| 1. |
Toptchieva A,
Sisson G,
Bryden LJ,
Taylor DE,
Hoffman PS,
( 2003 ) An inducible tellurite-resistance operon in Proteus mirabilis. PMID : 12724390 : DOI : 10.1099/mic.0.25981-0 Abstract >>
Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.
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2. |
Visalli MA,
Murphy E,
Projan SJ,
Bradford PA,
( 2003 ) AcrAB multidrug efflux pump is associated with reduced levels of susceptibility to tigecycline (GAR-936) in Proteus mirabilis. PMID : 12543675 : DOI : 10.1128/aac.47.2.665-669.2003 PMC : PMC151746 Abstract >>
Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the PROTEEAE: A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.
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3. |
Naas T,
Zerbib M,
Girlich D,
Nordmann P,
( 2003 ) Integration of a transposon Tn1-encoded inhibitor-resistant beta-lactamase gene, bla(TEM-67) from Proteus mirabilis, into the Escherichia coli chromosome. PMID : 12499163 : DOI : 10.1128/aac.47.1.19-26.2003 PMC : PMC148959 Abstract >>
Proteus mirabilis NEL-1 was isolated from a urine sample of a patient hospitalized in a long-term care facility. Strain NEL-1 produced a beta-lactamase with a pI of 5.2 conferring resistance to amoxicillin and amoxicillin-clavulanic acid. Sequencing of a PCR amplicon by using TEM-specific primers revealed a novel bla(TEM) gene, bla(TEM-67). TEM-67 was an IRT-1-like TEM derivative related to TEM-65 (Lys39, Cys244) with an additional Leu21Ile amino acid substitution in the leader peptide. The biochemical properties of TEM-67 were equivalent to those described for TEM-65. Analysis of sequences surrounding bla(TEM-67) revealed that it was located on a transposon, Tn1, which itself was located on a 48-kb non-self-transferable plasmid, pANG-1. Electroporation of plasmid pANG-1 into Escherichia coli DH10B resulted in the integration of bla(TEM-67) into the chromosome, whereas it remained episomal in the P. mirabilis CIP103181 reference strain. Further characterization of pANG-1 revealed the presence of two identical sequences on both sides of Tn1 that contained an IS26 insertion sequence followed by a novel colicin gene, colZ, which had 20% amino acid identity with other colicin genes. The characterization of this novel TEM derivative provides further evidence for the large diversity of plasmid-encoded beta-lactamases produced in P. mirabilis and for their spread to other enterobacterial species through transposable-element-mediated events.
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4. |
Liaw SJ,
Lai HC,
Ho SW,
Luh KT,
Wang WB,
( 2003 ) Role of RsmA in the regulation of swarming motility and virulence factor expression in Proteus mirabilis. PMID : 12488561 : DOI : 10.1099/jmm.0.05024-0 Abstract >>
Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper-flagellated swarm cells that undergo cycles of rapid and co-ordinated population migration across surfaces and exhibit high levels of virulence gene expression. RsmA (repressor of secondary metabolites) and CsrA, its homologue in Escherichia coli, control many phenotypic traits, such as motility and pathogenesis in Erwinia species, glycogen biosynthesis, cell size and biofilm formation in Escherichia coli and swarming motility in Serratia marcescens. To investigate the role of RsmA in Proteus mirabilis, the rsmA gene from Proteus mirabilis (hereafter referred to as rsmA(Pm)) was cloned. RsmA(Pm) showed high sequence similarity to Escherichia coli CsrA and RsmA cloned from Erwinia carotovora subsp. carotovora, Serratia marcescens, Haemophilus influenzae and Bacillus subtilis and could complement an Escherichia coli csrA mutant in glycogen synthesis. A low-copy-number plasmid carrying rsmA(Pm) expressed from its native promoter caused suppression of swarming motility and expression of virulence factors in Proteus mirabilis. mRNA stability assays suggested that RsmA(Pm) inhibited virulence factor expression through promoting mRNA degradation. RsmA homologues cloned from Serratia marcescens and Erwinia carotovora subsp. carotovora could also inhibit swarming and virulence factor expression in Proteus mirabilis.
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5. |
Andreoletti P,
Sainz G,
Jaquinod M,
Gagnon J,
Jouve HM,
( 2003 ) High-resolution structure and biochemical properties of a recombinant Proteus mirabilis catalase depleted in iron. PMID : 12486720 : DOI : 10.1002/prot.10283 Abstract >>
Heme catalases are homotetrameric enzymes with a highly conserved complex quaternary structure, and their functional role is still not well understood. Proteus mirabilis catalase (PMC), a heme enzyme belonging to the family of NADPH-binding catalases, was efficiently overexpressed in E. coli. The recombinant catalase (rec PMC) was deficient in heme with one-third heme and two-thirds protoporphyrin IX as determined by mass spectrometry and chemical methods. This ratio was influenced by the expression conditions, but the enzyme-specific activity calculated relative to the heme content remained unchanged. The crystal structure of rec PMC was solved to a resolution of 2.0 A, the highest resolution obtained to date with PMC. The overall structure was quite similar to that of wild-type PMC, and it is surprising that the absence of iron had no effect on the structure of the active site. Met 53 close to the essential His 54 was found less oxidized in rec PMC than in the wild-type enzyme. An acetate anion was modeled in an anionic pocket, away from the heme group but important for the enzymatic reaction. An alternate conformation observed for Arg 99 could play a role in the formation of the H-bond network connecting two symmetrical subunits of the tetramer.
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6. |
Decré D,
Verdet C,
Raskine L,
Blanchard H,
Burghoffer B,
Philippon A,
Sanson-Le-Pors MJ,
Petit JC,
Arlet G,
( 2002 ) Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area. PMID : 12407124 : DOI : 10.1093/jac/dkf193 Abstract >>
We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.
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7. |
Izquierdo L,
Abitiu N,
Coderch N,
Hita B,
Merino S,
Gavin R,
Tomás JM,
Regué M,
( 2002 ) The inner-core lipopolysaccharide biosynthetic waaE gene: function and genetic distribution among some Enterobacteriaceae. PMID : 12427940 : DOI : 10.1099/00221287-148-11-3485 Abstract >>
To determine the function of the waaE gene in the biosynthesis of the inner-core LPS of Klebsiella pneumoniae, a waaE non-polar mutant has been constructed. Data obtained from the comparative chemical analysis of LPS samples obtained from the wild-type, the mutant strain and the complemented mutant demonstrated that the waaE gene is involved in substitution of alpha-L-glycero-D-manno-heptopyranose I (L,D-HeppI) at the O-4 position by a beta-D-glucopyranose (beta-D-Glcp) residue. In addition, DNA amplification and nucleotide sequence determination studies revealed that waaE homologues located between the waaA and coaD genes are present in clinical isolates of Enterobacteriaceae containing the structure beta-D-Glcp-(1-->4)-alpha-L,D-HeppI (K. pneumoniae, Proteus mirabilis and Yersinia enterocolitica), as well as in strains of Serratia marcescens and Enterobacter aerogenes of unknown LPS-core structures. Complementation studies using non-polar waaE mutants prove that all the waaE homologues perform the same function. Furthermore, K. pneumoniae, Ser. marcescens and P. mirabilis non-polar waaE mutants showed reduced adhesion and pathogenicity. In addition, the Ser. marcescens and P. murabilis waaE mutants showed reduced swarming motility and ability to form biofilms in vitro. All these characteristics were rescued by reintroduction of the waaE gene independently of its origin. An easy DNA amplification method to detect this gene was established, which also helps in finding the potential presence of this structural feature [beta-D-Glcp-(1-->4)-alpha-L,D-HeppI] in the inner-core LPS of Enterobacteriaceae members with unknown LPS-core structures.
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8. |
Weigel LM,
Anderson GJ,
Tenover FC,
( 2002 ) DNA gyrase and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis. PMID : 12121936 : DOI : 10.1128/aac.46.8.2582-2587.2002 PMC : PMC127365 Abstract >>
Mutations associated with fluoroquinolone resistance in clinical isolates of Proteus mirabilis were determined by genetic analysis of the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE. This study included the P. mirabilis type strain ATCC 29906 and 29 clinical isolates with reduced susceptibility (MIC, 0.5 to 2 microg/ml) or resistance (MIC, > or =4 microg/ml) to ciprofloxacin. Susceptibility profiles for ciprofloxacin, clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were correlated with amino acid changes in the QRDRs. Decreased susceptibility and resistance were associated with double mutations involving both gyrA (S83R or -I) and parC (S80R or -I). Among these double mutants, MICs of ciprofloxacin varied from 1 to 16 microg/ml, indicating that additional factors, such as drug efflux or porin changes, also contribute to the level of resistance. For ParE, a single conservative change of V364I was detected in seven strains. An unexpected result was the association of gyrB mutations with high-level resistance to fluoroquinolones in 12 of 20 ciprofloxacin-resistant isolates. Changes in GyrB included S464Y (six isolates), S464F (three isolates), and E466D (two isolates). A three-nucleotide insertion, resulting in an additional lysine residue between K455 and A456, was detected in gyrB of one strain. Unlike any other bacterial species analyzed to date, mutation of gyrB appears to be a frequent event in the acquisition of fluoroquinolone resistance among clinical isolates of P. mirabilis.
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9. |
Claret L,
Hughes C,
( 2002 ) Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy. PMID : 12144778 : DOI : 10.1016/s0022-2836(02)00600-9 Abstract >>
The transcriptional activator FlhD2C2 is the master regulator of bacterial flagellum biogenesis and swarming migration, activating the "early" class II promoters of the large flagellar gene hierarchy. Using primer extensions, band-shift assays, and enzymatic and chemical footprinting, we describe the binding of the FlhD2C2 heterotetramer to the promoter regions of four class II flagella operons, fliAZ, flhBA and the divergent flgAMN and flgBCD(EFGHIJ). Each of the promoter regions was bound by a single heterotetramer, i.e. the flgAMN and flgBCD operons are characterised by a single FlhD2C2 binding site. Binding affinity differed, and correlated with previously reported promoter strength and order of activation. Methylation protection and interference, and depurination and depyrimidation interference provided a detailed map of critical bases within a common 46-59bp DNaseI footprint overlapping the promoter -35 sequences. These data and compilation of the 12 known class II promoter sequences of Escherichia coli, Proteus mirabilis and Salmonella typhimurium allowed determination of a FlhD2C2 binding site with pseudo symmetry, comprising two 17-18bp inverted repeats, each a consensus FlhD2C2 box, separated by a 10-11bp spacer. DNaseI hypersensitivity indicated that binding may cause a conformational change in the promoter regions. Only the FlhC subunit can bind DNA independently, but the specificity and stability of the interaction is strengthened by FlhD. Here, photo-crosslinking established that both FlhC and the stabilising FlhD contact the DNA within the FlhD2C2 tetramer. Our data suggest that specificity of recognition and stability of the FlhD2C2/DNA complex require protein-protein interaction and interaction of both FlhC and FlhD subunits with DNA. These characteristics of the FlhD and FlhC subunits in the FlhD2C2/DNA complex are strikingly atypical of prokaryotic regulators.
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10. |
Arduino SM,
Roy PH,
Jacoby GA,
Orman BE,
Pineiro SA,
Centron D,
( 2002 ) blaCTX-M-2 is located in an unusual class 1 integron (In35) which includes Orf513. PMID : 12069995 : DOI : 10.1128/aac.46.7.2303-2306.2002 PMC : PMC127297 Abstract >>
Examination of the bla(CTX-M-2) gene in plasmid pMAR-12 by sequencing and PCR analysis revealed that the bla gene and the surrounding DNA, which is closely related (99% homology) to the Kluyvera ascorbata chromosomal DNA that contains the bla(KLUA-1) gene, are located in a complex sul1-type integron, termed In35, that includes Orf513. It is possible that bla(CTX-M-2) was acquired by plasmid pMAR-12 through an uncharacterized recombinational event in which Orf513 could be involved.
