| 1. |
Wouters J,
Fonzé E,
Vermeire M,
Frère JM,
Charlier P,
( 2003 ) Crystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue. PMID : 14521155 : Abstract >>
The structures of the class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a boronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 A, respectively. The structure of the enzyme resembles those of other class C beta-lactamases. The structure of the complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C beta-lactamase in complex with a transition-state analogue provides further insights into the mechanism of action of these hydrolases.
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2. |
Lee SH,
Kim JY,
Shin SH,
An YJ,
Choi YW,
Jung YC,
Jung HI,
Sohn ES,
Jeong SH,
Lee KJ,
( 2003 ) Dissemination of SHV-12 and characterization of new AmpC-type beta-lactamase genes among clinical isolates of enterobacter species in Korea. PMID : 12791868 : DOI : 10.1128/jcm.41.6.2477-2482.2003 PMC : PMC156513 Abstract >>
To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of Enterobacter species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the ampC genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four ampC genes (bla(EcloK992004.1), bla(EcloK995120.1), bla(EcloK99230), and bla(EareK9911729)) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal ampC gene (bla(EcloMHN1)) of Enterobacter cloacae MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two ampC genes (bla(EcloK9973) and bla(EcloK9914325)) showed close similarity to the chromosomal ampC gene (bla(EcloQ908R)) of E. cloacae Q908R (99.7% identity). The results from phylogenetic analysis suggested that six ampC genes could originate from bla(EcloMHN1) or bla(EcloQ908R).
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3. |
Schütz A,
Sandalova T,
Ricagno S,
Hübner G,
König S,
Schneider G,
( 2003 ) Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid. PMID : 12752451 : DOI : 10.1046/j.1432-1033.2003.03601.x Abstract >>
The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open alpha/beta topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20 degrees in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.
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4. |
Martinez-Garcia E,
Tormo A,
Navarro-Lloréns JM,
( 2003 ) Polymorphism in the yclC-rpoS region of enterobacteria. PMID : 12732965 : DOI : 10.1007/s00284-002-3814-6 Abstract >>
The nucleotide sequence downstream from the rpoS gene in Enterobacter cloacae and Kluyvera cryocrescens contains the slyA-pad1-yclC genes. The DNA sequence of Enterobacter cloacae CETC960 shows a 2.6-kb insertion of unknown origin between rpoS and slyA. This 2.6-kb sequence has also been detected in species of Salmonella and in Pseudomonas aeruginosa, but not in the same location. This insertion has been detected in all the Enterobacter cloacae clinical strains studied although the size of the rpoS-yclC region was highly variable, possibly owing to the presence of insertions and/or deletions. The study of the rpoS-mutS region in other enterobacteria also showed variability in size. Our results support the idea of a variational hot spot in the rpoS-mutS region that could be related to pathogenesis and horizontal transfer.
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5. |
Hoffmann H,
Roggenkamp A,
( 2003 ) Population genetics of the nomenspecies Enterobacter cloacae. PMID : 12957918 : DOI : 10.1128/aem.69.9.5306-5318.2003 PMC : PMC194928 Abstract >>
The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.
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6. |
Plante I,
Centrón D,
Roy PH,
( 2003 ) Direct sequencing and PCR mapping of integrons reveals multiple class 1 integrons in the multiresistant strain Enterobacter cloacae SCH88040794. PMID : 12694911 : DOI : 10.1016/S0378-1097(03)00163-0 Abstract >>
We have characterized the variable region of three integrons found in the multiresistant strain Enterobacter cloacae SCH88040794, using polymerase chain reaction and direct sequencing of amplicons. The first integron has the gene cassette arrangement dfrAI-aadA1 in its variable region and the second aadB-oxa9. The third integron is In40 (aacA4-qacF-cmlB-oxa9), which has recently been described. The multiresistance expressed by this strain to amikacin, gentamicin, trimethoprim, sulfonamides, oxacillin, chloramphenicol and quaternary ammonium compounds is due, at least in part, to these three integrons.
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7. |
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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8. |
Jeong SH,
Lee K,
Chong Y,
Yum JH,
Lee SH,
Choi HJ,
Kim JM,
Park KH,
Han BH,
Lee SW,
Jeong TS,
( 2003 ) Characterization of a new integron containing VIM-2, a metallo- beta-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae. PMID : 12562709 : DOI : 10.1093/jac/dkg047 Abstract >>
We report the first description of a new integron containing bla(VIM-2), a metallo-beta-lactamase gene from Enterobacter cloacae KU680, which was isolated from peritoneal fluid of a liver cirrhosis patient in South Korea. Antibiotic susceptibility testing, and modified Hodge and EDTA-disc synergy tests, were carried out to screen for metallo-beta-lactamase-producing strains. PCR and sequence analysis were used to identify and analyse the bla(VIM-2)-containing integron. The isolate was resistant to most beta-lactams, including imipenem, and demonstrated a positive modified Hodge and EDTA-disc synergy test, which are findings suggesting a metallo-beta-lactamase. Preliminary PCR-based experiments detected the metallo-beta-lactamase gene bla(VIM-2). Sequencing of the 4392 bp cloned PCR amplicon, containing the gene cassette bla(VIM-2), revealed the structure of the class 1 integron. The integron also contained additional insert gene cassettes, aadA, and unknown open reading frames 'orfII' and 'orfIII'. To the best of our knowledge, this is the first time that this metallo-beta-lactamase gene has been detected in E. cloacae.
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9. |
Valkova N,
Lépine F,
Bollet C,
Dupont M,
Villemur R,
( 2002 ) prbA, a gene coding for an esterase hydrolyzing parabens in enterobacter cloacae and Enterobacter gergoviae strains. PMID : 12193616 : DOI : 10.1128/jb.184.18.5011-5017.2002 PMC : PMC135325 Abstract >>
The new gene prbA encodes an esterase responsible for the hydrolysis of the ester bond of parabens in Enterobacter cloacae strain EM. This gene is located on the chromosome of strain EM and was cloned by several PCR approaches. The prbA gene codes for an immature protein of 533 amino acids, the first 31 of which represent a proposed signal peptide yielding a mature protein of a putative molecular mass of 54.6 kDa. This enzyme presents analogies with other type B carboxylesterases, mainly of eukaryotic origin. The cloning and expression of the prbA gene in a strain of Escherichia coli previously unable to hydrolyze parabens resulted in the acquisition of a hydrolytic capacity comparable to the original activity of strain EM, along with an increased resistance of the transformed strain to methyl paraben. The presence of homologues of prbA was tested in additional ubiquitous bacteria, which may be causative factors in opportunistic infections, including Enterobacter gergoviae, Enterobacter aerogenes, Pseudomonas agglomerans, E. coli, Pseudomonas aeruginosa, and Burkholderia cepacia. Among the 41 total strains tested, 2 strains of E. gergoviae and 1 strain of Burkholderia cepacia were able to degrade almost completely 800 mg of methyl paraben liter(-1). Two strains of E. gergoviae, named G1 and G12, contained a gene that showed high homology to the prbA gene of E. cloacae and demonstrated comparable paraben esterase activities. The significant geographical distance between the locations of the isolated E. cloacae and E. gergoviae strains suggests the possibility of an efficient transfer mechanism of the prbA gene, conferring additional resistance to parabens in ubiquitous bacteria that represent a common source of opportunistic infections.
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10. |
Lohrke SM,
Dery PD,
Li W,
Reedy R,
Kobayashi DY,
Roberts DR,
( 2002 ) Mutation of rpiA in Enterobacter cloacae decreases seed and root colonization and biocontrol of damping-off caused by Pythium ultimum on cucumber. PMID : 12182339 : DOI : 10.1094/MPMI.2002.15.8.817 Abstract >>
Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.
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11. |
Patten CL,
Glick BR,
( 2002 ) Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. PMID : 12147474 : DOI : 10.1128/aem.68.8.3795-3801.2002 PMC : PMC124051 Abstract >>
Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.
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12. |
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.
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13. |
Haynes CA,
Koder RL,
Miller AF,
Rodgers DW,
( 2002 ) Structures of nitroreductase in three states: effects of inhibitor binding and reduction. PMID : 11805110 : DOI : 10.1074/jbc.M111334200 Abstract >>
The crystal structure of the nitroreductase enzyme from Enterobacter cloacae has been determined for the oxidized form in separate complexes with benzoate and acetate inhibitors and for the two-electron reduced form. Nitroreductase is a member of a group of enzymes that reduce a broad range of nitroaromatic compounds and has potential uses in chemotherapy and bioremediation. The monomers of the nitroreductase dimer adopt an alpha+beta fold and together bind two flavin mononucleotide prosthetic groups at the dimer interface. In the oxidized enzyme, the flavin ring system adopts a strongly bent (16 degrees) conformation, and the bend increases (25 degrees) in the reduced form of the enzyme, roughly the conformation predicted for reduced flavin free in solution. Because free oxidized flavin is planar, the induced bend in the oxidized enzyme may favor reduction, and it may also account for the characteristic inability of the enzyme to stabilize the one electron-reduced semiquinone flavin, which is also planar. Both inhibitors bind over the pyrimidine and central rings of the flavin in partially overlapping sites. Comparison of the two inhibitor complexes shows that a portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for accommodating varied substrates.
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14. |
Arpin C,
Labia R,
Dubois V,
Noury P,
Souquet M,
Quentin C,
( 2002 ) TEM-80, a novel inhibitor-resistant beta-lactamase in a clinical isolate of Enterobacter cloacae. PMID : 11959543 : DOI : 10.1128/aac.46.5.1183-1189.2002 PMC : PMC127146 Abstract >>
Enterobacter cloacae Ecl261 was isolated with Escherichia coli Ec257 from the urine of a patient living in a nursing home. Both isolates were resistant to ticarcillin (MICs, 1,024 microg/ml), without significant potentiation of its activity by 2 microg of clavulanate per ml (MICs, 512 microg/ml), and susceptible to naturally active cephalosporins. This inhibitor-resistant phenotype was conferred in both strains by similar conjugative plasmids of 40 kb (Ecl261) and 30 kb (Ec257), which also conveyed resistance to sulfonamides and trimethoprim. Clinical and transconjugant strains produced a beta-lactamase with a pI of 5.2 which belonged to the TEM family, as indicated by specific PCR amplification. Compared with TEM-1, this enzyme exhibited lower catalytic efficiencies (14- and 120-fold less for amoxicillin and ticarcillin, respectively), and higher concentrations of beta-lactamase inhibitors were required to yield a 50% reduction in benzylpenicillin hydrolysis (750-, 82-, and 50-fold higher concentrations for clavulanate, sulbactam, and tazobactam, respectively). Gene sequencing revealed four nucleotide differences with the nucleotide sequence of bla(TEM-1A). The first replacement (T32C), located in the promoter region, was described as being responsible for the increase in the level of beta-lactamase production. The three other changes led to amino acid substitutions that define a new inhibitor-resistant TEM (IRT) beta-lactamase, TEM-80 (alternate name, IRT-24). Two of them, Met69Leu and Asn276Asp, have previously been related to inhibitor resistance. The additional mutation, Ile127Val, was demonstrated by site-directed mutagenesis to have a very weak effect, at least alone, on the IRT phenotype. This is the first description of an IRT beta-lactamase in E. cloacae. The horizontal transfer of bla(TEM-80) may have occurred either from Ec257 to Ecl261 or in the reverse order.
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15. |
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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16. |
Khan H,
Harris RJ,
Barna T,
Craig DH,
Bruce NC,
Munro AW,
Moody PC,
Scrutton NS,
( 2002 ) Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives. PMID : 11923299 : DOI : 10.1074/jbc.M200637200 Abstract >>
The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.
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17. |
Linde HJ,
Notka F,
Irtenkauf C,
Decker J,
Wild J,
Niller HH,
Heisig P,
Lehn N,
( 2002 ) Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae. PMID : 11909836 : DOI : 10.1093/jac/49.4.625 Abstract >>
The mechanisms of fluoroquinolone resistance in two isolates of Enterobacter cloacae, Ecl#1 and Ecl#2, from the same patient and with identical pulsed-field gel electrophoresis patterns, have been analysed. MICs of ciprofloxacin were 0.25 and 1 mg/L for Ecl#1 and Ecl#2, respectively. Ecl#2 was also more resistant to chloramphenicol and organic solvents. The quinolone resistance determining regions of gyrA/B and parC/E, and the marORA and acrB genes, were sequenced. Expression of marR, acrB, soxS, robA, ramA and fis was analysed by northern blotting. The activity of a 90 bp E. cloacae mar promoter fragment was examined with the reporter plasmid pIGJ-1mar. Sequencing the gyrAB and parCE genes revealed a single amino acid substitution in GyrA (corresponding to position 83 in GyrA of Escherichia coli) in Ecl#1 and Ecl#2 (Phe83) compared with reference strain E. cloacae DSMZ 3264 (Thr83). Ecl#2 accumulated significantly less norfloxacin and displayed higher levels of expression of marR and acrB than Exl#1, indicative of greater fluoroquinolone efflux activity. Sequencing gyrB, parC/E and marORA, and northern blotting of robA, ramA and fis, did not reveal any further differences between the two strains. No homologue of soxRS was detected in E. cloacae. Expression of GFP from pIGJ1-mar in Ecl#2 was higher than in Ecl#1. In these two closely related clinical isolates of E. cloacae, a target mutation in GyrA (Ecl#1 and Ecl#2) and increased fluoroquinolone efflux by AcrAB (Ecl#2) contribute to the resistance phenotypes, corroborating findings in vitro and in vivo about the sequential development of fluoroquinolone resistance.
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18. |
Naas T,
Massuard S,
Garnier F,
Nordmann P,
( 2001 ) AmpD is required for regulation of expression of NmcA, a carbapenem-hydrolyzing beta-lactamase of Enterobacter cloacae. PMID : 11557489 : DOI : 10.1128/AAC.45.10.2908-2915.2001 PMC : PMC90751 Abstract >>
To further elucidate the induction process of the carbapenem-hydrolyzing beta-lactamase of Ambler class A, NmcA, ampD genes of the wild-type (WT) strain and of ceftazidime-resistant mutants of Enterobacter cloacae NOR-1 were cloned and tested in transcomplementation experiments. Ceftazidime-resistant E. cloacae NOR-1 mutants exhibited derepressed expression of the AmpC-type cephalosporinase and of the carbapenem-hydrolyzing beta-lactamase NmcA. The ampD genes of Escherichia coli and E. cloacae WT NOR-1 transcomplemented the ceftazidime-resistant E. cloacae NOR-1 mutants to the WT level of beta-lactamase expression, while the mutated ampD alleles of E. cloacae NOR-1 failed to do so. The deduced E. cloacae NOR-1 WT AmpD protein exhibited 95 and 91% amino acid identity with the E. cloacae O29 and E. cloacae 14 WT AmpD proteins, respectively. Of the 12 ceftazidime-resistant E. cloacae NOR-1 strains, 3 had AmpD proteins with amino acid changes, while the others had truncated AmpD proteins. Most of these mutations were located outside the conserved regions that link the AmpD proteins to the cell wall hydrolases. AmpD from E. cloacae NOR-1 is involved in the regulation of expression of both beta-lactamases (NmcA and AmpC), suggesting that structurally unrelated genes may be under the control of an identical genetic system.
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19. |
Li J,
Shah S,
Moffatt BA,
Glick BR,
( 2001 ) Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4. PMID : 11827211 : DOI : 10.1023/a:1013067707126 Abstract >>
A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.
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20. |
Barna TM,
Khan H,
Bruce NC,
Barsukov I,
Scrutton NS,
Moody PC,
( 2001 ) Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme. PMID : 11428899 : DOI : 10.1006/jmbi.2001.4779 Abstract >>
Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.
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21. |
Crichlow GV,
Nukaga M,
Doppalapudi VR,
Buynak JD,
Knox JR,
( 2001 ) Inhibition of class C beta-lactamases: structure of a reaction intermediate with a cephem sulfone. PMID : 11371184 : DOI : 10.1021/bi010131s Abstract >>
The crystallographic structure of the Enterobacter cloacae GC1 extended-spectrum class C beta-lactamase, inhibited by a new 7-alkylidenecephalosporin sulfone, has been determined by X-ray diffraction at 100 K to a resolution of 1.6 A. The crystal structure was solved by molecular replacement using the unliganded structure [Crichlow et al. (1999) Biochemistry 38, 10256-10261] and refined to a crystallographic R-factor equal to 0.183 (R(free) 0.208). Cryoquenching of the reaction of the sulfone with the enzyme produced an intermediate that is covalently bound via Ser64. After acylation of the beta-lactam ring, the dihydrothiazine dioxide ring opened with departure of the sulfinate. Nucleophilic attack of a side chain pyridine nitrogen atom on the C6 atom of the resultant imine yielded a bicyclic aromatic system which helps to stabilize the acyl enzyme to hydrolysis. A structural assist to this resonance stabilization is the positioning of the anionic sulfinate group between the probable catalytic base (Tyr150) and the acyl ester bond so as to block the approach of a potentially deacylating water molecule. Comparison of the liganded and unliganded protein structures showed that a major movement (up to 7 A) and refolding of part of the Omega-loop (215-224) accompanies the binding of the inhibitor. This conformational flexibility in the Omega-loop may form the basis of an extended-spectrum activity of class C beta-lactamases against modern cephalosporins.
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22. |
Núñez B,
De La Cruz F,
( 2001 ) Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation. PMID : 11251827 : DOI : 10.1046/j.1365-2958.2001.02308.x Abstract >>
The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT-mobB-mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5'-GGGTG/GTCGGG-3' by primer extension and sequencing reactions. Analysis of Mob- insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which nic cleavage results in a free nicked-DNA intermediate.
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23. |
Kato J,
Nagata C,
Yang L,
Kuroda A,
Ikeda T,
Takiguchi N,
Ohtake H,
( 2001 ) Isolation and characterization of the Enterobacter cloacae cheR mutant defective in phosphate taxis. PMID : 11302189 : DOI : 10.1271/bbb.65.456 Abstract >>
A chemotaxis-defective mutant of Enterobacter cloacae IFO3320, designated EC1, was isolated after N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis. Computer-assisted capillary assays showed that EC1 failed to show chemotactic responses to peptone and inorganic phosphate (Pi). Cloning and sequence analysis showed that EC1 is a cheR mutant, suggesting that Pi taxis by E. cloacae is dependent on a methyl-accepting chemotaxis protein(s) (MCP). EC1 was further mutagenized with NTG to construct cheR pstS and cheR pstA double mutants. A recombinant plasmid pECT01.2, which contained the E. cloacae cheR gene, restored the ability of these double mutants to show chemotaxis toward peptone but not Pi. These results suggest that the phosphate-specific transport (Pst) system, together with a MCP(s), is required for detecting Pi in E. cloacae.
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24. |
Barnaud G,
Labia R,
Raskine L,
Sanson-Le Pors MJ,
Philippon A,
Arlet G,
( 2001 ) Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. PMID : 11179650 : DOI : 10.1111/j.1574-6968.2001.tb10519.x Abstract >>
Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICs> or =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.
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25. |
van Dijk K,
Nelson EB,
( 2000 ) Fatty acid competition as a mechanism by which Enterobacter cloacae suppresses Pythium ultimum sporangium germination and damping-off. PMID : 11097912 : DOI : 10.1128/aem.66.12.5340-5347.2000 PMC : PMC92466 Abstract >>
Interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. While competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. We present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological control bacterium Enterobacter cloacae and the seed-rotting oomycete, Pythium ultimum, results in disease suppression. Since fatty acids from seeds and roots are required to elicit germination responses of P. ultimum, we generated mutants of E. cloacae to evaluate the role of E. cloacae fatty acid metabolism on the suppression of Pythium sporangium germination and subsequent plant infection. Two mutants of E. cloacae EcCT-501R3, Ec31 (fadB) and EcL1 (fadL), were reduced in beta-oxidation and fatty acid uptake, respectively. Both strains failed to metabolize linoleic acid, to inactivate the germination-stimulating activity of cottonseed exudate and linoleic acid, and to suppress Pythium seed rot in cotton seedling bioassays. Subclones containing fadBA or fadL complemented each of these phenotypes in Ec31 and EcL1, respectively. These data provide strong evidence for a competitive exclusion mechanism for the biological control of P. ultimum-incited seed infections by E. cloacae where E. cloacae prevents the germination of P. ultimum sporangia by the efficient metabolism of fatty acid components of seed exudate and thus prevents seed infections.
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26. |
Giakkoupi P,
Tzouvelekis LS,
Tsakris A,
Loukova V,
Sofianou D,
Tzelepi E,
( 2000 ) IBC-1, a novel integron-associated class A beta-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain. PMID : 10952563 : DOI : 10.1128/aac.44.9.2247-2253.2000 PMC : PMC90053 Abstract >>
A transferable beta-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. The bla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including aac(6')-Ib. The encoded beta-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with beta-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica, a carbenicillinase from Proteus mirabilis GN79, the species-specific beta-lactamases of Klebsiella oxytoca, and the carbapenemase Sme-1. However, a phylogenetic association with established beta-lactamase clusters could not be conclusively shown.
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27. |
Okamoto R,
Kuga A,
( 2000 ) ampR gene mutations that greatly increase class C beta-lactamase activity in Enterobacter cloacae. PMID : 10681318 : DOI : 10.1128/aac.44.3.561-567.2000 PMC : PMC89726 Abstract >>
The ampC and ampR genes of Enterobacter cloacae GN7471 were cloned into pMW218 to yield pKU403. Four mutant plasmids derived from pKU403 (pKU404, pKU405, pKU406, and pKU407) were isolated in an AmpD mutant of Escherichia coli ML4953 by selection with ceftazidime or aztreonam. The beta-lactamase activities expressed by pKU404, pKU405, pKU406, and pKU407 were about 450, 75, 160, and 160 times higher, respectively, than that expressed by the original plasmid, pKU403. These mutant plasmids all carried point mutations in the ampR gene. In pKU404 and pKU405, Asp-135 was changed to Asn and Val, respectively. In both pKU406 and pKU407, Arg-86 was changed to Cys. The ease of selection of AmpR mutations at a frequency of about 10(-6) in this study strongly suggests that derepressed strains, such as AmpD or AmpR mutants, could frequently emerge in the clinical setting.
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28. |
Mobashery S,
Miller MJ,
O'Brien M,
Kotra LP,
Bellettini J,
Bulychev A,
( 1999 ) Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams. PMID : 10464248 : DOI : 10.1074/jbc.274.36.25260 Abstract >>
beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.
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29. |
Forst S,
( 1999 ) Identification of a conserved N-terminal sequence involved in transmembrane signal transduction in EnvZ. PMID : 10464234 : PMC : PMC94069 Abstract >>
To determine whether N-terminal sequences are involved in the transmembrane signaling mechanism of EnvZ, the nucleotide sequences of envZ genes from several enteric bacteria were determined. Comparative analysis revealed that the amino acid sequence between Pro41 and Glu53 was highly conserved. To further analyze the role of the conserved sequence, envZ of Escherichia coli was subjected to random PCR mutagenesis and mutant alleles that produced a high-osmolarity phenotype, in which ompF was repressed, were isolated. The mutations identified clustered within, as well as adjacent to, the Pro41-to-Glu53 sequence. These findings suggest that the conserved Pro41-to-Glu53 sequence is involved in the signal transduction mechanism of EnvZ.
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30. |
Dery PD,
Yucel I,
Buyer J,
( 1999 ) Role of pfkA and general carbohydrate catabolism in seed colonization by Enterobacter cloacae. PMID : 10347036 : DOI : 10113/31322 PMC : PMC91371 Abstract >>
Enterobacter cloacae A-11 is a transposon mutant of strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol. 38:1128-1134, 1992). In vitro growth of strain A-11 was reduced or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (P
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31. |
Keeney D,
Ruzin A,
Bradford PA,
( 2007 ) RamA, a transcriptional regulator, and AcrAB, an RND-type efflux pump, are associated with decreased susceptibility to tigecycline in Enterobacter cloacae. PMID : 17536927 : DOI : 10.1089/mdr.2006.9990 Abstract >>
Tigecycline, a novel broad-spectrum glycylcycline antibiotic, is active against many gram-positive and gram-negative bacterial pathogens including most strains of Enterobacter cloacae. Recently, however, a few clinical strains of E. cloacae with decreased susceptibility to tigecycline were isolated. In this study, two tigecycline-susceptible mutants of E. cloacae, GC7696 and GC7697, were obtained by transposon mutagenesis of a tigecycline-resistant clinical isolate G946. Transposon insertions were mapped to either the acrA or acrB genes. Restoration of the original resistant phenotype occurred when GC7696 and GC7697 were transcomplemented with a plasmid harboring the intact acrAB region amplified from G946. Northern blot analysis of G946 and several other E. cloacae clinical strains that exhibited decreased susceptibility to tigecycline, revealed increased levels of the acrAB transcript. In addition, overexpression of acrAB correlated with increased expression of the ramA gene, whereas the expression of another transcriptional activator, marA, was not changed. These results suggest that decreased susceptibility to tigecycline in E. cloacae is the result of RamA-mediated overexpression of the AcrAB efflux pump.
