence of 150 bp of the MIR-1 gene proved to be 90% identical to the sequence of ampC from Enterobacter cloacae but only 71% identical to that of E. coli, thus explaining the lack of hybridization to the E. coli ampC probe. Plasmid profiles of the 11 K. pneumoniae clinical isolates were not identical, but each contained a plasmid from 40 to 60 kb that hybridized with the cloned MIR-1 gene. Both transfer-proficient and transfer-deficient MIR-1 plasmids belonged to the N incompatibility group. Thus, the resistance of these K. pneumoniae strains was the result of plasmid acquisition of a class I beta-lactamase, a new resistance determinant that expands the kinds of beta-lactam resistance capable of spread by plasmid dissemination among clinical isolates.
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292. |
Shen P,
Wei Z,
Jiang Y,
Du X,
Ji S,
Yu Y,
Li L,
( 2009 ) Novel genetic environment of the carbapenem-hydrolyzing beta-lactamase KPC-2 among Enterobacteriaceae in China. PMID : 19620332 : DOI : 10.1128/AAC.00260-09 PMC : PMC2764158 Abstract >>
Thirty-nine bla(KPC)-producing isolates of the family Enterobacteriaceae with carbapenem resistance or reduced carbapenem susceptibility were obtained from inpatients from eight hospitals in six cities of three provinces in eastern China. The pulsed-field gel electrophoresis analysis of all 36 Klebsiella pneumoniae isolates revealed six major patterns. The resistant plasmids of most isolates were successfully transferred by conjugation and evaluated experimentally to be 40 to 180 kb in size. A 20.2-kb bla(KPC)-surrounding nucleotide sequence from plasmid pKP048 has been obtained and contains an integration structure of a Tn3-based transposon and partial Tn4401 segment, with the gene order Tn3-transposase, Tn3-resolvase, ISKpn8, the bla(KPC-2) gene, and the ISKpn6-like element. The chimera of several transposon-associated elements indicated a novel genetic environment of the K. pneumoniae carbapenemase beta-lactamase gene in isolates from China.
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293. |
Schneider I,
Queenan AM,
Markovska R,
Markova B,
Keuleyan E,
Bauernfeind A,
( 2009 ) New variant of CTX-M-type extended-spectrum beta-lactamases, CTX-M-71, with a Gly238Cys substitution in a Klebsiella pneumoniae isolate from Bulgaria. PMID : 19620330 : DOI : 10.1128/AAC.00461-09 PMC : PMC2764165 Abstract >>
A single Klebsiella pneumoniae strain isolated in a Bulgarian hospital was found to produce CTX-M-71, a new CTX-M variant characterized by one amino acid substitution from glycine to cysteine at position 238 in comparison to CTX-M-15. This exchange decreased the hydrolytic activity of the beta-lactamase for cefotaxime, ceftazidime, and cefepime.
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294. |
Leinberger DM,
Grimm V,
Rubtsova M,
Weile J,
Schröppel K,
Wichelhaus TA,
Knabbe C,
Schmid RD,
Bachmann TT,
( 2010 ) Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. PMID : 20007393 : DOI : 10.1128/JCM.00765-09 PMC : PMC2815585 Abstract >>
Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).
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295. |
Cheng J,
Wang Q,
Chen Y,
Ye Y,
Li H,
Li X,
Li JB,
( 2009 ) Phenotypic and molecular characterization of a novel beta-lactamase carried by Klebsiella pneumoniae, CTX-M-72, derived from CTX-M-3. PMID : 19590148 : Abstract >>
This study reports phenotypic and molecular characterization of a novel CTX-M beta-lactamase carried by two Klebsiella pneumoniae isolates collected from two hospitals in China. Conjugation experiment, Southern hybridization, susceptibility testing, isoelectric focusing, PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization of the novel CTX-M-type enzyme. The analyses of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were performed to investigate epidemiology. The PCR products had 967 nucleotides and a novel CTX-M enzyme with a pI of 8.5 was implicated in this resistance: CTX-M-72. Two strains exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. The amino acid sequence of the CTX-M-72 beta-lactamase differed from that of the CTX-M-3 beta-lactamase by the Arg-->Gly change at position 164. The novel enzyme was susceptible to ceftazidime, the same response being observed for other CTX-M enzymes. The substrates of the beta-lactamase were also characterized. Furthermore, two resistant genes of clinical strains were closely related. The emergence of a novel CTX-M-type extended-spectrum beta-lactamase was rarely described in other areas. This study illustrated the importance of molecular surveillance in tracking CTX-M-producing strains in large teaching hospitals, suggested the horizontal transfer of plasmid-borne bla(CTX-M) genes contributed to the dissemination of CTX-M enzymes in hospital environments, and emphasized the need for epidemiological monitoring.
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296. |
Landman D,
Bratu S,
Quale J,
( 2009 ) Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae. PMID : 19556371 : DOI : 10.1099/jmm.0.012575-0 PMC : PMC2887543 Abstract >>
Isolates of Klebsiella pneumoniae harbouring the carbapenemase KPC may have carbapenem MICs that remain in the susceptible range, and may therefore go unrecognized. To understand the mechanisms contributing to the variability in carbapenem MICs, 20 clinical isolates, all belonging to either of two clonal groups of KPC-possessing K. pneumoniae endemic to New York City, were examined. Expression of genes encoding KPC, the porins OmpK35 and OmpK36, and the efflux pump AcrAB was examined by real-time RT-PCR. Outer-membrane profiles of selected KPC-producing isolates were examined by SDS-PAGE, and proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The identification of SHV and TEM beta-lactamases and the genomic sequences of ompK35 and ompK36 were determined by PCR and DNA sequencing, respectively. For one clonal group, carbapenem MICs increased with decreasing expression of ompK36. A second clonal group also had carbapenem MICs that correlated with ompK36 expression. However, all of the isolates in this latter group continued to produce OmpK36, suggesting that porin configuration may affect entry of carbapenems. For isolates that had the greatest expression of ompK36, carbapenem MICs tended to be lower when determined by the broth microdilution technique, and scattered colonies were seen around the Etest zones of inhibition. All of the KPC-producing isolates were highly resistant to ertapenem, regardless of ompK36 expression. In conclusion, isolates of KPC-possessing K. pneumoniae that express ompK36 tend to have lower MICs to carbapenems and therefore may be more difficult to detect by clinical laboratories. Regardless of ompK36 expression, all of the KPC producers were consistently resistant to ertapenem.
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297. |
Mata C,
Miró E,
Rivera A,
Mirelis B,
Coll P,
Navarro F,
( 2010 ) Prevalence of acquired AmpC beta-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes at a Spanish hospital from 1999 to 2007. PMID : 19523051 : DOI : 10.1111/j.1469-0691.2009.02864.x Abstract >>
In 2007, a significant increase in acquired ampC genes in Enterobacteriaceae from 0.06% in 1999 to 1.3% was observed. Proteus mirabilis showed the highest prevalence (0.95%) and CMY-2 was the most prevalent AmpC enzyme (66.7%). Other enzymes such as CMY-4, DHA-1, ACC-1, and three new enzymes called CMY-25, CMY-27 and CMY-40 were detected. Seven out of the 117 isolates (6%) also produced an extended-spectrum beta-lactamase. As acquired AmpC enzymes are likely to become a serious public health issue worldwide, close surveillance is necessary to curb their spread.
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298. |
Lawrence JG,
Ochman H,
Hartl DL,
( 1991 ) Molecular and evolutionary relationships among enteric bacteria. PMID : 1955870 : DOI : 10.1099/00221287-137-8-1911 Abstract >>
Classification of bacterial species into genera has traditionally relied upon variation in phenotypic characteristics. However, these phenotypes often have a multifactorial genetic basis, making unambiguous taxonomic placement of new species difficult. By designing evolutionarily conserved oligonucleotide primers, it is possible to amplify homologous regions of genes in diverse taxa using the polymerase chain reaction and determine their nucleotide sequences. We have constructed a phylogeny of some enteric bacteria, including five species classified as members of the genus Escherichia, based on nucleotide sequence variation at the loci encoding glyceraldehyde-3-phosphate dehydrogenase and outer membrane protein 3A, and compared this genealogy with the relationships inferred by biotyping. The DNA sequences of these genes defined congruent and robust phylogenetic trees indicating that they are an accurate reflection of the evolutionary history of the bacterial species. The five species of Escherichia were found to be distantly related and, contrary to their placement in the same genus, do not form a monophyletic group. These data provide a framework which allows the relationships of additional species of enteric bacteria to be inferred. These procedures have general applicability for analysis of the classification, evolution, and epidemiology of bacterial taxa.
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299. |
Yang Q,
Wang H,
Sun H,
Chen H,
Xu Y,
Chen M,
( 2010 ) Phenotypic and genotypic characterization of Enterobacteriaceae with decreased susceptibility to carbapenems: results from large hospital-based surveillance studies in China. PMID : 19805565 : DOI : 10.1128/AAC.01099-09 PMC : PMC2798477 Abstract >>
The resistance mechanism of 49 Enterobacteriaceae isolates with decreased susceptibility to carbapenems collected from 2004 to 2008 at 16 teaching hospitals in China was investigated. Moderate- to high-level carbapenem resistance in most isolates was more closely associated with loss or decreased expression of both major porins combined with production of AmpC or extended-spectrum beta-lactamase enzymes, while KPC-2, IMP-4, and IMP-8 carbapenemase production may lead to a low to moderate level of carbapenem resistance in Enterobacteriaceae in China.
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300. |
Mshana SE,
Imirzalioglu C,
Hossain H,
Hain T,
Domann E,
Chakraborty T,
( 2009 ) Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany. PMID : 19534775 : DOI : 10.1186/1471-2334-9-97 PMC : PMC2708165 Abstract >>
Multi-drug-resistant, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital. We analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays. Examination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb. Our data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.
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301. |
Zhu WH,
Luo L,
Wang JY,
Zhuang XH,
Zhong L,
Liao K,
Zeng Y,
Lu YJ,
( 2009 ) Complete nucleotide sequence of pCTX-M360, an intermediate plasmid between pEL60 and pCTX-M3, from a multidrug-resistant Klebsiella pneumoniae strain isolated in China. PMID : 19752275 : DOI : 10.1128/AAC.00032-09 PMC : PMC2786365 Abstract >>
In this work we report the characterization of plasmid pCTX-M360, isolated from a Klebsiella pneumoniae strain from China and encoding the CTX-M-3 extended-spectrum beta-lactamase. Sequence analysis of pCTX-M360 revealed extensive similarity with pEL60 and pCTX-M3, two other enterobacterial plasmids of the IncL/M incompatibility group. Compared to pEL60, pCTX-M360 contains several insertions but lacks most of a 27-kb insert found in pCTX-M3, suggesting that it could be an evolutionary intermediate between pEL60 and pCTX-M3.
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302. |
Docquier JD,
Calderone V,
De Luca F,
Benvenuti M,
Giuliani F,
Bellucci L,
Tafi A,
Nordmann P,
Botta M,
Rossolini GM,
Mangani S,
( 2009 ) Crystal structure of the OXA-48 beta-lactamase reveals mechanistic diversity among class D carbapenemases. PMID : 19477418 : DOI : 10.1016/j.chembiol.2009.04.010 Abstract >>
Carbapenem-hydrolyzing class D beta-lactamases (CHDLs) are enzymes found in important Gram-negative pathogens (mainly Acinetobacter baumannii and Enterobacteriaceae) that confer resistance to beta-lactam antibiotics, and notably carbapenems. The crystal structure of the OXA-48 carbapenemase was determined at pH 7.5 and at a resolution of 1.9 A. Surprisingly, and by contrast with OXA-24, the only other CHDL of known crystal structure, the structure of OXA-48 was similar to OXA-10, an enzyme devoid of carbapenemase activity, indicating that the hydrolysis of these compounds could depend on subtle changes in the active site region. Moreover, the active site groove of OXA-48 was different from that of OXA-24 in shape, dimensions, and charge distribution. Molecular dynamics pointed to the functional relevance of residues located in or close to the beta5-beta6 loop and allowed us to propose a mechanism for carbapenem hydrolysis by OXA-48.