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11. |
Fraser GM,
Claret L,
Furness R,
Gupta S,
Hughes C,
( 2002 ) Swarming-coupled expression of the Proteus mirabilis hpmBA haemolysin operon. PMID : 12101306 : DOI : 10.1099/00221287-148-7-2191 PMC : PMC2528290 Abstract >>
The HpmA haemolysin toxin of Proteus mirabilis is encoded by the hpmBA locus and its production is upregulated co-ordinately with the synthesis and assembly of flagella during differentiation into hyperflagellated swarm cells. Primer extension identified a sigma(70) promoter upstream of hpmB that was upregulated during swarming. Northern blotting indicated that this promoter region was also required for concomitant transcription of the immediately distal hpmA gene, and that the unstable hpmBA transcript generated a stable hpmA mRNA and an unstable hpmB mRNA. Transcriptional luxAB fusions to the DNA regions 5' of the hpmB and hpmA genes confirmed that hpmB sigma(70) promoter activity increased in swarm cells, and that there was no independent hpmA promoter. Increased transcription of the hpmBA operon in swarm cells was dependent upon a 125 bp sequence 5' of the sigma(70) promoter -35 hexamer. This sequence spans multiple putative binding sites for the leucine-responsive regulatory protein (Lrp), and band-shift assays with purified Lrp confirmed the presence of at least two such sites. The influence on hpmBA expression of the key swarming positive regulators FlhD(2)C(2) (encoded by the flagellar master operon), Lrp, and the membrane-located upregulator of the master operon, UmoB, was examined. Overexpression of each of these regulators moderately increased hpmBA transcription in wild-type P. mirabilis, and the hpmBA operon was not expressed in any of the flhDC, lrp or umoB mutants. Expression in the mutants was not recovered by cross-complementation, i.e. by overexpression of FlhD(2)C(2), Lrp or UmoB. Expression of the zapA protease virulence gene, which like hpmBA is also upregulated in swarm cells, did not require Lrp, but like flhDC it was upregulated by UmoB. The results indicate intersecting pathways of control linking virulence gene expression and swarm cell differentiation.
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12. |
Saladin M,
Cao VT,
Lambert T,
Donay JL,
Herrmann JL,
Ould-Hocine Z,
Verdet C,
Delisle F,
Philippon A,
Arlet G,
( 2002 ) Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. PMID : 12007800 : DOI : 10.1111/j.1574-6968.2002.tb11126.x Abstract >>
Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.
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13. |
Neuwirth C,
Madec S,
Siebor E,
Pechinot A,
Duez JM,
Pruneaux M,
Fouchereau-Peron M,
Kazmierczak A,
Labia R,
( 2001 ) TEM-89 beta-lactamase produced by a Proteus mirabilis clinical isolate: new complex mutant (CMT 3) with mutations in both TEM-59 (IRT-17) and TEM-3. PMID : 11709345 : DOI : 10.1128/AAC.45.12.3591-3594.2001 PMC : PMC90874 Abstract >>
TEM-89 (CMT-3) is the first complex mutant beta-lactamase produced by a clinical strain of Proteus mirabilis (strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM beta-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.
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14. |
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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15. |
McCoy AJ,
Liu H,
Falla TJ,
Gunn JS,
( 2001 ) Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides. PMID : 11408219 : DOI : 10.1128/AAC.45.7.2030-2037.2001 PMC : PMC90596 Abstract >>
Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility.
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16. |
Andreoletti P,
Franzetti B,
Nussaume L,
Andrieu JP,
Gagnon J,
Luche S,
Rabilloud T,
Jouve H,
( 2001 ) Comparison of the PR mutant with the wild-type strain of proteus mirabilis brings insight into peroxide resistance factors and regulation of catalase expression. PMID : 11261492 : Abstract >>
The peroxide resistant mutant (PR) of Proteus mirabilis was characterized by an increased constitutive catalase activity concomitant with a large production of specific mRNA. Survival toward hydrogen peroxide during exponential phase was increased by H2O2 pretreatment in the wild type but not in the mutant, although the catalase of both strains was not inducible under these conditions. In the mutant, besides catalase, over-produced proteins comprised two different alkyl hydroperoxide reductase subunit C (AhpC) proteins and a protein homologous to the stationary phase transcription factor SspA of Escherichia coli. Conversely, the flagellin A (FlaA) of P. mirabilis was repressed in the PR mutant. Genomic DNA fragments of 2.9 kb carrying the catalase gene (katA) together with the 5' and 3' flanking regions were isolated from both strains and found to be identical. Upstream of katA, a Fur box-like sequence was found, but surprisingly, restricting iron in the culture medium caused a decrease in catalase production. The PR mutant presents similarities with other peroxide resistant mutants, but the regulation of catalase biosynthesis in P. mirabilis seems somewhat different from other close species such as E. coli.
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17. |
Perilli M,
Segatore B,
de Massis MR,
Riccio ML,
Bianchi C,
Zollo A,
Rossolini GM,
Amicosante G,
( 2000 ) TEM-72, a new extended-spectrum beta-lactamase detected in Proteus mirabilis and Morganella morganii in Italy. PMID : 10952610 : DOI : 10.1128/aac.44.9.2537-2539.2000 PMC : PMC90100 Abstract >>
A new natural TEM-2 derivative, named TEM-72, was identified in a Proteus mirabilis strain and in a Morganella morganii strain isolated in Italy in 1999. Compared to TEM-1, TEM-72 contains the following amino acid substitutions: Q39K, M182T, G238S, and E240K. Kinetic analysis showed that TEM-72 exhibits an extended-spectrum activity, including activity against oxyimino-cephalosporins and aztreonam. Expression of bla(TEM-72) in Escherichia coli was capable of decreasing the host susceptibility to the above drugs.
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18. |
Bakare OO,
Coker C,
( 2000 ) H-NS is a repressor of the Proteus mirabilis urease transcriptional activator gene ureR. PMID : 10762273 : DOI : 10.1128/jb.182.9.2649-2653.2000 PMC : PMC111335 Abstract >>
Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A(6)TA(2)CA(2)TGGTA(5)GA(6)TGA(5), is located 16 bp upstream of the sigma(70)-like ureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured beta-galactosidase activity of wild-type Escherichia coli MC4100 (H-NS(+)) and its isogenic derivative ATM121 (hns::Tn10) (H-NS(-)) harboring a ureR-lacZ operon fusion plasmid (pLC9801). beta-Galactosidase activity in the H-NS(-) host strain was constitutive and sevenfold greater (P < 0.0001) than that in the H-NS(+) host. A recombinant plasmid containing cloned P. mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS(-) host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in beta-galactosidase activity in the H-NS(+) host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS(-) host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS(-) background and the H-NS(+) background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS(-) background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction.
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19. |
Belas R,
Moghaddame-Jafari S,
Lockatell CV,
Johnson D,
( 1999 ) ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells. PMID : 10361285 : DOI : 10.1046/j.1365-2958.1999.01401.x Abstract >>
The IgA-degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co-ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild-type cells and beta-galactosidase measurements using a zapA:lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co-ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's-eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA- strains produce wild-type colony patterns. ZapA- strains fail to degrade IgA and show decreased survival compared with the wild-type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA-degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA-specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.
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20. |
Hay NA,
Hughes C,
Gygi D,
( 1999 ) A novel membrane protein influencing cell shape and multicellular swarming of Proteus mirabilis. PMID : 10094676 : PMC : PMC93611 Abstract >>
Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type P. mirabilis; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a ccmA null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. mirabilis to become enlarged and ellipsoidal.
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21. |
Zhao H,
Mobley HL,
( 1999 ) Identification of protease and rpoN-associated genes of uropathogenic Proteus mirabilis by negative selection in a mouse model of ascending urinary tract infection. PMID : 10206698 : DOI : 10.1099/13500872-145-1-185 Abstract >>
Proteus mirabilis, a motile gram-negative bacterium, is a principal cause of urinary tract infections in patients with functional or anatomical abnormalities of the urinary tract or those with urinary catheters in place. Thus far, virulence factors including urease, flagella, haemolysin, various fimbriae, IgA protease and a deaminase have been characterized based on the phenotypic traits conferred by these proteins. In this study, an attempt was made to identify new virulence genes of P. mirabilis that may not have identifiable phenotypes using the recently described technique of signature-tagged mutagenesis. A pool of chromosomal transposon mutants was made through conjugation and kanamycin/tetracycline selection; random insertion was confirmed by Southern blotting of chromosomal DNA isolated from 16 mutants using the aphA gene as a probe. From the total pool, 2.3% (9/397) auxotrophic mutants and 3.5% (14/397) swarming mutants were identified by screening on minimal salts agar and Luria agar plates, respectively. Thirty per cent of the mutants, found to have either no tag or an unamplifiable tag, were removed from the input pool. Then 10(7) c.f.u. from a 96-mutant pool (approximately 10(5) c.f.u. of each mutant) were used as an input pool to transurethrally inoculate seven CBA mice. After 2 d infection, bacteria were recovered from the bladders and kidneys and yielded about 10(5) c.f.u. as an output pool. Dot blot analysis showed that two of the 96 mutants, designated B2 and B5, could not be hybridized by signature tags amplified from the bladder output pool. Interrupted genes from these two mutants were cloned and sequenced. The interrupted gene in B2 predicts a polypeptide of 37.3 kDa that shares amino acid similarity with a putative protease or collagenase precursor. The gene in B5 predicts a polypeptide of 32.6 kDa that is very similar to that encoded by ORF284 of the rpoN operon controlling expression of nitrogen-regulated genes from several bacterial species. The virulence of the two mutants was tested further by co-challenging CBA mice with each mutant and the parental strain. After 1 week of infection, the B2 and B5 mutants were recovered in numbers 100-fold and 1000-fold less than the parental strain, respectively. Using an in vitro assay, it was shown that the B2 mutant had significantly less (P = 0.0001) extracellular protease activity than the wild-type strain. These findings demonstrate that signature-tagged mutagenesis is a viable approach to identify bacterial genes associated with the ability to infect the urinary tract.
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22. |
Nordmann P,
Chaibi EB,
Labia R,
Naas T,
( 1999 ) Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene. PMID : 10049269 : PMC : PMC89162 Abstract >>
A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.
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23. |
O'Halloran JA,
McGrath BM,
Pembroke JT,
( 2007 ) The orf4 gene of the enterobacterial ICE, R391, encodes a novel UV-inducible recombination directionality factor, Jef, involved in excision and transfer of the ICE. PMID : 17504243 : DOI : 10.1111/j.1574-6968.2007.00747.x Abstract >>
The enterobacterial mobile genetic element R391, the prototype ICE (integrating-conjugative element) of the SXT/R391 family, shows increased conjugative transfer following UV irradiation. This is dependent on a functioning R391 orf4 gene, which is adjacent to the element encoded integrase gene, int. orf4 mutants fail to form a detectable circular transfer intermediate, do not show UV induced transfer and show a much reduced general transfer ability. The orf4 gene product, termed Jef (IncJ excision factor), shows little homology to anything currently in the nucleotide or protein databases. It is predicted to encode a 66 amino acid, 8.03 kDa, basic, DNA-binding protein with an iso-electric point of pH 8.1: these characteristics being similar to those of recombinational directionality factors involved in excision. Jef expression is up-regulated upon UV irradiation as demonstrated by real-time reverse transcriptase PCR and is controlled by two element encoded genes orf90 and orf91, which show similarity to the transcriptional activator complex FlhC and FlhD. orf4, orf90 and orf91 are conserved in all the SXT/R391-like elements sequenced to date including SXT, ICESpuPO1 and ICEVchMex1. orf4 is also conserved in other SXT/R391 family members such as R997, R392, R705 and pMERPH as shown by sequencing amplicons from these ICEs generated using orf4 specific primers.