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32. |
Liu S,
Hu X,
Lohrke SM,
Baker CJ,
Buyer JS,
de Souza JT,
Roberts DP,
( 2007 ) Role of sdhA and pfkA and catabolism of reduced carbon during colonization of cucumber roots by Enterobacter cloacae. PMID : 17768262 : DOI : 10.1099/mic.0.2006/005538-0 Abstract >>
We have been using a mutational approach to determine how plant-beneficial bacteria such as Enterobacter cloacae 501R3 obtain carbon and energy for colonization of subterranean portions of cucumber and other plants. Reduced carbon detected in cucumber root exudate consisted of 73.3 % amino acids, 22.2 % organic acids and 4.4 % carbohydrate. Ent. cloacae M2, a mini-Tn5 Km transposon mutant of strain 501R3, was severely reduced in in vitro growth relative to strain 501R3 on the mixture of amino acids and organic acids detected in cucumber root exudate when these compounds were supplied as the sole source of carbon and energy, but was similar in growth on the mixture of carbohydrates detected in this exudate. Molecular and biochemical characterization of Ent. cloacae M2 indicated that the transposon was inserted in sdhA, which encodes a subunit of succinate dehydrogenase. Ent. cloacae A-11, a mutant of strain 501R3 with a mini-Tn5 Km insertion in pfkA, was severely reduced in in vitro growth relative to strain 501R3 on the mixture of carbohydrates detected in cucumber root exudate, but similar in growth on the mixture of amino acids and organic acids. When strains A-11 and M2 were coapplied with strain 501R3 to cucumber seeds above carrying capacity in competitive root colonization assays, populations of strains A-11 and M2 were roughly one order of magnitude lower than those of strain 501R3 in cucumber rhizosphere, while populations of strains A-11 and M2 were similar to one other when coapplied to cucumber seeds. When Ent. cloacae strains were coapplied to cucumber seeds below carrying capacity, populations of A-11 and M2 were roughly two to three orders of magnitude lower than those of 501R3 in cucumber rhizosphere, and populations of A-11 were significantly lower than those of M2 when these two strains were coapplied to cucumber seed. The experiments reported here indicate an important role for pfkA and sdhA and the catabolism of carbohydrates, and of amino acids and organic acids, respectively, in the colonization of cucumber roots by Ent. cloacae. The results reported here also indicate that catabolism of carbohydrates by this bacterium is more important than catabolism of amino acids and organic acids at lower population densities, despite the much higher relative quantities of amino acids and organic acids detected in cucumber root exudate.
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33. |
Castanheira M,
Sader HS,
Jones RN,
Debbia E,
Picão RC,
Gales AC,
( 2007 ) In71, an Enterobacter cloacae blaVIM-1-carrying integron related to In70.2 from Italian Pseudomonas aeruginosa isolates: a SENTRY Antimicrobial Surveillance Program report. PMID : 17650966 : DOI : 10.1089/mdr.2007.728 Abstract >>
An Enterobacter cloacae strain showing decreased susceptibility to carbapenems was isolated from a blood culture of a patient hospitalized in Genoa, Italy, and screened for the presence of metallo-beta-lactamase (MBL) genes as part of the SENTRY Antimicrobial Surveillance Program. A bla(VIM-1)-carrying integron named In71 nearly identical to In70.2 reported in Pseudomonas aeruginosa strains isolated from various Italian cities since 2001 was identified in this strain. Interestingly, the In71 did not carry aadA1 nor possess the ISPa7 usually found in the P. aeruginosa integron In70.2. Mobilization of MBL genes from P. aeruginosa to members of the Enterobacteriaceae family is very worrisome because the rapid and wide dissemination of these potent antimicrobial resistance mechanisms could jeopardize the clinical use of carbapenems for the treatment of multidrug-resistant Enterobacteriaceae infections.
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34. |
Pham HN,
Ohkusu K,
Mishima N,
Noda M,
Monir Shah M,
Sun X,
Hayashi M,
Ezaki T,
( 2007 ) Phylogeny and species identification of the family Enterobacteriaceae based on dnaJ sequences. PMID : 17368802 : DOI : 10.1016/j.diagmicrobio.2006.12.019 Abstract >>
Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.
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35. |
Chen L,
Chen ZL,
Liu JH,
Zeng ZL,
Ma JY,
Jiang HX,
( 2007 ) Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China. PMID : 17353219 : DOI : 10.1093/jac/dkm065 Abstract >>
To investigate the occurrence of 16S rRNA methylases conferring high-level resistance to aminoglycosides in Enterobacteriaceae isolated from two pig farms in China. Enterobacteriaceae isolated from 151 pig rectal swab samples and 9 environmental samples were screened for the presence of the rmtA, rmtB, armA and rmtC genes by PCR and sequencing. Conjugation experiments were carried out to study the transferability of the 16S rRNA methylase genes. All isolates and their transconjugants were tested for susceptibility to antimicrobial agents. The clonal relatedness of RmtB-producing Escherichia coli was assessed by PFGE with XbaI. Of 152 Enterobacteriaceae isolates recovered from pigs, 49 (32%) were positive for the rmtB gene, including 48 E. coli and a single isolate of Enterobacter cloacae. Of the nine Enterobacteriaceae isolates from environmental samples, no 16S rRNA methylase gene was identified. The 49 rmtB-positive isolates showed resistance to ampicillin, tetracycline and trimethoprim and also carried a bla(TEM) gene. Transfer of the rmtB and bla(TEM) genes by conjugation experiments of all 49 isolates was successful, suggesting that the rmtB-containing plasmids in the E. coli and E. cloacae isolates were self-transmissible. Conjugative transfer frequencies varied from 2.2 x 10(-10) to 1.3 x 10(-6) transconjugants per recipient. The transfer of non-aminoglycoside antimicrobial resistance traits was also observed in most cases. Forty-four rmtB-positive E. coli showed 30 different PFGE types. The rmtB gene was detected on conjugative plasmids of porcine E. coli and E. cloacae isolates. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene. The emergence of 16S rRNA methylases in Enterobacteriaceae isolates is described for the first time in China. This is also the first report of rmtB-positive Enterobacteriaceae among healthy food-producing animals.
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36. |
Wu JJ,
Ko WC,
Tsai SH,
Yan JJ,
( 2007 ) Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital. PMID : 17242140 : DOI : 10.1128/AAC.01195-06 PMC : PMC1855486 Abstract >>
The prevalence of three plasmid-mediated quinolone resistance determinants, QnrA, QnrB, and QnrS, among 526 nonreplicate clinical isolates of Enterobacter cloacae collected at a Taiwanese university hospital in 2004 was determined by PCR and colony hybridization, and the association of Qnr with the IMP-8 metallo-beta-lactamase was investigated. Eighty-six (16.3%) of all isolates were qnr positive, and the qnrA1-like, qnrB2-like, and qnrS1-like genes were detected alone or in combination in 3 (0.6%), 53 (10.1%), and 34 (6.5%) isolates, respectively. Among 149 putative extended-spectrum-beta-lactamase-producing isolates, 59 (39.6%) isolates, all of which were SHV-12 producers, harbored qnrA (0.7%; 1 isolate), qnrB (28.9%; 43 isolates), or qnrS (12.1%; 18 isolates). Forty-four (78.6%) of 56 IMP-8 producers carried qnrB (58.9%; 33 isolates), qnrS (25.0%; 14 isolates), or both. PCR and sequence analysis revealed that qnrA1 was located in a complex sul1-type integron that contains dhr15, aadA2, qacEDelta1, sul1, orf513, qnrA1, ampR, and qacEDelta1. Conjugation experiments revealed the coexistence of qnrB and bla(IMP-8) on the transferred plasmids and the absence of beta-lactamase content on the transferred qnrS-positive plasmids. The transferred bla(IMP-8)-positive plasmids with and without qnrB had very similar restriction patterns, suggesting the horizontal mobility of qnrB. Pulsed-field gel electrophoresis showed six major patterns among the 44 qnr-positive IMP-8-producing isolates. Thus, the extremely high prevalence of qnr among the metallo-beta-lactamase-producing E. cloacae isolates in the hospital may be due mainly to the intrahospital spread of a few clones and the dissemination of plasmids containing both qnrB and blaIMP-8.
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37. |
Yee N,
Ma J,
Dalia A,
Boonfueng T,
Kobayashi DY,
( 2007 ) Se(VI) reduction and the precipitation of Se(0) by the facultative bacterium Enterobacter cloacae SLD1a-1 are regulated by FNR. PMID : 17261520 : DOI : 10.1128/AEM.02542-06 PMC : PMC1828800 Abstract >>
The fate of selenium in the environment is controlled, in part, by microbial selenium oxyanion reduction and Se(0) precipitation. In this study, we identified a genetic regulator that controls selenate reductase activity in the Se-reducing bacterium Enterobacter cloacae SLD1a-1. Heterologous expression of the global anaerobic regulatory gene fnr (fumarate nitrate reduction regulator) from E. cloacae in the non-Se-reducing strain Escherichia coli S17-1 activated the ability to reduce Se(VI) and precipitate insoluble Se(0) particles. Se(VI) reduction by E. coli S17-1 containing the fnr gene occurred at rates similar to those for E. cloacae, with first-order reaction constants of k = 2.07 x 10(-2) h(-1) and k = 3.36 x 10(-2) h(-1), respectively, and produced elemental selenium particles with identical morphologies and short-range atomic orders. Mutation of the fnr gene in E. cloacae SLD1a-1 resulted in derivative strains that were deficient in selenate reductase activity and unable to precipitate elemental selenium. Complementation by the wild-type fnr sequence restored the ability of mutant strains to reduce Se(VI). Our findings suggest that Se(VI) reduction and the precipitation of Se(0) by facultative anaerobes are regulated by oxygen-sensing transcription factors and occur under suboxic conditions.
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38. |
Kosti? T,
Weilharter A,
Rubino S,
Delogu G,
Uzzau S,
Rudi K,
Sessitsch A,
Bodrossy L,
( 2007 ) A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens. PMID : 17123456 : DOI : 10.1016/j.ab.2006.09.026 Abstract >>
A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.
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39. |
Poirel L,
Cattoir V,
Soares A,
Soussy CJ,
Nordmann P,
( 2007 ) Novel Ambler class A beta-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1. PMID : 17116662 : DOI : 10.1128/AAC.01082-06 PMC : PMC1797722 Abstract >>
The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum beta-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A beta-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related beta-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of beta-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1 gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.
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40. |
Minarini LA,
Gales AC,
Darini AL,
( 2007 ) First report of plasmid-mediated resistance to quinolones and cefotaxime in an Enterobacter cloacae strain isolated from an outpatient in Brazil. PMID : 17060528 : DOI : 10.1128/AAC.00935-06 PMC : PMC1797657 Abstract >>
N/A
|
41. |
Poirel L,
Leviandier C,
Nordmann P,
( 2006 ) Prevalence and genetic analysis of plasmid-mediated quinolone resistance determinants QnrA and QnrS in Enterobacteriaceae isolates from a French university hospital. PMID : 16982792 : DOI : 10.1128/AAC.00597-06 PMC : PMC1694006 Abstract >>
The spread of plasmid-mediated quinolone resistance determinants QnrA and QnrS was evaluated in a collection of 186 extended-spectrum beta-lactamase (ESBL)-positive enterobacterial isolates from 2002 to 2005 and 185 nalidixic acid-resistant strains isolated during the first 6 months of 2005 at the Bic?tre hospital, France. Out of these 186 ESBL-positive isolates, 2.2 and 1.6% carried a QnrA1 and a QnrS1 determinant, respectively. The ESBLs associated with QnrA1 were VEB-1, SHV-12, and CTX-M-1, whereas those associated with QnrS1 were TEM-52, SHV-12, and CTX-M-1. Among the 185 nalidixic acid-resistant strains isolated in 2005, 0.5 and 2.7% had a QnrA1 determinant and a QnrS1 determinant, respectively. The genetic environments of the qnrA1 gene differed but were always associated with sul1 type integrons. In contrast, qnrS1 genes were not embedded in class 1 integrons but located often (but not systematically) downstream of the insertion sequence ISEcl2 on plasmids that often carried a novel beta-lactamase gene, bla(LAP-1). This is the first study identifying the QnrS resistance determinant in Europe and indicating that this determinant might also be widespread.
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42. |
Stoorvogel J,
van Bussel MJ,
van de Klundert JA,
( 1991 ) Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX). PMID : 1702778 : DOI : 10.1128/jb.173.1.161-167.1991 PMC : PMC207170 Abstract >>
We have described a gene coding for an Enterobacter cloacae protein, provisionally called OmpX (J. Stoorvogel, M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert, J. Bacteriol. 173:156-160, 1991). In the work reported here, OmpX was localized in the cell envelope by means of sucrose gradient fractionation of membrane vesicles. Overproduction of OmpX in Escherichia coli from a multicopy plasmid resulted in a reduction in the amount of OmpF. No accumulation of OmpF, of its uncleft precursor, or of its degradation products could be detected in various cell fractions by Western immunoblot analysis using monoclonal antibodies produced in response to OmpF. A decrease in the rate of synthesis of ompF mRNA was indicated by a beta-galactosidase assay in an ompF-lacZ fusion strain containing the cloned ompX gene and by Northern (RNA) blot analysis. These results indicate that the inhibition is at the level of transcription. Colony hybridization, using an internal ompX fragment as a probe, showed a widespread distribution of the ompX gene among clinical isolates of members of the family Enterobacteriaceae. To study the function of the OmpX protein and its role in the regulation of porin protein synthesis, the ompX gene was deleted from the Enterobacter cloacae chromosome and replaced by the aphA gene. The absence of the ompX gene had no apparent effect on cell growth or on the regulation of the porin proteins.
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43. |
Paauw A,
Fluit AC,
Verhoef J,
Leverstein-van Hall MA,
( 2006 ) Enterobacter cloacae outbreak and emergence of quinolone resistance gene in Dutch hospital. PMID : 16704842 : DOI : 10.3201/eid1205.050910 PMC : PMC3374434 Abstract >>
An outbreak of Enterobacter cloacae infections with variable susceptibility to fluoroquinolones occurred in the University Medical Center Utrecht in the Netherlands in 2002. Our investigation showed that a qnrA1 gene was present in 78 (94%) of 83 outbreak isolates and that a qnrA1-encoding plasmid transferred to other strains of the same species and other species. The earliest isolate carrying this same plasmid was isolated in 1999. qnrA1 was located in a complex integron consisting of the intI1, aadB, qacEDelta1, sul1, orf513, qnrA1, ampR, qacEDelta1, and sul1 genes that were not described previously. On the same plasmid, 2 other class 1 integrons were present. One was a new integron associated with the bla(CTX-M-9) extended-spectrum beta-lactamase.
|
44. |
Matsui T,
Yoshida T,
Hayashi T,
Nagasawa T,
( 2006 ) Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240. PMID : 16758158 : DOI : 10.1007/s00203-006-0117-5 Abstract >>
We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its Km value for 4-hydroxybenzoate was 596 microM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the Km value was 6.80 mM. In the presence of 3 M KHCO3 and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The Km value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96-97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.
|
45. |
Wang CX,
Cai PQ,
Guo Y,
Huang ZM,
Mi ZH,
( 2006 ) Emerging plasmid-mediated quinolone resistance associated with the qnrA gene in Enterobacter cloacae clinical isolates in China. PMID : 16713016 : DOI : 10.1016/j.jhin.2006.02.013 Abstract >>
N/A
|
46. |
Delmas J,
Breysse F,
Devulder G,
Flandrois JP,
Chomarat M,
( 2006 ) Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene. PMID : 16626902 : DOI : 10.1016/j.diagmicrobio.2006.02.003 Abstract >>
Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioM?rieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.
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47. |
Bochtler M,
Szczepanowski RH,
Tamulaitis G,
Grazulis S,
Czapinska H,
Manakova E,
Siksnys V,
( 2006 ) Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. PMID : 16628220 : DOI : 10.1038/sj.emboj.7601096 PMC : PMC1462983 Abstract >>
Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV-DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson-Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.
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48. |
Ridley H,
Watts CA,
Richardson DJ,
Butler CS,
( 2006 ) Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions. PMID : 16885262 : DOI : 10.1128/AEM.00568-06 PMC : PMC1538730 Abstract >>
Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.
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49. |
Yu YS,
Du XX,
Zhou ZH,
Chen YG,
Li LJ,
( 2006 ) First isolation of blaIMI-2 in an Enterobacter cloacae clinical isolate from China. PMID : 16569898 : DOI : 10.1128/AAC.50.4.1610-1611.2006 PMC : PMC1426974 Abstract >>
N/A
|
50. |
Ling BD,
Liu G,
Xie YE,
Zhou QX,
Zhao TK,
Li CQ,
Yu X,
Lei J,
( 2006 ) Characterisation of a novel extended-spectrum beta-lactamase, SHV-70, from a clinical isolate of Enterobacter cloacae in China. PMID : 16563703 : DOI : 10.1016/j.ijantimicag.2006.02.003 Abstract >>
N/A
|
51. |
Kang SH,
Cho KK,
Bok JD,
Kim SC,
Cho JS,
Lee PC,
Kang SK,
Lee HG,
Woo JH,
Lee HJ,
Lee SC,
Choi YJ,
( 2006 ) Cloning, sequencing and characterization of a novel phosphatase gene, phoI, from soil bacterium Enterobacter sp. 4. PMID : 16550460 : DOI : 10.1007/s00284-005-4467-z Abstract >>
A gene, phoI, coding for a phosphatase from Enterobacter sp. 4 was cloned in Escherichia coli and sequenced. Analysis of the sequence revealed one open reading frame (ORF) that encodes a 269-amino acid protein with a calculated molecular mass of 29 kDa. PhoI belongs to family B acid phosphatase and exhibits 49.4% identity and 62.4% homology to the hel gene from Heamophilus influenzae, which encoded an outer membrane protein (P4). The optimum pH and temperature for phosphatase activity were pH 5.5 and 40 degrees C, respectively. Its specific activity on rho-nitrophenyl phosphatate was 70 U/mg at pH 5.5 and 40 degrees C. Enzyme activity was inhibited by Al3+, EDTA, and DTT, but fivefold activated by Cu2+ ion (350 U/mg). PhoI showed a strong synergistic effect when used with a purified E. coli phytase, AppA, to estimate combination effects.
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52. |
Hoffmann H,
Stindl S,
Ludwig W,
Stumpf A,
Mehlen A,
Heesemann J,
Monget D,
Schleifer KH,
Roggenkamp A,
( 2005 ) Reassignment of enterobacter dissolvens to Enterobacter cloacae as E. cloacae subspecies dissolvens comb. nov. and emended description of Enterobacter asburiae and Enterobacter kobei. PMID : 15900966 : DOI : 10.1016/j.syapm.2004.12.010 Abstract >>
The taxonomic position of Enterobacter dissolvens was re-evaluated based on the analysis of the type strain ATCC 23373T and three clinical isolates. The strains were assigned to the genetic cluster of the species by phylogenetic sequence analysis in the frame of a recent population genetic study. The relatedness of E. dissolves to the other species of the E. cloacae complex was analyzed by DNA-DNA hybridization studies based on melting profiles in microplates. The genetic cluster of E. dissolvens fell into the same DNA-relatedness group like E. cloacae with mean deltaTm-values of 3.9 degrees C confirming the hybridization results of three former studies. Phenotypic analysis of the E. cloacae and E. dissolvens strains, respectively, based on 115 biochemical reactions yielded the esculin test as the only one differentiating between them by being positive for E. dissolvens and negative for E. cloacae strains. The name E. cloacae subsp. dissolvens comb. nov. is proposed for the group of organisms formerly referred to as E. dissolvens, and the name E. cloacae subsp. cloacae comb. nov. for the group of organisms formerly referred to as E. cloacae. The species descriptions of Enterobacter kobei and Enterobacter asburiae were emended based on the data collected on 17 and 15 strains, respectively. The strains were assigned to the respective species by a combination of phylogenetic sequence analyzes and DNA-DNA hybridizations. Phenotypic analyzes of 115 reactions gave detailed species profiles with new differentiating phenotypic properties.
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53. |
Kaneko K,
Okamoto R,
Nakano R,
Kawakami S,
Inoue M,
( 2005 ) Gene mutations responsible for overexpression of AmpC beta-lactamase in some clinical isolates of Enterobacter cloacae. PMID : 15956430 : DOI : 10.1128/JCM.43.6.2955-2958.2005 PMC : PMC1151923 Abstract >>
AmpC regulatory genes in 21 ceftazidime-resistant clinical isolates of Enterobacter cloacae (MICs of > or = 16 microg/ml) were characterized. All isolates exhibited AmpC overproduction due to AmpD mutation. Additionally, we found two AmpR mutants among the isolates. This is the first report of chromosomal ampR mutation in clinical isolates of E. cloacae.
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54. |
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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55. |
Szabó D,
Melan MA,
Hujer AM,
Bonomo RA,
Hujer KM,
Bethel CR,
Kristóf K,
Paterson DL,
( 2005 ) Molecular analysis of the simultaneous production of two SHV-type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping. PMID : 16251316 : DOI : 10.1128/AAC.49.11.4716-4720.2005 PMC : PMC1280125 Abstract >>
Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of bla(SHV) gene products gave a "double peak" at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different bla(SHV) genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.
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56. |
Herter T,
Berezina OV,
Zinin NV,
Velikodvorskaya GA,
Greiner R,
Borriss R,
( 2006 ) Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity. PMID : 16193276 : DOI : 10.1007/s00253-005-0024-8 Abstract >>
Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg(-1) protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg(-1) of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.
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57. |
Davis IJ,
Richards H,
Mullany P,
( 2005 ) Isolation of silver- and antibiotic-resistant Enterobacter cloacae from teeth. PMID : 15836522 : DOI : 10.1111/j.1399-302X.2005.00218.x Abstract >>
Antibiotic-resistant bacteria pose a serious threat to human health; hence the mechanisms that lead to their selection need to be investigated. One possible mechanism is that the silver and mercury in amalgam dental restorations may select for bacteria that contain heavy metal and antibiotic-resistance determinants, leading to the spread of these resistances, particularly if they are contained on the same mobile genetic element. The incidence of silver-resistant bacteria on teeth is investigated in this work. Two silver-resistant Enterobacter cloacae isolates were isolated from infected teeth containing dental restorations. Both isolates were also resistant to ampicillin, erythromycin, and clindamycin. The silE gene, which is encoded on the silver resistance operon, has been sequenced from both isolates. Results suggest that the silver resistance operon is encoded on plasmid DNA.
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58. |
Galani I,
Souli M,
Chryssouli Z,
Orlandou K,
Giamarellou H,
( 2005 ) Characterization of a new integron containing bla(VIM-1) and aac(6')-IIc in an Enterobacter cloacae clinical isolate from Greece. PMID : 15761066 : DOI : 10.1093/jac/dki073 Abstract >>
A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production. MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of beta-lactamases. PCR assays with primers specific for the bla(VIM) gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-beta-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the bla(VIM-1) gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the bla(VIM-1) gene as a probe, amplified by PCR. The isolate was resistant to extended-spectrum beta-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10(8) cfu/mL was used. Isoelectric focusing detected a beta-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the bla(VIM-1) gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The bla(VIM-1) probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene. We describe a novel class 1 integron containing bla(VIM-1) and aac(6')-IIc genes in an E. cloacae clinical isolate.
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59. |
Gacar GG,
Midilli K,
Kolayli F,
Ergen K,
Gundes S,
Hosoglu S,
Karadenizli A,
Vahaboglu H,
( 2005 ) Genetic and enzymatic properties of metallo-beta-lactamase VIM-5 from a clinical isolate of Enterobacter cloacae. PMID : 16189133 : DOI : 10.1128/AAC.49.10.4400-4403.2005 PMC : PMC1251544 Abstract >>
A VIM-5-producing Enterobacter cloacae isolate (EDV/1) was identified in a collection of clinical strains stored before 2002. The gene, bla(VIM-5), was located on a 2,712-bp BamHI-HindIII fragment of a 23-kbp (approximately) nonconjugative plasmid (pEDV5) in a class 1 integron as a single gene cassette.
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60. |
Stumpf AN,
Roggenkamp A,
Hoffmann H,
( 2005 ) Specificity of enterobacterial repetitive intergenic consensus and repetitive extragenic palindromic polymerase chain reaction for the detection of clonality within the Enterobacter cloacae complex. PMID : 16182074 : DOI : 10.1016/j.diagmicrobio.2005.04.003 Abstract >>
An increasing number of clonal outbreaks caused by members of the E. cloacae complex is being reported. For the detection of clonality, pulsed-field gel electrophoresis (PFGE) is considered the golden standard, but PCR-based methods are cheaper, easier to perform, and provide faster results. One hundred ninety-five isolates of the E. cloacae complex isolated at the university hospital Grosshadern, Munich, Germany, were assigned to their respective genetic cluster by partial hsp60 sequencing. All study isolates belonging to genetic clusters III and VI were selected to evaluate the specificity of the enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) for the identification of clonal isolates belonging to the E. cloacae complex. For these 56 isolates, PFGE was performed, yielding 3 pairs of isolates with indistinguishable patterns. ERIC PCR resulted in 7 groups with identical patterns, together encompassing 49 study isolates. Comparing the ERIC PCR with the PFGE, a specificity of 14% considering the detection of "clonal" isolates was calculated. In this respect, REP PCR performed much better, yielding a specificity of 90%. An unweighted pair-group method with arithmetic averages tree based on ERIC PCR patterns allowed an accurate classification of the isolates to the respective genovars, suggesting that the ERIC PCR differentiates between genovars rather than between strains. In contrast, REP PCR differentiates better on the strain level. A proposed diagnostic system for the detection of subsumed outbreak strains of the E. cloacae complex is presented. It is based on an initial REP PCR, which should be confirmed by PFGE in cases of identical patterns, whereas ERIC PCR does not seem to be useful for the detection of outbreak strains when dealing with isolates of the E. cloacae complex.