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303. |
Chen LM,
Maloy S,
( 1991 ) Regulation of proline utilization in enteric bacteria: cloning and characterization of the Klebsiella put control region. PMID : 1987164 : DOI : 10.1128/jb.173.2.783-790.1991 PMC : PMC207072 Abstract >>
Enteric bacteria can grow on proline as the sole nitrogen and carbon source. Expression of the proline utilization (put) operon in Klebsiella strains and Escherichia coli is responsive to nitrogen regulation. In contrast, Salmonella typhimurium cannot activate put operon expression when growing in medium with glucose as a carbon source and proline as the sole nitrogen source. To compare nitrogen regulatory sites in the control regions of the put operons in these three closely related genera, we cloned the Klebsiella put operon onto a plasmid. The putA and putP genes were localized on the plasmid by transposon mutagenesis. The DNA sequence of the put control region was determined and compared with those of the put control regions from S. typhimurium and E. coli. The overall size and organization of the put control region were very similar in all three bacteria. However, no obvious ntr regulatory sites were found in this region, and transcription of the put genes started at the same sites during growth with limiting or excess nitrogen. These results strongly suggested that the Klebsiella put operon may not be directly regulated by the ntr system.
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304. |
Gruber TD,
Borrok MJ,
Westler WM,
Forest KT,
Kiessling LL,
( 2009 ) Ligand binding and substrate discrimination by UDP-galactopyranose mutase. PMID : 19500588 : DOI : 10.1016/j.jmb.2009.05.081 PMC : PMC2771219 Abstract >>
Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5'-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 A resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the consequences of these differences, we derived a model for the productive UGM-substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination.
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305. |
Turner MS,
Andersson P,
Bell JM,
Turnidge JD,
Harris T,
Giffard PM,
( 2009 ) Plasmid-borne blaSHV genes in Klebsiella pneumoniae are associated with strong promoters. PMID : 19749204 : DOI : 10.1093/jac/dkp338 Abstract >>
Extended-spectrum beta-lactamases (ESBLs) belonging to the SHV family remain a major cause of ESBL-positive phenotypes in Klebsiella pneumoniae. The bla(SHV) gene is a normal constituent of the K. pneumoniae chromosome. However, most ESBL-encoding bla(SHV) genes found in K. pneumoniae are plasmid borne. The objective was to determine the contribution of promoter variants to the expression of plasmid-borne bla(SHV) genes. K. pneumoniae clinical isolates were analysed for the presence of IS26 insertions characteristic of plasmid-borne bla(SHV), and differences in their bla(SHV) promoter sequences and expression levels. A high resolution melting (HRM)-based method for rapid promoter analysis was developed. An IS26 insertion characteristic of the plasmid-borne bla(SHV-1)/bla(SHV-2)/bla(SHV-5) family was 100% linked to a promoter mutated in the -10 region, a mutation previously only found on the chromosome. The mutation was shown by real-time reverse transcriptase PCR to be associated with increased bla(SHV) expression. Plasmid-borne bla(SHV) is associated with strong promoters. It is likely that an SHV-dependent ESBL-positive phenotype requires both a strong promoter and a coding sequence mutation. An HRM assay can indicate bla(SHV) expression.
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306. |
Kim HB,
Wang M,
Park CH,
Kim EC,
Jacoby GA,
Hooper DC,
( 2009 ) oqxAB encoding a multidrug efflux pump in human clinical isolates of Enterobacteriaceae. PMID : 19528276 : DOI : 10.1128/AAC.01574-08 PMC : PMC2715617 Abstract >>
The genes for multidrug efflux pump OqxAB, which is active on fluoroquinolones, were found in human clinical isolates on a plasmid in Escherichia coli and on the chromosome of Klebsiella pneumoniae. IS26-like sequences flanked the plasmid-mediated oqxAB genes, suggesting that they had been mobilized as part of a composite transposon.
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307. |
Chang CY,
Fang YT,
Tsai SM,
Chang LL,
Yu WL,
( 2009 ) Characterization of class 1 integrons and gene cassettes in clinical isolates of Klebsiella pneumoniae from Taiwan. PMID : 19748438 : DOI : 10.1016/j.diagmicrobio.2009.06.005 Abstract >>
We surveyed the prevalence and contents of class 1 integrons in clinical Klebsiella pneumoniae isolates collected from Kaohsiung, Taiwan, during 2 periods (1993 and 2004). Class 1 integrons were present in 78 isolates (34.2%) from 1993 (n = 228) and 129 (32.9%) from 2004 (n = 392) and contained varied gene cassette number, type, and array. We found 2 atypical sul3-associated class 1 integrons and identified 26 different gene cassettes, including an aac(6')-Ib-cr cassette here firstly described in K. pneumoniae from Taiwan. The continuing evolution of class 1 integrons is threatening to undermine the effectiveness of antimicrobial therapy for K. pneumoniae.
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308. |
Hennequin C,
Forestier C,
( 2009 ) oxyR, a LysR-type regulator involved in Klebsiella pneumoniae mucosal and abiotic colonization. PMID : 19786563 : DOI : 10.1128/IAI.00837-09 PMC : PMC2786449 Abstract >>
Colonization of the gastrointestinal tract is the first event in Klebsiella pneumoniae nosocomial infections, followed by colonization of the bladder or respiratory tract or entry into the bloodstream. To survive in the host, bacteria must harbor specific traits and overcome multiple stresses. OxyR is a conserved bacterial transcription factor with a key role both in the upregulation of defense mechanisms against oxidative stress and in pathogenesis by enhancing biofilm formation, fimbrial expression, and mucosal colonization. A homolog of oxyR was detected in silico in the K. pneumoniae sequenced genome and amplified from the LM21 wild-type strain. To determine the role of oxyR in K. pneumoniae host-interaction processes, an oxyR isogenic mutant was constructed, and its behavior was assessed. At concentrations lower than 10(7) ml(-1), oxyR-deficient organisms were easily killed by micromolar concentrations of H(2)O(2) and exhibited typical aerobic phenotypes. The oxyR mutant was impaired in biofilm formation and types 1 and 3 fimbrial gene expression. In addition, the oxyR mutant was unable to colonize the murine gastrointestinal tract, and in vitro assays showed that it was defective in adhesion to Int-407 and HT-29 intestinal epithelial cells. The behavior of the oxyR mutant was also determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment: deletion of oxyR resulted in higher sensitivity to bile and acid stresses but not to osmotic stress. These results show the pleiotropic role of oxyR in K. pneumoniae gastrointestinal colonization.
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309. |
Dropa M,
Balsalobre LC,
Lincopan N,
Mamizuka EM,
Cassettari VC,
Matté GR,
Matté MH,
( 2010 ) Emergence of Klebsiella pneumoniae carrying the novel extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and the class 1 integron-associated bla(GES-7) in Brazil. PMID : 19689462 : DOI : 10.1111/j.1469-0691.2009.02944.x Abstract >>
A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.
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310. |
Yong D,
Toleman MA,
Giske CG,
Cho HS,
Sundman K,
Lee K,
Walsh TR,
( 2009 ) Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. PMID : 19770275 : DOI : 10.1128/AAC.00774-09 PMC : PMC2786356 Abstract >>
A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary tract infection caused by a carbapenem-resistant Klebsiella pneumoniae strain that typed to the sequence type 14 complex. The isolate, Klebsiella pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. As well as possessing unique residues near the active site, NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation. The broad resistance carried on these plasmids is a further worrying development for India, which already has high levels of antibiotic resistance.
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311. |
Shu HY,
Fung CP,
Liu YM,
Wu KM,
Chen YT,
Li LH,
Liu TT,
Kirby R,
Tsai SF,
( 2009 ) Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates. PMID : 19744990 : DOI : 10.1099/mic.0.029017-0 Abstract >>
Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 5' end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 3' end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.
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312. |
Brisse S,
Fevre C,
Passet V,
Issenhuth-Jeanjean S,
Tournebize R,
Diancourt L,
Grimont P,
( 2009 ) Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization. PMID : 19319196 : DOI : 10.1371/journal.pone.0004982 PMC : PMC2656620 Abstract >>
Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedl?nder's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23(K1) and clone CC82(K1), were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82(K1) indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23(K1) remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent clones associated with specific infections and provide an evolutionary framework for research into the links between clones, virulence and other genomic features in K. pneumoniae.
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313. |
Gruber TD,
Westler WM,
Kiessling LL,
Forest KT,
( 2009 ) X-ray crystallography reveals a reduced substrate complex of UDP-galactopyranose mutase poised for covalent catalysis by flavin. PMID : 19719175 : DOI : 10.1021/bi901437v PMC : PMC2785223 Abstract >>
The flavoenzyme uridine 5'-diphosphate galactopyranose mutase (UGM or Glf) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. The latter is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin-galactose iminium. Here, we describe 2.3 and 2.5 A resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, C1 of the substrate is 3.6 A from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin.
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314. |
Struve C,
Bojer M,
Krogfelt KA,
( 2009 ) Identification of a conserved chromosomal region encoding Klebsiella pneumoniae type 1 and type 3 fimbriae and assessment of the role of fimbriae in pathogenicity. PMID : 19703972 : DOI : 10.1128/IAI.00585-09 PMC : PMC2772557 Abstract >>
Type 3 fimbriae are expressed by most clinical Klebsiella pneumoniae isolates and mediate adhesion to host structures in vitro. However, the role of type 3 fimbriae in K. pneumoniae virulence has not been evaluated by use of in vivo infection models. In this study, the type 3 fimbrial gene cluster (mrk) of the clinical isolate C3091 is described in detail. The mrk gene cluster was revealed to be localized in close proximity to the type 1 fimbrial gene cluster. Thus, a 20.4-kb fimbria-encoding region was identified and found to be highly conserved among different K. pneumoniae isolates. Interestingly, a homologue to PecS, known as a global regulator of virulence in Erwinia chrysanthemi, was identified in the fimbria-encoding region. Comparison to the previously characterized plasmid encoded mrk gene cluster revealed significant differences, and it is established here that the putative regulatory gene mrkE is not a part of the chromosomally encoded type 3 fimbrial gene cluster. To evaluate the role of type 3 fimbriae in virulence, a type 3 fimbria mutant and a type 1 and type 3 fimbria double mutant was constructed. Type 3 fimbria expression was found to strongly promote biofilm formation. However, the fimbria mutants were as effective at colonizing the intestine as the wild type, and their virulence was not attenuated in a lung infection model. Also, in a urinary tract infection model, type 3 fimbriae did not influence the virulence, whereas type 1 fimbriae were verified as an essential virulence factor. Thus, type 3 fimbriae were established not to be a virulence factor in uncomplicated K. pneumoniae infections. However, since type 3 fimbriae promote biofilm formation, their role in development of infections in catheterized patients needs to be elucidated.