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24. |
Salerno A,
Delétoile A,
Lefevre M,
Ciznar I,
Krovacek K,
Grimont P,
Brisse S,
( 2007 ) Recombining population structure of Plesiomonas shigelloides (Enterobacteriaceae) revealed by multilocus sequence typing. PMID : 17693512 : DOI : 10.1128/JB.00796-07 PMC : PMC2168737 Abstract >>
Plesiomonas shigelloides is an emerging pathogen that is widespread in the aquatic environment and is responsible for intestinal diseases and extraintestinal infections in humans and other animals. Virtually nothing is known about its genetic diversity, population structure, and evolution, which severely limits epidemiological control. We addressed these questions by developing a multilocus sequence typing (MLST) system based on five genes (fusA, leuS, pyrG, recG, and rpoB) and analyzing 77 epidemiologically unrelated strains from several countries and several ecological sources. The phylogenetic position of P. shigelloides within family Enterobacteriaceae was precisely defined by phylogenetic analysis of the same gene portions in other family members. Within P. shigelloides, high levels of nucleotide diversity (average percentage of nucleotide differences between strains, 1.49%) and genotypic diversity (64 distinct sequence types; Simpson's index, 99.7%) were found, with no salient internal phylogenetic structure. We estimated that homologous recombination in housekeeping genes affects P. shigelloides alleles and nucleotides 7 and 77 times more frequently than mutation, respectively. These ratios are similar to those observed in the naturally transformable species Streptococcus pneumoniae with a high rate of recombination. In contrast, recombination within Salmonella enterica, Escherichia coli, and Yersinia enterocolitica was much less frequent. P. shigelloides thus stands out among members of the Enterobacteriaceae. Its high rate of recombination results in a lack of association between genomic background and O and H antigenic factors, as observed for the 51 serotypes found in our sample. Given its robustness and discriminatory power, we recommend MLST as a reference method for population biology studies and epidemiological tracking of P. shigelloides strains.
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25. |
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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26. |
Fernández A,
Gil E,
Cartelle M,
Pérez A,
Beceiro A,
Mallo S,
Tomás MM,
Pérez-Llarena FJ,
Villanueva R,
Bou G,
( 2007 ) Interspecies spread of CTX-M-32 extended-spectrum beta-lactamase and the role of the insertion sequence IS1 in down-regulating bla CTX-M gene expression. PMID : 17332005 : DOI : 10.1093/jac/dkm030 Abstract >>
To characterize the extended-spectrum beta-lactamases (ESBLs) as well as their genetic environment in different isolates of Enterobacteriaceae from a patient with repeated urinary tract infections. Two isolates of Escherichia coli and one Proteus mirabilis, all with ESBL phenotypes, were studied. Conjugation experiments and restriction fragment length polymorphisms (RFLPs) were performed. Cloning of the bla genes was by plasmid restriction and fragments ligation. Antibiotic susceptibility testing was by Etest. The genetic environment was analysed by direct sequencing of the DNA surrounding the bla gene. RT-PCR was performed to study the differences in the bla(CTX-M) gene expression. The bla gene was transferred by conjugation from the three clinical isolates, which by RFLP showed the same plasmid. The bla gene and surrounding sequences were cloned, an approximately 9 kbp AccI fragment was sequenced and the bla(CTX-M-32) gene was identified. The MICs of ceftazidime for transconjugants and transformants bearing the bla(CTX-M-32) gene were lower than those previously reported. Analysis of the DNA surrounding the ESBL gene revealed a new genetic structure with two insertion sequences, IS5 and IS1, located immediately upstream of the bla(CTX-M-32) gene; IS1 was located between the bla gene and IS5, and within the -10 and -35 promoter boxes of the bla(CTX-M-32) gene. Microbiological and biochemical studies revealed lower bla(CTX-M-32) gene expression in bacterial isolates with IS1 between the promoter boxes. Data suggest putative in vivo horizontal bla(CTX-M-32) gene transfer between two different genera of Enterobacteriaceae. A new complex structure, IS5-IS1, was detected upstream of the bla gene and IS1 negatively modulated expression of the bla(CTX-M-32) gene because its location modified the bla promoter region.
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27. |
Marrero J,
Waldor MK,
( 2007 ) The SXT/R391 family of integrative conjugative elements is composed of two exclusion groups. PMID : 17307849 : DOI : 10.1128/JB.01902-06 PMC : PMC1855829 Abstract >>
Conjugative elements often encode entry exclusion systems that convert host cells into poor recipients for identical or similar elements. The diversity of exclusion systems within families of conjugative elements has received little attention. We report here the most comprehensive study to date of the diversity of exclusion determinants within a single family of conjugative elements. Unexpectedly, our analyses indicate that there are only two exclusion groups among the diverse members of the SXT/R391 family of integrative conjugative elements.
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28. |
Galani I,
Souli M,
Koratzanis E,
Koratzanis G,
Chryssouli Z,
Giamarellou H,
( 2007 ) Emerging bacterial pathogens: Escherichia coli, Enterobacter aerogenes and Proteus mirabilis clinical isolates harbouring the same transferable plasmid coding for metallo-beta-lactamase VIM-1 in Greece. PMID : 17255145 : DOI : 10.1093/jac/dkl508 Abstract >>
N/A
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29. |
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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30. |
Ahmed AM,
Hussein AI,
Shimamoto T,
( 2007 ) Proteus mirabilis clinical isolate harbouring a new variant of Salmonella genomic island 1 containing the multiple antibiotic resistance region. PMID : 17114173 : DOI : 10.1093/jac/dkl471 Abstract >>
A clinical isolate of Proteus mirabilis strain 18306, which displayed the multidrug resistance phenotype of Salmonella genomic island 1 (SGI1), was examined for the presence of this island including its multiple antibiotic resistance genomic region. P. mirabilis 18306 was isolated in March 2006 from a patient in Palestine with diabetic foot infection. Antibiotic susceptibility tests and various molecular techniques, including PCR, cloning and DNA sequencing were used for detection and characterization of SGI1 in P. mirabilis 18306. P. mirabilis 18306 showed the typical multidrug resistance phenotype of SGI1 as it was resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline, in addition to trimethoprim and nalidixic acid. Molecular characterization showed that P. mirabilis 18306 harboured a structure similar to SGI1, except that the aadA2 gene, which confers resistance to streptomycin and spectinomycin, of standard SGI1 had been replaced with dfrA15, which confers resistance to trimethoprim. Furthermore, the nucleotide sequence of the extrachromosomal circular form of SGI1 in P. mirabilis was found to be identical to that of Salmonella Typhimurium DT104. However, PCR results showed that P. mirabilis 18306 was negative for the left and right junctions which represent the integration sites of SGI1 into Salmonella enterica chromosome. Hence, this new variant of SGI1 may be integrated at a different site into the chromosome of P. mirabilis 18306. Tn1826-derived class 2 integron, which carries only two gene cassettes, sat2 and aadA1, was also identified in this strain. In this study, we identified a new variant SGI1 containing the multiple resistance genomic region in a multidrug-resistant strain of P. mirabilis. This is the first report for SGI1 in a genus other than Salmonella.
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31. |
Bahrani FK,
Johnson DE,
Robbins D,
Mobley HL,
( 1991 ) Proteus mirabilis flagella and MR/P fimbriae: isolation, purification, N-terminal analysis, and serum antibody response following experimental urinary tract infection. PMID : 1680106 : PMC : PMC258923 Abstract >>
Urinary tract infection with Proteus mirabilis may lead to serious complications, including cystitis, acute pyelonephritis, fever, bacteremia, and death. In addition to the production of hemolysin and the enzyme urease, fimbriae and flagellum-mediated motility have been postulated as virulence factors for this species. We purified mannose-resistant/proteuslike (MR/P) fimbriae and flagella from strains CFT322 and HU2450, respectively. Electron microscopy revealed highly concentrated preparations of fimbriae and flagella. Fimbrial and flagellar structural subunits were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18.5 and 41 kDa, respectively. N-terminal sequencing revealed that 10 of the first 20 amino acids of the major MR/P subunit matched the sequence of the P. mirabilis uroepithelial cell adhesin N terminus and 11 of 20 amino acids matched the predicted amino acid sequence of the Escherichia coli P fimbriae structural subunit, PapA. In addition, 90 and 80% homologies were found between the first 20 amino acids of P. mirabilis flagellin and those of Salmonella typhimurium phase-1 flagellin and the E. coli hag gene product, respectively. An enzyme-linked immunosorbent assay using purified antigens showed a strong reaction between the MR/P fimbriae or flagella and sera of CBA mice challenged transurethrally with P. mirabilis. A possible role for MR/P fimbriae in the pathogenesis of urinary tract infection is supported by (i) a strong immune response to the antigen in experimentally infected animals, (ii) amino acid sequence similarity to other enteric surface structure, and (iii) our previously reported observation that MR/P fimbriae are expressed preferentially as the sole fimbrial type in human pyelonephritis isolates.
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32. |
Cambau E,
Lascols C,
Sougakoff W,
Bébéar C,
Bonnet R,
Cavallo JD,
Gutmann L,
Ploy MC,
Jarlier V,
Soussy CJ,
Robert J,
( 2006 ) Occurrence of qnrA-positive clinical isolates in French teaching hospitals during 2002-2005. PMID : 16961639 : DOI : 10.1111/j.1469-0691.2006.01529.x Abstract >>
Bacteria harbouring the novel qnrA plasmid-mediated mechanism of quinolone resistance have been described in different countries, but the frequency of their occurrence has not been investigated. In total, 1,468 clinical isolates of Enterobacteriaceae with quinolone resistance or extended-spectrum beta-lactamase (ESBL) phenotypes were collected from eight teaching hospitals in France during 2002-2005 and screened for qnrA. Overall, 28 isolates (22 Enterobacter cloacae, three Klebsiella pneumoniae, one Citrobacter freundii, one Klebsiella oxytoca and one Proteus mirabilis) were positive for qnrA, representing 1.9% of all isolates, 3.3% of ESBL-producing isolates (22% of the E. cloacae isolates) and 0% of non-ESBL-producing isolates. The prevalence of qnrA among consecutive ESBL-producing isolates in 2004 from the eight hospitals was 2.8% (18/639). Of the qnrA-positive isolates, 100% were intermediately-resistant or resistant to nalidixic acid, and 75% to ciprofloxacin. Twenty-one of the 22 qnrA-positive E. cloacae isolates were obtained from two hospitals in the Paris area, and molecular typing and plasmid content analysis showed clonal relationships for five, three and two isolates, respectively. The qnrA genetic environment was similar to that of the In36 integron. The remaining two isolates had qnrA variants (30 and 29 nucleotide differences, respectively, compared with the original sequence) and an unknown genetic environment. The ESBL gene associated with qnrA was bla(SHV-12) in most of the isolates, but bla(PER-1) and bla(SHV-2a) were found in two isolates. In France, it appears that qnrA-positive isolates are predominantly E. cloacae isolates producing SHV-12, and may be associated with the dissemination of an In36-like integron.