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61. |
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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62. |
Khan H,
Barna T,
Bruce NC,
Munro AW,
Leys D,
Scrutton NS,
( 2005 ) Proton transfer in the oxidative half-reaction of pentaerythritol tetranitrate reductase. Structure of the reduced enzyme-progesterone complex and the roles of residues Tyr186, His181, His184. PMID : 16156787 : DOI : 10.1111/j.1742-4658.2005.04875.x Abstract >>
The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme-progesterone complex. Residues His181 and His184 are important in the binding of coenzyme, steroids, nitroaromatic ligands and the substrate 2-cyclohexen-1-one. The H181A and H184A enzymes retain activity in reductive and oxidative half-reactions, and thus do not play an essential role in catalysis. Ligand binding and catalysis is not substantially impaired in Y186F PETN reductase, which contrasts with data for the equivalent mutation (Y196F) in Old Yellow Enzyme. The structure of Y186F PETN reductase is identical to wild-type enzyme, with the obvious exception of the mutation. We show in PETN reductase that Tyr186 is not a key proton donor in the reduction of alpha/beta unsaturated carbonyl compounds. The structure of two electron-reduced PETN reductase bound to the inhibitor progesterone mimics the catalytic enzyme-steroid substrate complex and is similar to the structure of the oxidized enzyme-inhibitor complex. The reactive C1-C2 unsaturated bond of the steroid is inappropriately orientated with the flavin N5 atom for hydride transfer. With steroid substrates, the productive conformation is achieved by orientating the steroid through flipping by 180 degrees , consistent with known geometries for hydride transfer in flavoenzymes. Our data highlight mechanistic differences between Old Yellow Enzyme and PETN reductase and indicate that catalysis requires a metastable enzyme-steroid complex and not the most stable complex observed in crystallographic studies.
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63. |
Jiang X,
Ni Y,
Jiang Y,
Yuan F,
Han L,
Li M,
Liu H,
Yang L,
Lu Y,
( 2005 ) Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China. PMID : 15695687 : DOI : 10.1128/JCM.43.2.826-831.2005 PMC : PMC548041 Abstract >>
Over a 4-month period from November 2002 to February 2003, 27 ceftazidime-resistant or cefotaxime-resistant nonrepetitive Enterobacter cloacae isolates were collected from 27 patients hospitalized at HuaShan Hospital, Shanghai, People's Republic of China. The Etest did not detect extended-spectrum beta-lactamases (ESBLs) in those 27 isolates; however, screening by the NCCLS ESBL disk test and confirmatory tests detected ESBLs in 4 of 27 isolates and PCR detected ESBLs in 23 of 27 isolates. The majority of ESBL producers exhibited the same repetitive extragenic palindromic PCR pattern but harbored different ESBL genes. CTX-M-3 was the most prevalent ESBL in our study. Interestingly, 12 clonally related E. cloacae isolates possessed a novel bla(VEB)-type beta-lactamase, bla(VEB-3). Bla(VEB-3) was encoded by the chromosome and was located in an integron. Nine of the 12 isolates harbored both the bla(VEB-3) and the bla(CTX-M-3)-like ESBLs. This is the first report of a VEB-1-like ESBL in China and the first report of the simultaneous presence of VEB-1 and CTX-M-3-like ESBLs in an isolate.
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64. |
Bratu S,
Landman D,
Alam M,
Tolentino E,
Quale J,
( 2005 ) Detection of KPC carbapenem-hydrolyzing enzymes in Enterobacter spp. from Brooklyn, New York. PMID : 15673765 : DOI : 10.1128/AAC.49.2.776-778.2005 PMC : PMC547228 Abstract >>
Enterobacter spp. are rarely resistant to carbapenems. In this report, one Enterobacter sp. isolate possessed bla(KPC-3) and two possessed bla(KPC-2). For all three strains, the imipenem MICs were dependent on the inoculum and testing method; two were reported by the clinical laboratories to be carbapenem susceptible. Improved detection methods will be necessary to identify these enzymes.
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65. |
Stoebel DM,
( 2005 ) Lack of evidence for horizontal transfer of the lac operon into Escherichia coli. PMID : 15563718 : DOI : 10.1093/molbev/msi056 Abstract >>
The idea that Escherichia coli gained the lac operon via horizontal transfer, allowing it to invade a new niche and form a new species, has become a paradigmatic example of bacterial nonpathogenic adaptation and speciation catalyzed by horizontal transfer. Surprisingly, empirical evidence for this event is essentially nonexistent. To see whether horizontal transfer occurred, I compared a phylogeny of 14 Enterobacteriaceae based on two housekeeping genes to a phylogeny of a part of their lac operon. Although several species in this clade appear to have acquired some or all of the operon via horizontal transfer, there is no evidence of horizontal transfer into E. coli. It is not clear whether the horizontal transfer events for which there is evidence were adaptive because those species which have acquired the operon are not thought to live in high lactose environments. I propose that vertical transmission from the common ancestor of the Enterobacteriaceae, with subsequent loss of these genes in many species can explain much of the patchy distribution of lactose use in this clade. Finally, I argue that we need new, well-supported examples of horizontal transfer spurring niche expansion and speciation, particularly in nonpathogenic cases, before we can accept claims that horizontal transfer is a hallmark of bacterial adaptation.
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66. |
Venkatesan AM,
Gu Y,
Dos Santos O,
Abe T,
Agarwal A,
Yang Y,
Petersen PJ,
Weiss WJ,
Mansour TS,
Nukaga M,
Hujer AM,
Bonomo RA,
Knox JR,
( 2004 ) Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates. PMID : 15588091 : DOI : 10.1021/jm049680x Abstract >>
The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine beta-lactamase inhibitors is described. These compounds proved to be very potent inhibitors of the TEM-1 and AmpC beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria. Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A, respectively, and refined to R-factors equal 0.163 and 0.145. In both beta-lactamases, a seven-membered 1,4-thiazepine ring has formed. The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate. Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes. The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.
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67. |
Ojo KK,
Tung D,
Luis H,
Bernardo M,
Leitao J,
Roberts MC,
( 2004 ) Gram-positive merA gene in gram-negative oral and urine bacteria. PMID : 15358427 : DOI : 10.1016/j.femsle.2004.08.004 Abstract >>
Clinical mercury resistant (Hg(r)) Gram-negative bacteria carrying Gram-positive mercury reductase (merA)-like genes were characterized using DNA-DNA hybridization, PCR and sequencing. A PCR assay was developed which discriminated between the merA genes related to Staphylococcus and those related to the Bacillus/Streptococcus merA genes by the difference in size of the PCR product. DNA sequence analysis correlated with the PCR assay. The merA genes from Acinetobacter junii, Enterobacter cloacae and Escherichia coli were sequenced and shared 98-99% identical nucleotide (nt) and 99.6-100% amino acid identity with the Staphylococcus aureus MerA protein. A fourth merA gene, from Pantoeae agglomerans, was partially sequenced (60%) and had 99% identical nt and 100% amino acid identity with the Streptococcus oralis MerA protein. All the Hg(r) Gram-negative bacteria transferred their Gram-positive merA genes to a Gram-positive Enterococcus faecalis recipient with the resulting transconjugants expressing mercury resistance. These Gram-positive merA genes join Gram-positive tetracycline resistance and Gram-positive macrolide resistance genes in their association with mobile elements which are able to transfer and express in Gram-negative bacteria.
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68. |
Mishra J,
Khurana S,
Kumar N,
Ghosh AK,
Das D,
( 2004 ) Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08. PMID : 15474481 : DOI : 10.1016/j.bbrc.2004.09.108 Abstract >>
Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster.
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69. |
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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70. |
Khan H,
Barna T,
Harris RJ,
Bruce NC,
Barsukov I,
Munro AW,
Moody PC,
Scrutton NS,
( 2004 ) Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation. PMID : 15128738 : DOI : 10.1074/jbc.M403541200 Abstract >>
The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.
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71. |
Roggenkamp A,
Hoffmann H,
Hornef MW,
( 2004 ) Growth control of small-colony variants by genetic regulation of the hemin uptake system. PMID : 15039350 : DOI : 10.1128/iai.72.4.2254-2262.2004 PMC : PMC375170 Abstract >>
Small-colony variants (SCVs) are slow-growing variants of human bacterial pathogens. They are associated with chronic persistent infections, and their biochemical identification and antimicrobial treatment are impaired by altered metabolic properties. To contribute to the understanding of SCV-mediated infections, we analyzed a clinical SCV isolate derived from a chronic prosthetic hip infection. A sequence analysis of housekeeping genes identified an Enterobacter hormaechei-like organism. The SCV phenotype, with growth as microcolonies, was caused by a block within the heme biosynthesis pathway through deletion of the hemB locus, as shown by hybridization and complementation experiments. At a low frequency, large-colony variants (LCVs) arose that were dependent on exogenous hemin. To investigate this phenomenon, we cloned and sequenced the 5.8-kb hemin uptake system, denoted ehu. Gene expression analysis indicated regulation of this locus in wild-type bacteria by the global iron regulator Fur. Inactivation of Fur in LCVs caused the derepression of ehu expression and facilitated bacterial growth. Genetic alterations of the fur locus in LCVs were identified as insertions of IS1A elements and point mutations. In contrast, SCVs could utilize exogenous hemin only in the absence of iron. Thus, we provide the first molecular characterization of the growth properties of a clinical SCV isolate, which may help to improve the diagnostic and therapeutic management of patients with chronic persistent infections.
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72. |
Williams RE,
Rathbone DA,
Scrutton NS,
Bruce NC,
( 2004 ) Biotransformation of explosives by the old yellow enzyme family of flavoproteins. PMID : 15184158 : DOI : 10.1128/AEM.70.6.3566-3574.2004 PMC : PMC427764 Abstract >>
Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.
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73. |
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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74. |
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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75. |
Nukaga M,
Abe T,
Venkatesan AM,
Mansour TS,
Bonomo RA,
Knox JR,
( 2003 ) Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate. PMID : 14609325 : DOI : 10.1021/bi034986b Abstract >>
A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases. The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM). High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively. The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively. Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety. The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent. In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.
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76. |
Obeng AS,
Rickard H,
Ndi O,
Sexton M,
Barton M,
( 2012 ) Antibiotic resistance, phylogenetic grouping and virulence potential of Escherichia coli isolated from the faeces of intensively farmed and free range poultry. PMID : 21856098 : DOI : 10.1016/j.vetmic.2011.07.010 Abstract >>
Antibiotic use in poultry production is a risk factor for promoting the emergence of resistant Escherichia coli. To ascertain differences in different classes of chickens, the resistance profile, some virulence genes and phylogenetic grouping on 251 E. coli isolates from intensive meat (free range and indoor commercial) and free range egg layer chickens collected between December 2008 and June 2009 in South Australia were performed. Among the 251 strains, 102 (40.6%) and 67 (26.7%) were found to be resistant to tetracycline and ampicillin respectively. Resistance was also observed to trimethoprim-sulfamethoxazole (12.4%), streptomycin (10.8%), spectinomycin (9.6%), neomycin (6.0%) and florfenicol (2.0%) but no resistance was found to ceftiofur, ciprofloxacin or gentamicin. Amplification of DNA of the isolates by polymerase chain reaction revealed the presence of genes that code for resistant determinants: tetracycline (tet(A), tet(B) and tet(C)), ampicillin (bla(TEM) and bla(SHV)), trimethoprim (dhfrV and dhfrXIII), sulphonamide (sulI and sulII), neomycin (aph(3)-Ia(aphA1)), and spectinomycin-streptinomycin (aadA2). In addition, 32.3-39.4% of the isolates were found to belong to commensal groups (A and B1) and 11.2-17.1% belonged to the virulent groups (B2 and D). Among the 251 E. coli isolates, 25 (10.0%) carried two or more virulence genes typical of Extraintestinal pathogenic E. coli (ExPEC). Furthermore, 17 of the isolates with multi-resistance were identified to be groups B2 and D. Although no significant difference was observed between isolates from free range and indoor commercial meat chickens (P>0.05), significant differences was observed between the different classes of meat chickens (free range and indoor commercial) and egg layers (P<0.05). While this study assessed the presence of a limited number of virulence genes, our study re emphasises the zoonotic potential of poultry E. coli isolates.
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77. |
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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78. |
Brenner V,
Venetianer P,
Kiss A,
( 1990 ) Cloning and nucleotide sequence of the gene encoding the Ecal DNA methyltransferase. PMID : 2183182 : DOI : 10.1093/nar/18.2.355 PMC : PMC330275 Abstract >>
The gene coding for the GGTNACC specific Ecal DNA methyltransferase (M.Ecal) has been cloned in E. coli from Enterobacter cloacae and its nucleotide sequence has been determined. The ecalM gene codes for a protein of 452 amino acids (Mr: 51,111). It was determined that M.Ecal is an adenine methyltransferase. M.Ecal shows limited amino acid sequence similarity to other adenine methyltransferases. A clone that expresses Ecal methyltransferase at high level was constructed.
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79. |
Xu H,
Miao V,
Kwong W,
Xia R,
Davies J,
( 2011 ) Identification of a novel fosfomycin resistance gene (fosA2) in Enterobacter cloacae from the Salmon River, Canada. PMID : 21392044 : DOI : 10.1111/j.1472-765X.2011.03016.x Abstract >>
To investigate the occurrence of fosfomycin-resistant (fos(R)) bacteria in aquatic environments. A fos(R) strain of Enterobacter cloacae was isolated from a water sample collected at a site (50�X41'33�P44?N, 119�X19'49�P50?W) near the mouth of the Salmon River at Salmon Arm, in south-central British Columbia, Canada. The strain was identified by PCR screening for plasmid-borne, fosA-family amplicons, followed by selective plating. Sequencing of the resistance gene cloned using PCR primers to conserved flanking DNA revealed a new allele (95% amino acid identity to fosA), and I-Ceu I PFGE showed that it was chromosomally located. In Escherichia coli, the cloned DNA conferred a greater resistance to fosfomycin than its fosA counterpart. Gene fosA2 conferred fosfomycin resistance in an environmental isolate of Ent. cloacae. The repurposing of older antibiotics should be considered in the light of existing reservoirs of resistance genes in the environment.
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80. |
Poirel L,
Castanheira M,
Carrër A,
Rodriguez CP,
Jones RN,
Smayevsky J,
Nordmann P,
( 2011 ) OXA-163, an OXA-48-related class D �]-lactamase with extended activity toward expanded-spectrum cephalosporins. PMID : 21422200 : DOI : 10.1128/AAC.00022-11 PMC : PMC3101449 Abstract >>
Two bla(OXA-48)-like-positive isolates (Klebsiella pneumoniae and Enterobacter cloacae) were recovered in Argentina in 2008 as part of a large-scale survey focused on multidrug resistance in Enterobacteriaceae. In both cases, sequencing identified �]-lactamase OXA-163, differing from OXA-48 by a single amino substitution and a 4-amino-acid deletion. OXA-163 hydrolyzed penicillins, ceftazidime, and cefotaxime, whereas OXA-48 did not. However, OXA-163 had a much lower ability to hydrolyze carbapenems than OXA-48, therefore barely being considered a carbapenemase. In both isolates, the bla(OXA-163) gene was located on plasmids that differed in structure and size. However, a detailed genetic analysis revealed a similar genetic context in those isolates, with the bla(OXA-163) gene being bracketed by novel transposase genes, making this genetic environment different from that reported for the bla(OXA-48) gene. This study identified the first class D �]-lactamase compromising both extended-spectrum cephalosporin and carbapenem activities.
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81. |
Fernández A,
Pereira MJ,
Suárez JM,
Poza M,
Treviño M,
Villalón P,
Sáez-Nieto JA,
Regueiro BJ,
Villanueva R,
Bou G,
( 2011 ) Emergence in Spain of a multidrug-resistant Enterobacter cloacae clinical isolate producing SFO-1 extended-spectrum beta-lactamase. PMID : 21227991 : DOI : 10.1128/JCM.01872-10 PMC : PMC3067750 Abstract >>
Between February 2006 and October 2009, 38 patients in different wards at the A Coru?a University Hospital (northwest Spain) were either infected with or colonized by an epidemic, multidrug-resistant (MDR), and extended-spectrum-�]-lactamase (ESBL)-producing strain of Enterobacter cloacae (EbSF), which was susceptible only to carbapenems. Semiautomated repetitive extragenic palindromic sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) analysis revealed that all of the E. cloacae isolates belonged to the same clone. Cloning and sequencing enabled the detection of the SFO-1 ESBL in the epidemic strain and the description of its genetic environment. The presence of the ampR gene was detected upstream of bla(SFO-1), and two complete sequences of IS26 surrounding ampR and ampA were detected. These IS26 sequences are bordered by complete left and right inverted repeats (IRL and IRR, respectively), which suggested that they were functional. The whole segment flanked by two IS26 copies may be considered a putative large composite transposon. A gene coding for aminoglycoside acetyltransferase (gentamicin resistance gene [aac3]) was found downstream of the 3' IS26. Despite the implementation of strict infection control measures, strain EbSF spread through different areas of the hospital. A case-control study was performed to assess risk factors for EbSF acquisition. A multivariate analysis revealed that the prior administration of �]-lactam antibiotics, chronic renal failure, tracheostomy, and prior hospitalization were statistically associated with SFO-1-producing E. cloacae acquisition. This study describes for the first time an outbreak in which an SFO-1-producing E. cloacae strain was involved. Note that so far, this �]-lactamase has previously been isolated in only a single case of E. cloacae infection in Japan.
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82. |
Toogood HS,
Fryszkowska A,
Hulley M,
Sakuma M,
Mansell D,
Stephens GM,
Gardiner JM,
Scrutton NS,
( 2011 ) A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity. PMID : 21374779 : DOI : 10.1002/cbic.201000662 Abstract >>
We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of �\,�]-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to C?C reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.
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83. |
Shlaes DM,
Currie-McCumber C,
Hull A,
Behlau I,
Kron M,
( 1990 ) OHIO-1 beta-lactamase is part of the SHV-1 family. PMID : 2121093 : DOI : 10.1128/aac.34.8.1570 PMC : PMC171875 Abstract >>
The OHIO-1 beta-lactamase gene was subcloned in a 1.16-kilobase TaqI fragment in the 2.4-kilobase chimeric plasmid pSK04. After directional subcloning into M13, the DNA sequence of this fragment was determined. The results showed an open reading frame of 858 base pairs (bp) encoding a protein of 286 amino acids. The structural gene showed 95, 87, and 60% DNA sequence identity with SHV-1, LEN-1, and TEM-1, respectively, and 93, 85, and 62% predicted amino acid sequence identity, respectively. The 87 bp upstream of the OHIO-1 structural gene had 96% identity with the upstream flanking sequence of SHV-1, including the -35 and -10 consensus sequences and the putative ribosomal binding site. A 223-bp DNA probe derived from a PstI-HaeII fragment in the C-terminal sequence of OHIO-1 had predicted 96, 88, and 61% sequence identity with SHV-1, LEN-1, and TEM-1, respectively. This probe hybridized to SHV-1 and poorly to LEN-1, but not to TEM-1 or a variety of other plasmid-mediated beta-lactamase genes, under stringent conditions. Screening of plasmid DNA derived from 40 ampicillin-resistant clinical isolates by Southern hybridization with the 223-bp probe uncovered no strains encoding OHIO-1. Isoelectric focusing of the same collection did identify two strains producing enzymes resembling SHV-1, however. We have also performed a kinetic comparison of OHIO-1, SHV-1, and TEM-1. OHIO-1 and SHV-1 were indistinguishable from each other but could be distinguished from TEM-1. These data clearly place OHIO-1 within the SHV-1 family of beta-lactamases.
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84. |
Chu YW,
Tung VW,
Cheung TK,
Chu MY,
Cheng N,
Lai C,
Tsang DN,
Lo JY,
( 2011 ) Carbapenemases in enterobacteria, Hong Kong, China, 2009. PMID : 21192875 : DOI : 10.3201/eid1701.101443 PMC : PMC3204654 Abstract >>
N/A
|
85. |
Mehnaz S,
Baig DN,
Lazarovits G,
( 2010 ) Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in pakistan. PMID : 21193815 : Abstract >>
Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41degrees C and at pH 11.
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86. |
Burgos Y,
Beutin L,
( 2010 ) Common origin of plasmid encoded alpha-hemolysin genes in Escherichia coli. PMID : 20637130 : DOI : 10.1186/1471-2180-10-193 PMC : PMC2918590 Abstract >>
Alpha (alpha)-hemolysin is a pore forming cytolysin and serves as a virulence factor in intestinal and extraintestinal pathogenic strains of E. coli. It was suggested that the genes encoding alpha-hemolysin (hlyCABD) which can be found on the chromosome and plasmid, were acquired through horizontal gene transfer. Plasmid-encoded alpha-hly is associated with certain enterotoxigenic (ETEC), shigatoxigenic (STEC) and enteropathogenic E. coli (EPEC) strains. In uropathogenic E. coli (UPEC), the alpha-hly genes are located on chromosomal pathogenicity islands. Previous work suggested that plasmid and chromosomally encoded alpha-hly may have evolved independently. This was explored in our study. We have investigated 11 alpha-hly plasmids from animal and human ETEC, STEC and EPEC strains. The size of alpha-hly plasmids ranges from 48-157 kb and eight plasmids are conjugative. The regulatory gene (hlyR) located upstream of the hlyCABD gene operon and an IS911 element located downstream of hlyD are conserved. Chromosomally-encoded alpha-hly operons lack the hlyR and IS911 elements. The DNA sequence of hlyC and hlyA divided the plasmid- and chromosomally-encoded alpha-hemolysins into two clusters. The plasmid-encoded alpha-hly genes could be further divided into three groups based on the insertion of IS1 and IS2 in the regulatory region upstream of the alpha-hly operon. Transcription of the hlyA gene was higher than the housekeeping icdA gene in all strains (rq 4.8 to 143.2). Nucleotide sequence analysis of a chromosomally located alpha-hly determinant in Enterobacter cloacae strain indicates that it originates from an E. coli alpha-hly plasmid. Our data indicate that plasmids encoding alpha-hly in E. coli descended from a common ancestor independent of the plasmid size and the origin of the strains. Conjugative plasmids could contribute to the spread of the alpha-hly determinant to Enterobacter cloacae. The presence of IS-elements flanking the plasmid-encoded alpha-hly indicate that they might be mobile genetic elements.
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87. |
Hulley ME,
Toogood HS,
Fryszkowska A,
Mansell D,
Stephens GM,
Gardiner JM,
Scrutton NS,
( 2010 ) Focused directed evolution of pentaerythritol tetranitrate reductase by using automated anaerobic kinetic screening of site-saturated libraries. PMID : 21064170 : DOI : 10.1002/cbic.201000527 Abstract >>
This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and �\-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against �\,�]-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.
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88. |
Tijet N,
Andres P,
Chung C,
Lucero C,
N/A N/A,
Low DE,
Galas M,
Corso A,
Petroni A,
Melano RG,
( 2011 ) rmtD2, a new allele of a 16S rRNA methylase gene, has been present in Enterobacteriaceae isolates from Argentina for more than a decade. PMID : 21078935 : DOI : 10.1128/AAC.00962-10 PMC : PMC3028771 Abstract >>
The first allele of a 16S rRNA methyltransferase gene, rmtD2, conferring very high resistance to all clinically available aminoglycosides, was detected in 7/1,064 enterobacteria collected in 2007. rmtD2 was located on a conjugative plasmid in a Tn2670-like element inside a structure similar to that of rmtD1 but probably having an independent assembly. rmtD2 has been found since 1996 to 1998 mainly in Enterobacter and Citrobacter isolates, suggesting a possible reservoir in these genera. This presumption deserves monitoring by continuous surveillance.