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315. |
Roche C,
Cotter M,
O Connell N,
Crowley B,
( 2009 ) First identification of class A carbapenemase-producing Klebsiella pneumoniae in the Republic of Ireland. PMID : 19341609 : Abstract >>
The Klebsiella pneumoniae carbapenemase (KPC) was detected in a carbapenem-resistant respiratory isolate of Klebsiella pneumoniae in an Irish hospital. This is the first report of a KPC-producing isolate in the Republic of Ireland. The isolate was resistant to all beta-lactams. Furthermore, it had reduced susceptibility to three other classes of non-beta-lactam antibiotics. The isolate was not associated with travel abroad. Detection of KPC-producing bacteria has important infection control and public health implications.
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316. |
Ben Achour N,
Mercuri PS,
Ben Moussa M,
Galleni M,
Belhadj O,
( 2009 ) Characterization of a novel extended-spectrum TEM-type beta-lactamase, TEM-164, in a clinical strain of Klebsiella pneumoniae in Tunisia. PMID : 19728777 : DOI : 10.1089/mdr.2009.0900 Abstract >>
Klebsiella pneumoniae ML1708 exhibited a multiresistance phenotype, including resistance to all beta-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The double-disk synergy test was positive. ML1708 harbored a 50 kb conjugative plasmid that encoded a beta-lactamase of pI 5.5. The corresponding bla gene was identified by polymerase chain reaction and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new variant of TEM-1 beta-lactamase designated TEM-164. TEM-164 contains the unusual following mutations: L40V and I279T. These modifications may result in a change of the pI to 5.5 and hydrolyze cefotaxime and ceftazidime.
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317. |
Ozgumus OB,
Sandalli C,
Sevim A,
Celik-Sevim E,
Sivri N,
( 2009 ) Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey. PMID : 19229487 : DOI : 10.1007/s12275-008-0206-z Abstract >>
We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla(oxA-30), and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.
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318. |
Zhang XX,
Zhang T,
Zhang M,
Fang HH,
Cheng SP,
( 2009 ) Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants. PMID : 19224208 : DOI : 10.1007/s00253-009-1886-y Abstract >>
Class 1 integrons and gene cassettes containing antibiotic resistance genes (ARGs) in five different sewage treatment plants (STPs) were characterized and quantified using polymerase chain reaction (PCR), sequencing, and quantitative real-time PCR (qRT-PCR) in this study. Class 1 integronase gene (intI1) was found commonly occurring in all of activated sludge samples from the five STPs, as well as in influent and effluent of two STPs at Hong Kong. One hundred and nine lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from activated sludge of Shatin STP. Among them, 36 strains (33.0%) were found to carry class 1 integrons. PCR assays showed that 11 of the 36 intI1-carrying isolates harbored a common type of gene cassette array of about 1,600 bps, as well as the static genes (sulI and qacEDelta1) on class 1 integrons. This gene cassette array was found phylogenetically close to antibiotic resistance genes dfr17 and aadA5, encoding dihydrofolate reductase conferring resistance to trimethoprim and adenylyltransferase conferring resistance to spectinomycin/streptomycin, respectively. Antimicrobial susceptibility analysis demonstrated that all the 11 LFEs carrying gene cassette were multi-resistant, especially having common resistance to trimethoprim and streptomycin. qRT-PCR assay showed that genes copies of both class 1 integron and the gene cassette varied significantly among the activated sludge sampled from different STPs, at different time points or different treatment steps. More than 90% of class 1 integrons and the gene cassette were removed by activated sludge processes in two STPs, while the disinfection process removed 94% integron and 77% gene cassette in one STP.
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319. |
Liu W,
Chen L,
Li H,
Duan H,
Zhang Y,
Liang X,
Li X,
Zou M,
Xu L,
Hawkey PM,
( 2009 ) Novel CTX-M {beta}-lactamase genotype distribution and spread into multiple species of Enterobacteriaceae in Changsha, Southern China. PMID : 19297379 : DOI : 10.1093/jac/dkp068 Abstract >>
The aim of this study was to undertake a survey of the occurrence of CTX-M and SHV extended-spectrum beta-lactamase (ESBL) genotypes in Enterobacteriaceae from Hunan Province, China. Clinical isolates (425) from three major hospitals in Changsha, Hunan Province, were collected between October 2004 and July 2005, and their antimicrobial susceptibilities of the genotype of bla(CTX-M) and bla(SHV) were determined. Random amplified polymorphic DNA was used to characterize the clonality of all of the isolates. The overall rate of ESBL-positive isolates was 33.4% (142/425). The dominant ESBLs were CTX-M types, and were found in 109/142 (76.8%) isolates comprising seven different genera/species, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris and Providencia stuartii. The most common bla(CTX-M) genotypes were bla(CTX-M-14) (47.7%), bla(CTX-M-3) (29.4%) and bla(CTX-M-15) (17.4%). A novel gene derived from bla(CTX-M-15), bla(CTX-M-82) (Ala-40-->Pro), was identified. The dominant ESBL genotype in Hunan Province was bla(CTX-M). The high prevalence (17.4%) of bla(CTX-M-15) has not previously been reported from China. Our results identify that an epidemic of bla(CTX-M) in Changsha, Hunan Province, has evolved with the appearance and spread of bla(CTX-M-15) against the dominant genotypes bla(CTX-M-14) and bla(CTX-M-3.) The worldwide dominance of bla(CTX-M-15) could be poised to spread to China, displacing the current prevailing genotypes.
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320. |
Hammad AM,
Ishida Y,
Shimamoto T,
( 2009 ) Prevalence and molecular characterization of ampicillin-resistant Enterobacteriaceae isolated from traditional Egyptian Domiati cheese. PMID : 19343954 : DOI : 10.4315/0362-028x-72.3.624 Abstract >>
The aim of this study was to address the prevalence and the molecular characteristics of antibiotic-resistant enteric bacteria isolated from one of the most popular types of Egyptian cheese. A total of 215 ampicillin-resistant enterobacterial isolates were obtained from 80 samples of Domiati cheese, and they were screened by PCR for a large pool of antibiotic resistance markers, including extended-spectrum beta-lactamases (ESBLs), class 1 and class 2 integrons, and plasmid-mediated quinolone resistance genes. It was determined that the most frequent mechanism of ampicillin resistance was from a TEM-1-type beta-lactamase. As well, SHV beta-lactamases, including SHV-1, SHV-25, and SHV-26, showed a high prevalence, and two novel SHV beta-lactamases, SHV-110 and SHV-111, were identified. Type CTX-M-14, OXY-1, OXA-1, and CMY-4 beta-lactamases were also detected in a few isolates. In addition, a novel AmpC beta-lactamase was detected that was designated CMY-41. Sequencing results of class 1 integrons revealed that the uncommon aminoglycoside resistance gene cassette aadA22 was found for the first time in an Escherichia coli strain. The other class 1 integrons harbored various common gene cassettes, including aadA1, aadA1a, aadA2, aadA12, dfr5, dfr7, dfr12, and dfr15. The only isolate that carried a class 2 integron contained dfrA1, sat2, and aadA1. Plasmid-mediated quinolone resistance determinants qnrS and qnrB showed a low prevalence. This study provides meaningful data on high antimicrobial resistance contained in Domiati cheese samples and reports for the first time the presence of beta-lactamases, plasmid-mediated quinolone resistance, and integrons in isolates from food of Egyptian animal origin.
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321. |
Pavez M,
Mamizuka EM,
Lincopan N,
( 2009 ) Early dissemination of KPC-2-producing Klebsiella pneumoniae strains in Brazil. PMID : 19332672 : DOI : 10.1128/AAC.00089-09 PMC : PMC2687248 Abstract >>
N/A
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322. |
Gootz TD,
Lescoe MK,
Dib-Hajj F,
Dougherty BA,
He W,
Della-Latta P,
Huard RC,
( 2009 ) Genetic organization of transposase regions surrounding blaKPC carbapenemase genes on plasmids from Klebsiella strains isolated in a New York City hospital. PMID : 19258268 : DOI : 10.1128/AAC.01355-08 PMC : PMC2681555 Abstract >>
Carbapenem-resistant Klebsiella strains carrying Klebsiella pneumoniae carbapenemases (KPC) are endemic to New York City and are spreading across the United States and internationally. Recent studies have indicated that the KPC structural gene is located on a 10-kb plasmid-borne element designated Tn4401. Fourteen Klebsiella pneumoniae strains and one Klebsiella oxytoca strain isolated at a New York City hospital in 2005 carrying either bla(KPC-2) or bla(KPC-3) were examined for isoforms of Tn4401. Ten of the Klebsiella strains contained a 100-bp deletion in Tn4401, corresponding to the Tn4401a isoform. The presence of this deletion adjacent to the upstream promoter region of bla(KPC) in Tn4401a resulted in a different -35 promoter sequence of TGGAGA than that of CTGATT present in isoform Tn4401b. Complete sequencing of one plasmid carrying bla(KPC) from each of three nonclonal isolates indicated the presence of genes encoding other types of antibiotic resistance determinants. The 70.6-kb plasmid from K. pneumoniae strain S9 carrying bla(KPC-2) revealed two identical copies of Tn4401b inserted in an inverse fashion, but in this case, one of the elements disrupted a group II self-splicing intron. In K. pneumoniae strain S15, the Tn4401a element carrying bla(KPC-2) was found on both a large 120-kb plasmid and a smaller 24-kb plasmid. Pulsed-field gel electrophoresis results indicate that the isolates studied represent a heterogeneous group composed of unrelated as well as closely related Klebsiella strains. Our results suggest that endemic KPC-positive Klebsiella strains constitute a generally nonclonal population comprised of various alleles of bla(KPC) on several distinct plasmid genetic backgrounds. This study increases our understanding of the genetic composition of the evolving and expanding role of KPC-producing, healthcare-associated, gram-negative pathogens.
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323. |
Endimiani A,
Hujer AM,
Perez F,
Bethel CR,
Hujer KM,
Kroeger J,
Oethinger M,
Paterson DL,
Adams MD,
Jacobs MR,
Diekema DJ,
Hall GS,
Jenkins SG,
Rice LB,
Tenover FC,
Bonomo RA,
( 2009 ) Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA. PMID : 19155227 : DOI : 10.1093/jac/dkn547 PMC : PMC2640158 Abstract >>
The emergence of bla(KPC)-containing Klebsiella pneumoniae (KPC-Kp) isolates is attracting significant attention. Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed. We analysed 42 KPC-Kp recovered during 2006-07 from five institutions located in the Eastern USA. Antimicrobial susceptibility tests, analytical isoelectric focusing (aIEF), PCR and sequencing of bla genes, PFGE and rep-PCR were performed. Results By in vitro testing, KPC-Kp isolates were highly resistant to all non-carbapenem beta-lactams (MIC(90)s >or= 128 mg/L). Among carbapenems, MIC(50/90)s were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 for ertapenem. Combinations of clavulanate or tazobactam with a carbapenem or cefepime did not significantly lower the MIC values. Genetic analysis revealed that the isolates possessed the following bla genes: bla(KPC-2) (59.5%), bla(KPC-3) (40.5%), bla(TEM-1) (90.5%), bla(SHV-11) (95.2%) and bla(SHV-12) (50.0%). aIEF of crude beta-lactamase extracts from these strains supported our findings, showing beta-lactamases at pIs of 5.4, 7.6 and 8.2. The mean number of beta-lactamases was 3.5 (range 3-5). PFGE demonstrated that 32 (76.2%) isolates were clonally related (type A). Type A KPC-Kp isolates (20 bla(KPC-2) and 12 bla(KPC-3)) were detected in each of the five institutions. rep-PCR showed patterns consistent with PFGE. We demonstrated the complex beta-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.