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33. |
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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34. |
Saito R,
Sato K,
Kumita W,
Inami N,
Nishiyama H,
Okamura N,
Moriya K,
Koike K,
( 2006 ) Role of type II topoisomerase mutations and AcrAB efflux pump in fluoroquinolone-resistant clinical isolates of Proteus mirabilis. PMID : 16870650 : DOI : 10.1093/jac/dkl297 Abstract >>
We conducted a study to determine the role played by amino acid mutations in DNA gyrase and topoisomerase IV, and the AcrAB efflux pump in resistance to fluoroquinolones in clinical isolates of Proteus mirabilis. Nine clinical isolates of P. mirabilis containing eight fluoroquinolone-resistant isolates and one fluoroquinolone-susceptible isolate as the causative pathogen were collected from different patients with urinary tract infections. Fluoroquinolone resistance was characterized by PCR and DNA sequencing. The role of the AcrAB efflux pump was investigated by semi-quantifying the transcriptional expression of the acrB gene. Double mutations were found in GyrA, at S83I and E87K, and single mutations in GyrB (S464F) and ParC (S80I) in four isolates with ciprofloxacin MICs of 16 to >128 mg/L. In three isolates (ciprofloxacin MICs of >128 mg/L), the level of acrB expression was 2.1- to 3.2-fold higher than that in the wild-type control strain (ciprofloxacin MIC of < or =0.12 mg/L) and these isolates also had increased MICs of minocycline (>64 versus 8-16 mg/L) and chloramphenicol (>256 versus 4-8 mg/L) compared with the five other fluoroquinolone-resistant isolates. Our findings demonstrate that two mechanisms--mutations in GyrA (at S83I and E87K), GyrB and ParC, and overproduction of the AcrAB efflux pump--might synergistically contribute to a highest level of resistance to fluoroquinolones in clinical isolates of P. mirabilis.
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35. |
D'Andrea MM,
Nucleo E,
Luzzaro F,
Giani T,
Migliavacca R,
Vailati F,
Kroumova V,
Pagani L,
Rossolini GM,
( 2006 ) CMY-16, a novel acquired AmpC-type beta-lactamase of the CMY/LAT lineage in multifocal monophyletic isolates of Proteus mirabilis from northern Italy. PMID : 16436718 : DOI : 10.1128/AAC.50.2.618-624.2006 PMC : PMC1366893 Abstract >>
We report multifocal detection (four different cities in northern Italy) of Proteus mirabilis isolates resistant to both oxyimino- and 7-alpha-methoxy-cephalosporins and producing a novel acquired AmpC-like beta-lactamase. The enzyme, named CMY-16, is a variant of the CMY/LAT lineage, which differs from the closest homologues, CMY-4 and CMY-12, by a single amino acid substitution (A171S or N363S, respectively) and from CMY-2 by two substitutions (A171S and W221R). Expression of the cloned bla(CMY-16) gene in Escherichia coli decreased susceptibility to penicillins, cephalosporins, and aztreonam. Tazobactam was more effective than clavulanate at antagonizing the enzyme activity. Genotyping, by random amplification of polymorphic DNA and pulsed-field gel electrophoresis of genomic DNA digested with SfiI, showed that isolates were clonally related to each other, although not identical. The bla(CMY-16) gene was not transferable to E. coli by conjugation or transformation. In all isolates, it was chromosomally located and inserted in a conserved genetic environment. PCR mapping experiments revealed that the bla(CMY-16) was flanked by ISEcp1 and the blc gene, similar to other genes of this lineage from plasmids of Salmonella enterica, Klebsiella spp., and E. coli. Overall, these results revealed multifocal spreading of a CMY-16-producing P. mirabilis clone in northern Italy. This finding represents the first report of an acquired AmpC-like beta-lactamase in Proteus mirabilis from Italy and underscores the emergence of similar resistance determinants in the European setting.
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36. |
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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37. |
Wachino J,
Yamane K,
Shibayama K,
Kurokawa H,
Shibata N,
Suzuki S,
Doi Y,
Kimura K,
Ike Y,
Arakawa Y,
( 2006 ) Novel plasmid-mediated 16S rRNA methylase, RmtC, found in a proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides. PMID : 16377684 : DOI : 10.1128/AAC.50.1.178-184.2006 PMC : PMC1346777 Abstract >>
Proteus mirabilis ARS68, which demonstrated a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. The aminoglycoside resistance of this strain could not be transferred to recipient strains Escherichia coli CSH-2 and E. coli HB101 by a general conjugation experiment, but E. coli DH5alpha was successfully transformed by electroporation with the plasmid of the parent strain, ARS68, and acquired an unusually high degree of resistance against aminoglycosides. Cloning and sequencing analyses revealed that the presence of a novel 16S rRNA methylase gene, designated rmtC, was responsible for resistance in strain ARS68 and its transformant. The G+C content of rmtC was 41.1%, and the deduced amino acid sequences of the newly identified 16S rRNA methylase, RmtC, shared a relatively low level of identity (< or = 29%) to other plasmid-mediated 16S rRNA methylases, RmtA, RmtB, and ArmA, which have also been identified in pathogenic gram-negative bacilli. Also, RmtC shared a low level of identity (< or = 28%) with the other 16S rRNA methylases found in aminoglycoside-producing actinomycetes. The purified histidine-tagged RmtC clearly showed methyltransferase activity against E. coli 16S rRNA in vitro. rmtC was located downstream of an ISEcp1-like element containing tnpA. Several plasmid-mediated 16S rRNA methylases have been identified in pathogenic gram-negative bacilli belonging to the family Enterobacteriaceae, and some of them are dispersing worldwide. The acceleration of aminoglycoside resistance among gram-negative bacilli by producing plasmid-mediated 16S rRNA methylases, such as RmtC, RmtB, and RmtA, may indeed become an actual clinical hazard in the near future.
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38. |
Eckert C,
Gautier V,
Arlet G,
( 2006 ) DNA sequence analysis of the genetic environment of various blaCTX-M genes. PMID : 16291869 : DOI : 10.1093/jac/dki398 Abstract >>
Over a 3 year period (2000-2003) 21 Escherichia coli, 5 Klebsiella pneumoniae, 1 Serratia marcescens and 1 Proteus mirabilis producing CTX-M-type beta-lactamase were collected from five different hospitals in Paris, France. This study was conducted to analyse the genetic environment of these 28 bla(CTX-M) genes. Antimicrobial susceptibility testing was performed by the disc diffusion method and MICs of various beta-lactams were determined by an agar dilution method. PCR was used to detect and sequence alleles encoding CTX-M, TEM, SHV and CMY enzymes. The genetic environment was analysed by amplification and direct sequencing using various set of PCR primers or cloning in pBK-CMV. Sequence analysis revealed that these isolates contained seven different bla(CTX-M) genes: bla(CTX-M-1) (4 strains), bla(CTX-M-2) (2 strains), bla(CTX-M-3) (4 strains), bla(CTX-M-9) (1 strain), bla(CTX-M-14) (5 strains), bla(CTX-M-15) (11 strains), bla(CTX-M-24) (1 strain). TEM-1 was associated with CTX-M-type enzymes in 15 isolates. Two strains produced both CTX-M-15 and SHV-2 or CTX-M-14 and CMY-2. In 25 strains the insertion sequence ISEcp1 was located upstream of the 5' end of the bla(CTX-M) gene. Among these strains, in five isolates, ISEcp1 was disrupted by insertion sequences such as IS26 (in three of them) or IS1 or IS10. Insertion sequence IS903 was found downstream of bla(CTX-M-14) or bla(CTX-M-24). Examination of the other three bla(CTX-M) genes (two bla(CTX-M-2) and one bla(CTX-M-9)) by cloning, sequencing and PCR analysis revealed the presence of complex Class 1 integrons, In35, an integron similar to In60 and a novel integron. This work further confirmed the predominant role of ISEcp1 in the mobilization of bla(CTX-M) genes of the CTX-M-1 cluster and the presence of In35, of an integron similar to In60 and a novel complex Class 1 integron.
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39. |
Ho PL,
Ho AY,
Chow KH,
Wong RC,
Duan RS,
Ho WL,
Mak GC,
Tsang KW,
Yam WC,
Yuen KY,
( 2005 ) Occurrence and molecular analysis of extended-spectrum {beta}-lactamase-producing Proteus mirabilis in Hong Kong, 1999-2002. PMID : 15857942 : DOI : 10.1093/jac/dki135 Abstract >>
A study was conducted to evaluate the occurrence and characterization of extended-spectrum beta-lactamases (ESBLs) among blood isolates of Proteus mirabilis collected over a 4 year period in Hong Kong. Production of ESBLs among 99 consecutive and non-duplicate isolates was evaluated by the double-disc synergy test. The ESBLs were characterized by isoelectric focusing and PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by the Dienes test and PFGE. ESBLs were identified in 13 isolates, from none in 1999-2000 and up to 18.5% (5/27) in 2001 and 25.8% (8/31) in 2002. The ESBL-producing isolates were more resistant to ceftriaxone than to ceftazidime, and were more likely than non-ESBL-producers to have resistance to ciprofloxacin (76.9% versus 14%) and gentamicin (38.5% versus 9.3%). The ESBL content included CTX-M-13 (n=8), CTX-M-14 (n=3), SHV-5 (n=2), TEM-11 (n=1), and an unidentified ESBL with a pI of 7.5. The Dienes test revealed that the genetic background in the 99 isolates was highly heterogeneous, with 54 distinct types among 92 isolates and seven were non-typeable. Among the 13 ESBL-producing isolates, five different backgrounds, including one cluster (Dienes-pulsotype A) with nine isolates, were identified by both Dienes test and PFGE, thus suggesting both clonal and multi-clonal spread of the CTX-M enzymes. Our findings indicate the emergence of CTX-M enzymes among P. mirabilis in Hong Kong. More ESBL screening of this species is required to improve their recognition.
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40. |
Literacka E,
Empel J,
Baraniak A,
Sadowy E,
Hryniewicz W,
Gniadkowski M,
( 2004 ) Four variants of the Citrobacter freundii AmpC-Type cephalosporinases, including novel enzymes CMY-14 and CMY-15, in a Proteus mirabilis clone widespread in Poland. PMID : 15504832 : DOI : 10.1128/AAC.48.11.4136-4143.2004 PMC : PMC525428 Abstract >>
Twenty-nine Proteus mirabilis isolates from 17 Polish hospitals were analyzed. The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to beta-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers. Indeed, beta-lactamases with a pI of approximately 9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants. The enzymes were of Citrobacter freundii origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones. The bla(CMY) genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of approximately 10 kb. In the CMY-12- and -15-producing isolates, an additional fragment of approximately 4.5 kb hybridized with the bla(CMY) probe as well, which could have arisen from a duplication event during the evolution of the genes. In all of the isolates, the ISEcp1 mobile element, which most probably is involved in mobilization of the C. freundii ampC gene, was placed at the same distance from the 5' ends of the bla(CMY) genes, and sequences located between them were identical in isolates carrying each of the four genes. These data suggested that a single chromosome-to-chromosome transfer of the ampC gene from C. freundii to P. mirabilis could have initiated the spread and evolution of the AmpC-producing P. mirabilis in Poland. The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the P. mirabilis isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates.