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89. |
Roberts DP,
Lohrke SM,
McKenna L,
Lakshman DK,
Kong H,
Lydon J,
( 2011 ) Mutation of a degS homologue in Enterobacter cloacae decreases colonization and biological control of damping-off on cucumber. PMID : 20942652 : DOI : 10.1094/PHYTO-03-10-0076 Abstract >>
We have been using mutagenesis to determine how biocontrol bacteria such as Enterobacter cloacae 501R3 deal with complex nutritional environments found in association with plants. E. cloacae C10, a mutant of 501R3 with a transposon insertion in degS, was diminished in growth on synthetic cucumber root exudate (SRE), colonization of cucumber seed and roots, and control of damping-off of cucumber caused by Pythium ultimum. DegS, a periplasmic serine protease in the closely related bacterium Escherichia coli K12, is required for the RpoE-mediated stress response. C10 containing wild-type degS from 501R3 or from E. coli K12 on pBeloBAC11 was significantly increased in growth on SRE, colonization of cucumber roots, and control of P. ultimum relative to C10 containing pBeloBAC11 alone. C10 and 501R3 were similar in sensitivity to acidic conditions, plant-derived phenolic compounds, oxidative stress caused by hydrogen peroxide, dessication, and high osmoticum; stress conditions potentially associated with plants. This study demonstrates a role for degS in the spermosphere and rhizosphere during colonization and disease control by Enterobacter cloacae. This study implicates, for the first time, the involvement of DegS and, by extension, the RpoE-mediated stress response, in reducing stress on E. cloacae resulting from the complex nutritional environments in the spermosphere and rhizosphere.
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90. |
Terán FJ,
Suárez JE,
Mendoza MC,
( 1991 ) Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile. PMID : 2069376 : DOI : 10.1128/aac.35.4.714 PMC : PMC245084 Abstract >>
A gene coding for an aminoglycoside 6'-N-acetyltransferase that was able to modify amikacin was cloned from a plasmid isolated from a clinical strain of Enterobacter cloacae. Sequencing of a 955-bp segment which mediates the modifying activity revealed a single open reading frame of 432 nucleotides that predicted a polypeptide of 144 amino acid residues with a molecular weight of 16,021. Putative ribosomal binding sites and -10 and -35 sequences were located at the 5' end of the gene. The size of the polypeptide was confirmed through minicell analysis of the expression products of plasmids containing the sequence. The use of the gene as a molecular probe revealed its specificity toward strains harboring genes coding for related enzymes. This probe is therefore useful for epidemiological studies.
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91. |
Koga J,
Adachi T,
Hidaka H,
( 1991 ) Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae. PMID : 2034209 : DOI : 10.1007/bf00273581 Abstract >>
Although indole-3-acetic acid (IAA) is a well-known plant hormone, the main IAA biosynthetic pathway from L-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) has yet to be elucidated. Previous studies have suggested that IAA is produced by Enterobacter cloacae isolated from the rhizosphere of cucumbers and its biosynthetic pathway may possibly be the same as that in plants. To elucidate this pathway, the IAA biosynthetic gene was isolated from a genomic library of E. cloacae by assaying for the ability to convert Trp to IAA. DNA sequence analysis showed that this gene codes for only one enzyme and its predicted protein sequence has extensive homology with pyruvate decarboxylase in yeast and Zymomonas mobilis. Cell-free extracts prepared from Escherichia coli harboring this gene could convert IPyA to indole-3-acetaldehyde (IAAld). These results clearly show that this pathway is mediated only by indolepyruvate decarboxylase, which catalyzes the conversion of IPyA to IAAld.
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92. |
Hornsey M,
Ellington MJ,
Doumith M,
Scott G,
Livermore DM,
Woodford N,
( 2010 ) Emergence of AcrAB-mediated tigecycline resistance in a clinical isolate of Enterobacter cloacae during ciprofloxacin treatment. PMID : 20189357 : DOI : 10.1016/j.ijantimicag.2010.01.011 Abstract >>
Tigecycline resistance remains rare amongst Enterobacteriaceae in the UK, as elsewhere, but has been associated with upregulation of the AcrAB efflux system. Using isolates of an Enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator RamA in tigecycline resistance. Laboratory mutants were selected from a susceptible clinical isolate in vitro by exposure to increasing concentrations of tigecycline. Expression of the acrAB operon and the ramA gene was monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). Overexpression of ramA was achieved using the pBAD expression vector, whilst insertional inactivation of acrB with a gentamicin resistance cassette was achieved with the bacteriophage lambda Red recombination system. Increased tigecycline minimum inhibitory concentrations in the clinical isolate and a laboratory mutant were associated with increases in acrAB and ramA transcripts. Induction of increased ramA expression resulted in increased acrAB expression, whilst insertional inactivation of acrB restored full susceptibility to tigecycline. Treatment with ciprofloxacin, a substrate of AcrAB in E. cloacae, possibly selected for cross-resistance to tigecycline as a result of RamA-mediated AcrAB upregulation.
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93. |
Bryant C,
Hubbard L,
McElroy WD,
( 1991 ) Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae. PMID : 1999406 : Abstract >>
The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.
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94. |
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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95. |
Bryant C,
DeLuca M,
( 1991 ) Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. PMID : 1999405 : Abstract >>
The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.
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96. |
Garza-Ramos U,
Davila G,
Gonzalez V,
Alpuche-Aranda C,
López-Collada VR,
Alcantar-Curiel D,
Newton O,
Silva-Sanchez J,
( 2009 ) The blaSHV-5 gene is encoded in a compound transposon duplicated in tandem in Enterobacter cloacae. PMID : 19519856 : DOI : 10.1111/j.1469-0691.2009.02790.x Abstract >>
The presence of bla(SHV-5) is described in a compound transposon, duplicated in tandem and flanked by IS26 copies on a 70-kb conjugative plasmid (pHNM1), in an Enterobacter cloacae strain associated with a nosocomial outbreak that occurred in Mexico.
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97. |
Ramírez MS,
Piñeiro S,
N/A N/A,
Centrón D,
( 2010 ) Novel insights about class 2 integrons from experimental and genomic epidemiology. PMID : 19917745 : DOI : 10.1128/AAC.01392-08 PMC : PMC2812161 Abstract >>
In order to contribute to the knowledge of the architecture and epidemiology of class 2 integrons, we performed a class 2 integron molecular survey in which we analyzed 726 isolates in two bacterial populations from environmental and nonepidemiologically related clinical samples, respectively, collected from 1982 to 2007. We recovered the intI2 gene from 130 of 726 isolates, most of which were clinical isolates, and only 1 (a psychrophilic Pseudomonas sp.) was from a water sample. Unlike the widespread distribution of class 1 integrons within Gram-negative bacilli, only Acinetobacter baumannii and Enterobacter cloacae harbored class 2 integrons at a high frequency in our collection. Class 2 integrons with six novel cassette arrays were documented. Characterization of the transposition module of Tn7, the genetic platform in which class 2 integrons have always been reported, showed tns modules with a mosaic genetic structure. A bioinformatic analysis performed with the tns genes present in sequence databases, the finding of intI2 not associated with tns genes, and the genetic examination of novel tns-like genes found in three isolates indicated the possibility of the independent evolution of the two components related to horizontal gene transfer, the class 2 integrons and the Tn7 transposons.
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98. |
Bourouis A,
Dubois V,
Coulange L,
André C,
Bejhadj C,
Ben Moussa M,
Quentin C,
Belhadj O,
( 2011 ) First report of CTX-M-9 in a clinical isolate of Enterobacter cloacae in a Tunisian hospital. PMID : 19481370 : DOI : 10.1016/j.patbio.2009.03.008 Abstract >>
�]-lactamases are one of several mechanisms of bacterial resistance to �]-lactam antibiotics. The aim of this study was to analyze the resistance to extended-spectrum cephalosporins of a multidrug-resistant isolate of Enterobacter cloacae, BF5011. This strain was isolated from a stool culture, during the nosocomials infections occurring in the intensive care unit of the Military Hospital of Tunis in 2005. Analysis of E. cloacae BF5011 by double-disk synergy test yielded a positive result suggesting the production of extended-spectrum-�]-lactamases. Cell sonicate of this isolate is very active against cefotaxime and showed a specific activity (AS) of 7,54 U/mg for the same antibiotic. This activity was inhibited by the sulbactam and the clavulanic acid. By polymerase chain reaction and sequencing, the isolate was found to produce extended-spectrum-�]-lactamase, CTX-M-9. The bla(CTX-M-9) gene was transferred from E. cloacae by conjugation. Isoelectric focusing method revealed one band with a pI of about 8. This is the first report of CTX-M-9 in Tunisia.
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99. |
Pudney CR,
Hay S,
Levy C,
Pang J,
Sutcliffe MJ,
Leys D,
Scrutton NS,
( 2009 ) Evidence to support the hypothesis that promoting vibrations enhance the rate of an enzyme catalyzed H-tunneling reaction. PMID : 19891489 : DOI : 10.1021/ja908469m Abstract >>
In recent years there has been a shift away from transition state theory models for H-transfer reactions. Models that incorporate tunneling as the mechanism of H-transfer are now recognized as a better description of such reactions. Central to many models of H-tunneling is the notion that specific vibrational modes of the protein and/or substrate can increase the probability of a H-tunneling reaction, modes that are termed promoting vibrations. Thus far there has been limited evidence that promoting vibrations can increase the rate of H-transfer. In the present communication we examine the single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR). We find that there is a significant promoting vibration with NADPH but not with NADH and that the observed rate of hydride transfer is significantly (approximately 15x) faster with NADPH. We rule out differences in rate due to variation in driving force and the donor-acceptor distance, suggesting it is the promoting vibration with NADPH that is the origin of the increased observed rate. This study therefore provides direct evidence that promoting vibrations can lead to an increase in rate.
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100. |
Eaton RW,
Karns JS,
( 1991 ) Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains. PMID : 1991731 : DOI : 10.1128/jb.173.3.1363-1366.1991 PMC : PMC207267 Abstract >>
DNA encoding the catabolism of the s-triazines ammelide and cyanuric acid was cloned from Pseudomonas sp. strain NRRLB-12228 and Klebsiella pneumoniae 99 with, as a probe, a 4.6-kb PstI fragment from a third strain, Pseudomonas sp. strain NRRLB-12227, which also encodes these activities. In strains NRRLB-12228 and 99 the ammelide aminohydrolase (trzC) and cyanuric acid amidohydrolase (trzD) genes are located on identical 4.6-kb PstI fragments which are part of a 12.4-kb DNA segment present in both strains. Strain NRRLB-12227 also carries this 12.4-kb DNA segment, except that a DNA segment of 0.8 to 1.85 kb encoding a third enzyme, ammeline aminohydrolase (trzB), has been inserted next to the ammelide aminohydrolase gene with the accompanying deletion of 1.1 to 2.15 kb of DNA. In addition, the s-triazine catabolic genes are flanked in strain NRRLB-12227 by apparently identical 2.2-kb segments that are not present in the other two strains and that seem to cause rearrangements in adjacent DNA.
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101. |
Stoorvogel J,
van Bussel MJ,
Tommassen J,
van de Klundert JA,
( 1991 ) Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX). PMID : 1987115 : DOI : 10.1128/jb.173.1.156-160.1991 PMC : PMC207169 Abstract >>
A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The ompX gene was located by insertions of the gamma delta sequence into the recombinant plasmid. The polarity of the gene was determined by in vitro transcription and translation of the gamma delta-containing plasmids. The nucleotide sequence of the ompX gene was elucidated by using both inverted terminal repeats of the gamma delta sequence as starting points for M13 dideoxy sequencing. The gene was found to encode a precursor of the OmpX protein consisting of 172 amino acid residues with a molecular mass of 18.6 kDa. The protein contains an N-terminal signal sequence of 23 amino acid residues. The exact cleavage point was established by sequencing the N-terminal part of the mature protein. The OmpX protein has several characteristics in common with outer membrane proteins of gram-negative bacteria. The protein is rather hydrophilic and is devoid of long hydrophobic stretches. On the basis of these results, we present a model for the OmpX protein folding in an outer membrane.
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102. |
Minarini LA,
Poirel L,
Trevisani NA,
Darini AL,
Nordmann P,
( 2009 ) Predominance of CTX-M-type extended-spectrum beta-lactamase genes among enterobacterial isolates from outpatients in Brazil. PMID : 19748435 : DOI : 10.1016/j.diagmicrobio.2009.05.021 Abstract >>
Two hundred fifty-seven nalidixic acid-resistant enterobacterial isolates were collected in a Brazilian community from January 2000 to May 2005 to determine the prevalence of plasmid-encoded extended-spectrum beta-lactamases. The bla(CTX-M) genetic environment was determined by polymerase chain reaction and sequencing. Eleven isolates (4.2%) harbored a bla(CTX-M-2) gene, 3 isolates bla(CTX-M-9), 2 isolates bla(CTX-M-8), and 6 isolates bla(SHV-5). Two novel bla(CTX-M-2) variants, namely, bla(CTX-M-74) and bla(CTX-M-75), were identified.
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103. |
Xu H,
Su Z,
Wang S,
Dai X,
Chen J,
Kong F,
Li Y,
Peng S,
Shao Q,
Lu L,
Ezaki T,
( 2009 ) Four novel resistance integron gene-cassette occurrences in bacterial isolates from zhenjiang, china. PMID : 19365688 : DOI : 10.1007/s00284-009-9405-z Abstract >>
Integrons, which are widely distributed among bacteria and are strongly associated with resistance, are specialized genetic elements that are capable of capturing, integrating, and mobilizing gene cassette. In this work, we investigated classes 1, 2, and 3 integrons associated integrases genes in 365 bacteria isolates, amplified and analyzed the structure of class 1 integron, detected 8 resistant gene cassettes [dfr17, aadA5, aadA1, aadA2, dhfrI, aadB, aac(6')-II, and pse-I], and found four novel gene-cassette arrays. We also found that commensal bacteria in the common microenvironment had the same integron gene cassette, which provided direct evidence that integron was an important horizontal transmission element.
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104. |
Kuhnert P,
Korczak BM,
Stephan R,
Joosten H,
Iversen C,
( 2009 ) Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA). PMID : 19321218 : DOI : 10.1016/j.ijfoodmicro.2009.02.022 Abstract >>
Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.
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105. |
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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106. |
Poirel L,
Carrër A,
Pitout JD,
Nordmann P,
( 2009 ) Integron mobilization unit as a source of mobility of antibiotic resistance genes. PMID : 19332679 : DOI : 10.1128/AAC.00033-09 PMC : PMC2687222 Abstract >>
Antibiotic resistance genes are spread mostly through plasmids, integrons (as a form of gene cassettes), and transposons in gram-negative bacteria. We describe here a novel genetic structure, named the integron mobilization unit (IMU), that has characteristics similar to those of miniature inverted transposable elements (MITEs). Two IMUs (288 bp each) were identified from a carbapenem-resistant Enterobacter cloacae isolate that formed a composite structure encompassing a defective class 1 integron containing the carbapenem resistance gene bla(GES-5). This beta-lactamase gene was located on a 7-kb IncQ-type plasmid named pCHE-A, which was sequenced completely. The plasmid pCHE-A was not self conjugative but was mobilizable, and it was successfully transferred from E. cloacae to Pseudomonas aeruginosa. The in silico analysis of the extremities of the IMU elements identified similarities with those of insertion sequence ISSod9 from Shewanella oneidensis MR-1. The mobilization of the IMU composite structure was accomplished by using the transposase activity of ISSod9 that was provided in trans. This is the first identification of MITE-type structures as a source of gene mobilization, implicating here a clinically relevant antibiotic resistance gene.
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107. |
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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108. |
Chen YT,
Liao TL,
Liu YM,
Lauderdale TL,
Yan JJ,
Tsai SF,
( 2009 ) Mobilization of qnrB2 and ISCR1 in plasmids. PMID : 19075060 : DOI : 10.1128/AAC.00970-08 PMC : PMC2650544 Abstract >>
The DNA sequences of two IncHI2 plasmids, pEC-IMP and pEC-IMPQ, from metallo-beta-lactamase-producing Enterobacter cloacae clinical isolates were determined. The two conjugative plasmids are almost identical, but pEC-IMPQ carries an additional segment containing an orf513 (ISCR1), a truncated 3' conserved sequence, and a qnrB2. Comparative analyses provide support for the proposed ISCR1-mediated gene mobilization.
|
109. |
Petrella S,
Ziental-Gelus N,
Mayer C,
Renard M,
Jarlier V,
Sougakoff W,
( 2008 ) Genetic and structural insights into the dissemination potential of the extremely broad-spectrum class A beta-lactamase KPC-2 identified in an Escherichia coli strain and an Enterobacter cloacae strain isolated from the same patient in France. PMID : 18625772 : DOI : 10.1128/AAC.00163-08 PMC : PMC2565876 Abstract >>
Two clinical strains of Escherichia coli (2138) and Enterobacter cloacae (7506) isolated from the same patient in France and showing resistance to extended-spectrum cephalosporins and low susceptibility to imipenem were investigated. Both strains harbored the plasmid-contained bla(TEM-1) and bla(KPC-2) genes. bla(KLUC-2), encoding a mutant of the chromosomal beta-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of bla(KLUC) from the chromosome. Determination of the KPC-2 structure at 1.6 A revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 beta-lactamase by the inhibitory protein BLIP. Determination of the 1.23-A structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6 alpha-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand beta 3.
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110. |
Pasteran FG,
Otaegui L,
Guerriero L,
Radice G,
Maggiora R,
Rapoport M,
Faccone D,
Di Martino A,
Galas M,
( 2008 ) Klebsiella pneumoniae Carbapenemase-2, Buenos Aires, Argentina. PMID : 18598660 : DOI : 10.3201/eid1407.070826 PMC : PMC2600346 Abstract >>
N/A
|
111. |
Huang Z,
Mi Z,
Wang C,
( 2008 ) A novel beta-lactamase gene, LAP-2, produced by an Enterobacter cloacae clinical isolate in China. PMID : 18550213 : DOI : 10.1016/j.jhin.2008.04.012 Abstract >>
N/A
|
112. |
Ma J,
Kobayashi DY,
Yee N,
( 2009 ) Role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12. PMID : 18811645 : DOI : 10.1111/j.1462-2920.2008.01749.x Abstract >>
In this study, we investigated the role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12. A mini-Tn5 transposon mutant of E. cloacae SLD1a-1, designated as 4E6, was isolated that had lost the ability to reduce Se(VI) to Se(0). Genetic analysis of mutant strain 4E6 showed that the transposon was inserted within a menD gene among a menFDHBCE gene cluster that encodes for proteins required for menaquinone biosynthesis. A group of E. coli K12 strains with single mutations in the menF, menD, menC and menE genes were tested for loss of selenate reduction activity. The results showed that E. coli K12 carrying a deletion of either the menD, menC or menE gene was unable to reduce selenate. Complementation using wild-type sequences of the E. cloacae SLD1a-1 menFDHBCE sequence successfully restored the selenate reduction activity in mutant strain 4E6, and E. coli K12 menD and menE mutants. Selenate reduction activity in 4E6 was also restored by chemical complementation using the menaquinone precursor compound 1,4-dihydroxy-2-nathphoic acid. The results of this work suggest that menaquinones are an important source of electrons for the selenate reductase, and are required for selenate reduction activity in E. cloacae SLD1a-1 and E. coli K12.
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113. |
Labbate M,
Roy Chowdhury P,
Stokes HW,
( 2008 ) A class 1 integron present in a human commensal has a hybrid transposition module compared to Tn402: evidence of interaction with mobile DNA from natural environments. PMID : 18502858 : DOI : 10.1128/JB.00199-08 PMC : PMC2493286 Abstract >>
In a survey of class 1 integrons from human stools, an unusual class 1 integron from a strain of Enterobacter cloacae was isolated and characterized in detail. Sequence analysis of a fosmid containing the class 1 integron revealed a complex set of transposons which included two Tn402-like transposons. One of these transposons, Tn6007, included a class 1 integron with two non-antibiotic-resistance-type gene cassettes and a complete transposition module. This tni module is a hybrid with a boundary within the res site compared to Tn402, implying that a site-specific recombination event generated either Tn6007 or Tn402. The second Tn402-like transposon, Tn6008, possesses neither a mer operon nor an integron, and most of its tni module has been deleted. Tn6007, Tn6008, and the 2,478 bases between them, collectively designated Tn6006, have transposed into a Tn5036/Tn3926-like transposon as a single unit. Tn6006, Tn6007, and Tn6008 could all transpose as discrete entities. Database analysis also revealed that a version of Tn6008 was present in the genome of Xanthomonas campestris pv. vesicatoria. Overall, the E. cloacae isolate further demonstrated that functional class 1 integrons/transposons are probably common in bacterial communities and have the potential to add substantially to the problem of multidrug-resistant nosocomial infections.
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114. |
Gillings M,
Boucher Y,
Labbate M,
Holmes A,
Krishnan S,
Holley M,
Stokes HW,
( 2008 ) The evolution of class 1 integrons and the rise of antibiotic resistance. PMID : 18487337 : DOI : 10.1128/JB.00152-08 PMC : PMC2447024 Abstract >>
Class 1 integrons are central players in the worldwide problem of antibiotic resistance, because they can capture and express diverse resistance genes. In addition, they are often embedded in promiscuous plasmids and transposons, facilitating their lateral transfer into a wide range of pathogens. Understanding the origin of these elements is important for the practical control of antibiotic resistance and for exploring how lateral gene transfer can seriously impact on, and be impacted by, human activities. We now show that class 1 integrons can be found on the chromosomes of nonpathogenic soil and freshwater Betaproteobacteria. Here they exhibit structural and sequence diversity, an absence of antibiotic resistance genes, and a phylogenetic signature of lateral transfer. Some examples are almost identical to the core of the class 1 integrons now found in pathogens, leading us to conclude that environmental Betaproteobacteria were the original source of these genetic elements. Because these elements appear to be readily mobilized, their lateral transfer into human commensals and pathogens was inevitable, especially given that Betaproteobacteria carrying class 1 integrons are common in natural environments that intersect with the human food chain. The strong selection pressure imposed by the human use of antimicrobial compounds then ensured their fixation and global spread into new species.
|
115. |
Ryu RJ,
Patten CL,
( 2008 ) Aromatic amino acid-dependent expression of indole-3-pyruvate decarboxylase is regulated by TyrR in Enterobacter cloacae UW5. PMID : 18757531 : DOI : 10.1128/JB.00804-08 PMC : PMC2580706 Abstract >>
The plant growth-promoting rhizobacterium Enterobacter cloacae UW5 synthesizes the plant growth hormone indole-3-acetic acid (IAA) via the indole-3-pyruvate pathway utilizing the enzyme indole-3-pyruvate decarboxylase that is encoded by ipdC. In this bacterium, ipdC expression and IAA production occur in stationary phase and are induced by an exogenous source of tryptophan, conditions that are present in the rhizosphere. The aim of this study was to identify the regulatory protein that controls the expression of ipdC. We identified a sequence in the promoter region of ipdC that is highly similar to the recognition sequence for the Escherichia coli regulatory protein TyrR that regulates genes involved in aromatic amino acid transport and metabolism. Using a tyrR insertional mutant, we demonstrate that TyrR is required for IAA production and for induction of ipdC transcription. TyrR directly induces ipdC expression, as was determined by real-time quantitative reverse transcription-PCR, by ipdC promoter-driven reporter gene activity, and by electrophoretic mobility shift assays. Expression increases in response to tryptophan, phenylalanine, and tyrosine. This suggests that, in addition to its function in plant growth promotion, indolepyruvate decarboxylase may be important for aromatic amino acid uptake and/or metabolism.
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116. |
Paauw A,
Caspers MP,
Schuren FH,
Leverstein-van Hall MA,
Delétoile A,
Montijn RC,
Verhoef J,
Fluit AC,
( 2008 ) Genomic diversity within the Enterobacter cloacae complex. PMID : 18716657 : DOI : 10.1371/journal.pone.0003018 PMC : PMC2515634 Abstract >>
Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome. This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogeneous. Genetic markers were identified that could discriminate between the two clades and cluster 1. Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.
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117. |
Dortet L,
Radu I,
Gautier V,
Blot F,
Chachaty E,
Arlet G,
( 2008 ) Intercontinental travels of patients and dissemination of plasmid-mediated carbapenemase KPC-3 associated with OXA-9 and TEM-1. PMID : 18032422 : DOI : 10.1093/jac/dkm455 Abstract >>
N/A
|
118. |
Ma J,
Kobayashi DY,
Yee N,
( 2007 ) Chemical kinetic and molecular genetic study of selenium oxyanion reduction by Enterobacter cloacae SLD1a-1. PMID : 18075090 : DOI : 10.1021/es0712672 Abstract >>
Microbial processes play an important role in the redox transformations of toxic selenium oxyanions. In this study, we employed chemical kinetic and molecular genetic techniques to investigate the mechanisms of Se(IV) and Se-(VI) reduction by the facultative anaerobe Enterobacter cloacae SLD1a-1. The rates of microbial selenium oxyanion reduction were measured as a function of initial selenium oxyanion concentration (0-1.0 mM) and temperature (10-40 degrees C), and mutagenesis studies were performed to identify the genes involved in the selenium oxyanion reduction pathway. The results indicate that Se(IV) reduction is significantly more rapid than the reduction of Se(VI). The kinetics of the reduction reactions were successfully quantified using the Michaelis-Menten kinetic equation. Both the rates of Se(VI) and Se(IV) reduction displayed strong temperature-dependence with E(a) values of 121 and 71.2 kJ/ mol, respectively. X-ray absorption near-edge spectra collected for the precipitates formed by Se(VI) and Se(IV) reduction confirmed the formation of Se(0). A miniTn5 transposon mutant of E. cloacae SLD1a-1 was isolated that had lost the ability to reduce Se(VI) but was not affected in Se(IV) reduction activity. Nucleotide sequence analysis revealed the transposon was inserted within a tatC gene, which encodes for a central protein in the twin arginine translocation system. Complementation by the wild-type tatC sequence restored the ability of mutant strains to reduce Se(VI). The results suggest that Se(VI) reduction activity is dependent on enzyme export across the cytoplasmic membrane and that reduction of Se(VI) and Se(IV) are catalyzed by different enzymatic systems.