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324. |
Cheng J,
Gao W,
Yin J,
Sun Z,
Ye Y,
Gao YF,
Li X,
Li JB,
( 2010 ) Phenotypic and molecular characterization of two novel CTX-M enzymes carried by Klebsiella pneumoniae. PMID : 19294528 : DOI : 10.1007/s11033-009-9499-1 Abstract >>
Two clinical strains (Klebsiella pneumoniae 516 and K. pneumoniae 1335) collected in September 2006 from different hospitals in Anhui Province (China) harboured two novel plasmid-mediated bla(CTX-M) genes, designated bla(CTX-M-80) and bla(CTX-M-81), respectively. Both CTX-M-80 with pI of 9.0 and CTX-M-81 with pI of 8.4 were extended-spectrum beta-lactamases (ESBLs). The results of susceptibility testing demonstrated two enzymes were highly activity against broad spectrum beta-lactams, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-80) gene was detected on a 110-kb plasmid and the bla(CTX-M-81) gene existed on a 120-kb plasmid. The deduced amino acid sequence of CTX-M-80 differed from that of CTX-M-3 by the substitution Ala-27-->Val, and CTX-M-81 possessed the Lys-->Glu, Lys-->Gln, and Asn-->His changes at respective position 82, 98, and 132 in compassion with CTX-M-14. The enzymatic properties showed CTX-M-80 and CTX-M-81 had higher affinities for penicillin G (lower Km values) than for cephalosporins. The activities of novel enzymes against ceftazidime were undetectable or limited, as indicated by MICs data, the same response being observed for many other CTX-M enzymes. This report was evidence of the diversity of CTX-M-type ESBLs in China.
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325. |
Coudeyras S,
Nakusi L,
Charbonnel N,
Forestier C,
( 2008 ) A tripartite efflux pump involved in gastrointestinal colonization by Klebsiella pneumoniae confers a tolerance response to inorganic acid. PMID : 18644883 : DOI : 10.1128/IAI.00356-08 PMC : PMC2546844 Abstract >>
The colonization of the gastrointestinal tract of patients by the opportunistic gram-negative bacillus Klebsiella pneumoniae generally occurs prior to the development of nosocomial infections. Mutant strain C-81 was isolated owing to its reduced capacity to colonize the digestive tract in a murine model following transposon mutagenesis (N. Maroncle, D. Balestrino, C. Rich, and C. Forestier, Infect. Immun. 70:4729-4734, 2002). Nucleotide sequence analysis showed that the transposon had inserted into the first open reading frame, eefA, of a three-gene locus (eefABC) whose homologue encodes a tripartite efflux pump in Enterobacter aerogenes (M. Masi, J. M. Pages, C. Villard, and E. Pradel, J. Bacteriol. 187:3894-3897, 2005), and this operon includes an additional short (183-bp) potential open reading frame, eefX, upstream of eefA. In vivo assays showed that a DeltaeefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the DeltaeefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.
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326. |
Jones CH,
Ruzin A,
Tuckman M,
Visalli MA,
Petersen PJ,
Bradford PA,
( 2009 ) Pyrosequencing using the single-nucleotide polymorphism protocol for rapid determination of TEM- and SHV-type extended-spectrum beta-lactamases in clinical isolates and identification of the novel beta-lactamase genes blaSHV-48, blaSHV-105, and blaTEM-155. PMID : 19075050 : DOI : 10.1128/AAC.01155-08 PMC : PMC2650538 Abstract >>
TEM- and SHV-type extended-spectrum beta-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the bla(TEM) and bla(SHV) genes. Three novel beta-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing bla(SHV-105) were >128, 128, and >128 microg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing bla(TEM-155) were >128, 64, and > 128 microg/ml, respectively. Pyrosequence analysis determined the true identity of the beta-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.
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327. |
Zioga A,
Whichard JM,
Kotsakis SD,
Tzouvelekis LS,
Tzelepi E,
Miriagou V,
( 2009 ) CMY-31 and CMY-36 cephalosporinases encoded by ColE1-like plasmids. PMID : 19104021 : DOI : 10.1128/AAC.01284-08 PMC : PMC2650574 Abstract >>
Two CMY-2 derivatives, CMY-31 (Gln(215)-->Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala(77)-->Cys and Gln(193)-->Glu) from Klebsiella pneumoniae, were characterized. Both cephalosporinases functionally resembled CMY-2. bla(CMY) alleles occurred as parts of a putative transposon comprising ISEcp1B and a Citrobacter freundii-derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca.
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328. |
Schubert S,
Darlu P,
Clermont O,
Wieser A,
Magistro G,
Hoffmann C,
Weinert K,
Tenaillon O,
Matic I,
Denamur E,
( 2009 ) Role of intraspecies recombination in the spread of pathogenicity islands within the Escherichia coli species. PMID : 19132082 : DOI : 10.1371/journal.ppat.1000257 PMC : PMC2606025 Abstract >>
Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI-encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI-flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli.
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329. |
Liu Y,
Zhang B,
Cao Q,
Huang W,
Shen L,
Qin X,
( 2009 ) Two clinical strains of Klebsiella pneumoniae carrying plasmid-borne blaIMP-4, blaSHV-12, and armA isolated at a Pediatric Center in Shanghai, China. PMID : 19164142 : DOI : 10.1128/AAC.01325-08 PMC : PMC2663070 Abstract >>
Two cases of pulmonary infection due to strains of multidrug-resistant Klebsiella pneumoniae were investigated. Beta-lactamase determinants, such as bla(IMP-4) and bla(SHV-12), and the 16S rRNA methyltransferase-encoding gene armA were detected in these plasmid-bearing organisms. The integron-borne bla(IMP-4) and armA contained intervening sequences highly related to those of a Vibrio cholerae O139 plasmid found in Hangzhou, China.
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330. |
Stahlhut SG,
Chattopadhyay S,
Struve C,
Weissman SJ,
Aprikian P,
Libby SJ,
Fang FC,
Krogfelt KA,
Sokurenko EV,
( 2009 ) Population variability of the FimH type 1 fimbrial adhesin in Klebsiella pneumoniae. PMID : 19151141 : DOI : 10.1128/JB.00601-08 PMC : PMC2648365 Abstract >>
FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli, suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae, our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae.
|
331. |
Nelson K,
Whittam TS,
Selander RK,
( 1991 ) Nucleotide polymorphism and evolution in the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) in natural populations of Salmonella and Escherichia coli. PMID : 1862091 : DOI : 10.1073/pnas.88.15.6667 PMC : PMC52149 Abstract >>
Nucleotide sequences of the gapA gene, encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, were determined for 16 strains of Salmonella and 13 strains of Escherichia coli recovered from natural populations. Pairs of sequences from strains representing the eight serovar groups of Salmonella differed, on average, at 3.8% of nucleotide sites and 1.1% of inferred amino acids, and comparable values for E. coli were an order of magnitude smaller (0.2% and 0.1%, respectively). The rate of substitution at synonymous sites was significantly higher for codons specifying the catalytic domain of the enzyme than for those encoding the NAD(+)-binding domain, but the nonsynonymous substitution rate showed the opposite relationship. For Salmonella, statistical tests for nonrandom clustering of polymorphic sites failed to provide evidence that intragenic recombination or gene conversion has contributed to the generation of allelic diversity. The topology of a tree constructed from the gapA sequences was generally similar to that of phylogenetic trees of the strains based on multilocus enzyme electrophoresis, but the level of divergence of gapA in Salmonella group V from other Salmonella and E. coli strains is much greater than that indicated by DNA hybridization for the genome as a whole.
|
332. |
Hammad AM,
Ahmed AM,
Ishida Y,
Shimamoto T,
( 2008 ) First characterization and emergence of SHV-60 in raw milk of a healthy cow in Japan. PMID : 19057150 : DOI : 10.1292/jvms.70.1269 Abstract >>
During monitoring of raw milk samples from healthy cows for the presence of antibiotic resistant bacteria, one isolate of Klebsiella pneumoniae strain HUF-100 was found to be resistant to oxyimino-cephalosporins and aztreonam. It was found to carry a chromosomally-encoded extended-spectrum beta-lactamase that has not been described previously, namely SHV-60. Thus, it must be expected that this strain will spread further among food-producing animals and thereby constitute a reservoir of this resistant strain and resistance gene that can transfer to and cause treatment problems for humans. The present study confirms the hypothesis that some of novel multiple antibiotic resistant zoonotic bacterial pathogens may initially emerge from food animals and reports, for the first time, this type of emergence in Japan.
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333. |
Shi W,
Qin J,
Mi Z,
( 2008 ) A Klebsiella pneumoniae sputum culture isolate from China carrying blaOXA-1, blaCTX-M-55 and aac(6')-Ib-cr. PMID : 19018035 : DOI : 10.1099/jmm.0.2008/000950-0 Abstract >>
N/A
|
334. |
Abbassi MS,
Torres C,
Achour W,
Vinué L,
Sáenz Y,
Costa D,
Bouchami O,
Ben Hassen A,
( 2008 ) Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia. PMID : 18620848 : DOI : 10.1016/j.ijantimicag.2008.04.009 Abstract >>
Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.
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335. |
Dubois V,
Poirel L,
Demarthe F,
Arpin C,
Coulange L,
Minarini LA,
Bezian MC,
Nordmann P,
Quentin C,
( 2008 ) Molecular and biochemical characterization of SHV-56, a novel inhibitor-resistant beta-lactamase from Klebsiella pneumoniae. PMID : 18663019 : DOI : 10.1128/AAC.00387-08 PMC : PMC2565901 Abstract >>
A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla(SHV-56), encoding a new inhibitor-resistant beta-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.
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336. |
Källman O,
Motakefi A,
Wretlind B,
Kalin M,
Olsson-Liljequist B,
Giske CG,
( 2008 ) Cefuroxime non-susceptibility in multidrug-resistant Klebsiella pneumoniae overexpressing ramA and acrA and expressing ompK35 at reduced levels. PMID : 18647746 : DOI : 10.1093/jac/dkn296 Abstract >>
The aims were to study if efflux and down-regulation of porins contribute to cefuroxime resistance in Klebsiella pneumoniae and to co-resistance to unrelated antibiotics. Ten cefuroxime-non-susceptible but cefotaxime-susceptible blood culture isolates of K. pneumoniae and one multiply antibiotic-resistant (MAR) laboratory strain (selected by chloramphenicol) were examined. Transcription of the genes acrA, ompK35, ramA, marA and soxS was determined with quantitative RT-PCR. All clinical isolates and the MAR laboratory strain had similar antibiograms with non-susceptibility to cefuroxime, tigecycline, chloramphenicol and nalidixic acid. Phenylalanine arginine beta-naphthylamide (PAbetaN) increased susceptibility to tigecycline, chloramphenicol and nalidixic acid, but not to cefuroxime. Increased acrA transcription and decreased ompK35 transcription was seen in all strains. Increased ramA transcription was seen in all strains except one clinical isolate. This multidrug-resistant phenotype of K. pneumoniae is associated with increased acrA and ramA transcription and decreased ompK35 transcription. Since the cefuroxime resistance was not reversed by PAbetaN, it was probably attributable to decreased levels of OmpK35, rather than to efflux.
|
337. |
Post V,
Hall RM,
( 2009 ) Insertion sequences in the IS1111 family that target the attC recombination sites of integron-associated gene cassettes. PMID : 19025573 : DOI : 10.1111/j.1574-6968.2008.01412.x Abstract >>
Members of the recently identified IS1111 family differ from the majority of insertion sequences (IS) in that they target specific sites in an orientation-specific manner. However, the way in which target selection is achieved is not known. ISKpn4 is representative of a new subgroup of the IS1111 family whose members are found in the attC sites (59-be) of the gene cassettes associated with integrons. The transposases of this subgroup are closely related (over 75% identity), confirming that closely related IS usually share a common target. However, among more distant relatives encoding a transposase <45% identical to those of the ISKpn4 group, one IS, ISPa25, was found that also targets attC sites. It appears that the targeting determinant of the ISKpn4 group has become associated with a transposase gene from a different group, and this allowed us to localize the region that is likely to be required for target selection to a long noncoding region found downstream of the transposase gene in all IS1111 family members. This region may determine an RNA used to guide the IS to its specific target.