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41. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. PMID : 15289485 : DOI : 10.1101/gr.2649204 PMC : PMC509277 Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
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42. |
De Champs C,
Chanal C,
Sirot D,
Baraduc R,
Romaszko JP,
Bonnet R,
Plaidy A,
Boyer M,
Carroy E,
Gbadamassi MC,
Laluque S,
Oules O,
Poupart MC,
Villemain M,
Sirot J,
( 2004 ) Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study. PMID : 15282240 : DOI : 10.1093/jac/dkh395 Abstract >>
To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region. During 2001-2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR. ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1. ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.
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43. |
McGrath BM,
Pembroke JT,
( 2004 ) Detailed analysis of the insertion site of the mobile elements R997, pMERPH, R392, R705 and R391 in E. coli K12. PMID : 15268933 : DOI : 10.1016/j.femsle.2004.06.009 Abstract >>
The IncJ group of mobile elements have not been extensively studied until recently, due to the inability to isolate extrachromosomal DNA from IncJ-strains. Sequence analysis of the prototype IncJ element, R391, revealed it to be a mosaic structure, integrated into the prfC gene in E. coli. Using inverse PCR (iPCR), we localised the other available IncJ elements (R392, R705, R997 and pMERPH) site of insertion to a 17-bp sequence, within the 5' end of prfC at 99.31 min on the E. coli chromosome, and confirmed this for R391. Despite disrupting prfC, the IncJ's encode novel promoter and 5' sequences, restoring function of the disrupted prfC. Sequence analysis of the elements ends revealed that they contain integrase genes, which share extensive homologies among the group, despite being isolated from broad geographic locations. The elements excise from the host chromosome by recombination between their attL and attR sites, with subsequent recombination between the attP sites on the circular forms and the attB sites in the host genomes. The attB site is highly conserved and found in many different bacteria, suggesting a possible broad host range.
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44. |
Sturgill G,
Rather PN,
( 2004 ) Evidence that putrescine acts as an extracellular signal required for swarming in Proteus mirabilis. PMID : 14756784 : DOI : 10.1046/j.1365-2958.2003.03835.x Abstract >>
In a search for Proteus mirabilis genes that were regulated by cell-to-cell signalling, a lacZ fusion (cmr437::mini-Tn5lacZ) was identified that was repressed 10-fold by a self-produced extracellular signal from wild-type cells. However, the cmr437::mini-Tn5lacZ insertion itself led to a marked reduction in this extracellular repressing signal. The cmr437::mini-Tn5lacZ insertion was mapped to a speA homologue in P. mirabilis. Sequence analysis indicated that a speB homologue was encoded downstream of speA. Products of the SpeA and SpeB enzymes (agmatine and putrescine) were tested for repression of cmr437::lacZ. Agmatine did not have repressing activity. However, putrescine was an effective repressing molecule at concentrations down to 30 microM. A second prominent phenotype of the cmr437 (speA)::mini-Tn5lacZ insertion was a severe defect in swarming motility. This swarming defect was also observed in a strain containing a disruption of the downstream speB gene. Differentiation of the speB mutant to swarmer cells was delayed by two hours relative to wild-type cells. Furthermore, the speB mutant was unable to migrate effectively across agar surfaces and formed very closely spaced swarming rings. Exogenous putrescine restored both the normal timing of swarmer cell differentiation and the ability to migrate to speB mutants.
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45. |
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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46. |
Böltner D,
Osborn AM,
( 2004 ) Structural comparison of the integrative and conjugative elements R391, pMERPH, R997, and SXT. PMID : 14711525 : Abstract >>
R391 and SXT are members of a group of eleven chromosome-borne conjugative elements found in the gamma-proteobacteria, whose members carry different antibiotic resistance traits. Recent genomic analysis of R391 and SXT revealed a highly conserved 'backbone' encoding integration/excision, conjugative transfer, and regulation functions, augmented by an array of phenotypic traits and transposable elements. In this study, PCR amplification and sequence analysis were employed to investigate the genomic structure of two further MGE of the R391 family, pMERPH (HgR) and R997 (ApR, SmR, SuR). R997 and pMERPH were found to be structurally related to R391 and SXT and share a number of virtually identical regions with them-including putative integration, conjugative transfer, and regulatory determinants-interrupted by variable DNA segments and transposable elements. The presence of a highly conserved backbone in the four elements strongly suggests their origin in a common ancestral element, which itself was a mosaic of sequences related to phages and plasmids. Subsequent genetic recombination and the acquisition of transposable elements resulted in the possession of variable phenotypic traits among the four MGE, and diversification into two distinct lineages, the first one including R391 and pMERPH, the second one containing SXT and R997.
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47. |
Drieux L,
Bourgeois-Nicolaos N,
Cremniter J,
Lawrence C,
Jarlier V,
Doucet-Populaire F,
Sougakoff W,
( 2011 ) Accumulation of carbapenemase-producing Gram-negative bacteria in a single patient linked to the acquisition of multiple carbapenemase producers and to the in vivo transfer of a plasmid encoding VIM-1. PMID : 21570257 : DOI : 10.1016/j.ijantimicag.2011.03.017 Abstract >>
N/A
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48. |
Siebor E,
Neuwirth C,
( 2011 ) The new variant of Salmonella genomic island 1 (SGI1-V) from a Proteus mirabilis French clinical isolate harbours blaVEB-6 and qnrA1 in the multiple antibiotic resistance region. PMID : 21846670 : DOI : 10.1093/jac/dkr335 Abstract >>
The clinical strain of Proteus mirabilis VB1248 isolated from a blood culture in August 2009 was multiresistant (i.e. resistant to �]-lactams, fluoroquinolones, aminoglycosides and sulphonamides). We searched for the presence of a Salmonella genomic island 1 (SGI1). The whole genetic structure surrounding the genes involved in antibiotic resistance was characterized by PCR or gene walking followed by DNA sequencing. The new variant SGI1-V (42.9 kb) was located downstream of the thdF chromosomal gene. Genes sharing homology with phage-related genes were detected on a structure of 8.3 kb located between the right junction of the SGI1-V and the hipB/hipA genes. Some genetic rearrangements occurred in the SGI1-V backbone: an insertion of 2349 bp within the open reading frame (ORF) S014, and a deletion of 3766 bp in the region spanning from ORFs S021 to S025 leading to the lack of ORFs S023 and S024. The multidrug resistance (MDR) region of 17.1 kb was located on a complex class 1 integron extremely different from those described so far. The cassette array included aacA4, aadB and dhfrA1. Adjacent to this classical structure, bla(VEB-6) was found flanked by 135 bp elements and bracketed by two 3'-conserved segments (3'-CS). Downstream of the second copy of 3'-CS, the qnrA1 gene was associated with common region 1. We have identified in P. mirabilis the new variant SGI1-V containing the bla(VEB-6) and qnrA1 genes in the MDR region. This is the first report of an extended-spectrum �]-lactamase-encoding gene and a qnr determinant conferring resistance to quinolones on an SGI1-like structure. It might constitute a source of spread of resistance to other bacterial species.
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49. |
Petrovski S,
Stanisich VA,
( 2011 ) Embedded elements in the IncPbeta plasmids R772 and R906 can be mobilized and can serve as a source of diverse and novel elements. PMID : 21393370 : DOI : 10.1099/mic.0.047761-0 Abstract >>
IncP plasmids are important contributors to bacterial adaptation. Their phenotypic diversity is due largely to accessory regions located in one or two specific parts of the plasmid. The accessory regions are themselves diverse, as judged from sequenced plasmids mostly isolated from non-clinical sources. To further understand the diversity, evolutionary history and functional attributes of the accessory regions, we compared R906 and R772, focusing on the oriV-trfA accessory region. These IncP�] plasmids were from porcine and clinical sources, respectively. We found that the accessory regions formed potentially mobile elements, Tn510 (from R906) and Tn511 (from R772), that differed internally but had identical borders. Both elements appeared to have evolved from a TnAO22-like mer transposon that had inserted into an ancestral IncP�] plasmid and then accrued additional transposable elements and genes from various proteobacteria. Structural comparisons suggested that Tn510 (and a descendent in pB10), Tn511 and the mer element in pJP4 represent three lineages that evolved from the same widely dispersed IncP�] carrier. Functional studies on Tn511 revealed that its mer module is inactive due to a merT mutation, and that its aphAI region is prone to deletion. More significantly, we showed that by providing a suitable transposase gene in trans, the defective Tn510 and Tn511 could transpose intact or in part, and could also generate new elements (stable cointegrates and novel transposons). The ingredients for assisted transposition events similar to those observed here occur in natural microcosms, providing non-self-mobile elements with avenues for dispersal to new replicons and for structural diversification. This work provides an experimental demonstration of how the complex embedded elements uncovered in IncP plasmids and in other plasmid families may have been generated.
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50. |
Bi S,
Yan H,
Chen M,
Zhang Z,
Shi L,
Wang H,
( 2011 ) New variant Salmonella genomic island 1-U in Proteus mirabilis clinical and food isolates from South China. PMID : 21393148 : DOI : 10.1093/jac/dkr030 Abstract >>
N/A
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51. |
D'Andrea MM,
Literacka E,
Zioga A,
Giani T,
Baraniak A,
Fiett J,
Sadowy E,
Tassios PT,
Rossolini GM,
Gniadkowski M,
Miriagou V,
( 2011 ) Evolution and spread of a multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases in Europe. PMID : 21402851 : DOI : 10.1128/AAC.01736-10 PMC : PMC3101460 Abstract >>
Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.
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52. |
Baek JO,
Seo JW,
Kwon O,
Seong SI,
Kim IH,
Kim CH,
( 2011 ) Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli. PMID : 21298676 : DOI : 10.1002/jobm.201000086 Abstract >>
L-amino acid deaminases catalyze the deamination of natural L-amino acids. Two types of L-amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L-amino acids, typically L-phenylalanine, whereas the other acts on a relatively narrow range of basic L-amino acids, typically L-histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L-amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L-histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the V(max) and K(m) values of Pm1 were 119.7 (�gg phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and �\-oxoglutarate.
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53. |
Skovgaard O,
( 1990 ) Nucleotide sequence of a Proteus mirabilis DNA fragment homologous to the 60K-rnpA-rpmH-dnaA-dnaN-recF-gyrB region of Escherichia coli. PMID : 2172087 : DOI : 10.1016/0378-1119(90)90131-a Abstract >>
A 6.5-kb DNA fragment from Proteus mirabilis hybridized to the Escherichia coli dnaA gene. This DNA fragment was cloned and the nucleotide (nt) sequence determined. The fragment is homologous to a region of the E. coli chromosome containing a part of the gene encoding a 60-kDa membrane-associated protein (60K), the rnpA-rpmH-dnaA-dnaN-recF genes, and the N-terminal part of the gyrB gene. The degree of homology is variable: the amino-acid (aa) sequence of a part of the 60K protein and a part of the DnaA protein is only minimally conserved, whereas the C-terminal 148 aa of DnaA are identical in the two species. The conservation of the nt sequence between the rnpA gene and the gene encoding the 60K protein suggests that this region encodes a hitherto unrecognized protein. The ORF for this protein partially overlaps the 3' end of the rnpA structural gene, and the degree of conservation suggests that this gene is important for these bacteria.