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119. |
Ikonomidis A,
Spanakis N,
Poulou A,
Pournaras S,
Markou F,
Tsakris A,
( 2007 ) Emergence of carbapenem-resistant Enterobacter cloacae carrying VIM-4 metallo-beta-lactamase and SHV-2a extended-spectrum beta-lactamase in a conjugative plasmid. PMID : 18184047 : DOI : 10.1089/mdr.2007.768 Abstract >>
Enterobacter cloacae strains are still infrequently resistant to carbapenems. A carbapenem-resistant clinical isolate of E. cloacae producing a plasmid-mediated metallo-beta-lactamase (MBL), VIM-4, was recovered from a Greek hospitalized patient. The bla(VIM-4) gene was located in the variable array of a class 1 integron structure repeatedly detected among bla(VIM-1)-bearing Gram-negative pathogens in Greece. The isolate possessed also on the same conjugative plasmid an extended-spectrum beta-lactamase (ESBL), SHV-2a, which contributed to the beta-lactam-resistant phenotype. This is the first report showing co-transfer of an ESBL with a VIM-type MBL. It suggests also that different VIM-type gene cassettes have been incorporated in a common integron structure, which seems indigenous of Gram-negative pathogens in our region.
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120. |
Pérez A,
Canle D,
Latasa C,
Poza M,
Beceiro A,
Tomás Mdel M,
Fernández A,
Mallo S,
Pérez S,
Molina F,
Villanueva R,
Lasa I,
Bou G,
( 2007 ) Cloning, nucleotide sequencing, and analysis of the AcrAB-TolC efflux pump of Enterobacter cloacae and determination of its involvement in antibiotic resistance in a clinical isolate. PMID : 17638702 : DOI : 10.1128/AAC.00072-07 PMC : PMC2043211 Abstract >>
Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcB-TolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (EcDeltaacrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.
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121. |
Liu G,
Ling BD,
Xie YE,
Lin L,
Zeng Y,
Zhang X,
Lei J,
( 2007 ) Characterization of CTX-M-22 and TEM-141 Encoded by a single plasmid from a clinical isolate of Enterobacter cloacae in China. PMID : 17881870 : Abstract >>
We analyzed the resistance to expanded-spectrum cephalosporins of an Enterobacter cloacae clinical isolate, EC002, by transconjugation, isoelectric-focusing analysis, and cloning experiments. It produced two beta-lactamases with isoelectric point values of 5.4 and 8.7, corresponding to TEM-141, a novel variant of TEM-1, and CTX-M-22, encoded by a transferable plasmid.
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122. |
Quiroga MP,
Andres P,
Petroni A,
Soler Bistué AJ,
Guerriero L,
Vargas LJ,
Zorreguieta A,
Tokumoto M,
Quiroga C,
Tolmasky ME,
Galas M,
Centrón D,
( 2007 ) Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina. PMID : 17938184 : DOI : 10.1128/AAC.00726-07 PMC : PMC2167984 Abstract >>
Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6')-Ib-cr within the variable region of integrons. The three arrays differed from bla(CTX-M-2)-bearing integrons, which are broadly distributed in Argentina.
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123. |
Cattoir V,
Poirel L,
Rotimi V,
Soussy CJ,
Nordmann P,
( 2007 ) Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. PMID : 17561500 : DOI : 10.1093/jac/dkm204 Abstract >>
To develop a rapid and reliable single-tube-based PCR technique for detecting simultaneously the plasmid-mediated quinolone resistance qnrA, qnrB and qnrS genes. After multiple alignments, primers were designed to detect known qnr variants (six for qnrA-, six for qnrB- and two for qnrS-like genes). They were used for screening a collection of 64 expanded-spectrum beta-lactamase (ESBL)-producing enterobacterial isolates from Kuwait, collected from 2002 to 2004, as ESBL genes have been often associated with qnr genes. Sequencing was performed to identify qnr and associated ESBL genes. In optimized conditions, all positive controls (used separately or mixed) confirmed the specificity of the PCR primers. Out of 64 isolates, only 3 isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like and qnrS-like gene was detected. A qnrB2 gene was detected in an Enterobacter cloacae K34 (SHV-12+) isolate, whereas qnrB1-like (termed qnrB7) and qnrB6-like (termed qnrB8) genes were identified from E. cloacae K37 (SHV-12+) and Citrobacter freundii K70 (VEB-1b+) isolates, respectively. We report here a fast and reliable technique for rapid screening of qnr-positive strains to be used for epidemiological surveys. A low prevalence of Qnr determinants among ESBL-producing Enterobacteriaceae was identified in the study with Kuwaiti isolates.
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124. |
Cookson AL,
Biggs PJ,
Marshall JC,
Reynolds A,
Collis RM,
French NP,
Brightwell G,
( 2017 ) Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure. PMID : 28404985 : DOI : 10.1038/s41598-017-00890-6 PMC : PMC5429811 Abstract >>
Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. 'Animal' was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.
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125. |
Boyd DA,
Mataseje LF,
Davidson R,
Delport JA,
Fuller J,
Hoang L,
Lefebvre B,
Levett PN,
Roscoe DL,
Willey BM,
Mulvey MR,
( 2017 ) Enterobacter cloacae Complex Isolates Harboring blaNMC-A or blaIMI-Type Class A Carbapenemase Genes on Novel Chromosomal Integrative Elements and Plasmids. PMID : 28223374 : DOI : 10.1128/AAC.02578-16 PMC : PMC5404547 Abstract >>
Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A blaNMC-A or blaIMI-type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, blaNMC-A was highly associated with Enterobacter ludwigii Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A (n = 10), IMI-1 (n = 5), and IMI-9 (n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, blaIMI-5 and blaIMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring blaNMC-A/IMI-type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential.
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126. |
Liang Q,
Yin Z,
Zhao Y,
Liang L,
Feng J,
Zhan Z,
Wang H,
Song Y,
Tong Y,
Wu W,
Chen W,
Wang J,
Jiang L,
Zhou D,
( 2017 ) Sequencing and comparative genomics analysis of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA. PMID : 28390961 : DOI : 10.1016/j.ijantimicag.2017.01.021 Abstract >>
Incompatibility group IncHI plasmids are important vectors of antibiotic resistance in Enterobacteriaceae. In this study, a scheme for typing IncHI into five separately clustering subgroups, including previously designated IncHI1-3 as well as IncHI4-5, was proposed based on sequenced IncHI plasmids. The complete nucleotide sequences of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA from clinical Enterobacter cloacae, Citrobacter freundii and Raoultella ornithinolytica isolates, respectively, were determined and were compared with IncHI2 and IncHI5 reference plasmids. Considerable genetic conservation was observed within the backbone sequences of each of the IncHI2 and IncHI5 subgroups, but the backbone sequences of the two subgroups were dramatically different from each other. However, the conjugal transfer regions tra1 and tra2 as well as the tellurium resistance gene cluster ter were present in all five plasmids. A number of accessory regions associated with integrons, transposons and insertion sequence-based mobile elements have been inserted at various sites of the plasmid backbones, among which were several large regions harbouring genes conferring resistance to multiple classes of antibiotics. Data generated from this study provide us with a deeper understanding of the diversification of IncHI-type resistance plasmids.
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127. |
Kwon H,
Smith O,
Raven EL,
Moody PC,
( 2017 ) Combining X-ray and neutron crystallography with spectroscopy. PMID : 28177310 : DOI : 10.1107/S2059798316016314 PMC : PMC5297917 Abstract >>
X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H+ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction.
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128. |
Defez R,
Andreozzi A,
Bianco C,
( 2017 ) The Overproduction of Indole-3-Acetic Acid (IAA) in Endophytes Upregulates Nitrogen Fixation in Both Bacterial Cultures and Inoculated Rice Plants. PMID : 28197647 : DOI : 10.1007/s00248-017-0948-4 Abstract >>
Endophytic bacteria from roots and leaves of rice plants were isolated and identified in order to select the diazotrophs and improve their nitrogen-fixing abilities. The nitrogen-fixing endophytes were identified by PCR amplification of the nifH gene fragment. For this purpose, two isolates, Enterobacter cloacae RCA25 and Klebsiella variicola RCA26, and two model bacteria (Herbaspirillum seropedicae z67 and Sinorhizobium fredii NGR234) were transformed to increase the biosynthesis of the main plant auxin indole-3-acetic acid (IAA). A significant increase in the production of IAA was observed for all strains. When the expression of nifH gene and the activity of the nitrogenase enzyme were analyzed in liquid cultures, we found that they were positively affected in the IAA-overproducing endophytes as compared to the wild-type ones. Rice plants inoculated with these modified strains showed a significant upregulation of the nitrogenase activity when plants infected with the wild-type strains were used as reference. Similar results were obtained too with common bean plants infected with the S. fredii NGR234 strain. These findings suggest that IAA overproduction improves nitrogen-fixing apparatus of endophytic bacteria both in liquid cultures and in inoculated host plants. The present study highlights new perspectives to enhance nitrogen-fixing ability in non-legume crops. These strains could be used as bioinoculants to improve the growth and the yield of agricultural crops, offering an alternative to the use of chemical nitrogen fertilizers.
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129. |
Di Luca MC,
Skaare D,
Aasnaes B,
Sundsfjord A,
Samuelsen ?,
( 2016 ) Identification of a novel IMI carbapenemase variant (IMI-9) in Enterobacter cloacae complex. PMID : 27742202 : DOI : 10.1016/j.ijantimicag.2016.09.004 Abstract >>
N/A
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130. |
Arunmanee W,
Pathania M,
Solovyova AS,
Le Brun AP,
Ridley H,
Baslé A,
van den Berg B,
Lakey JH,
( 2016 ) Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis. PMID : 27493217 : DOI : 10.1073/pnas.1602382113 PMC : PMC5003275 Abstract >>
The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion.
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131. |
Wailan AM,
Sidjabat HE,
Yam WK,
Alikhan NF,
Petty NK,
Sartor AL,
Williamson DA,
Forde BM,
Schembri MA,
Beatson SA,
Paterson DL,
Walsh TR,
Partridge SR,
( 2016 ) Mechanisms Involved in Acquisition of blaNDM Genes by IncA/C2 and IncFIIY Plasmids. PMID : 27114281 : DOI : 10.1128/AAC.00368-16 PMC : PMC4914633 Abstract >>
blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family.
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132. |
Stojanoski V,
Adamski CJ,
Hu L,
Mehta SC,
Sankaran B,
Zwart P,
Prasad BV,
Palzkill T,
( 2016 ) Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine �]-Lactamases by Relieving Steric Strain. PMID : 27073009 : DOI : 10.1021/acs.biochem.6b00056 PMC : PMC5124363 DOI : 10.1021/acs.biochem.6b00056 PMC : PMC5124363 Abstract >>
Serine �]-lactamases are bacterial enzymes that hydrolyze �]-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine �]-lactamases. Six different enzymes comprising representatives from each of the three classes of serine �]-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the C�] atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability.
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133. |
Du H,
Chen L,
Chavda KD,
Pandey R,
Zhang H,
Xie X,
Tang YW,
Kreiswirth BN,
( 2016 ) Genomic Characterization of Enterobacter cloacae Isolates from China That Coproduce KPC-3 and NDM-1 Carbapenemases. PMID : 26787700 : DOI : 10.1128/AAC.03053-15 PMC : PMC4808189 Abstract >>
Here, we report twoEnterobacter cloacaesequence type 231 isolates coproducing KPC-3 and NDM-1 that have caused lethal infections in a tertiary hospital in China. TheblaNDM-1-harboring plasmids carry IncA/C2and IncR replicons, showing a mosaic plasmid structure, and theblaNDM-1is harbored on a novel class I integron-like element.blaKPC-3is located on a Tn3-�GblaTEM-1-blaKPC-3-�GTn1722element, flanked by two 9-bp direct-repeat sequences and harbored on an IncX6 plasmid.
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134. |
Thomson GK,
Snyder JW,
McElheny CL,
Thomson KS,
Doi Y,
( 2016 ) Coproduction of KPC-18 and VIM-1 Carbapenemases by Enterobacter cloacae: Implications for Newer �]-Lactam-�]-Lactamase Inhibitor Combinations. PMID : 26719440 : DOI : 10.1128/JCM.02739-15 PMC : PMC4767958 Abstract >>
Enterobacter cloacae strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes, bla(KPC-18) and bla(VIM-1). Whole-genome sequencing localized bla(KPC-18) to the chromosome and bla(VIM-1) to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-�]-lactamase with KPC-type carbapenemase has implications for the use of next-generation �]-lactam-�]-lactamase inhibitor combinations.
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135. |
Fursova NK,
Astashkin EI,
Knyazeva AI,
Kartsev NN,
Leonova ES,
Ershova ON,
Alexandrova IA,
Kurdyumova NV,
Sazikina SY,
Volozhantsev NV,
Svetoch EA,
Dyatlov IA,
( 2015 ) The spread of bla OXA-48 and bla OXA-244 carbapenemase genes among Klebsiella pneumoniae, Proteus mirabilis and Enterobacter spp. isolated in Moscow, Russia. PMID : 26526183 : DOI : 10.1186/s12941-015-0108-y PMC : PMC4630924 Abstract >>
The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a great problem of healthcare worldwide. Study of the spread for bla OXA-48-like genes coding epidemically significant carbapenemases among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance. Antibacterial resistant isolates of Klebsiella pneumoniae (n = 95) from 54 patients, P. mirabilis (n = 32) from 20 patients, Enterobacter aerogenes (n = 6) from four patients, and Enterobacter cloacae (n = 4) from four patients were collected from January, 2013 to October, 2014 in neurosurgical intensive care unit (ICU) of the Burdenko Neurosurgery Institute, Moscow. Characteristics of the isolates were done using susceptibility tests, PCR detection of the resistance genes, genotyping, conjugation, DNA sequencing, and bioinformatic analysis. Major strains under study were multi drug resistant (MDR), resistant to three or more functional classes of drugs simultaneously-98.9 % K. pneumoniae, 100 % P. mirabilis, one E. aerogenes isolate, and one E. cloacae isolate. Molecular-genetic mechanism of MDR in K. pneumoniae and P. mirabilis isolates were based on carrying of epidemic extended-spectrum beta-lactamase bla CTX-M-15 gene (87.2 and 90.6 % accordingly), carbapenemase bla OXA-48-like gene (55.3 and 23.3 % accordingly), and class 1 (54.8 and 31.3 % accordingly) and class 2 (90.6 % P. mirabilis) integrons. The bla OXA-48-like-positive K. pneumoniae were collected during whole two-year surveillance period, while P. mirabilis and Enterobacter spp. carrying bla OXA-48-like genes were detected only after four and 18 months after the research start, respectively. The bla OXA-48-like gene acquisition was shown for P. mirabilis isolates collected from five patients and for E. cloacae isolate collected from one patient during their stay in the ICU, presumably from bla OXA-48-like-positive K. pneumoniae. The source of the bla OXA-244 gene acquired by E. aerogenes isolates and the time of this event were not recognized. The expanding of CPE in the surveyed ICU was associated with the spread of bla OXA-48 and bla OXA-244 carbapenemase genes documented not only among K. pneumoniae, well-known bacterial host for such genes, but among P. mirabilis, E. aerogenes, and E. cloacae.
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136. |
Tijet N,
Richardson D,
MacMullin G,
Patel SN,
Melano RG,
( 2015 ) Characterization of multiple NDM-1-producing Enterobacteriaceae isolates from the same patient. PMID : 25845877 : DOI : 10.1128/AAC.04862-14 PMC : PMC4432124 Abstract >>
A male patient was admitted to a community hospital in Ontario, Canada, with an infected sacral ulcer after returning from India, where he was hospitalized. Carbapenem-resistant Escherichia coli (isolated from blood cultures), Enterobacter cloacae, and Providencia stuartii (from urine samples), all positive for bla(NDM-1), were recovered. Comparative NDM-1 plasmid analysis suggests both lateral plasmid transfer and independent acquisition of the bla(NDM-1) gene in these clinical isolates.
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137. |
Bourouis A,
Ben Moussa M,
Belhadj O,
( 2015 ) Multidrug-resistant phenotype and isolation of a novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae. PMID : 25888770 : DOI : 10.1186/s12929-015-0131-5 PMC : PMC4407307 Abstract >>
ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species. E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum �]-lactam and the change of the isoelectric point of the protein. These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.
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138. |
Babouee Flury B,
Ellington MJ,
Hopkins KL,
Turton JF,
Doumith M,
Loy R,
Staves P,
Hinic V,
Frei R,
Woodford N,
( 2016 ) Association of Novel Nonsynonymous Single Nucleotide Polymorphisms in ampD with Cephalosporin Resistance and Phylogenetic Variations in ampC, ampR, ompF, and ompC in Enterobacter cloacae Isolates That Are Highly Resistant to Carbapenems. PMID : 26856839 : DOI : 10.1128/AAC.02835-15 PMC : PMC4808197 Abstract >>
InEnterobacter cloacae, the genetic lesions associated with derepression of the AmpC �]-lactamase include diverse single nucleotide polymorphisms (SNPs) and/or indels in theampDandampRgenes and SNPs inampC, while diverse SNPs in the promoter region or SNPs/indels within the coding sequence of outer membrane proteins have been described to alter porin production leading to carbapenem resistance. We sought to define the underlying mechanisms conferring cephalosporin and carbapenem resistance in a collection ofE. cloacaeisolates with unusually high carbapenem resistance and no known carbapenemase and, in contrast to many previous studies, considered the SNPs we detected in relation to the multilocus sequence type (MLST)-based phylogeny of our collection. Whole-genome sequencing was applied on the most resistant isolates to seek novel carbapenemases, expression ofampCwas measured by reverse transcriptase PCR, and porin translation was detected by SDS-PAGE. SNPs occurring inampC,ampR,ompF, andompCgenes (and their promoter regions) were mostly phylogenetic variations, relating to the isolates' sequence types, whereas nonsynonymous SNPs inampDwere associated with derepression of AmpC and cephalosporin resistance. The additional loss of porins resulted in high-level carbapenem resistance, underlining the clinical importance of chromosomal mutations among carbapenem-resistantE. cloacae.
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139. |
Huang L,
Wang X,
Feng Y,
Xie Y,
Xie L,
Zong Z,
( 2015 ) First identification of an IMI-1 carbapenemase-producing colistin-resistant Enterobacter cloacae in China. PMID : 26607057 : DOI : 10.1186/s12941-015-0112-2 PMC : PMC4658791 Abstract >>
Carbapenem resistance among the Enterobacteriaceae is a serious healthcare challenge. bla IMI is a carbapenemase gene mediating resistance to carbapenems but has not been commonly found. A bla IMI-carrying Enterobacter cloacae, which was also resistant to colistin, is reported here. E. cloacae strain WCHECl-1060 was recovered from a blood sample of a leukemia patient, who was not previously exposed to colistin. Strain WCHECl-1060 belongs to a new sequence type, ST410, and was resistant to carbapenems and colistin but was susceptible to third-generation cephalosporins. A new allelic variant of bla IMI-1, which has two silent mutations compared to the original bla IMI-1 variant, was found in strain WCHECl-1060. Conjugation and transformation experiments failed to transfer bla IMI-1, suggesting a likely chromosome origin. To our knowledge, this is the first report of an IMI-1 carbapenemase-producing colistin-resistant E. cloacae in China. Microbiological laboratories should be aware of the unusual carbapenem-resistant but third-generation cephalosporin-susceptible profiles of these IMI-producing isolates. The trend of colistin resistance among the Enterobacteriaceae should be also monitored.
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140. |
Girlich D,
Poirel L,
Nordmann P,
( 2015 ) Clonal distribution of multidrug-resistant Enterobacter cloacae. PMID : 25680336 : DOI : 10.1016/j.diagmicrobio.2015.01.003 Abstract >>
A multilocus sequence typing (MLST) scheme including 7 housekeeping genes was used to evaluate whether the current spread of multidrug-resistant Enterobacter cloacae isolates worldwide might be associated to specific successful clones. Fifty E. cloacae clinical isolates of worldwide origin, with various �]-lactamase content, and recovered at different periods of time were studied. Forty-four sequence types were identified, highlighting a high clonal diversity with 3 main lineages. This study revealed that a precise identification of the isolates by sequencing of the chromosomal ampC gene of E. cloacae would provide a significant added value to improve the reliability of the MLST scheme.
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141. |
Zamorano L,
Miró E,
Juan C,
Gómez L,
Bou G,
González-López JJ,
Martínez-Martínez L,
Aracil B,
Conejo MC,
Oliver A,
Navarro F,
( 2015 ) Mobile genetic elements related to the diffusion of plasmid-mediated AmpC �]-lactamases or carbapenemases from Enterobacteriaceae: findings from a multicenter study in Spain. PMID : 26077249 : DOI : 10.1128/AAC.00562-15 PMC : PMC4538544 Abstract >>
We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 of 640 Enterobacteriaceae isolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74 bla AmpC genes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The bla CMY-2-like genes were associated with ISEcp1; the surrounding bla DHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the bla ACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (bla VIM-1, bla IMP-22, and bla IMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.
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142. |
Papagiannitsis CC,
Izdebski R,
Baraniak A,
Fiett J,
Herda M,
Hrabák J,
Derde LP,
Bonten MJ,
Carmeli Y,
Goossens H,
Hryniewicz W,
Brun-Buisson C,
Gniadkowski M,
N/A N/A,
N/A N/A,
( 2015 ) Survey of metallo-�]-lactamase-producing Enterobacteriaceae colonizing patients in European ICUs and rehabilitation units, 2008-11. PMID : 25759034 : DOI : 10.1093/jac/dkv055 Abstract >>
The objective of this study was to perform a multinational survey of patients' colonization by metallo-�]-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. Patients in 18 hospital units across Europe and Israel (n = 17 945) were screened between mid-2008 and mid-2011. MBL-producing isolates were typed by PFGE and MLST. MBL genes were amplified and sequenced within their integrons. Plasmids with MBL genes were analysed by nuclease S1 plus hybridization profiling, mating and transformation assays, and by PCR-based replicon typing. Ninety-one patients in nine centres (six countries), including 62 patients in two Greek ICUs, carried 94 non-duplicate MBL-producing organisms. Klebsiella pneumoniae isolates from Greece dominated (n = 57) and belonged mainly to ST147, ST36 and ST383. All but one of the isolates expressed VIM-1-type MBLs. Isolates of Greek origins produced five enzymes, including new VIM-39, encoded by class 1 integrons of four types. In-e541-like elements prevailed, comprising six variants located on IncR, IncFIIK, IncR + FIIK, IncR + A/C or non-typeable plasmids. The other group were new In4873 and In4863, being the first In416-like elements identified in Greece, which were present on IncA/C or non-typeable plasmids. Isolates from other countries produced only VIM-1 and the major integron was In916, identified in 16 organisms from France, Italy and Spain. In916 was carried by four plasmid types, including IncA/C, IncFIIK and IncHI2. Other integrons included a new element, In3103, in Spain and In110 identified only in Latvia. This study provided fully comparable data on the occurrence and molecular characteristics of VIM-producing Enterobacteriaceae in a group of hospital units across Europe, documenting recent changes in their epidemiology.
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143. |
Vliegenthart JS,
Ketelaar-van Gaalen PA,
van de Klundert JA,
( 1989 ) Nucleotide sequence of the aacC2 gene, a gentamicin resistance determinant involved in a hospital epidemic of multiply resistant members of the family Enterobacteriaceae. PMID : 2552900 : DOI : 10.1128/aac.33.8.1153 PMC : PMC172616 Abstract >>
A gentamicin resistance determinant of a conjugative plasmid from Enterobacter cloacae was cloned on a 3.2-kilobase fragment in the PstI site of pBR322. Substrate profiles for eight aminoglycosides at three concentrations showed that the resistance was due to aminoglycoside-(3)-N-acetyltransferase isoenzyme II. Insertion mapping by the gamma-delta transposon revealed that the size of the gene was approximately 1 kilobase. Nucleotide sequencing of the aacC2 gene identified an open reading frame of 858 base pairs, preceded by a promoter and a ribosome-binding site. From these data the molecular mass of the protein was calculated to be 30.6 kilodaltons. A comparison of the nucleotide sequence of the aacC2 gene with those published for the aacC3 and aacC4 genes showed complete homology of the aacC2 gene and the presumed aacC3 gene. An internal restriction fragment of the gene used as a probe in colony hybridization demonstrated the presence of the aacC2 gene in 86% of 86 multiply resistant isolates of the family Enterobacteriaceae obtained during an 18-month hospital epidemic. This corroborates our earlier data on the enzyme identification by the susceptibility profiling method.