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338. |
Monteiro J,
Santos AF,
Asensi MD,
Peirano G,
Gales AC,
( 2009 ) First report of KPC-2-producing Klebsiella pneumoniae strains in Brazil. PMID : 19015350 : DOI : 10.1128/AAC.00736-08 PMC : PMC2612176 Abstract >>
N/A
|
339. |
Diestra K,
Juan C,
Curiao T,
Moyá B,
Miró E,
Oteo J,
Coque TM,
Pérez-Vázquez M,
Campos J,
Cantón R,
Oliver A,
Navarro F,
N/A N/A,
( 2009 ) Characterization of plasmids encoding blaESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae. PMID : 18988679 : DOI : 10.1093/jac/dkn453 Abstract >>
The aim of the study was to characterize plasmids that harbour blaESBL genes and their genetic environment in Escherichia coli and Klebsiella pneumoniae clones circulating in Spain. The incompatibility group of plasmids within 58 strains harbouring blaCTX-M (n=45) and blaSHV (n=15) genes was determined by rep-typing-PCR and hybridization. The blaESBL genetic environment was determined by PCR and sequencing. The blaCTX-M-9 genes (n=14) were linked to In60 located in IncI1 (50%) or IncHI2 plasmids (28%). All blaCTX-M-14 genes (n=13) were flanked by ISEcp1 and IS903 and 12 were associated with IncK plasmids. One of two blaCTX-M-10 genes was present in an IncK plasmid, but both genes were linked to a phage-related element. Five of seven blaCTX-M-1 (71%), all three blaCTX-M-32 and one of two blaCTX-M-3 genes were linked to IncN plasmids. The other blaCTX-M-3 gene was linked to IncA/C and the remaining two blaCTX-M-1 genes to IncFII plasmids. Three blaCTX-M-15 genes were associated with IncF (repFIA) and one with IncFII plasmids. All these genes from blaCTX-M group-1 showed the ISEcp1 upstream truncated by different insertion sequences. Forty-three percent of blaSHV-12 genes (n=14) were located in IncI1 plasmids, all flanked by the IS26 and DEOR region. The only detected blaSHV-5 gene was located in an IncFII plasmid and flanked by recF and DEOR regions. A diversity of the plasmid incompatibility groups that harbour blaESBL genes was observed, except for the blaCTX-M-14 gene. Moreover, a high variability was confirmed in the genetic environment of these genes as a result of insertion and deletion events.
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340. |
Struve C,
Bojer M,
Krogfelt KA,
( 2008 ) Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence. PMID : 18559432 : DOI : 10.1128/IAI.00494-08 PMC : PMC2519443 Abstract >>
Klebsiella pneumoniae is recognized as an important gram-negative opportunistic pathogen. The ability of bacteria to adhere to host structures is considered essential for the development of infections; however, few studies have examined the influence of adhesion factors on K. pneumoniae virulence. In this study, we cloned and characterized the type 1 fimbria gene cluster of a clinical K. pneumoniae isolate. Although this cluster was not identical to the Escherichia coli type 1 fimbria gene cluster, an overall high degree of structural resemblance was demonstrated. Unique to the K. pneumoniae fim gene cluster is the fimK gene, whose product contains an EAL domain, suggesting that it has a role in regulation of fimbrial expression. Like expression of type 1 fimbriae in E. coli, expression of type 1 fimbriae in K. pneumoniae was found to be phase variable, and an invertible DNA element (fim switch) was characterized. An isogenic type 1 fimbria mutant was constructed and used to evaluate the influence of type 1 fimbriae in different infection models. Type 1 fimbriae did not influence the ability of K. pneumoniae to colonize the intestine or infect the lungs, but they were determined to be a significant virulence factor in K. pneumoniae urinary tract infection. By use of a PCR-based assay, the orientation of the fim switch during colonization and infection was investigated and was found to be all "off" in the intestine and lungs but all "on" in the urinary tract. Our results suggest that during colonization and infection, there is pronounced selective pressure in different host environments for selection of either the type 1 fimbriated or nonfimbriated phenotype of K. pneumoniae.
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341. |
Teo JW,
Ng KY,
Lin RT,
( 2009 ) Detection and genetic characterisation of qnrB in hospital isolates of Klebsiella pneumoniae in Singapore. PMID : 18993034 : DOI : 10.1016/j.ijantimicag.2008.08.019 Abstract >>
Polymerase chain reaction (PCR) screening of 116 ciprofloxacin-resistant Klebsiella pneumoniae hospital isolates for the presence of qnr genes that mediate plasmid quinolone resistance revealed that none were positive for qnrA or qnrS. However, qnrB was detected in ca. 5.2% of the isolates. Southern hybridisation demonstrated that the qnrB-hybridising plasmids were large (>70kb) and capable of transferring quinolone resistance by conjugation. Sequence analysis of the qnrB genes detected in this study showed that they were identical to previously identified qnrB1, qnrB4 and qnrB6 genes, although a novel variant designated qnrB20 was also identified. Analysis of the genetic environment around the cloned qnrB genes showed that they were present in diverse plasmid backbones, sometimes within novel genetic contexts, but always associated with mobile or transposable elements.
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342. |
Soge OO,
Beck NK,
White TM,
No DB,
Roberts MC,
( 2008 ) A novel transposon, Tn6009, composed of a Tn916 element linked with a Staphylococcus aureus mer operon. PMID : 18583328 : DOI : 10.1093/jac/dkn255 PMC : PMC2536709 Abstract >>
The aim of this study was to characterize a novel conjugative transposon Tn6009 composed of a Tn916 linked to a Staphylococcus aureus mer operon in representative Gram-positive and Gram-negative bacteria isolated in Nigeria and Portugal. Eighty-three Gram-positive and 34 Gram-negative bacteria were screened for the presence of the Tn6009 using DNA-DNA hybridization, PCR, hybridization of PCR products, sequencing and mating experiments by established procedures. Forty-three oral and 23 urine Gram-negative and Gram-positive isolates carried the Tn6009. Sequencing was performed to verify the direct linkage between the mer resistance genes and the tet(M) gene. A Nigerian Klebsiella pneumoniae, isolated from a urinary tract infection patient, and one commensal isolate from each of the other Tn6009-positive genera, Serratia liquefaciens, Pseudomonas sp., Enterococcus sp. and Streptococcus sp. isolated from the oral and urine samples of healthy Portuguese children, were able to act as donors and conjugally transfer the Tn6009 to the Enterococcus faecalis JH2-2 recipient, resulting in tetracycline- and mercury-resistant E. faecalis transconjugants. This study reports a novel non-composite conjugative transposon Tn6009 containing a Tn916 element linked to an S. aureus mer operon carrying genes coding for inorganic mercury resistance (merA), an organic mercury resistance (merB), a regulatory protein (merR) and a mercury transporter (merT). This transposon was identified in 66 isolates from two Gram-positive and three Gram-negative genera and is the first transposon in the Tn916 family to carry the Gram-positive mer genes directly linked to the tet(M) gene.
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343. |
Rice LB,
Carias LL,
Hutton RA,
Rudin SD,
Endimiani A,
Bonomo RA,
( 2008 ) The KQ element, a complex genetic region conferring transferable resistance to carbapenems, aminoglycosides, and fluoroquinolones in Klebsiella pneumoniae. PMID : 18573935 : DOI : 10.1128/AAC.00493-08 PMC : PMC2533511 Abstract >>
The bla(KPC-3) and qnrB19 determinants of transferable Klebsiella pneumoniae plasmid pLRM24 reside within a complex region consisting of a Tn1331 backbone into which a Tn4401-like element and qnrB19 mobilized by an adjacent ISEcp1 insertion sequence have been inserted. This novel element represents a coalescence of genes conferring multidrug resistance in K. pneumoniae.
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344. |
Li JB,
Cheng J,
Wang Q,
Chen Y,
Ye Y,
Zhang XJ,
( 2009 ) A novel SHV-type beta-lactamase variant (SHV-89) in clinical isolates in China. PMID : 18587684 : DOI : 10.1007/s11033-008-9290-8 Abstract >>
Two clinical strains of Klebsiella pneumoniae (K. pneumoniae) and one isolate of Escherichia coli (E. coli) were collected from two large general hospitals in China. Conjugation experiment, susceptibility testing, isoelectric focusing, PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization of the novel SHV-tpye enzyme. The analysis of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were performed to investigate epidemiology. These isolates had CTX-M-14 and SHV-89 beta-lactamases. SHV-89 beta-lactamase of pI 7.6 is a novel variant with two substitutions compared with the sequence of SHV-1: Leu35Gln and Met129Val. Its gene also had two silent mutations at positions 369 and 774, respectively. The results of substrate profiles and MIC determinations showed the activity of the novel enzyme was insufficient for the enzyme to count as an extended-spectrum beta-lactamase (ESBL). The substrates of the enzyme were also characterized. Furthermore, the three novel SHV enzyme-producing strains were epidemiologically unrelated. The emergence of a novel SHV-type beta-lactamase is rarely described in other areas. This study illustrates the importance of molecular survelliance in tracking SHV-producing strains in large teaching hospitals and emphasizes the need for epidemiological monitoring.
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345. |
de la Riva L,
Badia J,
Aguilar J,
Bender RA,
Baldoma L,
( 2008 ) The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation. PMID : 18849434 : DOI : 10.1128/JB.01022-08 PMC : PMC2593211 Abstract >>
Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO, and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on sigma(54), is mediated by the Ntr system. In contrast, neither NtrC nor the nitrogen assimilation control protein is involved in the nitrogen control of hpxDE, which is dependent on sigma(70) for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N(4)-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.
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346. |
Wang CX,
Huang ZM,
Mi ZH,
Chen GQ,
( 2009 ) A Klebsiella pneumoniae clinical isolate producing the LAP-2 beta-lactamase in China. PMID : 19013676 : DOI : 10.1016/j.jhin.2008.09.003 Abstract >>
N/A
|
347. |
Lascols C,
Podglajen I,
Verdet C,
Gautier V,
Gutmann L,
Soussy CJ,
Collatz E,
Cambau E,
( 2008 ) A plasmid-borne Shewanella algae Gene, qnrA3, and its possible transfer in vivo between Kluyvera ascorbata and Klebsiella pneumoniae. PMID : 18515416 : DOI : 10.1128/JB.00243-08 PMC : PMC2493261 Abstract >>
The plasmid-borne quinolone resistance gene qnrA1 is prevalent in multidrug-resistant Enterobacteriaceae. A chromosomally encoded homologue in Shewanella algae, qnrA3, has been described. We isolated two qnrA3-positive strains, one of Klebsiella pneumoniae (He96) and one of Kluyvera ascorbata (Kas96), from the feces of an immunocompromised outpatient. The qnrA3 allele was identical to that of S. algae except for 5 nucleotides and differed from qnrA1 by 29 nucleotides affecting three amino acids. The analysis of the qnrA3 genetic environment showed that qnrA3 was inserted downstream from an ISCR1 element at a recombination crossover site described for other resistance genes, including qnrA1, and immediately upstream from IS26, a situation not described before. IS26 preceded an incomplete class 1 integron which contained, among other genes, aac(6')-Ib-cr, another transferable quinolone resistance gene, and the beta-lactamase gene bla(OXA-1/30). The 10-kb fragment encompassing qnrA3 was compared to previously described qnrA1-containing plasmids and multidrug-resistant plasmids; it shares identical sequences with pC15a, pHSH2, pQR1, pQKp311H, and pSAL-1 but with rearrangements, deletions, and mutations. Conjugal transfer of qnrA3 was highly efficient (10(-2)) from K. pneumoniae He96 or K. ascorbata Kas96 to Escherichia coli J53 but less so (10(-5)) from either donor to a clinical strain of Enterobacter cloacae. This first description of a plasmid-borne copy and of the in vitro transfer of qnrA3 is taken to illustrate its likely in vivo transfer from S. algae to the Enterobacteriaceae.