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54. |
Song W,
Kim J,
Bae IK,
Jeong SH,
Seo YH,
Shin JH,
Jang SJ,
Uh Y,
Shin JH,
Lee MK,
Lee K,
( 2011 ) Chromosome-encoded AmpC and CTX-M extended-spectrum �]-lactamases in clinical isolates of Proteus mirabilis from Korea. PMID : 21282448 : DOI : 10.1128/AAC.01835-09 PMC : PMC3067170 Abstract >>
Among 222 Proteus mirabilis clinical isolates collected from 17 hospitals in Korea in 2008, 28 (12.6%) and 8 (3.6%) isolates exhibited extended-spectrum �]-lactamase (ESBL) and AmpC phenotypes, respectively. The most common type of ESBL gene identified by PCR and sequencing experiments was bla(CTX-M-14a) (n = 12). The bla(CTX-M-90) (n = 4), bla(CTX-M-15) (n = 3), bla(CTX-M-12) (n = 3), bla(CTX-M-2) (n = 2), bla(CTX-M-14b) (n = 1), bla(TEM-52) (n = 5), and bla(SHV-12) (n = 1) genes were also detected. Eight isolates carried an AmpC �]-lactamase gene, such as bla(CMY-2) (n = 6) or bla(DHA-1) (n = 2). All bla genes encoding CTX-M-1- and CTX-M-9-type enzymes and all bla(CMY-2) genes were preceded by ISEcp1-like elements. The bla(CTX-M-2) gene found in two isolates was located on a complex class 1 integron. The bla(DHA-1) gene was preceded by a transcriptional regulator gene and was followed by phage shock protein genes. The bla(CTX-M) genes were located on the chromosome in 21 isolates. A plasmid location for the bla(CTX-M) gene was found in only four isolates: the bla(CTX-M-14a) gene was located on ?150-kbp IncA/C plasmids in three isolates and on a ?50-kbp IncN plasmid in one isolate. The bla(TEM-52) gene was located on ?50-kbp IncN plasmids in all five isolates. The AmpC �]-lactamase genes were located on the chromosome in seven of eight isolates; one isolate carried the bla(CMY-2) gene on a ?150-kbp IncA/C plasmid. Our results show that a chromosomal location of CTX-M ESBL and AmpC �]-lactamase genes in P. mirabilis is no longer an unusual phenomenon in hospital environments.
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55. |
Aquilini E,
Azevedo J,
Jimenez N,
Bouamama L,
Tomás JM,
Regué M,
( 2010 ) Functional identification of the Proteus mirabilis core lipopolysaccharide biosynthesis genes. PMID : 20622068 : DOI : 10.1128/JB.00494-10 PMC : PMC2937370 Abstract >>
In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster.
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56. |
Harada S,
Ishii Y,
Saga T,
Tateda K,
Yamaguchi K,
( 2010 ) Chromosomally encoded blaCMY-2 located on a novel SXT/R391-related integrating conjugative element in a Proteus mirabilis clinical isolate. PMID : 20566768 : DOI : 10.1128/AAC.00111-10 PMC : PMC2934980 Abstract >>
Integrating conjugative elements (ICEs) are mobile genetic elements that can transfer from the chromosome of a host to the chromosome of a new host through the process of excision, conjugation, and integration. Although SXT/R391-related ICEs, originally demonstrated in Vibrio cholerae O139 isolates, have become prevalent among V. cholerae isolates in Asia, the prevalence of the ICEs among gram-negative bacteria other than Vibrio spp. remains unknown. In addition, SXT/R391-related ICEs carrying genes conferring resistance to extended-spectrum cephalosporins have never been described. Here we carried out a genetic analysis of a cefoxitin-resistant Proteus mirabilis clinical isolate, TUM4660, which revealed the presence of a novel SXT/R391-related ICE, ICEPmiJpn1. ICEPmiJpn1 had a core genetic structure showing high similarity to that of R391 and carried xis and int genes completely identical to those of R391, while an IS10-mediated composite transposon carrying bla(CMY-2) was integrated into the ICE. A nucleotide sequence identical to the 3' part of ISEcp1 was located upstream of the bla(CMY-2) gene, and other genes observed around bla(CMY-2) in earlier studies were also present. Furthermore, the nucleotide sequences of hot spot 2 and hot spot 4 in ICEPmiJpn1 showed high similarity to that of hot spot 2 in SXT(MO10) and with a part of the nucleotide sequence found in P. mirabilis ATCC 29906, respectively. ICEPmiJpn1 was successfully transferred to Escherichia coli, Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Citrobacter koseri in conjugation experiments. These observations suggest that ICEs may contribute to the dissemination of antimicrobial resistance genes among clinically relevant Enterobacteriaceae, which warrants careful observation of the prevalence of ICEs, including SXT/R391-related ICEs.
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57. |
Aquilini E,
Azevedo J,
Merino S,
Jimenez N,
Tomás JM,
Regué M,
( 2010 ) Three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide. PMID : 20959463 : DOI : 10.1074/jbc.M110.168385 PMC : PMC3000955 Abstract >>
The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (�\HexN-(1,4)-�\GalA) attached by an �\1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-�\-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase.
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58. |
Malekjamshidi MR,
Shahcheraghi F,
Feizabadi MM,
( 2010 ) Detection and PFGE analysis of ESBL-producing isolates of Proteus species isolated from patients at Tehran hospitals. PMID : 20885345 : Abstract >>
The production of extended-spectrum �]-lactamases (ESBLs), in particular TEM and CTX-M type, by the strains of Proteus spp. is a contributing factor to the emergence of bacterial drug resistance. The aim of this study was to screen and determine of ESBLs genes among strains of Proteus spp. at Tehran hospitals. A total of 100 clinical isolates of Proteus species and 1 isolate of Morganella morganii were collected from hospitals in Tehran. Susceptibility of these isolates to various antibiotics was tested by disk diffusion method. Microbroth dilution assay was then used to determine the minimum inhibitory concentration (MIC) of ceftazidime. The phenotypic confirmatory test (PCT) was used for detection of ESBLs. Isolates showing MIC ?4 ?g/ml for ceftazidime were screened for detection of ESBLs genes by PCR. The genomic DNA from ESBL-producing isolates was extracted and analyzed by PFGE. During the study, 11.8% of the isolates were positive for ESBLs genes. The MICs of ceftazidime-resistant isolates were in the range of 4 to 8 ?g/ml. The Frequencies of different genes encoding the ESBLs were as follows: blaCTX-M (n=13), blaTEM (n=10), blaVEB (n=7), blaSHV (n=1), and blaPER (n=0). Analysis of 12 ESBL-producing isolates by PFGE produced 5 distinct clusters designated as I-V. The Detection of ESBL-producing isolates of Proteus spp. with similar PFGE pattern is alarming and suggests the clonal outbreaks with these strains at Tehran hospitals is likely, necessitating control over prescription of new generation cephalosporins.
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59. |
Wang Q,
Torzewska A,
Ruan X,
Wang X,
Rozalski A,
Shao Z,
Guo X,
Zhou H,
Feng L,
Wang L,
( 2010 ) Molecular and genetic analyses of the putative Proteus O antigen gene locus. PMID : 20581173 : DOI : 10.1128/AEM.02946-09 PMC : PMC2918944 Abstract >>
Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups.
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60. |
Wachino J,
Shibayama K,
Kimura K,
Yamane K,
Suzuki S,
Arakawa Y,
( 2010 ) RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms. PMID : 20722735 : DOI : 10.1111/j.1574-6968.2010.02068.x Abstract >>
Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance.
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61. |
Doublet B,
Poirel L,
Praud K,
Nordmann P,
Cloeckaert A,
( 2010 ) European clinical isolate of Proteus mirabilis harbouring the Salmonella genomic island 1 variant SGI1-O. PMID : 20682564 : DOI : 10.1093/jac/dkq283 Abstract >>
N/A
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62. |
Giammanco GM,
Grimont PA,
Grimont F,
Lefevre M,
Giammanco G,
Pignato S,
( 2011 ) Phylogenetic analysis of the genera Proteus, Morganella and Providencia by comparison of rpoB gene sequences of type and clinical strains suggests the reclassification of Proteus myxofaciens in a new genus, Cosenzaea gen. nov., as Cosenzaea myxofaciens comb. nov. PMID : 20709916 : DOI : 10.1099/ijs.0.021964-0 Abstract >>
Phylogenetic analysis of partial rpoB gene sequences of type and clinical strains belonging to different 16S rRNA gene-fingerprinting ribogroups within 11 species of enterobacteria of the genera Proteus, Morganella and Providencia was performed and allowed the definition of rpoB clades, supported by high bootstrap values and confirmed by ?2.5 % nucleotide divergence. None of the resulting clades included strains belonging to different species and the majority of the species were confirmed as discrete and homogeneous. However, more than one distinct rpoB clade could be defined among strains belonging to the species Proteus vulgaris (two clades), Providencia alcalifaciens (two clades) and Providencia rettgeri (three clades), suggesting that some strains represent novel species according to the genotypes outlined by rpoB gene sequence analysis. Percentage differences between the rpoB gene sequence of the type strain of Proteus myxofaciens and other members of the same genus (17.3-18.9 %) were similar to those calculated amongst strains of the genus Providencia (16.4-18.7 %), suggesting a genetic distance at the genus-level between Proteus myxofaciens and the rest of the Proteus-Providencia group. Proteus myxofaciens therefore represents a member of a new genus, for which the name Cosenzaea gen. nov., is proposed.
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63. |
Jiang SS,
Lin TY,
Wang WB,
Liu MC,
Hsueh PR,
Liaw SJ,
( 2010 ) Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance, swarming, and virulence. PMID : 20160049 : DOI : 10.1128/AAC.01384-09 PMC : PMC2863647 Abstract >>
Proteus mirabilis is known to be highly resistant to the action of polymyxin B (PB). However, the mechanism underlying PB resistance is not clear. In this study, we used Tn5 transposon mutagenesis to identify genes that may affect PB resistance in P. mirabilis. Two genes, ugd and galU, which may encode UDP-glucose dehydrogenase (Ugd) and UDP-glucose pyrophosphorylase (GalU), respectively, were identified. Knockout mutants of ugd and galU were found to be extremely sensitive to PB, presumably because of alterations in lipopolysaccharide (LPS) structure and cell surface architecture in these mutants. These mutants were defective in swarming, expressed lower levels of virulence factor hemolysin, and had lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd or galU gene, respectively, led to the restoration of wild-type phenotypic traits. Interestingly, we found that the expression of Ugd and GalU was induced by PB through RppA, a putative response regulator of the bacterial two-component system that we identified previously. Mutation in either ugd or galU led to activation of RpoE, an extracytoplasmic function sigma factor that has been shown to be activated by protein misfolding and alterations in cell surface structure in other bacteria. Activation of RpoE or RpoE overexpression was found to cause inhibition of FlhDC and hemolysin expression. To our knowledge, this is the first report describing the roles and regulation of Ugd and GalU in P. mirabilis.
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64. |
Jiang SS,
Liu MC,
Teng LJ,
Wang WB,
Hsueh PR,
Liaw SJ,
( 2010 ) Proteus mirabilis pmrI, an RppA-regulated gene necessary for polymyxin B resistance, biofilm formation, and urothelial cell invasion. PMID : 20123999 : DOI : 10.1128/AAC.01219-09 PMC : PMC2849355 Abstract >>
Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn5 inserted into the PpmrI (Proteus pmrI) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the PpmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the PpmrI knockout mutant. Moreover, the PpmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the PpmrI mutant with the full-length PpmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis. Here we showed that RppA could mediate the induction of PpmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative PpmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis.