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144. |
Kupper MB,
Herzog K,
Bennink S,
Schlömer P,
Bogaerts P,
Glupczynski Y,
Fischer R,
Bebrone C,
Hoffmann KM,
( 2015 ) The three-dimensional structure of VIM-31--a metallo-�]-lactamase from Enterobacter cloacae in its native and oxidized form. PMID : 25825035 : DOI : 10.1111/febs.13283 Abstract >>
The metallo-�]-lactamase VIM-31 differs from VIM-2 by only two Tyr224His and His252Arg substitutions. Located close to the active site, the Tyr224His substitution is also present in VIM-1, VIM-4, VIM-7 and VIM-12. The VIM-31 variant was reported in 2012 from Enterobacter cloacae and kinetically characterized. It exhibits globally lower catalytic efficiencies than VIM-2. In the present study, we report the three-dimensional structures of VIM-31 in its native (reduced) and oxidized forms. The so-called 'flapping-loop' (loop 1) and loop 3 of VIM-31 were not positioned as in VIM-2 but instead were closer to the active site as in VIM-4, resulting in a narrower active site in VIM-31. Also, the presence of His224 in VIM-31 disrupts hydrogen-bonding networks close to the active site. Moreover, a third zinc-binding site, which also exists in VIM-2 structures, could be identified as a structural explanation for the decreased activity of VIM-MBLs at high zinc concentrations.
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145. |
Manageiro V,
Pinto M,
Caniça M,
( 2015 ) Complete Sequence of a blaOXA-48-Harboring IncL Plasmid from an Enterobacter cloacae Clinical Isolate. PMID : 26383652 : DOI : 10.1128/genomeA.01076-15 PMC : PMC4574381 Abstract >>
We report a 63,584-bp conjugative IncL plasmid (pUR17313-1) from an Enterobacter cloacae clinical isolate, containing a blaOXA-48 gene. The plasmid sequence also carried important mobile genetic elements involved in the spread of antibiotic resistance, namely, the Tn1999.2 composite transposon, which enclosed blaOXA-48-, integrase-, and transposase-encoding genes.
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146. |
Wang Y,
Lo WU,
Lai EL,
Chow KH,
Ho PL,
( 2015 ) Complete sequence of the multidrug-resistant IncL/M plasmid pIMP-HB623 Cocarrying bla IMP-34 and fosC2 in an Enterobacter cloacae strain associated with medical travel to China. PMID : 26100693 : DOI : 10.1128/AAC.00375-15 PMC : PMC4538467 Abstract >>
N/A
|
147. |
Porres-Osante N,
Sáenz Y,
Somalo S,
Torres C,
( 2015 ) Characterization of Beta-lactamases in Faecal Enterobacteriaceae Recovered from Healthy Humans in Spain: Focusing on AmpC Polymorphisms. PMID : 25501887 : DOI : 10.1007/s00248-014-0544-9 Abstract >>
The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.
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148. |
Wendel AF,
MacKenzie CR,
( 2015 ) Characterization of a novel metallo-�]-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany. PMID : 25547359 : DOI : 10.1128/AAC.05062-14 PMC : PMC4325787 Abstract >>
N/A
|
149. |
Wu W,
Feng Y,
Carattoli A,
Zong Z,
( 2015 ) Characterization of an Enterobacter cloacae Strain Producing both KPC and NDM Carbapenemases by Whole-Genome Sequencing. PMID : 26248381 : DOI : 10.1128/AAC.01275-15 PMC : PMC4576032 Abstract >>
A carbapenem-resistant Enterobacter cloacae strain, WCHECl-14653, causing a fatal bloodstream infection, was characterized by genome sequencing and conjugation experiments. The strain carried two carbapenemase genes, blaNDM-1 and blaKPC-2, on separate IncF plasmids. The coexistence of blaNDM-1 and blaKPC-2 conferred slightly higher-level carbapenem resistance compared with that of blaNDM-1 or blaKPC-2 alone, and the coexistence of two IncF plasmids may generate new platforms for spreading carbapenemase genes.
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150. |
Yang L,
Wu AW,
Su DH,
Lin YP,
Chen DQ,
Qiu YR,
( 2014 ) resistome analysis of Enterobacter cloacae CY01, an extensively drug-resistant strain producing VIM-1 metallo-�]-lactamase from China. PMID : 25114139 : DOI : 10.1128/AAC.03060-14 PMC : PMC4187898 Abstract >>
Resistome analysis of clinical VIM-1-producing Enterobacter cloacae strain CY01 from China revealed the presence of multiple resistance determinants. Two resistance plasmids were identified in CY01. The pCY-VIM plasmid was 14 kb in size and possessed a replicase gene (repA), a gene cluster encoding the partitioning function (parABC), and a carbapenemase gene (blaVIM-1). Another 5.9-kb plasmid, pCY-MdT, with an aac(6')-Ib gene, was very closely related (13 nucleotide differences) to pMdT1, a ColE1 plasmid carrying aac(6')-Ib-cr4.
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151. |
Manageiro V,
Ferreira E,
Pinto M,
Caniça M,
( 2014 ) First description of OXA-48 carbapenemase harbored by Escherichia coli and Enterobacter cloacae from a single patient in Portugal. PMID : 25246399 : DOI : 10.1128/AAC.02961-14 PMC : PMC4249543 Abstract >>
N/A
|
152. |
Yang Q,
Fang L,
Fu Y,
Du X,
Shen Y,
Yu Y,
( 2015 ) Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid. PMID : 26047502 : DOI : 10.1371/journal.pone.0129454 PMC : PMC4457825 Abstract >>
The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of blaNDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including blaKPC, blaIMP, blaVIM, blaOXA-48 and blaNDM-1 were screened and sequenced. Ninety isolates were identified as harboring the blaKPC-2 genes, and five blaNDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three blaNDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the blaNDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around blaNDM-1 (blaNDM-1-trpF- dsbC-cutA1-groEL-�GInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller blaNDM-1 plasmids contained a common gene environment around blaNDM-1 (IS5-blaNDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the blaNDM-1 gene among the CRE.
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153. |
Stojanoski V,
Chow DC,
Fryszczyn B,
Hu L,
Nordmann P,
Poirel L,
Sankaran B,
Prasad BV,
Palzkill T,
( 2015 ) Structural Basis for Different Substrate Profiles of Two Closely Related Class D �]-Lactamases and Their Inhibition by Halogens. PMID : 25938261 : DOI : 10.1021/acs.biochem.5b00298 PMC : PMC5123777 Abstract >>
OXA-163 and OXA-48 are closely related class D �]-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active-site �]5 strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the �]5 and �]6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities, we performed enzyme kinetic analysis, inhibition assays, X-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72 ? resolution reveals an expanded active site compared to that of OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized under another condition that included iodide. The crystal structure determined at 2.87 ? resolution revealed iodide in the active site accompanied by several significant conformational changes, including a distortion of the �]5 strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active-site configuration and thereby affect the substrate profile of an enzyme.
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154. |
Probert WS,
McQuaid C,
Schrader K,
( 2014 ) Isolation and identification of an Enterobacter cloacae strain producing a novel subtype of Shiga toxin type 1. PMID : 24759708 : DOI : 10.1128/JCM.00338-14 PMC : PMC4097712 Abstract >>
We describe here the isolation and identification of a Shiga toxin 1 (Stx1)-producing Enterobacter cloacae strain, M12X01451, from a human clinical specimen. The bacterial isolate was identified as E. cloacae using a polyphasic approach that included phenotypic, genetic, and proteomic analyses. The M12X01451 stx1 was sequenced, and the holotoxin was found to share only 87% amino acid sequence identity with the nearest Stx1 subtype reference sequence. Sequence analysis of the regions immediately flanking stx1 displayed similarities with bacteriophage-related sequences, suggesting a prophage origin. The stx1 gene was a stable element within the M12X01451 genome, as demonstrated by real-time PCR detection following successive subculturing of the bacterial isolate. Culture supernatant from M12X01451 was cytotoxic to Vero cells but was not neutralized by an anti-Stx1 monoclonal antibody. In addition, Stx1 from M12X01451 demonstrated limited antigenicity with two commercially available lateral flow immunoassays. The M12X01451 Stx represents a new Stx1 subtype based on the degree of sequence dissimilarity with Stx1 subtype reference sequences and its limited reactivity with anti-Stx1 antibodies.
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155. |
Literak I,
Manga I,
Wojczulanis-Jakubas K,
Chroma M,
Jamborova I,
Dobiasova H,
Sedlakova MH,
Cizek A,
( 2014 ) Enterobacter cloacae with a novel variant of ACT AmpC beta-lactamase originating from glaucous gull (Larus hyperboreus) in Svalbard. PMID : 24629772 : DOI : 10.1016/j.vetmic.2014.02.015 Abstract >>
We aimed at Escherichia coli and Enterobacter cloacae isolates resistant to cephalosporins and fluoroquinolones and Salmonella isolates in wild birds in Arctic Svalbard, Norway. Cloacal swabs of little auks (Alle alle, n=215) and samples of faeces of glaucous gulls (Larus hyperboreus, n=15) were examined. Inducible production of AmpC enzyme was detected in E. cloacae KW218 isolate. Sequence analysis of the 1146 bp PCR product of the ampC gene from this isolate revealed 99% sequence homology with the blaACT-14 and blaACT-5 AmpC beta-lactamase genes. Four, respectively six of the identified single nucleotide polymorphisms generated amino acid substitutions in the amino acid chain. As the ampC sequence polymorphism in the investigated E. cloacae strain was identified as unique, we revealed a novel variant of the ampC beta-lactamase gene blaACT-23.
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156. |
Bariši? I,
Mitteregger D,
Hirschl AM,
Noehammer C,
Wiesinger-Mayr H,
( 2014 ) High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis. PMID : 25159028 : DOI : 10.1016/j.meegid.2014.08.014 Abstract >>
The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 �]-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe's largest hospitals, the General Hospital Vienna, were screened for the presence of 31 �]-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of �]-lactamase genes was estimated using biostatistical models. The carbapenemase genes blaKPC-2, blaKPC-3, and blaVIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, blaOXA-72 in an extended-spectrum �]-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired blaDHA-1 and blaCMY AmpC �]-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all �]-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated �]-lactamases and other resistance genes in one of Europe's largest hospitals.
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157. |
Ohad S,
Block C,
Kravitz V,
Farber A,
Pilo S,
Breuer R,
Rorman E,
( 2014 ) Rapid identification of Enterobacter hormaechei and Enterobacter cloacae genetic cluster III. PMID : 24428402 : DOI : 10.1111/jam.12439 Abstract >>
Enterobacter cloacae complex bacteria are of both clinical and environmental importance. Phenotypic methods are unable to distinguish between some of the species in this complex, which often renders their identification incomplete. The goal of this study was to develop molecular assays to identify Enterobacter hormaechei and Ent. cloacae genetic cluster III which are relatively frequently encountered in clinical material. The molecular assays developed in this study are qPCR technology based and served to identify both Ent. hormaechei and Ent. cloacae genetic cluster III. qPCR results were compared to hsp60 sequence analysis. Most clinical isolates were assigned to Ent. hormaechei subsp. steigerwaltii and Ent. cloacae genetic cluster III. The latter was proportionately more frequently isolated from bloodstream infections than from other material (P < 0�P05). The qPCR assays detecting Ent. hormaechei and Ent. cloacae genetic cluster III demonstrated high sensitivity and specificity. The presented qPCR assays allow accurate and rapid identification of clinical isolates of the Ent. cloacae complex. The improved identifications obtained can specifically assist analysis of Ent. hormaechei and Ent. cloacae genetic cluster III in nosocomial outbreaks and can promote rapid environmental monitoring. An association was observed between Ent. cloacae cluster III and systemic infection that deserves further attention.
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158. |
Cheng C,
Zheng F,
Rui Y,
( 2014 ) Rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria by loop-mediated isothermal amplification. PMID : 25000338 : DOI : 10.1089/mdr.2014.0040 Abstract >>
A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of blaKPC, blaNDM, blaIMP, and blaVIM carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with blaNDM, 11 strains with blaKPC, 11 strains with blaVIM, and 2 strains with blaIMP obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria.
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159. |
Lahiri SD,
Johnstone MR,
Ross PL,
McLaughlin RE,
Olivier NB,
Alm RA,
( 2014 ) Avibactam and class C �]-lactamases: mechanism of inhibition, conservation of the binding pocket, and implications for resistance. PMID : 25022578 : DOI : 10.1128/AAC.03057-14 PMC : PMC4187909 Abstract >>
Avibactam is a novel non-�]-lactam �]-lactamase inhibitor that inhibits a wide range of �]-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of �]-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the �]-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first �]-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used �]-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C �]-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C �]-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam.
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160. |
Wendel AF,
Brodner AH,
Wydra S,
Ressina S,
Henrich B,
Pfeffer K,
Toleman MA,
Mackenzie CR,
( 2013 ) Genetic characterization and emergence of the metallo-�]-lactamase GIM-1 in Pseudomonas spp. and Enterobacteriaceae during a long-term outbreak. PMID : 23877696 : DOI : 10.1128/AAC.00118-13 PMC : PMC3811479 Abstract >>
Since the first isolation in 2002, the metallo-�]-lactamase GIM-1 has not been detected outside Germany. The data presented here, for 50 clinical blaGIM-1-positive isolates, including Pseudomonas spp. and Enterobacteriaceae (Enterobacter cloacae, Klebsiella oxytoca, Serratia marcescens, Escherichia coli, and Citrobacter freundii), collected between 2007 and 2012 at the original site in an ongoing outbreak, demonstrate a diverse genetic background and dissemination of the gene conferring resistance to enteric bacteria.
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161. |
Zheng F,
Sun J,
Cheng C,
Rui Y,
( 2013 ) The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in bacteria. PMID : 24143953 : DOI : 10.1186/1476-0711-12-30 PMC : PMC3816589 Abstract >>
The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-�]-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of blaNDM and blaKPC with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for blaNDM (R2 = 0.971; slope, -3.273) and blaKPC (R2 = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.
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162. |
Chen W,
Ai L,
Yang J,
Ren J,
Li Y,
Guo B,
( 2013 ) Development of a PCR assay for rapid detection of Cronobacter spp. from food. PMID : 24102218 : DOI : 10.1139/cjm-2013-0243 Abstract >>
The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 �XC. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.
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163. |
Bogaerts P,
Bebrone C,
Huang TD,
Bouchahrouf W,
Degheldre Y,
Deplano A,
Hoffmann K,
Glupczynski Y,
( 2012 ) Detection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in a clinical isolate of Enterobacter cloacae. PMID : 22391550 : DOI : 10.1128/AAC.06249-11 PMC : PMC3370733 Abstract >>
We report the first description of the metallo-�]-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. bla(VIM-31) was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested beta-lactam agents (except for ertapenem), resulting from lower k(cat) (except for ertapenem) and higher K(m) values for VIM-31.
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164. |
Kristóf K,
Nobilis A,
Damjanova I,
Juhász E,
( 2012 ) Emergence of VIM-4- and SHV-12-producing Enterobacter cloacae in a neonatal intensive care unit. PMID : 22721811 : DOI : 10.1016/j.ijmm.2012.05.003 Abstract >>
In order to reveal colonization with multidrug-resistant bacteria early, routine screening is done on samples of all patients of the neonatal intensive care units at Semmelweis University, Hungary. Due to the extended-spectrum �]-lactamase (ESBL) screening examinations, emergence of multidrug-resistant Enterobacter cloacae isolates was found with suspicion of clonal transmission, therefore active microbiological surveillance was initiated. The aim of our study was to characterize 60 E. cloacae isolates recovered in a 7-month period in 2010. MIC values of antibiotics were determined and ESBL and carbapenemase production was tested. Metallo-�]-lactamase (MBL) genes, ESBL genes, and class-1 integrons were characterized, and the possible clonal relationship between isolates was investigated. The isolates showed increased MIC values for carbapenems and cephalosporins. All 60 E. cloacae strains recovered from 16 neonates proved to be VIM-4 MBL producers. Fifty-three strains were SHV-12 ESBL producers also. In all cases, the bla(VIM-4) gene was a part of class-1 integron, In238a. XbaI-macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) revealed identical patterns for the isolates. Our study supports the importance of active microbiological surveillance as well as molecular epidemiology at the NICUs as a part of infection control.
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165. |
Komatsu T,
Ohta M,
Kido N,
Arakawa Y,
Ito H,
Mizuno T,
Kato N,
( 1990 ) Molecular characterization of an Enterobacter cloacae gene (romA) which pleiotropically inhibits the expression of Escherichia coli outer membrane proteins. PMID : 2193928 : DOI : 10.1128/jb.172.7.4082-4089.1990 PMC : PMC213395 Abstract >>
The introduction of a newly cloned Enterobacter cloacae chromosomal gene romA, into Escherichia coli and E. cloacae resulted in enhancement of resistance to quinolones, beta-lactams, chloramphenicol, and tetracycline. The primary effect of romA on a multicopy vector in E. coli was almost complete inhibition of OmpF expression in the outer membrane. From the experiments with ompR and envZ mutants or with ompF-lacZ and ompC-lacZ fusion plasmids, it was concluded that this inhibition is posttranscriptional. The introduction of romA on a multicopy vector into strains with micF deletion elicited only a moderate decrease in OmpF protein expression. This indicates that reduction of OmpF expression by romA is partly mediated posttranscriptionally by the activation of micF. Moreover, the overexpression of RomA protein from an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter resulted in nearly complete inhibition of expression of OmpC and OmpA, as well as OmpF. Taken together with an observation in a recent study that overexpressed OmpC inhibited the synthesis of OmpA and LamB, a possible inhibitory mechanism at the translational stage of the synthesis of outer membrane proteins should also be considered. By Southern hybridization, romA was generally detected in the chromosomes of all E. cloacae strains tested but not in the E. coli K-12 chromosome. Sequence data show that there is an open reading frame specifying 368 amino acids residues including a putative signal peptide. RomA appears to belong to the outer membrane protein family since it was extractable from an outer membrane preparation, but no sequence homology to other outer membrane proteins was detected.
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166. |
Wang YG,
Xia QY,
Gu WL,
Sun JB,
Zhang H,
Lu XH,
Lu J,
Peng M,
Zhang X,
( 2012 ) Isolation of a strong promoter fragment from endophytic Enterobacter cloacae and verification of its promoter activity when its host strain colonizes banana plants. PMID : 22080347 : DOI : 10.1007/s00253-011-3684-6 Abstract >>
To engineer endophytic Enterobacter cloacae as a biocontrol agent against banana fusarium wilt, a promoter-probe plasmid pUCK was constructed to identify a strong promoter to express disease resistance genes. Using a kanamycin resistance gene for selection, 10 fragments with strong promoter activity were identified from the genome of the E. cloacae KKWB-10 strain. The regions of these 10 fragments that were the primary contributors to the promoter function were identified, and their promoter activities were further evaluated using green fluorescent protein (GFP) as a reporter gene. Fragment 132a? drove the highest level of GFP activity when the bacteria bearing the fragments were cultured in Luria-Bertani and banana stem extract media. The GFP-expressing strain harboring fragment 132a? (K-pUCK7-132a?-GT) was then inoculated into banana plantlets (about 1 �� 10(7) CFU per plant) to verify the activity of fragment 132a? in planta. Ten days after inoculation, tissue sections of these banana plantlets were observed by laser confocal scanning microscope. Green fluorescence was observed in the tissues of banana plantlets inoculated with K-pUCK7-132a?-GT but not in uninoculated controls. These results suggest that fragment 132a? possesses strong promoter activity when its host strain colonizes the banana plants and can be used to engineer endophytic E. cloacae KKWB-10 for biocontrol.
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167. |
Glupczynski Y,
Huang TD,
Bouchahrouf W,
Rezende de Castro R,
Bauraing C,
Gérard M,
Verbruggen AM,
Deplano A,
Denis O,
Bogaerts P,
( 2012 ) Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals. PMID : 22115539 : DOI : 10.1016/j.ijantimicag.2011.10.005 Abstract >>
During a polymerase chain reaction (PCR)-based surveillance study of �]-lactam resistance, 19 OXA-48-positive enterobacterial isolates were detected at nine Belgian hospitals from January 2010 to April 2011. Most cases were presumed to have been locally acquired and were detected in patients who had not travelled abroad. Clonally related outbreaks occurred in two different cities. The majority of isolates co-produced several �]-lactamases as well as non-�]-lactam resistance genes. This report highlights the rapid emergence and spread of OXA-48-producing Enterobacteriaceae in Belgium.
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168. |
Rodríguez-Martínez JM,
Nordmann P,
Poirel L,
( 2012 ) Group IIC intron with an unusual target of integration in Enterobacter cloacae. PMID : 22020643 : DOI : 10.1128/JB.05786-11 PMC : PMC3256610 Abstract >>
A potential role of group IIC-attC introns in integron gene cassette formation, that is, the way in which they could provide the attC sequence essential for recombination, has been proposed. Group IIC introns usually target the attC site of gene cassettes and more specifically their inverse core. Here we characterized a novel group IIC intron targeting the core site of the aadA1 gene cassette attC site (aadA1-qacE�G1 gene cassette junction) from enterobacterial isolates. Intron mobility (retrohoming) was analyzed using a two-plasmid assay performed in Escherichia coli. Intron mobility assays confirmed the mobilization-integration of the group II intron into the core site of the aadA2, bla(VIM-2), bla(CARB-2), aac(6')-Ib, dfrXVb, arr2, cmlA4, and aadB gene cassettes but not into the attI site. This mobility was dependent on maturase activity. Reverse transcriptase PCR showed that this intron was transcriptionally active, and an intermediate circular form was detected by inverse PCR. This element was linked to the bla(VEB-1) extended-spectrum �]-lactamase gene in a high number of enterobacterial isolates. A phylogenetic tree showed that the identified element was located in a branch separate from group IIC-attC introns, being an IIC intron possessing the ability to integrate using the core site of the attC sites as target.
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169. |
( 1997 ) Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes. PMID : 9317027 : PMC : PMC175603 Abstract >>
Enterobacter cloacae has been implicated as one of the causative agents in neonatal infection and causes a septicemia thought to be initiated via the gastrointestinal tract. The adhesion of radiolabeled E. cloacae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrations of alpha-mannosides and micromolar concentrations of high-mannose oligosaccharides. A variety of well-characterized mannose oligosaccharides were tested as inhibitors of adhesion. The best inhibitor was the Man9(GlcNAc)2-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man7(GlcNAc)2, Man6(GlcNAc)2 or Man5(GlcNAc)2. Further evidence that the bacteria preferred Man9(GlcNAc)2 structures was obtained by growing HT-29 cells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man9(GlcNAc)2 at the cell surface. Such cells bound 1.5- to 2-fold more bacteria than did control cells. The adhesin involved in binding to high-mannose structures was purified from isolated pili. On sodium dodecyl sulfate-gels, a 35-kDa protein was identified by its specific binding to a mannose-containing biotinylated albumin. The amino acid sequences of several peptides from the 35-kDa subunit showed over 85% identity to FimH, the mannose-specific adhesin of Salmonella typhimurium. Pili were labeled with 125I and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man9(GlcNAc)2-tyrosinamide but not by oligosaccharides with fewer mannose residues. Polyclonal antibody against this 35-kDa protein also effectively blocked adhesion of pili or E. cloacae, but no effect was observed with nonspecific antibody. These studies demonstrate that the 35-kDa pilus subunit is a lectin whose specificity is directed toward Man, (GlcNAc)2 oligosaccharides.
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170. |
( 1997 ) Isolation and characterization of Enterobacter cloacae mutants which are defective in chemotaxis toward inorganic phosphate. PMID : 9324271 : DOI : 10.1128/jb.179.19.6192-6195.1997 PMC : PMC179527 Abstract >>
Enterobacter cloacae IFO3320 is attracted to Pi when cells are starved for Pi. Two Tn1737KH-induced mutants, which were constitutive for alkaline phosphatase, failed to exhibit Pi taxis even under conditions of Pi limitation. Both of the mutant strains exhibited normal chemotactic responses to peptone, suggesting that they are specifically defective in Pi taxis. Cloning and sequence analysis showed that the TN1737KH insertions were located in either the pstA or pstB genes which encode the channel-forming proteins of the Pi-specific transport (Pst) system in E. cloacae. These results suggest that the E. cloacae Pst system is required for Pi chemoreception.