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348. |
Fu Y,
Guo L,
Xu Y,
Zhang W,
Gu J,
Xu J,
Chen X,
Zhao Y,
Ma J,
Liu X,
Zhang F,
( 2008 ) Alteration of GyrA amino acid required for ciprofloxacin resistance in Klebsiella pneumoniae isolates in China. PMID : 18505849 : DOI : 10.1128/AAC.00151-08 PMC : PMC2493132 Abstract >>
Resistance to ciprofloxacin was detected in 111 (48.1%) isolates of Klebsiella pneumoniae from China. GyrA alterations were identified in the ciprofloxacin-resistant and ciprofloxacin-susceptible isolates. The results, including previously published data, indicate that the single substitution Ser83-->Ile and three types of double mutations at Ser83 and Asp87 were required for ciprofloxacin resistance (P < 0.05).
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349. |
Penteado AP,
Castanheira M,
Pignatari AC,
Guimarães T,
Mamizuka EM,
Gales AC,
( 2009 ) Dissemination of bla(IMP-1)-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23. PMID : 18990526 : DOI : 10.1016/j.diagmicrobio.2008.09.013 Abstract >>
The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in S?o Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(IMP-1), aac(6')-31, and aadA1. Eight strains from the same hospital also carried another class 1 integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in S?o Paulo. These isolates appear to be the source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance.
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350. |
Ma J,
Zeng Z,
Chen Z,
Xu X,
Wang X,
Deng Y,
Lü D,
Huang L,
Zhang Y,
Liu J,
Wang M,
( 2009 ) High prevalence of plasmid-mediated quinolone resistance determinants qnr, aac(6')-Ib-cr, and qepA among ceftiofur-resistant Enterobacteriaceae isolates from companion and food-producing animals. PMID : 18936192 : DOI : 10.1128/AAC.00886-08 PMC : PMC2630616 Abstract >>
Three kinds of plasmid-mediated quinolone resistance (PMQR) determinants have been discovered and have been shown to be widely distributed among clinical isolates: qnr genes, aac(6')-Ib-cr, and qepA. Few data on the prevalence of these determinants in strains from animals are available. The presence of PMQR genes in isolates from animals was determined by PCR amplification and DNA sequencing. The production of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases in the strains was detected, and their genotypes were determined. The genetic environment of PMQR determinants in selected plasmids was analyzed. All samples of ceftiofur-resistant (MICs > or = 8 microg/ml) isolates of the family Enterobacteriaceae were selected from 36 companion animals and 65 food-producing animals in Guangdong Province, China, between November 2003 and April 2007, including 89 Escherichia coli isolates, 9 Klebsiella pneumoniae isolates, and isolates of three other genera. A total of 68.3% (69/101) of the isolates produced ESBLs and/or AmpC beta-lactamases, mainly those of the CTX-M and CMY types. Of the 101 strains, PMQR determinants were present in 35 (34.7%) isolates, with qnr, aac(6')-Ib-cr, and qepA detected alone or in combination in 8 (7.9%), 19 (18.8%), and 16 (15.8%) strains, respectively. The qnr genes detected included one qnrB4 gene, four qnrB6 genes, and three qnrS1 genes. Five strains were positive for both aac(6')-Ib-cr and qepA, while one strain was positive for qnrS1, aac(6')-Ib-cr, and qepA. qnrB6 was flanked by two copies of ISCR1 with an intervening dfr gene downstream and sul1 and qacEDelta1 genes upstream. In another plasmid, aac(6')-Ib-cr followed intI1 and arr-3 was downstream. PMQR determinants are highly prevalent in ceftiofur-resistant Enterobacteriaceae strains isolated from animals in China. This is the first report of the occurrence of PMQR determinants among isolates from companion animals.
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351. |
Marçal D,
Rêgo AT,
Carrondo MA,
Enguita FJ,
( 2009 ) 1,3-Propanediol dehydrogenase from Klebsiella pneumoniae: decameric quaternary structure and possible subunit cooperativity. PMID : 19011020 : DOI : 10.1128/JB.01077-08 PMC : PMC2631990 DOI : 10.1128/JB.01077-08 PMC : PMC2631990 Abstract >>
Klebsiella pneumoniae is a nosocomial pathogen frequently isolated from opportunistic infections, especially in clinical environments. In spite of its potential pathogenicity, this microorganism has several metabolic potentials that could be used in biotechnology applications. K. pneumoniae is able to metabolize glycerol as a sole source of carbon and energy. 1,3-Propanediol dehydrogenase is the core of the metabolic pathway for the use of glycerol. We have determined the crystallographic structure of 1,3-propanediol dehydrogenase, a type III Fe-NAD-dependent alcohol dehydrogenase, at 2.7-A resolution. The structure of the enzyme monomer is closely related to that of other alcohol dehydrogenases. The overall arrangement of the enzyme showed a decameric structure, formed by a pentamer of dimers, which is the catalytic form of the enzyme. Dimers are associated by strong ionic interactions that are responsible for the highly stable in vivo packing of the enzyme. Kinetic properties of the enzyme as determined in the article would suggest that this decameric arrangement is related to the cooperativity between monomers.
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352. |
Turton JF,
Baklan H,
Siu LK,
Kaufmann ME,
Pitt TL,
( 2008 ) Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. PMID : 18507682 : DOI : 10.1111/j.1574-6968.2008.01208.x Abstract >>
A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.
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353. |
Ko KS,
Lee MY,
Song JH,
Lee H,
Jung DS,
Jung SI,
Kim SW,
Chang HH,
Yeom JS,
Kim YS,
Ki HK,
Chung DR,
Kwon KT,
Peck KR,
Lee NY,
( 2008 ) Prevalence and characterization of extended-spectrum beta-lactamase-producing Enterobacteriaceae isolated in Korean hospitals. PMID : 18482815 : DOI : 10.1016/j.diagmicrobio.2008.03.005 Abstract >>
Prevalence and characteristics of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Korean hospitals were assessed. A total of 1484 clinical Enterobacteriaceae isolates were collected from 8 tertiary-care hospitals in various regions of Korea over a 3-month period (June to August) in 2005. Among 546 Klebsiella pneumoniae isolates, 123 isolates (22.4%) showed ESBL-producing activity, and 47 (10.2%) of 460 isolates of Escherichia coli were ESBL producers. Of the Enterobacter cloacae isolates, 16.2% (17/105) evidenced ESBL-producing activity. The most prevalent ESBLs were SHV-12 and CTX-M-14 in K. pneumoniae and E. coli, respectively. In E. cloacae, SHV-12 was also the most prevalent. Prevalence of ESBL production differed among the specimens. Although the K. pneumoniae isolates from urine and aspirates evidenced high ESBL production rates (35.4% and 57.1%, respectively), those from sputum, blood, and pus showed relatively low ESBL production rates (17.0%, 14.8%, and 5.3%, respectively). However, E. coli isolates obtained from sputum showed significantly higher ESBL production rates (37.5%) than were seen in samples obtained from other sources, but those obtained from urine showed lower ESBL production rates (8.3%). These significant differences in ESBL-producing K. pneumoniae and E. coli isolates among the isolated specimens should be examined further, with an eye toward the implications of this research in clinical settings.
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354. |
Rosen DA,
Pinkner JS,
Walker JN,
Elam JS,
Jones JM,
Hultgren SJ,
( 2008 ) Molecular variations in Klebsiella pneumoniae and Escherichia coli FimH affect function and pathogenesis in the urinary tract. PMID : 18474655 : DOI : 10.1128/IAI.00340-08 PMC : PMC2446687 Abstract >>
Type 1 pili mediate binding, invasion, and biofilm formation of uropathogenic Escherichia coli (UPEC) in the host urothelium during urinary tract infection (UTI) via the adhesin FimH. In this study, we characterized the molecular basis of functional differences between FimH of the UPEC isolate UTI89 and the Klebsiella pneumoniae cystitis isolate TOP52. Type 1 pili characteristically mediate mannose-sensitive hemagglutination of guinea pig erythrocytes. Although the adhesin domain of K. pneumoniae TOP52 FimH (FimH(52)) is highly homologous to that of E. coli, with an identical mannose binding pocket and surrounding hydrophobic ridge, it lacks the ability to agglutinate guinea pig erythrocytes. In addition, FimH-dependent biofilm formation in K. pneumoniae is inhibited by heptyl mannose, but not methyl mannose, suggesting the need for contacts outside of the mannose binding pocket. The binding specificity differences observed for FimH(52) resulted in significant functional differences seen in the pathogenesis of K. pneumoniae UTI compared to E. coli UTI. Infections in a murine model of UTI demonstrated that although the K. pneumoniae strain TOP52 required FimH(52) for invasion and IBC formation in the bladder, FimH(52) was not essential for early colonization. This work reveals that a limited amount of sequence variation between the FimH of E. coli and K. pneumoniae results in significant differences in function and ability to colonize the urinary tract.
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355. |
Chen Y,
Cheng J,
Wang Q,
Ye Y,
Li JB,
Zhang XJ,
( 2009 ) ACT-3, a novel plasmid-encoded class C beta-lactamase in a Klebsiella pneumoniae isolate from China. PMID : 18789849 : DOI : 10.1016/j.ijantimicag.2008.06.026 Abstract >>
N/A
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356. |
Pan YJ,
Fang HC,
Yang HC,
Lin TL,
Hsieh PF,
Tsai FC,
Keynan Y,
Wang JT,
( 2008 ) Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. PMID : 18508935 : DOI : 10.1128/JCM.01716-07 PMC : PMC2446917 Abstract >>
Community-acquired pyogenic liver abscess caused by Klebsiella pneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype.
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357. |
Renault M,
Saurel O,
Czaplicki J,
Demange P,
Gervais V,
Löhr F,
Réat V,
Piotto M,
Milon A,
( 2009 ) Solution state NMR structure and dynamics of KpOmpA, a 210 residue transmembrane domain possessing a high potential for immunological applications. PMID : 18952100 : DOI : 10.1016/j.jmb.2008.10.021 Abstract >>
The three-dimensional structure of the outer membrane protein A from Klebsiella pneumoniae transmembrane domain was determined by NMR.This protein induces specific humoral and cytotoxic responses, and is a potent carrier protein. This is one of the largest integral membrane proteins(210 residues) for which nearly complete resonance assignment, including side chains, has been achieved so far. The methodology rested on the use of 900 MHz 3D and 4D TROSY experiments recorded on a uniformly 15N,13C,2H-labeled sample and on a perdeuterated methyl protonated sample. The structure was refined from 920 experimental constraints, giving an ensemble of 20 best structures with an r.m.s. deviation of 0.54 A for the main chain atoms in the core eight-stranded beta-barrel. The protein dynamics was assessed, in a residue-specific manner, by 1H-15N NOEs (pico- to nanosecond timescale), exchange broadening (millisecond to second) and 1H-2H chemical exchange (hour-weeks).