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65. |
McGettigan SE,
Hu B,
Andreacchio K,
Nachamkin I,
Edelstein PH,
( 2009 ) Prevalence of CTX-M beta-lactamases in Philadelphia, Pennsylvania. PMID : 19587301 : DOI : 10.1128/JCM.00319-09 PMC : PMC2738063 Abstract >>
CTX-M beta-lactamases were thought to be rare in the United States, but a recent study in Texas showed that up to 70% of extended-spectrum beta-lactamase (ESBL)-containing members of the Enterobacteriaceae family were CTX-M positive (J. S. Lewis, M. Herrera, B. Wickes, J. E. Patterson, and J. H. Jorgensen, Antimicrob. Agents Chemother. 51:4015-4021, 2007). We used PCR to detect CTX-M in all 291 extended-spectrum cephalosporin-resistant gram-negative bacteria isolated in our laboratory during 2007. Thirty (48%) Escherichia coli isolates, 6 (3%) Klebsiella sp. isolates, and 7 (100%) Proteus mirabilis isolates tested were CTX-M positive, with 15% of all Enterobacteriaceae tested being positive. The E. coli CTX-M groups were I (57%), IV (37%), II (3%), and not groupable (3%); three of the group IV isolates were positive for CTX-M-18, and three of the group I isolates were positive for CTX-M-15. One of seven positive P. mirabilis isolates was in group II, with the remainder being positive for a CTX-M-25-like beta-lactamase; and 33% of the Klebsiella sp. isolates were in group I or IV, with the remainder not being in groups I to IV. CTX-M-producing bacteria were isolated from urine (n = 13), blood (n = 13), wounds (n = 12), and the respiratory tract (n = 4). All 31 CTX-M-positive isolates tested for the presence of ESBL were confirmed to produce ESBLs by the use of tests recommended by the CLSI. Pulsed-field gel electrophoresis of the CTX-M-positive isolates showed that six P. mirabilis isolates were clonal and that there were seven different E. coli clusters. Five of seven P. mirabilis isolates were from blood cultures. The CLSI tests for the confirmation of ESBL production reliably detect these isolates if both cefotaxime and ceftazidime are tested, but only about half would be classified as a possible CTX-M producers on the basis of the antibiogram alone. A new panprimer set increases the ability to detect CTX-M-producing strains. CTX-M-positive bacteria are common in our geographic region, are often invasive, and, with the exception of P. mirabilis, are multiclonal.
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66. |
Verdet C,
Gautier V,
Chachaty E,
Ronco E,
Hidri N,
Decré D,
Arlet G,
( 2009 ) Genetic context of plasmid-carried blaCMY-2-like genes in Enterobacteriaceae. PMID : 19596889 : DOI : 10.1128/AAC.00753-08 PMC : PMC2737857 Abstract >>
Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of blaCMY-2-like genes reflected the replicon type, usually IncA/C or IncI1. These blaCMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.
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67. |
Wylie BA,
Koornhof HJ,
( 1991 ) Nucleotide sequence of dihydrofolate reductase type VI. PMID : 1941991 : DOI : 10.1099/00222615-35-4-214 Abstract >>
The complete sequence of the type VI dihydrofolate reductase (DHFR) gene from plasmid pUK672 was determined. The structural gene coded for a polypeptide of 157 amino acids which had a deduced mol. wt of 17,424. Comparison with amino-acid sequences of the type I, type V and Escherichia coli K12 chromosomal DHFRs showed that there was 63%, 61% and 31% homology respectively. Putative RNA polymerase and ribosomal binding sites were identified proximal to the initiation codon and a feature consistent with transcription termination was present distal to the coding region. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the enzyme had a subunit mol. wt of 17,500.
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68. |
Wang M,
Guo Q,
Xu X,
Wang X,
Ye X,
Wu S,
Hooper DC,
Wang M,
( 2009 ) New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis. PMID : 19258263 : DOI : 10.1128/AAC.01400-08 PMC : PMC2681562 Abstract >>
Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6')-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 microg/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.
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69. |
Jones CH,
Ruzin A,
Tuckman M,
Visalli MA,
Petersen PJ,
Bradford PA,
( 2009 ) Pyrosequencing using the single-nucleotide polymorphism protocol for rapid determination of TEM- and SHV-type extended-spectrum beta-lactamases in clinical isolates and identification of the novel beta-lactamase genes blaSHV-48, blaSHV-105, and blaTEM-155. PMID : 19075050 : DOI : 10.1128/AAC.01155-08 PMC : PMC2650538 Abstract >>
TEM- and SHV-type extended-spectrum beta-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the bla(TEM) and bla(SHV) genes. Three novel beta-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing bla(SHV-105) were >128, 128, and >128 microg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing bla(TEM-155) were >128, 64, and > 128 microg/ml, respectively. Pyrosequence analysis determined the true identity of the beta-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.
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70. |
Zong Z,
Partridge SR,
Iredell JR,
( 2009 ) A blaVEB-1 variant, blaVEB-6, associated with repeated elements in a complex genetic structure. PMID : 19139283 : DOI : 10.1128/AAC.01313-08 PMC : PMC2663121 Abstract >>
bla(VEB-6) was found on the Proteus mirabilis chromosome in a context similar to those of bla(VEB-1a) and bla(VEB-1b), in a truncated gene cassette flanked by 135-bp elements and duplications of the 3'-conserved segment of class 1 integrons. A linked aacA4-aadB-dfrA1-orfC cassette array includes components of Tn1331, illustrating the complex mosaicism of multiresistance regions.
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71. |
Gibbs KA,
Urbanowski ML,
Greenberg EP,
( 2008 ) Genetic determinants of self identity and social recognition in bacteria. PMID : 18621670 : DOI : 10.1126/science.1160033 PMC : PMC2567286 Abstract >>
The bacterium Proteus mirabilis is capable of movement on solid surfaces by a type of motility called swarming. Boundaries form between swarming colonies of different P. mirabilis strains but not between colonies of a single strain. A fundamental requirement for boundary formation is the ability to discriminate between self and nonself. We have isolated mutants that form boundaries with their parent. The mutations map within a six-gene locus that we term ids for identification of self. Five of the genes in the ids locus are required for recognition of the parent strain as self. Three of the ids genes are interchangeable between strains, and two encode specific molecular identifiers.
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72. |
Empel J,
Baraniak A,
Literacka E,
Mrówka A,
Fiett J,
Sadowy E,
Hryniewicz W,
Gniadkowski M,
N/A N/A,
( 2008 ) Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals. PMID : 18458126 : DOI : 10.1128/AAC.00043-08 PMC : PMC2443902 Abstract >>
The first national survey of resistance to newer beta-lactams in nosocomial populations of Enterobacteriaceae in Poland was performed. The study covered all nonrepetitive enterobacterial isolates cultured from specimens from inpatients in 13 regional secondary-care hospitals from November 2003 to January 2004. Among 2,388 isolates, the predominant species was Escherichia coli (59.6%), followed by Proteus mirabilis (14.5%) and Klebsiella spp. (8.5%). The frequency of extended-spectrum beta-lactamases (ESBLs) was very high, with ESBLs present in 11.1% of all isolates and 40.4% of Klebsiella pneumoniae isolates, the latter value greatly exceeding that for E. coli (2.5%). The contribution of outbreak isolates was significant, resulting, for example, in a particularly high rate of ESBL producers among Serratia marcescens isolates (70.8%). The pool of ESBL types was overwhelmingly dominated (81.7%) by CTX-M-like beta-lactamases CTX-M-3 (80.6%) and CTX-M-15, with SHV types (17.5%; SHV-2, SHV-5, and SHV-12) and sporadic TEM-like enzymes (0.7%; TEM-19 and TEM-48) being the next most frequent. Acquired AmpC-type cephalosporinases were observed exclusively in P. mirabilis, in 20.5% of the isolates of this species (compared with the frequency of ESBL producers of 11.5% of P. mirabilis isolates). All these cephalosporinases (CMY-12, CMY-15, and a novel variant, CMY-38) originated from Citrobacter freundii. Four isolates of E. coli (two isolates), K. pneumoniae (one isolate), and P. mirabilis (one isolate) produced class A inhibitor-resistant beta-lactamases (TEM-30, TEM-32, TEM-37, and SHV-49), being the first of such producers identified in Poland. The survey documented both specific and more global characteristics of the epidemiology of the beta-lactamase-mediated resistance in enterobacteria from Polish hospitals and demonstrated that the ESBL frequency has reached an alarming level.
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73. |
Navon-Venezia S,
Chmelnitsky I,
Leavitt A,
Carmeli Y,
( 2008 ) Dissemination of the CTX-M-25 family beta-lactamases among Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae and identification of the novel enzyme CTX-M-41 in Proteus mirabilis in Israel. PMID : 18487232 : DOI : 10.1093/jac/dkn182 Abstract >>
The CTX-M-25 family of beta-lactamases is a closely related family of enzymes found rarely in the world. We aimed to describe the occurrence and to understand the dissemination of this extended-spectrum beta-lactamase family among Enterobacteriaceae strains in our hospital. Fifty-four CTX-M-producing Enterobacteriaceae strains collected from 2000 to 2005 were screened for bla(CTX-M-25) genes by PCR and sequencing. Genetic relatedness was analysed by PFGE. Antibiotic susceptibilities were determined by VITEK-2. Plasmids encoding bla(CTX-M-25)-type genes were isolated, transformed and analysed by Southern blot using a bla(CTX-M-25) probe. Chromosomal location of bla(CTX-M-25)-type was studied by I-CeuI restriction analysis. The bla(CTX-M-25) genetic environment was characterized by PCR mapping and partial sequencing. Ten out of 54 CTX-M-producing isolates (18.5%) carried bla(CTX-M-25) genes, including Klebsiella pneumoniae (n = 4), Escherichia coli (n = 3), Enterobacter cloacae (n = 1) and Proteus mirabilis (n = 2). Isolates were genetically unrelated. Four beta-lactamases were found: CTX-M-25, CTX-M-26, CTX-M-39 and CTX-M-41, a new member of the family (accession no. DQ023162) that differed from CTX-M-25 in three amino acids, Ala80Val, Val106Ile and Ile126Ser. bla(CTX-M-25)-type genes were plasmid-mediated in all genera but P. mirabilis, organized in a class I integron and located downstream of an ISEcp1 element. The genes were encoded on different plasmids with varying degree of similarities. Several antibiotic-resistant determinants conferring resistance to trimethoprim and aminoglycosides existed on the same integron. bla(CTX-M-25) exists in Israel in different enteric species. Spread of these enzymes within and between species is due to transfer of plasmids with common regions and by dissemination of determinants encoding these genes. CTX-M-41, a novel member of this family, was identified in the chromosome of P. mirabilis.
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74. |
Sakurai Y,
Tsukamoto K,
Sawai T,
( 1991 ) Nucleotide sequence and characterization of a carbenicillin-hydrolyzing penicillinase gene from Proteus mirabilis. PMID : 1840585 : DOI : 10.1128/jb.173.21.7038-7041.1991 PMC : PMC209063 Abstract >>
The structural gene of a carbenicillinase was cloned from the chromosomal DNA of Proteus mirabilis GN79. This gene codes for a protein of 270 amino acids. Alignment of the amino acid sequence with those of known beta-lactamases revealed that the enzyme is a novel class A beta-lactamase with a unique conserved triad, RTG. By using a DNA fragment of the structural gene, a lack of cross hybridization was confirmed between the DNA probe and total DNAs from natural isolates of P. mirabilis, suggesting that the carbenicillinase may not be a species-specific beta-lactamase of P. mirabilis.