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171. |
( 1997 ) Detection of mutations in the gyrA and parC genes in quinolone-resistant clinical isolates of Enterobacter cloacae. PMID : 9372424 : DOI : 10.1093/jac/40.4.543 Abstract >>
We have determined partial sequences of the gyrA and parC genes of Enterobacter cloacae type strain including the regions analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene. The deduced 65- and 49-amino acid sequences of the determined regions of the E. cloacae gyrA and parC genes were identical to the corresponding regions of the E. coli GyrA and ParC proteins, respectively. We examined 40 clinical strains of E. cloacae isolated from patients with urinary tract infection for susceptibilities to nalidixic acid and ciprofloxacin. Based on the nalidixic acid and ciprofloxacin MICs, these isolates were divided into 19 quinolone-susceptible strains (MICs of nalidixic acid, 3.13-25 mg/L; MICs of ciprofloxacin, < or = 0.025 mg/L) and 21 quinolone-resistant strains (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-100 mg/L). We analysed five quinolone-susceptible and 21 quinolone-resistant strains for alterations in GyrA and ParC. The five quinolone-susceptible strains had amino acid sequences in GyrA and ParC identical to those of type strain. Of the 21 quinolone-resistant isolates, three (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-3.13 mg/L) had a single amino acid change at the position equivalent to Ser-83 in the E. coli GyrA protein and no alterations in ParC; one (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 3.13 mg/L) had a single amino acid change at Ser-83 in GyrA and a single amino acid change at the position equivalent to Glu-84 in the E. coli ParC protein; two (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 25 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and no alterations in ParC; and 15 (MICs of nalidixic acid, > 800 mg/L; MICs of ciprofloxacin, 25-100 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and a single amino acid change at Ser-80 or Glu-84 in ParC. This study suggests, that in clinical isolates of E. cloacae, DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to nalidixic acid and to decrease susceptibility to ciprofloxacin, and that the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing high-level resistance to ciprofloxacin.
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172. |
( 1997 ) Intercontinental spread of promiscuous mercury-resistance transposons in environmental bacteria. PMID : 9159519 : DOI : 10.1046/j.1365-2958.1997.3261688.x Abstract >>
We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic-resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site-specific recombination. Our data suggest that the mercury-resistance operons of Gram-negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.
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173. |
( 1996 ) Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. PMID : 8878585 : PMC : PMC163477 Abstract >>
In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.
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174. |
( 1996 ) Phylogenetic analysis of tnpR genes in mercury resistant soil bacteria: the relationship between DNA sequencing and RFLP typing approaches. PMID : 8870257 : DOI : 10.1111/j.1574-6968.1996.tb08514.x Abstract >>
The diversity of resolvase (tnpR) genes carried by a number of mercury resistant soil bacteria has been investigated by DNA sequencing. The resulting DNA sequence information was compared to previously published tnpR DNA sequences and to previously published restriction fragment length polymorphism (RFLP) data, permitting the relationships between DNA sequencing and RFLP approaches to be studied by the use of phylogenetic trees. DNA maximum likelihood and DNA parsimony were used to construct a variety of phylogenetic trees. DNA sequencing confirmed the validity of RFLP analysis and highlighted the importance of restriction endonuclease choice upon the resulting RFLP patterns and dendrogram topology. The tnpR genes of two previously uncharacterised mercury resistant bacteria, T2-7 and T2-12 were also studied. DNA sequence data placed T2-7 in a previously described gene class, tnpR-D and T2-12 in a new gene class, tnpR-F. The significance of this data with respect to the recombination and evolution events occurring within bacterial populations are discussed.
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175. |
( 1996 ) Sequencing and analysis of four new Enterobacter ampD Alleles. PMID : 8843314 : PMC : PMC163450 Abstract >>
Sequences of ampD genes from wild-type, temperature-sensitive, and stably derepressed mutants of the wild-type strain of Enterobacter cloacae 029 and the hyperinducible strain E. cloacae 1194E were determined and compared with the ampD gene of the wild-type strain E. cloacae 14. Seventy nucleotide differences were found between the wild-type sequences, resulting in 13 amino acid changes. The deduced amino acid changes do not correspond to published AmpC regulation mutations and expand the number of known mutations leading to altered AmpC beta-lactamase expression in members of the family Enterobacteriaceae.
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176. |
( 1996 ) Enterobacter cloacae producing a Shiga-like toxin II-related cytotoxin associated with a case of hemolytic-uremic syndrome. PMID : 8789041 : PMC : PMC228823 Abstract >>
Two Shiga-like toxin-producing organisms were isolated from the feces of an infant with hemolytic-uremic syndrome by PCR followed by colony blot hybridization. One strain was identified as Escherichia coli OR:H9, while the other was identified as Enterobacter cloacae. Both isolates were highly cytotoxic for Vero cells, and Southern hybridization analysis of chromosomal DNA indicated that both contained a single slt-II-related gene and that these genes were located on similarly sized restriction fragments. Nucleotide sequence analysis indicated that the toxin encoded by the E. cloacae slt-II-related gene was very similar to Shiga-like toxin II variants from E. coli, differing from the most closely related toxin by 3 residues in the A subunit.
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177. |
( 1996 ) Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2. PMID : 8932320 : DOI : 10.1128/jb.178.22.6623-6627.1996 PMC : PMC178550 Abstract >>
Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one.
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178. |
( 1996 ) Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. PMID : 8919782 : PMC : PMC167887 Abstract >>
A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.
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179. |
( 1994 ) Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a beta-lactamase transition-state analog. PMID : 8204611 : DOI : 10.1021/bi00188a004 Abstract >>
The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogous beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amino group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4A); Lys67 and Lys315 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr150 and Lys67 are more closely associated with Ser64O gamma (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.
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180. |
( 1994 ) The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae. PMID : 8208606 : DOI : 10.1093/nar/22.10.1821 PMC : PMC308080 Abstract >>
Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.
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181. |
( 1993 ) Sequences of wild-type and mutant ampD genes of Citrobacter freundii and Enterobacter cloacae. PMID : 8383940 : DOI : 10.1128/aac.37.2.224 PMC : PMC187643 Abstract >>
The ampD gene product regulates the expression of AmpC beta-lactamase in gram-negative bacteria and is proposed to be involved in peptidoglycan metabolism. In this study, we sequenced the ampD wild type and three mutant genes of Enterobacter cloacae and Citrobacter freundii. They exhibited a high degree of homology with the corresponding gene of Escherichia coli except in the carboxy termini, where, in the wild-type genes of E. cloacae and C. freundii, four additional amino acids yielding the Ser-X-X-Lys motif were found. Evidence that this C-terminal region of the ampD gene product is necessary for activity was shown by constructing a deletion of the last 16 amino acids. The spontaneous mutation of ampD02 is an out-of-frame insertion and yields an inactive AmpD protein. The single-base-pair substitution of Gly for Asp-121 in ampD05 is responsible for a hyperinducible phenotype. These results demonstrate regions of the ampD gene and the corresponding protein which have functional importance for the induction of AmpC beta-lactamase in E. cloacae.
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182. |
( 1993 ) Analysis of the aac(3)-VIa gene encoding a novel 3-N-acetyltransferase. PMID : 8257126 : DOI : 10.1128/aac.37.10.2074 PMC : PMC192231 Abstract >>
Biochemical analysis (G. A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N-acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it was fourfold lower than that of 6'-N-ethylnetilmicin, a resistance pattern which suggested 2'-acetylating activity. However, high-pressure liquid chromatography analysis demonstrated that the enzyme acetylated sisomicin in the 3 position. We have cloned the structural gene for this enzyme from a large (> 70-kb) conjugative plasmid present in E. cloacae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1-kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino acid identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-VII protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene and was present in an integron. In addition, the aac(3)-VIa gene is also downstream of a 59-base element often seen in an integron environment. Primer extension analysis has identified a promoter for the aac(3)-VIa gene downstream of both the aadA1 gene and a 59-base element.
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183. |
( 1993 ) Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. PMID : 8248237 : DOI : 10.1073/pnas.90.23.11257 PMC : PMC47961 Abstract >>
The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.
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184. |
( 1994 ) Analysis of a carbapenem-hydrolyzing class A beta-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein. PMID : 8052644 : DOI : 10.1073/pnas.91.16.7693 PMC : PMC44468 Abstract >>
Carbapenems such as imipenem are extended-spectrum beta-lactam antibiotics, which are not hydrolyzed by the beta-lactamases commonly found in Enterobacteriaceae. Here we report a gene encoding a carbapenemase, which was cloned from the chromosome of a clinical isolate of Enterobacter cloacae, strain NOR-1, into pACYC184 plasmid in Escherichia coli. Unlike all the sequenced carbapenemases, which are class B metallo-beta-lactamases, the mature protein (NmcA) is a class A serine beta-lactamase. NmcA shares the highest amino acid identity (50%) with the extended-spectrum class A beta-lactamase MEN-1 from E. coli. In the opposite orientation from the nmcA promoter, an overlapping and divergent promoter was detected, along with an open reading frame, which encoded a 33.5-kDa protein (NmcR). The NmcR amino acid sequence displays homology with LysR-type transcriptional regulatory proteins, including the conserved residues near its N terminus within a helix-turn-helix motif. Deletion of nmcR resulted in decreased carbapenem resistance and a loss of beta-lactamase inducibility, demonstrating a positive role of NmcR in NmcA expression.
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185. |
Nukaga M,
Haruta S,
Tanimoto K,
Kogure K,
Taniguchi K,
Tamaki M,
Sawai T,
( 1995 ) Molecular evolution of a class C beta-lactamase extending its substrate specificity. PMID : 7890700 : DOI : 10.1074/jbc.270.11.5729 Abstract >>
Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.
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186. |
Deng X,
Shen S,
( 1995 ) Structure and oxygen sensitivity of nifLA promoter of Enterobacter cloacae. PMID : 7695817 : Abstract >>
The nucleotide sequence of the nifLA promoter of Enterobacter cloacae E26 was determined, and the transcription start site of nifLA was mapped by the primer extension. Studies on the oxygen regulation of E. cloacae nifLA promoter with the nifL'-lacZ fusion showed that the nifLA promoter was sensitive to oxygen as Klebsiella pneumoniae nifLA promoter reported previously. Comparison between the nifLA promoter sequences of E. cloacae and K. pneumoniae revealed the presence of an 11-bp conserved sequence between the -24/-12 consensus of sigma 54-dependent promoter and NtrC binding motif. The 11-bp sequence is speculated to be involved in the oxygen regulation of the nifLA promoter of both enteric bacteria.
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187. |
Zimmer W,
Hundeshagen B,
Niederau E,
( 1994 ) Demonstration of the indolepyruvate decarboxylase gene homologue in different auxin-producing species of the Enterobacteriaceae. PMID : 7704833 : DOI : 10.1139/m94-170 Abstract >>
Different Enterobacteriaceae were assayed for their ability to produce the plant hormone indole-3-acetate with the aim to study the distribution of the indole-3-pyruvate pathway, which is known to be involved in the production of indole-3-acetate in a root-associated Enterobacter cloacae strain. Other E. cloacae strains, and also Enterobacter agglomerans strains, Pantoea agglomerans, Klebsiella aerogenes, and Klebsiella oxytoca were found to convert tryptophan into indole-3-acetate. As it was also intended to identify the conserved regions of the indole-3-pyruvate decarboxylase, which is involved in producing indole-3-acetate in the E. cloacae strain, oligonucleotide primers were synthesized for different regions of the corresponding gene. One pair of these primers allowed us to amplify a segment of the predicted size by the polymerase chain reaction with DNA of the seven different Enterobacteriaceae that produce indole-3-acetate. Segments of five strains were cloned and sequenced. All sequences showed significant homology to the indole-3-pyruvate decarboxylase gene. As in addition a positive DNA-DNA hybridization signal was detected in the seven strains using the E. cloacae or E. agglomerans segments as a probe, indole-3-acetate biosynthesis is suggested to be catalyzed via the indole-3-pyruvate pathway not only in E. cloacae but also in the other soil-living Enterobacteriaceae. Conserved regions were detected in the indole-3-decarboxylase by alignment of the now-available five different partial sequences. These regions should enable identification of the gene in other bacterial families or even in plants.
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188. |
Nagano H,
Kawaguchi T,
Omori M,
Shoji Z,
Arai M,
( 1994 ) Molecular cloning and nucleotide sequence of the beta-galactosidase gene from Enterobacter cloacae GAO. PMID : 7765512 : Abstract >>
The gene encoding a beta-galactosidase from Enterobacter cloacae GAO was cloned and expressed in Escherichia coli. The nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons. The amino acid sequence of beta-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed significant homology to beta-galactosidases of other microorganisms, E. coli, Klebsiella pneumoniae, Lactobacillus bulgaricus, and Clostridium acetobutylicum.
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189. |
Nakahigashi K,
Yanagi H,
Yura T,
( 1995 ) Isolation and sequence analysis of rpoH genes encoding sigma 32 homologs from gram negative bacteria: conserved mRNA and protein segments for heat shock regulation. PMID : 7501460 : PMC : PMC307394 Abstract >>
The rpoH genes encoding homologs of Escherichia coli sigma 32 (heat shock sigma factor) were isolated and sequenced from five gram negative proteobacteria (gamma or alpha subgroup): Enterobacter cloacae (gamma), Serratia marcescens (gamma), Proteus mirabilis (gamma), Agrobacterium tumefaciens (alpha) and Zymomonas mobilis (alpha). Comparison of these and three known genes from E.coli (gamma), Citrobacter freundii (gamma) and Pseudomonas aeruginosa (gamma) revealed marked similarities that should reflect conserved function and regulation of sigma 32 in the heat shock response. Both the sequence complementary to part of 16S rRNA (the 'downstream box') and a predicted mRNA secondary structure similar to those involved in translational control of sigma 32 in E.coli were found for the rpoH genes from the gamma, but not the alpha, subgroup, despite considerable divergence in nucleotide sequence. Moreover, a stretch of nine amino acid residues Q(R/K)(K/R)LFFNLR, designated the 'RpoH box', was absolutely conserved among all sigma 32 homologs, but absent in other sigma factors; this sequence overlapped with the segment of polypeptide thought to be involved in DnaK/DnaJ chaperone-mediated negative control of synthesis and stability of sigma 32. In addition, a putative sigma E (sigma 24)-specific promoter was found in front of all rpoH genes from the gamma, but not alpha, subgroup. These results suggest that the regulatory mechanisms, as well as the function, of the heat shock response known in E.coli are very well conserved among the gamma subgroup and partially conserved among the alpha proteobacteria.
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190. |
Sacerdot C,
Dessen P,
Hershey JW,
Plumbridge JA,
Grunberg-Manago M,
( 1984 ) Sequence of the initiation factor IF2 gene: unusual protein features and homologies with elongation factors. PMID : 6096856 : DOI : 10.1073/pnas.81.24.7787 PMC : PMC392237 Abstract >>
The gene for protein synthesis initiation factor IF2 in Escherichia coli, infB, is located downstream from nusA on the same operon. We sequenced about 3 kilobases of DNA beginning within nusA and including the entire infB structural gene plus another 392 bases downstream. This region contains no obvious strong promoter signals, but a possible transcriptional termination or pausing site occurs downstream from infB. The putative initiator codon for IF2 alpha (97,300 daltons) is AUG; that for IF2 beta (79,700 daltons) is GUG, located 471 bases downstream in the same reading frame. The codon usage for IF2 is typical of other highly expressed proteins in E. coli and suggests that IF2 mRNA is efficiently translated. IF2 alpha contains two adjacent regions (residues 104-155 and 167-214) that are rich in alanine and charged amino acids and that show striking periodicities in their sequences. These regions may alternate between flexible and helical conformations, thereby drawing together the NH2-terminal and COOH-terminal globular domains of the factor as IF2 interacts with ribosomes or tRNA. Certain regions of the DNA and protein sequences of IF2 share strong homologies with elongation factor EF-Tu and lesser homology with EF-G. In particular, a region of EF-Tu implicated in GTP binding contains sequences and secondary structure that are conserved in IF2. The homologies indicate that the genes for IF2 and the elongation factors are derived at least in part from a common ancestor.
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191. |
Dammel CS,
Noller HF,
( 1995 ) Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA. PMID : 7535280 : DOI : 10.1101/gad.9.5.626 Abstract >>
A novel 15-kDa protein, RbfA, has been identified by virtue of its ability to act as a high copy suppressor of a previously characterized dominant cold-sensitive mutation (C23U) in 16S rRNA. RbfA is found associated with free 30S ribosomal subunits, but not with 70S ribosomes or polysomes, and is essential for maximal cell growth, particularly at low temperatures. Cells lacking RbfA in a wild-type rRNA background exhibit a cold-sensitive phenotype that is strikingly similar to that of the cold-sensitive C23U rRNA mutant. The observed patterns of allele specificity of suppression and synthetic lethality in cells containing an RbfA knockout in combination with various 16S rRNA mutations suggests that RbfA interacts with the 5'-terminal helix region of 16S rRNA, possibly during a late step of 30S maturation.
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192. |
George AM,
Hall RM,
Stokes HW,
( 1995 ) Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli. PMID : 7551053 : DOI : 10.1099/13500872-141-8-1909 Abstract >>
Spontaneous multidrug-resistant (Mdr) mutants of Klebsiella pneumoniae strain ECL8 arose at a frequency of 2.2 x 10(-8) and showed increased resistance to a range of unrelated antibiotics, including chloramphenicol, tetracycline, nalidixic acid, ampicillin, norfloxacin, trimethoprim and puromycin. A chromosomal fragment from one such mutant was cloned, and found to confer an Mdr phenotype on Escherichia coli K12 cells that was essentially identical to that of the K. pneumoniae mutant. Almost complete loss of the OmpF porin in the E. coli transformant, and of the corresponding porin in the K. pneumoniae mutant, was observed. The presence of the Mdr mutation in K. pneumoniae or the cloned K. pneumoniae ramA (resistance antibiotic multiple) locus in E. coli also resulted in active efflux of tetracycline, and increased active efflux of chloramphenicol. After transformation of a ramA plasmid into E. coli, expression of chloramphenicol resistance occurred later than expression of resistance to tetracycline, puromycin, trimethoprim and nalidixic acid. The ramA gene was localized and sequenced. It encodes a putative positive transcriptional activator that is weakly related to the E. coli MarA and SoxS proteins. A ramA gene was also found to be present in an Enterobacter cloacae fragment that has previously been shown to confer an Mdr phenotype, and it appears that ramA, rather than the romA gene identified in that study, is responsible for multidrug resistance. The ramA gene from the wild-type K. pneumoniae was identical to that of the mutant strain and also conferred an Mdr phenotype on E. coli, indicating that the mutation responsible for Mdr in K. pneumoniae had not been cloned.
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193. |
Ishii S,
Ihara M,
Maekawa T,
Nakamura Y,
Uchida H,
Imamoto F,
( 1984 ) The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia coli. PMID : 6326058 : DOI : 10.1093/nar/12.7.3333 PMC : PMC318749 Abstract >>
The nucleotide sequence of the promoter-proximal portion of the nusA operon including the genes for tRNAMetf2, a 15 kilodalton protein and the initial portion of the nusA gene has been determined previously (1). Here, we report the sequence for the entire nusA gene and its flanking region. The open reading frame, consisting of 1,482 nucleotides, was identified as that of the nusA protein on the basis of agreement of the amino acid sequence deduced from the DNA sequence with the N-terminal sequence of the purified nusA protein. The molecular weight of 54,417 daltons calculated for the 494 amino acid polypeptide is significantly lower than that determined previously by SDS polyacrylamide gel analysis. The nusA gene is immediately followed by another open reading frame encoding a polypeptide of at least 22 amino acids, which was identified as the initial portion of the infB structural gene. In the spacer region of 24 base pairs between the nusA and infB structural genes there is no significant DNA sequence that fits the canonical transcriptional termination signal or promoter sequence. We suggest, therefore, that the genes for tRNAMetf2, a 15 kilodalton protein, the nusA protein and IF2 alpha, aligned in this order, are co-transcribed.
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194. |
Owens TW,
Taylor RJ,
Pahil KS,
Bertani BR,
Ruiz N,
Kruse AC,
Kahne D,
( 2019 ) Structural basis of unidirectional export of lipopolysaccharide to the cell surface. PMID : 30894747 : DOI : 10.1038/s41586-019-1039-0 PMC : PMC6629255 Abstract >>
Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.
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195. |
Kotsakis SD,
Flach CF,
Razavi M,
Larsson DGJ,
( 2019 ) Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity. PMID : 30397053 : DOI : 10.1128/AAC.01817-18 PMC : PMC6325193 Abstract >>
While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, �]-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D �]-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional �]-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural bla OXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the k cat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-�]-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.
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196. |
Iorgu AI,
Baxter NJ,
Cliff MJ,
Levy C,
Waltho JP,
Hay S,
Scrutton NS,
( 2018 ) Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes. PMID : 31119061 : DOI : 10.1021/acscatal.8b02810 PMC : PMC6516726 Abstract >>
Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR-coenzyme complexes. We find that mutagenesis of these second sphere "noncatalytic" residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR-NADH and PETNR-NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy-enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.
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197. |
Galleni M,
Lindberg F,
Normark S,
Cole S,
Honore N,
Joris B,
Frere JM,
( 1988 ) Sequence and comparative analysis of three Enterobacter cloacae ampC beta-lactamase genes and their products. PMID : 3260487 : DOI : 10.1042/bj2500753 PMC : PMC1148921 Abstract >>
The sequences of three Enterobacter cloacae ampC beta-lactamase genes have been determined. The deduced amino acid sequences are very similar: out of a total of 361 residues, only eight positions were found to be variable, and several mutations yielded residues with very similar properties. The kinetic properties of two of the enzymes were not significantly different. The three enzymes also exhibited a high degree of homology (greater than 70%) with the ampC beta-lactamases of Escherichia coli K12 and Citrobacter freundii, confirming the homogeneity of class-C beta-lactamases.
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198. |
Adhav A,
Harne S,
Bhide A,
Giri A,
Gayathri P,
Joshi R,
( 2019 ) Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae. PMID : 30657252 : DOI : 10.1111/febs.14760 Abstract >>
Energy metabolism in the diamondback moth Plutella xylostella is facilitated by trehalase, an enzyme which assists in trehalose hydrolysis, from the predominant gut bacterium Enterobacter cloacae. We report the biochemical and structural characterization of recombinant trehalase from E. cloacae (Px_EclTre). Px_EclTre showed KM of 1.47 (��0.05) mm, kcat of 6254.72 min-1 and Vmax 0.2 (��0.002) mm�Pmin-1 at 55 �XC and acidic pH. Crystal structures of Px_EclTre were determined in the ligand-free form and bound to the inhibitor Validoxylamine A. The crystal structure of the ligand-free form, unavailable until now for any other bacterial trehalases, enabled us to delineate the conformational changes accompanying ligand binding in trehalases. Multiple salt bridges were identified that potentially facilitated closure of a hood over the substrate-binding site. A cluster of five tryptophans lined the -1 substrate-binding subsite, interacted with crucial active site residues and contributed to both trehalase activity and stability. The importance of these residues in enzyme activity was further validated by mutagenesis studies. Many of these identified residues form part of signature motifs and other conserved sequences in trehalases. The structure analysis thus led to the assignment of the functional role to these conserved residues. This information can be further explored for the design of effective inhibitors against trehalases.
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199. |
Van der Ley P,
Bekkers A,
Van Meersbergen J,
Tommassen J,
( 1987 ) A comparative study on the phoE genes of three enterobacterial species. Implications for structure-function relationships in a pore-forming protein of the outer membrane. PMID : 3032618 : DOI : 10.1111/j.1432-1033.1987.tb11080.x Abstract >>
The cloned phoE genes from Enterobacter cloacae and Klebsiella pneumoniae are normally expressed and regulated in Escherichia coli K-12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell-surface-exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K. pneumoniae phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non-coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81% of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure-function relationships in PhoE protein are discussed.
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200. |
Kumar P,
Serpersu EH,
Cuneo MJ,
( 2018 ) A low-barrier hydrogen bond mediates antibiotic resistance in a noncanonical catalytic triad. PMID : 29632894 : DOI : 10.1126/sciadv.aas8667 PMC : PMC5884680 Abstract >>
One group of enzymes that confer resistance to aminoglycoside antibiotics through covalent modification belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. We show how a unique GNAT subfamily member uses a previously unidentified noncanonical catalytic triad, consisting of a glutamic acid, a histidine, and the antibiotic substrate itself, which acts as a nucleophile and attacks the acetyl donor molecule. Neutron diffraction studies allow for unambiguous identification of a low-barrier hydrogen bond, predicted in canonical catalytic triads to increase basicity of the histidine. This work highlights the role of this unique catalytic triad in mediating antibiotic resistance while providing new insights into the design of the next generation of aminoglycosides.
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201. |
Honoré N,
Nicolas MH,
Cole ST,
( 1986 ) Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. PMID : 3030737 : PMC : PMC1167415 Abstract >>
Cephalosporin hyper-resistant Enterobacter cloacae strains are isolated with increasing frequency from hospital infections. Resistance is principally due to the chromosomal ampC gene encoding a cephalosporinase. In contrast to Escherichia coli which expresses ampC constitutively from a promoter located in the upstream frdD gene, E. cloacae displays inducible ampC expression. By cloning the ampC gene it was shown that a linked genetic locus, ampR, mediated the induction by beta-lactams. In the absence of the antibiotic the 30,500 dalton AmpR protein represses ampC expression. The ampR gene shows a highly compact arrangement and is situated between the divergently expressed ampC gene and the frd operon from which it is separated by a bifunctional transcription terminator. The promoters for ampR and ampC substantially overlap and mRNA analyses showed that on induction transcription from the ampC promoter increased greatly whereas that from ampR did not. Two regions of sequence homology flank the ampR gene and it is proposed that a homologous recombination event between these in an ancestral enteric bacterium may have led to the deletion of ampR from the E. coli genome.