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358. |
Campos E,
de la Riva L,
Garces F,
Giménez R,
Aguilar J,
Baldoma L,
Badia J,
( 2008 ) The yiaKLX1X2PQRS and ulaABCDEFG gene systems are required for the aerobic utilization of L-ascorbate in Klebsiella pneumoniae strain 13882 with L-ascorbate-6-phosphate as the inducer. PMID : 18708499 : DOI : 10.1128/JB.00815-08 PMC : PMC2566198 Abstract >>
The capacity to both ferment and oxidize L-ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment L-ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes corresponding to the specific promoters indicated that L-ascorbate-6-phosphate is the effector molecule recognized by both regulators, since binding of the repressors to their recognition sites was impaired by the presence of this compound. We demonstrated that in K. pneumoniae cells L-ascorbate-6-phosphate is formed only by the action of the UlaABC phosphotransferase system. This finding explains why strains that lack the ula genetic system and therefore are unable to form the inducer intracellularly cannot efficiently use this vitamin as a carbon source under either anaerobic or aerobic conditions. Thus, efficient aerobic metabolism of L-ascorbate in K. pneumoniae is dependent on the presence of both the yiaK-S and ula systems. The expression of the yiaK-S operon, but not the expression of the ula regulon, is controlled by oxygen availability. Both systems are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and by IHF.
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359. |
Espedido BA,
Partridge SR,
Iredell JR,
( 2008 ) bla(IMP-4) in different genetic contexts in Enterobacteriaceae isolates from Australia. PMID : 18490506 : DOI : 10.1128/AAC.01634-07 PMC : PMC2493119 Abstract >>
The IMP-4 metallo-beta-lactamase, originally recognized in Acinetobacter spp. from Hong Kong, more recently appeared simultaneously in isolates of the family Enterobacteriaceae from Sydney and Melbourne, Australia. The bla(IMP-4)-qacG2-aacA4-catB3 cassette array was found in isolates from both cities, but in different wider genetic contexts and on different plasmids, suggesting movement of this array by homologous recombination.
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360. |
Shen P,
Jiang Y,
Zhou Z,
Zhang J,
Yu Y,
Li L,
( 2008 ) Complete nucleotide sequence of pKP96, a 67 850 bp multiresistance plasmid encoding qnrA1, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae. PMID : 18812424 : DOI : 10.1093/jac/dkn397 Abstract >>
The multiresistance plasmid pKP96 from Klebsiella pneumoniae was sequenced completely and analysed concerning its genetic environment and distributing of antimicrobial resistance genes. The complete sequence of the plasmid was determined using a whole-genome shotgun approach. MICs of 13 antimicrobial agents were determined using Etests. A conjugation experiment was performed in liquid medium. pKP96 is a circularly closed 67 850 bp multiresistance plasmid with an IncN incompatibility group. Seventy putative genes were identified according to the annotation of the finished sequence. The backbone region of the plasmid, comprising the conjugal transfer and plasmid replication regions, showed 91% identity to the IncN plasmid R46. Several mobile elements were found to be inserted into pKP96 together with antimicrobial resistance genes, including qnrA1, aac(6')-Ib-cr and bla(CTX-M-24). Plasmid pKP96 is a chimera that has acquired its multiple antimicrobial resistance determinants horizontally from different sources. It may have evolved from an ancestor plasmid similar to R46 through the stepwise events of integration or recombination.
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361. |
Shimizu-Ibuka A,
Bauvois C,
Sakai H,
Galleni M,
( 2008 ) Structure of the plasmid-mediated class C beta-lactamase ACT-1. PMID : 18453698 : DOI : 10.1107/S1744309108008531 PMC : PMC2376412 Abstract >>
The crystallographic structure of ACT-1, which is the first plasmid-mediated AmpC-type beta-lactamase to have been completely analyzed in terms of nucleotide sequence and which has a high degree of sequence similarity to the chromosomal AmpC enzymes of Enterobacter cloacae and the plasmid-encoded MIR-1, has been solved at 2.4 A resolution. The overall structure of ACT-1 is similar to those of other class C beta-lactamases, such as the AmpC enzymes from E. cloacae P99 and Escherichia coli.
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362. |
Isabel S,
Leblanc E,
Boissinot M,
Boudreau DK,
Grondin M,
Picard FJ,
Martel EA,
Parham NJ,
Chain PS,
Bader DE,
Mulvey MR,
Bryden L,
Roy PH,
Ouellette M,
Bergeron MG,
( 2008 ) Divergence among genes encoding the elongation factor Tu of Yersinia Species. PMID : 18790860 : DOI : 10.1128/JB.01067-08 PMC : PMC2576667 Abstract >>
Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.
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363. |
Hsieh PF,
Lin TL,
Lee CZ,
Tsai SF,
Wang JT,
( 2008 ) Serum-induced iron-acquisition systems and TonB contribute to virulence in Klebsiella pneumoniae causing primary pyogenic liver abscess. PMID : 18433330 : DOI : 10.1086/588383 Abstract >>
Klebsiella pneumoniae has become the predominant pathogen causing primary pyogenic liver abscess (PLA). K. pneumoniae was stimulated by human serum, and gene expression was analyzed by microarray. Three putative iron acquisition systems, Yersinia high-pathogenicity island (HPI), iucABCDiutA, and iroA(iroNDCB), that increased in expression and predominated in PLA-associated K. pneumoniae strains were identified. By use of siderophore uptake assays, these 3 systems were confirmed to be siderophore-dependent iron acquisition systems. Only the irp2-iuc-iroA triple mutant showed decreased virulence in mice. Full-genome analysis of K. pneumoniae strain NTUH-K2044 identified 10 putative iron uptake systems. Seven of these 10 systems were TonB dependent, including Yersinia HPI, iucABCDiutA, and iroA. A tonB deletion mutant was demonstrated to have profound attenuation of virulence. Immunization with the tonB mutant resulted in seroconversion of extracellular polysaccharide antibodies and protective efficacy against subsequent exposure to the parental strain. Iron uptake systems were the genes in K. pneumoniae that were highly up-regulated in response to sera. Although there are multiple iron transporter systems in NTUH-K2044, a mutation in all 3 loci (irp2, iuc, and iroA) is necessary to decrease virulence. The tonB mutant is a potential vaccine candidate because it can induce a significant protective immune response against challenge with a wild-type strain.
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364. |
Zong Z,
Partridge SR,
Thomas L,
Iredell JR,
( 2008 ) Dominance of blaCTX-M within an Australian extended-spectrum beta-lactamase gene pool. PMID : 18725449 : DOI : 10.1128/AAC.00107-08 PMC : PMC2573124 Abstract >>
bla(CTX-M) genes, particularly bla(CTX-M-15), are the dominant extended-spectrum beta-lactamase (ESBL) genes among clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sydney, Australia, where we also found one example of bla(CTX-M-62), encoding a novel enzyme conferring ceftazidime resistance. ESBL genes were present in diverse community isolates and in a variety of associated conjugative plasmids.
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365. |
Bowers TH,
Reid NM,
Lloyd-Jones G,
( 2008 ) Composition of nifH in a wastewater treatment system reliant on N(2) fixation. PMID : 18449537 : DOI : 10.1007/s00253-008-1486-2 Abstract >>
High levels of nitrogen fixation have been observed in the wastewaters of pulp and paper mills. In this study, we show that nitrogen fixation in a model pulp and paper wastewater treatment system is supported by a high density of nifH sequences that are of low diversity. Quantitative PCR revealed a ratio of nifH to 16S rDNA of 1.14 +/- 0.76 which shows that very high levels of the nifH gene were enriched to support the high rates of nitrogen fixation that occur in this wastewater. Changes in wastewater composition and dissolved oxygen levels did not affect the nifH levels and allowed stable wastewater treatment. The nifH sequences identified display a similar profile to those seen in forest soil environments where nifH sequences derived from alpha-proteobacteria and beta-proteobacteria are also prevalent.
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366. |
Márquez C,
Labbate M,
Raymondo C,
Fernández J,
Gestal AM,
Holley M,
Borthagaray G,
Stokes HW,
( 2008 ) Urinary tract infections in a South American population: dynamic spread of class 1 integrons and multidrug resistance by homologous and site-specific recombination. PMID : 18753343 : DOI : 10.1128/JCM.00835-08 PMC : PMC2566090 Abstract >>
One hundred four bacterial strains mediating urinary tract infections in separate individuals from a Uruguayan community were isolated. Forty-six strains conferred a multidrug resistance phenotype. All 104 strains were examined for the presence of class 1, 2, and 3 integrons. Class 1 integrons were found in 21 isolates across four distinct bacterial genera. A large class 1 integron in a Klebsiella pneumoniae strain was fully sequenced and was 29,093 bp in length. This integron probably arose by homologous recombination since it was embedded in a hybrid Tn21-like transposon backbone which comprised a Tn5036-like tnp transposition module at the IRi integron end and a Tn21 mer module at the IRt integron end. The parent integron/transposon that contributed the Tn5036 module was not related to Tn1696 since the integron insertion points in the transposon backbones were 16 bases apart. Examination of the other 20 class 1 integron-containing strains revealed further evidence of genetic exchange. This included a strain that possessed a Tn5036 module at the IRt end but not at the IRi end and another that possessed a tnp module beyond IRi that was a hybrid of Tn21 and Tn5051 and that is presumed to have arisen by site-specific recombination. This study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance in a community.
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367. |
Rosen DA,
Pinkner JS,
Jones JM,
Walker JN,
Clegg S,
Hultgren SJ,
( 2008 ) Utilization of an intracellular bacterial community pathway in Klebsiella pneumoniae urinary tract infection and the effects of FimK on type 1 pilus expression. PMID : 18411285 : DOI : 10.1128/IAI.00090-08 PMC : PMC2446714 Abstract >>
Klebsiella pneumoniae is an important cause of urinary tract infection (UTI), but little is known about its pathogenesis in vivo. The pathogenesis of the K. pneumoniae cystitis isolate TOP52 was compared to that of the uropathogenic Escherichia coli (UPEC) isolate UTI89 in a murine cystitis model. Bladder and kidney titers of TOP52 were lower than those of UTI89 at early time points but similar at later time points. TOP52, like UTI89, formed biofilm-like intracellular bacterial communities (IBCs) within the murine bladder, albeit at significantly lower levels than UTI89. Additionally, filamentation of TOP52 was observed, a process critical for UTI89 evasion of neutrophil phagocytosis and persistence in the bladder. Thus, the IBC pathway is not specific to UPEC alone. We investigated if differences in type 1 pilus expression may explain TOP52's early defect in vivo. The type 1 pilus operon is controlled by recombinase-mediated (fimE, fimB, and fimX) phase variation of an invertible promoter element. We found that K. pneumoniae carries an extra gene of unknown function at the 3' end of its type 1 operon, fimK, and the genome lacks the recombinase fimX. A deletion mutant of fimK was constructed, and TOP52 Delta fimK had higher titers and formed more IBCs in the murine cystitis model than wild type. The loss of fimK or expression of E. coli fimX from a plasmid in TOP52 resulted in a larger phase-ON population and higher expression levels of type 1 pili and gave TOP52 the ability to form type 1-dependent biofilms. Complementation with pfimK decreased type 1 pilus expression and biofilm formation of TOP52 Delta fimK and decreased UTI89 biofilm formation. Thus, K. pneumoniae appears programmed for minimal expression of type 1 pili, which may explain, in part, why K. pneumoniae is a less prevalent etiologic agent of UTI than UPEC.