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202. |
Teo JWP,
Kalisvar M,
Venkatachalam I,
Ng OT,
Lin RTP,
Octavia S,
( 2018 ) mcr-3 and mcr-4 Variants in Carbapenemase-Producing Clinical Enterobacteriaceae Do Not Confer Phenotypic Polymyxin Resistance. PMID : 29237785 : DOI : 10.1128/JCM.01562-17 PMC : PMC5824041 Abstract >>
N/A
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203. |
Phan HTT,
Stoesser N,
Maciuca IE,
Toma F,
Szekely E,
Flonta M,
Hubbard ATM,
Pankhurst L,
Do T,
Peto TEA,
Walker AS,
Crook DW,
Timofte D,
( 2018 ) Illumina short-read and MinION long-read WGS to characterize the molecular epidemiology of an NDM-1 Serratia marcescens outbreak in Romania. PMID : 29237003 : DOI : 10.1093/jac/dkx456 PMC : PMC5890751 Abstract >>
Serratia marcescens is an emerging nosocomial pathogen, and the carbapenemase blaNDM has been reported in several surveys in Romania. We aimed to investigate the molecular epidemiology of S. marcescens in two Romanian hospitals over 2010-15, including a neonatal NDM-1 S. marcescens outbreak. Isolates were sequenced using Illumina technology together with carbapenem-non-susceptible NDM-1-positive and NDM-1-negative Klebsiella pneumoniae and Enterobacter cloacae to provide genomic context. A subset was sequenced with MinION to fully resolve NDM-1 plasmid structures. Resistance genes, plasmid replicons and ISs were identified in silico for all isolates; an annotated phylogeny was reconstructed for S. marcescens. Fully resolved study NDM-1 plasmid sequences were compared with the most closely related publicly available NDM-1 plasmid reference. 44/45 isolates were successfully sequenced (S. marcescens, n = 33; K. pneumoniae, n = 7; E. cloacae, n = 4); 10 with MinION. The S. marcescens phylogeny demonstrated several discrete clusters of NDM-1-positive and -negative isolates. All NDM-1-positive isolates across species harboured a pKOX_NDM1-like plasmid; more detailed comparisons of the plasmid structures demonstrated a number of differences, but highlighted the largely conserved plasmid backbones across species and hospital sites. The molecular epidemiology is most consistent with the importation of a pKOX_NDM1-like plasmid into Romania and its dissemination amongst K. pneumoniae/E. cloacae and subsequently S. marcescens across hospitals. The data suggested multiple acquisitions of this plasmid by S. marcescens in the two hospitals studied; transmission events within centres, including a large outbreak on the Targu Mures neonatal unit; and sharing of the pKOX_NDM1-like plasmid between species within outbreaks.
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204. |
Azevedo PAA,
Furlan JPR,
Oliveira-Silva M,
Nakamura-Silva R,
Gomes CN,
Costa KRC,
Stehling EG,
Pitondo-Silva A,
( 2018 ) Detection of virulence and �]-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil. PMID : 29858139 : DOI : 10.1016/j.bjm.2018.04.009 PMC : PMC6328715 Abstract >>
Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several �]-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none.
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205. |
( 2013 ) An antibiotic-resistant class 3 integron in an Enterobacter cloacae isolate from hospital effluent. PMID : 23458448 : DOI : 10.1111/1469-0691.12186 Abstract >>
Hospital effluents are involved in dissemination of antibiotic-resistant integrons. We describe here a new class 3 integron, In3-5, detected in an Enterobacter cloacae isolate retrieved from a random French hospital effluent sample collected in 2009. In3-5 carries two gene cassettes: the new blaOXA -256 and an aac(6')-Ib variant, respectively conferring resistance to �]-lactams and aminoglycosides. In3-5 is located on an IncQ-like backbone plasmid. Class 3 integrons could thus be involved in the dissemination of antibiotic resistance in both clinical settings and the environment, and could participate in the exchange of antibiotic-resistance genes between these two ecosystems.
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206. |
( 2013 ) Emergence and spread of NDM-1 producer Enterobacteriaceae with contribution of IncX3 plasmids in the United Arab Emirates. PMID : 23579399 : DOI : 10.1099/jmm.0.059014-0 Abstract >>
Among 28 clinically relevant, carbapenem non-susceptible Enterobacteriaceae isolates collected in 2009-2011 in the United Arab Emirates three Klebsiella pneumoniae, two Escherichia coli, one Enterobacter cloacae and one Citrobacter freundi were identified to produce NDM-1 carbapenemase. Unexpectedly, with the exception of a K. pneumoniae strain, sequence type ST11, originally acquired in India and subsequently spread nosocomially in the UAE, the majority of the strains could not be directly linked to foreign travel. All isolates harboured the blaNDM-1 gene on plasmids of IncA/C, IncHI1b and IncX3 types, or were untypable. IncX3 type plasmids with a mass of 50 kb and with the same or highly similar restriction patterns, with regions flanking the blaNDM-1 gene identical to the IncX3 NDM plasmids described from China were present in three different species, Enterobacter cloacae, Escherichia coli and C. freundii. Our findings strongly support the assumptions that, beyond the Indian subcontinent, the Middle East is an important reservoir of NDM-producing organisms. Furthermore, we also provide evidence that IncX3 plasmids, recently implicated in the spread of blaNDM-1 in China, have been widely distributed and are important vehicles of the inter-species spread of the NDM-1 gene.
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207. |
Lund BA,
Thomassen AM,
Carlsen TJO,
Leiros HKS,
( 2017 ) Structure, activity and thermostability investigations of OXA-163, OXA-181 and OXA-245 using biochemical analysis, crystal structures and differential scanning calorimetry analysis. PMID : 28994407 : DOI : 10.1107/S2053230X17013838 Abstract >>
The first crystal structures of the class D �]-lactamases OXA-181 and OXA-245 were determined to 2.05 and 2.20 ? resolution, respectively; in addition, the structure of a new crystal form of OXA-163 was resolved to 2.07 ? resolution. All of these enzymes are OXA-48-like and have been isolated from different clinical Klebsiella pneumoniae strains and also from other human pathogens such as Pseudomonas aeruginosa and Escherichia coli. Here, enzyme kinetics and thermostability studies are presented, and the new crystal structures are used to explain the observed variations. OXA-245 had the highest melting point (Tm = 55.8�XC), as determined by differential scanning calorimetry, compared with OXA-163 (Tm = 49.4�XC) and OXA-181 (Tm = 52.6�XC). The differences could be explained by the loss of two salt bridges in OXA-163, and an overall decrease in the polarity of the surface of OXA-181 compared with OXA-245.
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208. |
( 2013 ) Biochemical characterization of CTX-M-15 from Enterobacter cloacae and designing a novel non-�]-lactam-�]-lactamase inhibitor. PMID : 23437273 : DOI : 10.1371/journal.pone.0056926 PMC : PMC3578935 Abstract >>
The worldwide dissemination of CTX-M type �]-lactamases is a threat to human health. Previously, we have reported the spread of bla(CTX-M-15) gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other �]-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5�\ transformed with bla(CTX-M-15) gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional �]-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC(50) value (6 nM), high affinity (K(i) = 0.017 ?M) and better acylation efficiency (k(+2)/K' = 0.44 ?M(-1)s(-1)). It forms an acyl-enzyme covalent complex, which is quite stable (k(+3) = 0.0057 s(-1)). Since increasing resistance has been reported against conventional �]-lactam antibiotic-inhibitor combinations, we aspire to design a non-�]-lactam core containing �]-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-�]-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it's IC(50) (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K(i) = 0.388 ?M). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-�]-lactam containing �]-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.
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209. |
Ahmad N,
Ali SM,
Khan AU,
( 2018 ) Detection of New Delhi Metallo-�]-Lactamase Variants NDM-4, NDM-5, and NDM-7 in Enterobacter aerogenes Isolated from a Neonatal Intensive Care Unit of a North India Hospital: A First Report. PMID : 28613981 : DOI : 10.1089/mdr.2017.0038 Abstract >>
A total 402 Enterobacteriaceae isolates were recovered from blood and rectal swabs of 1,000 infants admitted to the Neonatal Intensive Care Unit (NICU) of the Jawaharlal Medical College and Hospital Aligarh, India. Carbapenamase producers were determined by Carba NP phenotype biochemical assay. Out of 402 isolates, it was the first time three of the isolates were identified as Enterobacter aerogenes carrying blaNDM-4, blaNDM-5, and blaNDM-7 genes. These genes were identified by polymerase chain reaction (PCR) and sequence analysis. The isolates were further characterized to know the plasmid type and genetic environment features, including integron and IS elements. All the three E. aerogenes isolates (AK-93, AK-95, and AK-96) were resistant to all �]-lactams, including carbapenems. The �]-lactamase genes blaOXA-1, blaOXA-9, blaSHV-1, and blaVIM-2 were also found to be coassociated with blaNDM-4 in AK-93, blaOXA-1, blaOXA-9, and blaCMY-149 were found to be coexisted with blaNDM-5 in AK-95, and blaOXA-1; blaOXA-9, and blaCMY-145 were also found to be coassociated with blaNDM-7 in AK-96, identified by PCR analysis. Plasmid-based replicon typing revealed plasmids of different incompatibility in E. aerogenes in each of the isolates, AK-93 AK-95, and AK-96, respectively. ERIC-PCR was performed for the analysis of genetic relatedness of the strains. We found blaNDM-4, blaNDM-5, and blaNDM-7 producing three E. aerogenes strains, which were not clonally related. Genetic environment analysis revealed the presence of bleomycin resistance gene (bleMBL) to downstream of blaNDM and complete ISAba125 sequence were found upstream of blaNDM in all the three variants of these isolates. This is the first time we have identified blaNDM-4, blaNDM-5, and blaNDM-7 in E. aerogenes species, isolated from the NICU of a tertiary care hospital in India.
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210. |
Payer SE,
Marshall SA,
Bärland N,
Sheng X,
Reiter T,
Dordic A,
Steinkellner G,
Wuensch C,
Kaltwasser S,
Fisher K,
Rigby SEJ,
Macheroux P,
Vonck J,
Gruber K,
Faber K,
Himo F,
Leys D,
Pavkov-Keller T,
Glueck SM,
( 2017 ) Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase. PMID : 28857436 : DOI : 10.1002/anie.201708091 PMC : PMC5656893 Abstract >>
The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.
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211. |
( 2012 ) Effect of transcriptional activators SoxS, RobA, and RamA on expression of multidrug efflux pump AcrAB-TolC in Enterobacter cloacae. PMID : 23006750 : DOI : 10.1128/AAC.01085-12 PMC : PMC3497196 Abstract >>
Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
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212. |
( 2012 ) VIM-4 carbapenemase-producing Enterobacter cloacae in the United Arab Emirates. PMID : 23078093 : DOI : 10.1111/1469-0691.12051 Abstract >>
Screening 34 carbapenem non-susceptible Enterobacteriaceae recovered in Abu Dhabi hospitals identified an Enterobacter cloacae strain carrying bla(VIM-4) , bla(CMY-4) and bla(CTX-M-15) . It was isolated from the urine of an Egyptian patient repeatedly hospitalized and treated with broad-spectrum antibiotics, including carbapenems, in the United Arab Emirates. The bla(VIM-4) coding class I integron, highly similar to In416, was carried on a 175-kilobase non-conjugative incA/C type plasmid also hybridizing with the bla(CMY-4) probe. This is the first detailed report on the isolation of a Verona integron-encoded metallo-�]-lactamase (VIM) -producing enteric bacterium in the Arabian Peninsula with characteristics suggestive of spreading from the Mediterranean region.
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213. |
( 2013 ) Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth. PMID : 23212670 : DOI : 10.1007/s00253-012-4574-2 Abstract >>
Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rif(R)) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20 days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rif(R) variants systemically colonized the entire cacao seedlings in 15-20 days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings' height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rif(R) variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings/plantlets production systems was discussed.
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214. |
( 1999 ) Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae. PMID : 9925524 : PMC : PMC89069 Abstract >>
Enterobacter cloacae 8009 produced an inducible class A beta-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other beta-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. The bla gene was transferable to Escherichia coli by electroporation of plasmid DNA. The molecular mass of the beta-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A beta-lactamases like FEC-1. The gene encoding this beta-lactamase was cloned and sequenced. The deduced amino acid sequence of the beta-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A beta-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the beta-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal beta-lactamases from Citrobacter diversus (80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intact ampR produced a small amount of beta-lactamase constitutively, suggesting that AmpR works as an activator of ampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A beta-lactamase in gram-negative bacteria.
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215. |
( 2013 ) A mosaic transposon encoding OXA-48 and CTX-M-15: towards pan-resistance. PMID : 23027715 : DOI : 10.1093/jac/dks397 Abstract >>
N/A
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216. |
( 2012 ) Identification and characterization of two novel bla(KLUC) resistance genes through large-scale resistance plasmids sequencing. PMID : 23056610 : DOI : 10.1371/journal.pone.0047197 PMC : PMC3467222 Abstract >>
Plasmids are important antibiotic resistance determinant carriers that can disseminate various drug resistance genes among species or genera. By using a high throughput sequencing approach, two groups of plasmids of Escherichia coli (named E1 and E2, each consisting of 160 clinical E. coli strains isolated from different periods of time) were sequenced and analyzed. A total of 20 million reads were obtained and mapped onto the known resistance gene sequences. As a result, a total of 9 classes, including 36 types of antibiotic resistant genes, were identified. Among these genes, 25 and 27 single nucleotide polymorphisms (SNPs) appeared, of which 9 and 12 SNPs are nonsynonymous substitutions in the E1 and E2 samples. It is interesting to find that a novel genotype of bla(KLUC), whose close relatives, bla(KLUC-1) and bla(KLUC-2), have been previously reported as carried on the Kluyvera cryocrescens chromosome and Enterobacter cloacae plasmid, was identified. It shares 99% and 98% amino acid identities with Kluc-1 and Kluc-2, respectively. Further PCR screening of 608 Enterobacteriaceae family isolates yielded a second variant (named bla(KLUC-4)). It was interesting to find that Kluc-3 showed resistance to several cephalosporins including cefotaxime, whereas bla(KLUC-4) did not show any resistance to the antibiotics tested. This may be due to a positively charged residue, Arg, replaced by a neutral residue, Leu, at position 167, which is located within an omega-loop. This work represents large-scale studies on resistance gene distribution, diversification and genetic variation in pooled multi-drug resistance plasmids, and provides insight into the use of high throughput sequencing technology for microbial resistance gene detection.
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( 2013 ) Characterisation and clinical features of Enterobacter cloacae bloodstream infections occurring at a tertiary care university hospital in Switzerland: is cefepime adequate therapy? PMID : 23313399 : DOI : 10.1016/j.ijantimicag.2012.10.022 PMC : PMC4018813 Abstract >>
Despite many years of clinical experience with cefepime, data regarding the outcome of patients suffering from bloodstream infections (BSIs) due to Enterobacter cloacae (Ecl) are scarce. To address the gap in our knowledge, 57 Ecl responsible for 51 BSIs were analysed implementing phenotypic and molecular methods (microarrays, PCRs for bla and other genes, rep-PCR to analyse clonality). Only two E. cloacae (3.5%) were ESBL-producers, whereas 34 (59.6%) and 18 (31.6%) possessed inducible (Ind-Ecl) or derepressed (Der-Ecl) AmpC enzymes, respectively. All isolates were susceptible to imipenem, meropenem, gentamicin and ciprofloxacin. Der-Ecl were highly resistant to ceftazidime and piperacillin/tazobactam (both MIC???256 �gg/mL), whereas cefepime retained its activity (MIC?? of 3 �gg/mL). rep-PCR indicated that the isolates were sporadic, but Ecl collected from the same patients were indistinguishable. In particular, three BSIs initially due to Ind-Ecl evolved (under ceftriaxone or piperacillin/tazobactam treatment) into Der-Ecl because of mutations or a deletion in ampD or insertion of IS4321 in the promoter. These last two mechanisms have never been described in Ecl. Mortality was higher for BSIs due to Der-Ecl than Ind-Ecl (3.8% vs. 29.4%; P=0.028) and was associated with the Charlson co-morbidity index (P=0.046). Using the following directed treatments, patients with BSI showed a favourable treatment outcome: cefepime (16/18; 88.9%); carbapenems (12/13; 92.3%); ceftriaxone (4/7; 57.1%); piperacillin/tazobactam (5/7; 71.4%); and ciprofloxacin (6/6; 100%). Cefepime represents a safe therapeutic option and an alternative to carbapenems to treat BSIs due to Ecl when the prevalence of ESBL-producers is low.
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( 2013 ) KPC-4 Is encoded within a truncated Tn4401 in an IncL/M plasmid, pNE1280, isolated from Enterobacter cloacae and Serratia marcescens. PMID : 23070154 : DOI : 10.1128/AAC.01062-12 PMC : PMC3535906 Abstract >>
We describe the transfer of bla(KPC-4) from Enterobacter cloacae to Serratia marcescens in a single patient. DNA sequencing revealed that KPC-4 was encoded on an IncL/M plasmid, pNE1280, closely related to pCTX-M360. Further analysis found that KPC-4 was encoded within a novel Tn4401 element (Tn4401f) containing a truncated tnpA and lacking tnpR, ISKpn7 left, and Tn4401 IRL-1, which are conserved in other Tn4401 transposons. This study highlights the continued evolution of Tn4401 transposons and movement to multiple plasmid backbones that results in acquisition by multiple species of Gram-negative bacilli.
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( 2012 ) pEl1573 Carrying blaIMP-4, from Sydney, Australia, is closely related to other IncL/M plasmids. PMID : 22926566 : DOI : 10.1128/AAC.01189-12 PMC : PMC3486572 Abstract >>
Complete sequencing of pEl1573, a representative IncL/M plasmid carrying bla(IMP-4) from Sydney, Australia, revealed an ?60-kb backbone almost identical to those of IncL/M plasmids pCTX-M3, from Poland, and pCTX-M360, from China, and less closely related to pNDM-HK, pOXA-48a, and pEL60, suggesting different lineages. The ?28-kb Tn2-derived multiresistance region in pEl1573 is inserted in the same location as those in pCTX-M3 and pNDM-HK and shares some of the same components but has undergone rearrangements.
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( 2012 ) Novel ISCR1-linked resistance genes found in multidrug-resistant Gram-negative bacteria in southern China. PMID : 22890194 : DOI : 10.1016/j.ijantimicag.2012.06.016 Abstract >>
Non-duplicate multidrug-resistant (MDR) Gram-negative bacteria (n=1329) isolated from southern China between January 2008 and December 2009 were investigated for the presence of ISCR1 as well as characterisation of ISCR1-linked resistance genes. Of 433 ISCR1-positive strains, 151 appeared to carry ISCR1-linked resistance genes. Seven different ISCR1-linked resistance gene arrays were identified by restriction fragment length polymorphism (RFLP) and DNA sequencing analysis. Many of these arrays are reported in some species for the first time. A total of 12 genes, including a novel ABC transporter (GenBank accession no. GU944725), qnrA1, qnrB2, qnrB6, bla(DHA-1), ampR, bla(CTX-M-9), bla(PER-1), insB, sapA-like peptide transport periplasmic protein, putative glutathione S-transferase and short-chain dehydrogenase/reductase, were detected. This study was the first to employ PCR-RFLP using HinfI and RsaI to analyse ISCR1-linked genes. ISCR1 was widely disseminated among MDR Gram-negative bacteria and was in close association with quinolone resistance and �]-lactamase genes (class A and class C) in southern China.
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( 2012 ) Development of a model system for the study of spoilage associated secondary cucumber fermentation during long-term storage. PMID : 22924596 : DOI : 10.1111/j.1750-3841.2012.02845.x Abstract >>
Calcium chloride fermentations represent an alternative to reduce chloride concentrations in the wastewaters generated from commercial cucumber fermentations, currently performed in cover brine solutions containing 6% to 12% sodium chloride. However, preliminary attempts to commercially ferment the cucumbers in the presence of oxygen led to the development of a secondary cucumber fermentation or spoilage. The development of cucumber secondary fermentation has also been occasionally reported by processors using cover brine solutions containing sodium chloride. This study focused on the development of a model system to characterize CaCl(2) and NaCl secondary cucumber fermentations under conditions similar to those present on the commercial scale. Cucumber fruits mixed with cover brine solutions, containing 100 mM CaCl(2) or 1.03 M NaCl, and 25 mM acetic acid, were fermented in 2 L fermentation vessels subjected to air-purging at a rate of 5 mL/min. Microorganisms and selected biochemical changes detected in the experimental cucumber fermentations had been previously observed in commercial spoilage samples, suggesting the successful reproduction of the secondary fermentation in the laboratory. Experimental secondary fermentations were characterized by the rapid oxidation of the lactic acid produced during the primary fermentation, which, in turn, increased pH. Lactic acid disappearance seemed to be the result of yeast metabolism that also led to the chemical reduction of the environment to levels at which other bacteria could become established and produce butyric, propionic, and acetic acids. This model system will be applied for the identification of strategies to prevent the initiation of the cucumber secondary fermentation and reduce economic losses in the pickling industry. The study of secondary cucumber fermentation has represented a challenge for many years. The successful development of a model system for the study of this phenomenon in the laboratory is instrumental in furthering the study of the event and in optimizing the sodium-chloride-free fermentation at the commercial scale.
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( 1998 ) An extended-spectrum AmpC-type beta-lactamase obtained by in vitro antibiotic selection. PMID : 9711843 : DOI : 10.1111/j.1574-6968.1998.tb13131.x Abstract >>
A predictive approach was assayed to evaluate the possibility of mutant Amp-C beta-lactamase emergence with increased substrate spectrum (including new C-3' quaternary ammonium cephems). The ampC gene encoding the AmpC beta-lactamase from Enterobacter cloacae was cloned and expressed in an AmpC-defective strain of E. coli. After the AmpC containing strain was challenged with cefpirome, an ampC variant encoding an enzyme with increased resistance to cefpirome and cefepime was selected. In addition, this variant conferred increased resistance to penicillins and third generation cephalosporins. The complete nucleotide sequence of the gene was determined. The deduced peptide sequence showed a single change with respect to the wild-type gene: valine to glutamic acid at position 318 of the native protein (298 of the mature enzyme). The potential emergence and spread of this type of AmpC variants among pathogens should be considered.
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( 1998 ) Isolation and characterization of ACC deaminase genes from two different plant growth-promoting rhizobacteria. PMID : 9851025 : Abstract >>
We have recently proposed that one way that plant growth-promoting rhizobacteria (PGPR) stimulate plant growth is through the activity of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which causes a lowering of plant ethylene levels resulting in longer roots. As part of an effort to understand the role of this enzyme in PGPR, the genes for ACC deaminase from two PGPR, Enterobacter cloacae CAL2 and UW4, have been isolated. These genes are highly homologous to the ACC deaminase genes from Pseudomonas strains 6G5 and F17 and similar to the ACC deaminase gene from Pseudomonas sp. strain ACP. The region downstream (i.e., at the 3'-terminal end) of the strain UW4 ACC deaminase gene has a potential hairpin-like transcription termination site. The regions upstream of the strains UW4 and CAL2 ACC deaminase genes contain putative ribosome-binding sites; however, the promoter sequences have not yet been identified. Southern hybridization experiments suggest that there is a single copy of the ACC deaminase gene in Enterobacter cloacae strains UW4 and CAL2 and that there may be several different types of ACC deaminase genes in different microbes. The cloned ACC deaminase gene can be expressed in Escherichia coli enabling this bacterium to grow on ACC as a sole source of nitrogen and confers upon both Escherichia coli and Pseudomonas spp. strains that are transformed with this gene the ability to promote the elongation of the roots of canola seedlings.
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( 1998 ) gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. PMID : 9756773 : PMC : PMC105915 Abstract >>
Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 microg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.
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( 1997 ) E. coli translation initiation factor IF2--an extremely conserved protein. Comparative sequence analysis of the infB gene in clinical isolates of E. coli. PMID : 9428651 : DOI : 10.1016/s0014-5793(97)01472-5 Abstract >>
The functionally uncharacterised N-terminal of translation initiation factor IF2 has been found to be extremely variable when comparing different bacterial species. In order to study the intraspecies variability of IF2 the 2670 basepairs nucleotide sequence of the infB gene (encoding IF2) was determined in 10 clinical isolates of E. coli. The N-terminal domains (I, II and III) were completely conserved indicating a specific function of this region of IF2. Only one polymorphic position was found in the deduced 890 amino acid sequence. This Gln/Gly490 is located within the central GTP/GDP-binding domain IV of IF2. The results are further evidence that IF2 from E. coli has reached a highly defined level of structural and functional development.
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