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368. |
de Oliveira Garcia D,
Doi Y,
Szabo D,
Adams-Haduch JM,
Vaz TM,
Leite D,
Padoveze MC,
Freire MP,
Silveira FP,
Paterson DL,
( 2008 ) Multiclonal outbreak of Klebsiella pneumoniae producing extended-spectrum beta-lactamase CTX-M-2 and novel variant CTX-M-59 in a neonatal intensive care unit in Brazil. PMID : 18347108 : DOI : 10.1128/AAC.01440-07 PMC : PMC2346667 Abstract >>
An outbreak of cephalosporin-resistant Klebsiella pneumoniae occurred in a neonatal intensive care unit in S?o Paulo, Brazil. Of the 10 pulsotypes identified during the outbreak and follow-up periods, nine produced CTX-M-2 or its new variant CTX-M-59 and one produced SHV-5. bla(CTX-M-2/59) genes were located on closely related plasmids that were transferable.
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369. |
Hao J,
Wang W,
Tian J,
Li J,
Liu D,
( 2008 ) Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing dhaT gene in Klebsiella pneumoniae TUAC01. PMID : 18365261 : DOI : 10.1007/s10295-008-0340-y Abstract >>
Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain.
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370. |
Soler Bistué AJ,
Birshan D,
Tomaras AP,
Dandekar M,
Tran T,
Newmark J,
Bui D,
Gupta N,
Hernandez K,
Sarno R,
Zorreguieta A,
Actis LA,
Tolmasky ME,
( 2008 ) Klebsiella pneumoniae multiresistance plasmid pMET1: similarity with the Yersinia pestis plasmid pCRY and integrative conjugative elements. PMID : 18350140 : DOI : 10.1371/journal.pone.0001800 PMC : PMC2262945 Abstract >>
Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria. The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31). The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1)show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains.
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371. |
Doi Y,
Adams-Haduch JM,
Paterson DL,
( 2008 ) Genetic environment of 16S rRNA methylase gene rmtD. PMID : 18391044 : DOI : 10.1128/AAC.00037-08 PMC : PMC2415751 Abstract >>
The genetic environment of the 16S rRNA methylase gene rmtD was investigated. rmtD was flanked by a novel ISCR motif located downstream of class I integron In163 in the original Pseudomonas aeruginosa strain. rmtD found in Klebsiella pneumoniae appeared to have been mobilized from P. aeruginosa by an IS26-mediated event.
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372. |
Mendonça N,
Manageiro V,
Robin F,
Salgado MJ,
Ferreira E,
Caniça M,
Bonnet R,
( 2008 ) The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition. PMID : 18316518 : DOI : 10.1128/AAC.01381-07 PMC : PMC2346665 Abstract >>
The new beta-lactamase SHV-72 was isolated from clinical Klebsiella pneumoniae INSRA1229, which exhibited the unusual association of resistance to the amoxicillin-clavulanic acid combination (MIC, 64 microg/ml) and susceptibility to cephalosporins, aztreonam, and imipenem. SHV-72 (pI 7.6) harbored the three amino acid substitutions Ile8Phe, Ala146Val, and Lys234Arg. SHV-72 had high catalytic efficiency against penicillins (k(cat)/K(m), 35 to 287 microM(-1) x s(-1)) and no activity against oxyimino beta-lactams. The concentration of clavulanic acid necessary to inhibit the enzyme activity by 50% was 10-fold higher for SHV-72 than for SHV-1. Molecular-dynamics simulation suggested that the Lys234Arg substitution in SHV-72 stabilized an atypical conformation of the Ser130 side chain, which moved the O gamma atom of Ser130 around 3.5 A away from the key O gamma atom of the reactive serine (Ser70). This movement may therefore decrease the susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70.
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373. |
Roche C,
Boo TW,
Walsh F,
Crowley B,
( 2008 ) Detection and molecular characterisation of plasmidic AmpC beta-lactamases in Klebsiella pneumoniae isolates from a tertiary-care hospital in Dublin, Ireland. PMID : 18397332 : DOI : 10.1111/j.1469-0691.2008.01998.x Abstract >>
This study determined the types of AmpC enzymes produced by Klebsiella pneumoniae isolates resistant to third-generation cephalosporins and the clonality of these isolates. The presence of AmpC enzymes was identified by cephalosporin-cloxacillin synergy tests. Genes encoding AmpC enzymes were characterised by PCR and sequencing. Pulsed-field gel electrophoresis (PFGE) was used to type the isolates. Fifteen K. pneumoniae isolates were positive for bla(AmpC), 13 were positive for bla(ACC-1) and two were positive for bla(DHA-1). Production of the DHA-1 enzyme was inducible. The ampR gene was identified upstream of the bla(DHA-1) gene. PFGE demonstrated the polyclonal origin of the isolates carrying bla(ACC-1).
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374. |
Hu FP,
Xu XG,
Zhu DM,
Wang MG,
( 2008 ) Coexistence of qnrB4 and qnrS1 in a clinical strain of Klebsiella pneumoniae. PMID : 18298896 : DOI : 10.1111/j.1745-7254.2008.00757.x Abstract >>
To identify the location and the relationship, and to analyze the genetic background of 2 plasmid-mediated quinolone resistance genes, qnrB4 and qnrS1, carried by a clinical strain of Klebsiella pneumoniae (K pneumoniae). The plasmids carrying qnrB4 or qnrS1 were identified by Southern blotting. A HindIII fragment containing qnrB4 or qnrS1 was cloned into plasmid puc18 and sequenced. qnrB4 and qnrS1 were located on 2 different plasmids, pHS7 and pHS8, and were 180 and 45 kb in size, respectively. A transconjugant carrying plasmid pHS7 bearing qnrB4 and another transconjugant carrying pHS9 bearing qnrB4 and qnrS1 were obtained by conjugation. Plasmid pHS8 bearing qnrS1 was also transferred to J53 by transformation. The ciprofloxacin minimal inhibitory concentrations (MIC) for J53 transconjugants or the transformant carrying qnrB4 only, qnrS1 only, and both qnrB4 and qnrS1 were 0.19, 0.25, and 0.25 mg/L, respectively, while the parent clinical strain of K pneumoniae had a MIC of 0.75 mg/L. qnrB4 was located in a sul1-type integron with blaDHA-1, ampR and psp genes in upstream and insertion sequence IS26, and sap genes in downstream of qnrB4. qnrS1 was not located in an integron, but IS26 was found both upstream and downstream, and IS2 was found directly upstream of qnrS1. qnrB and qnrS can be harbored simultaneously by a single clinical strain of K pneumoniae. These 2 genes are carried by 2 different plasmids and have different genetic environments in plasmid DNA structure.
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375. |
Revilla C,
Garcillán-Barcia MP,
Fernández-López R,
Thomson NR,
Sanders M,
Cheung M,
Thomas CM,
de la Cruz F,
( 2008 ) Different pathways to acquiring resistance genes illustrated by the recent evolution of IncW plasmids. PMID : 18268088 : DOI : 10.1128/AAC.00982-07 PMC : PMC2292564 Abstract >>
DNA sequence analysis of five IncW plasmids (R388, pSa, R7K, pIE321, and pIE522) demonstrated that they share a considerable portion of their genomes and allowed us to define the IncW backbone. Among these plasmids, the backbone is stable and seems to have diverged recently, since the overall identity among its members is higher than 95%. The only gene in which significant variation was observed was trwA; the changes in the coding sequence correlated with parallel changes in the corresponding TrwA binding sites at oriT, suggesting a functional connection between both sets of changes. The present IncW plasmid diversity is shaped by the acquisition of antibiotic resistance genes as a consequence of the pressure exerted by antibiotic usage. Sequence comparisons pinpointed the insertion events that differentiated the five plasmids analyzed. Of greatest interest is that a single acquisition of a class I integron platform, into which different gene cassettes were later incorporated, gave rise to plasmids R388, pIE522, and pSa, while plasmids R7K and pIE321 do not contain the integron platform and arose in the antibiotic world because of the insertion of several antibiotic resistance transposons.
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376. |
Ong CL,
Ulett GC,
Mabbett AN,
Beatson SA,
Webb RI,
Monaghan W,
Nimmo GR,
Looke DF,
McEwan AG,
Schembri MA,
( 2008 ) Identification of type 3 fimbriae in uropathogenic Escherichia coli reveals a role in biofilm formation. PMID : 18055599 : DOI : 10.1128/JB.01523-07 PMC : PMC2223576 Abstract >>
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens.
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377. |
Naas T,
Cuzon G,
Villegas MV,
Lartigue MF,
Quinn JP,
Nordmann P,
( 2008 ) Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene. PMID : 18227185 : DOI : 10.1128/AAC.01451-07 PMC : PMC2292522 Abstract >>
Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.
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378. |
da Fonseca EL,
Freitas Fdos S,
de Amorim JC,
Vicente AC,
( 2008 ) Detection of new arr-4 and arr-5 gene cassettes in clinical Pseudomonas aeruginosa and Klebsiella pneumoniae strains from Brazil. PMID : 18299416 : DOI : 10.1128/AAC.00017-08 PMC : PMC2346643 Abstract >>
New arr alleles emerged in class 1 integrons from a clinical Pseudomonas aeruginosa strain (arr-4) and four Klebsiella pneumoniae strains (arr-5) in Brazil/American continent. arr-4 was preceded by aacA7-catB3, whereas arr-5 was the unique cassette. The putative proteins shared 75% (Arr-5) and 78% (Arr-4) identities with Arr-2.
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379. |
Adler H,
Fenner L,
Walter P,
Hohler D,
Schultheiss E,
Oezcan S,
Frei R,
( 2008 ) Plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: prevalence at a Swiss university hospital and occurrence of the different molecular types in Switzerland. PMID : 18065410 : DOI : 10.1093/jac/dkm472 Abstract >>
N/A
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380. |
Lavilla S,
González-López JJ,
Sabaté M,
García-Fernández A,
Larrosa MN,
Bartolomé RM,
Carattoli A,
Prats G,
( 2008 ) Prevalence of qnr genes among extended-spectrum beta-lactamase-producing enterobacterial isolates in Barcelona, Spain. PMID : 18029415 : DOI : 10.1093/jac/dkm448 Abstract >>
To evaluate the presence of qnr genes among enterobacterial isolates carrying extended-spectrum beta-lactamases (ESBLs) in Barcelona, Spain. Screening for the qnrA, qnrB and qnrS genes was carried out by PCR amplification with specific primers in 305 non-duplicate, clinically relevant ESBL-producing enterobacterial isolates obtained from February 2003 to August 2004. ESBLs from all qnr-positive isolates were characterized by isoelectric focusing, PCR amplification and DNA sequencing. Plasmid analysis was performed by S1 digestion and hybridization with specific probes for the qnr and bla genes. Plasmids containing qnr genes were transferred by conjugation or transformation. The genetic environment of qnrA1 in selected isolates was characterized by cloning experiments. Fifteen isolates, each from a different individual, carried qnr. Among them, 14 had qnrA1 (6 Klebsiella pneumoniae, 6 Enterobacter cloacae and 2 Escherichia coli isolates) and 1 had qnrS1 (K. pneumoniae). None of the isolates carried qnrB. Among the qnrA1-carrying isolates, 10 possessed both bla(CTX-M-9) and bla(SHV-12), 2 had both bla(CTX-M-9) and bla(SHV-92) and 2 had bla(CTX-M-9) alone. The isolate with qnrS1 possessed bla(SHV-12). The qnrA1 and ESBL genes were located together on plasmids ranging in size from 40 to 320 kb. qnrS1 and bla(SHV-12) were not loca |