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75. |
Wang WB,
Chen IC,
Jiang SS,
Chen HR,
Hsu CY,
Hsueh PR,
Hsu WB,
Liaw SJ,
( 2008 ) Role of RppA in the regulation of polymyxin b susceptibility, swarming, and virulence factor expression in Proteus mirabilis. PMID : 18316383 : DOI : 10.1128/IAI.01557-07 PMC : PMC2346679 Abstract >>
Proteus mirabilis, a human pathogen that frequently causes urinary tract infections, is intrinsically highly resistant to cationic antimicrobial peptides, such as polymyxin B (PB). To explore the mechanisms underlying P. mirabilis resistance to PB, a mutant which displayed increased (> 160-fold) sensitivity to PB was identified by transposon mutagenesis. This mutant was found to have Tn5 inserted into a novel gene, rppA. Sequence analysis indicated that rppA may encode a response regulator of the two-component system and is located upstream of the rppB gene, which may encode a membrane sensor kinase. An rppA knockout mutant of P. mirabilis had an altered lipopolysaccharide (LPS) profile. The LPS purified from the rppA knockout mutant could bind more PB than the LPS purified from the wild type. These properties of the rppA knockout mutant may contribute to its PB-sensitive phenotype. The rppA knockout mutant exhibited greater swarming motility and cytotoxic activity and expressed higher levels of flagellin and hemolysin than the wild type, suggesting that RppA negatively regulates swarming, hemolysin expression, and cytotoxic activity in P. mirabilis. PB could modulate LPS synthesis and modification, swarming, hemolysin expression, and cytotoxic activity in P. mirabilis through an RppA-dependent pathway, suggesting that PB could serve as a signal to regulate RppA activity. Finally, we demonstrated that the expression of rppA was up-regulated by a low concentration of PB and down-regulated by a high concentration of Mg2+. Together, these data highlight the essential role of RppA in regulating PB susceptibility and virulence functions in P. mirabilis.
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76. |
Aragón LM,
Mirelis B,
Miró E,
Mata C,
Gómez L,
Rivera A,
Coll P,
Navarro F,
( 2008 ) Increase in beta-lactam-resistant Proteus mirabilis strains due to CTX-M- and CMY-type as well as new VEB- and inhibitor-resistant TEM-type beta-lactamases. PMID : 18292096 : DOI : 10.1093/jac/dkn056 Abstract >>
The aim of this study was to characterize the different inhibitor-resistant TEM beta-lactamases, extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC beta-lactamases implicated in beta-lactam resistance in Proteus mirabilis, which has increased over recent years. From February 2000 to December 2005, 1423 clinical isolates of P. mirabilis were collected. The AmpC phenotype was checked by means of a double-disc synergy test using cloxacillin as an inhibitor of AmpC enzymes. The production of ESBL was assessed by the double-disc synergy method and by Etest ESBL. Analytical isoelectric focusing, determination of kinetic constants, conjugation, PCR and a sequencing strategy were used to characterize the enzymes. The possible relationships between isolates were analysed by PFGE. Twenty-five of 1423 isolates were found to display intermediate or full resistance to co-amoxiclav, cefotaxime or ceftazidime. Seventeen isolates had reduced susceptibility to co-amoxiclav; of these, seven produced TEM-110, eight produced the new TEM-159, one the new TEM-160 and one TEM-1. Five isolates producing TEM-110, TEM-159 or TEM-160 enzymes shared the same PFGE profile. Three isolates produced an ESBL, CTX-M-1, CTX-M-32 and the new variant, VEB-4. Finally, five isolates with an AmpC phenotype produced CMY-2, two with the same PFGE profile. Our data emphasize the diversity of beta-lactamases found in this species.
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77. |
Boyd DA,
Shi X,
Hu QH,
Ng LK,
Doublet B,
Cloeckaert A,
Mulvey MR,
( 2008 ) Salmonella genomic island 1 (SGI1), variant SGI1-I, and new variant SGI1-O in proteus mirabilis clinical and food isolates from China. PMID : 18025121 : DOI : 10.1128/AAC.00902-07 PMC : PMC2223879 Abstract >>
Salmonella genomic island 1 (SGI1) and variants (SGI1-I and the new variant SGI1-O) were mapped in five strains of Proteus mirabilis isolated from humans and food in China. Sequencing showed that SGI1 and variants were integrated at the 3' end of the chromosomal thdF gene as previously described for Salmonella strains.
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78. |
Zong Z,
Partridge SR,
Iredell JR,
( 2008 ) RmtC 16S rRNA methyltransferase in Australia. PMID : 18025117 : DOI : 10.1128/AAC.01399-07 PMC : PMC2224735 Abstract >>
N/A
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79. |
Hatt JK,
Rather PN,
( 2008 ) Characterization of a novel gene, wosA, regulating FlhDC expression in Proteus mirabilis. PMID : 18192389 : DOI : 10.1128/JB.01010-07 PMC : PMC2258889 Abstract >>
In this study, we describe wosA, a Proteus mirabilis gene identified by its ability to increase swarming motility when overexpressed. At various times during the swarming cycle, the increased expression of wosA resulted in a 4- to 16-fold upregulation of the transcription of flhDC, encoding the master regulator of the flagellar cascade. In turn, the expression of flaA, encoding flagellin, was substantially increased in wosA-overexpressing strains. The overexpression of wosA also resulted in constitutive swarmer cell differentiation in liquid medium, a normally nonpermissive condition. However, in wosA-overexpressing strains, the onset of swarming was not altered. A null wosA allele resulted in a slight decrease in swarming motility. The expression of wosA was growth phase dependent during growth in liquid and on agar plates during swarmer cell differentiation. Increasing the viscosity of liquid medium by the addition of polyvinylpyrrolidone induced swarmer cell differentiation and resulted in a fourfold increase in wosA transcription. A fliL mutation that results in constitutive swarmer cell elongation also increased wosA transcription. In this study, we discuss the possible role of the wosA gene product in signal transduction from solid surfaces to induce swarmer cell differentiation, possibly via alterations in the motor switch complex. This study also suggests that despite constitutive swarmer cell differentiation in wosA-overexpressing strains, there are additional regulatory and/or environmental conditions that may control the onset of swarming migration.
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80. |
Wu JJ,
Chen HM,
Ko WC,
Wu HM,
Tsai SH,
Yan JJ,
( 2008 ) Prevalence of extended-spectrum beta-lactamases in Proteus mirabilis in a Taiwanese university hospital, 1999 to 2005: identification of a novel CTX-M enzyme (CTX-M-66). PMID : 17913434 : DOI : 10.1016/j.diagmicrobio.2007.08.004 Abstract >>
A total of 1574 nonduplicate Proteus mirabilis isolates collected at a Taiwanese hospital during 1999 to 2005 were analyzed for production of extended-spectrum beta-lactamases (ESBLs). Forty-four ESBL-producing isolates including 22 CTX-M-14, 18 CTX-M-3, 2 CTX-M-24, and 2 CTX-M-66 producers were detected, and the proportion of ESBL producers increased from 0.7% in 1999 to approximately 6% after 2002. CTX-M-66 is a novel variant of CTX-M ESBLs that differs from CTX-M-3 by a Ser to Asn change at amino acid position 23. Coresistances to aminoglycosides and ciprofloxacin were very common in the CTX-M-3 producers. The presence of ArmA-type or RmtB-type 16S rRNA methylase that confers high-level aminoglycoside resistance was detected in 12 CTX-M-3 producers and 4 CTX-M-14 producers. Twenty-four clones including an endemic CTX-M-14-producing clone were observed among the 44 ESBL producers by pulsed-field gel electrophoresis, suggesting that both horizontal transfer and clonal spread contributed to the increased prevalence of bla(CTX-M) in P. mirabilis.
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81. |
Espinal P,
Miró E,
Ramoneda L,
Flores M,
Rivera A,
Coll P,
Navarro F,
( 2017 ) Characterization of the Genetic Environment of the blaVEB-4 Gene, Associated with a Transposable Region in a Proteus mirabilis Clinical Isolate. PMID : 28304213 : DOI : 10.1089/mdr.2016.0262 Abstract >>
Proteus mirabilis is the second most common cause of urinary tract infections and is also an important cause of nosocomial infections. TEM-type and CTX-M-type extended-spectrum �]-lactamases (ESBLs) are the most widely distributed in this bacterial species, but minor ESBLs such as the VEB-type have also been identified. The aim of this study was to analyze the genetic environment of the blaVEB-4 gene found in a P. mirabilis clinical isolate recovered in Spain. P. mirabilis N2231 showed resistance to penicillins, cephalosporins, and aminoglycosides, remaining susceptible to imipenem, cefoxitin, �]-lactamases inhibitors, and quinolones. Southern blot analysis revealed that blaVEB-4 was located in the chromosome. Analysis of the blaVEB-4 genetic context revealed a 15 kb segment 98% identical to the multidrug resistance (MDR) region of a Salmonella genomic island 1 (SGI1), which included a class 1 integron belonging to the In104 family, previously described in blaVEB-6-producing P. mirabilis VB1248. blaVEB-4 was surrounded by repeat elements, transposon Tn1721, and located on a class 1 integron containing aacA4-aadB-dfrA1-orfC genes. The blaVEB-4 gene was inserted in a complex structure of a class 1 integron, which is part of an MDR region of an SGI1, possibly involved in the mobilization of the gene and homologous recombination.
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82. |
Lange F,
Pfennigwerth N,
Gerigk S,
Gohlke F,
Oberdorfer K,
Purr I,
Wohanka N,
Roggenkamp A,
Gatermann SG,
Kaase M,
( 2017 ) Dissemination of blaOXA-58 in Proteus mirabilis isolates from Germany. PMID : 28093482 : DOI : 10.1093/jac/dkw566 Abstract >>
Characterization of Proteus mirabilis isolates harbouring bla OXA-58 with emphasis on the genetic environment of this resistance determinant. Strains of P. mirabilis (n = 37) isolated from different patients were tested for the presence of bla OXA-58 . The genetic context of bla OXA-58 was determined by WGS of two strains and Sanger sequencing. Clonality of the strains was assessed by PFGE. Susceptibility testing was performed by microdilution according to EUCAST. Four strains isolated in different geographical regions of Germany were positive for bla OXA-58 , and WGS showed that this resistance gene was harboured on a plasmid. Sanger sequencing confirmed the presence of two nearly identical plasmids, 6219 and 6208 bp in size, in all four strains. Upstream of bla OXA-58 an IS Aba 3-like transposase gene was located. The P. mirabilis strains were not clonally related according to PFGE. MICs of meropenem for three of the strains were only just above the EUCAST breakpoint and the Carba NP test was positive for only two of the strains. To our knowledge, this is the first description of bla OXA-58 in the species P. mirabilis . The resistance gene is harboured by almost identical plasmids in strains not clonally related and from different geographical regions. Apart from an IS Aba 3-like transposase gene upstream of bla OXA-58 the genetic context is different from bla OXA-58 harboured on plasmids in the genus Acinetobacter . With MICs of meropenem well below the EUCAST breakpoint or only just above it and equivocal or false negative results from the Carba NP test, bla OXA-58 can be easily overlooked in P. mirabilis .
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83. |
He D,
Liu L,
Guo B,
Wu S,
Chen X,
Wang J,
Zeng Z,
Liu JH,
( 2017 ) Chromosomal location of the fosA3 and blaCTX-M genes in Proteus mirabilis and clonal spread of Escherichia coli ST117 carrying fosA3-positive IncHI2/ST3 or F2:A-:B- plasmids in a chicken farm. PMID : 28238801 : DOI : 10.1016/j.ijantimicag.2016.12.009 Abstract >>
The aim of this study was to investigate the spread and location of the fosA3 gene among Enterobacteriaceae from diseased broiler chickens. Twenty-nine Escherichia coli and seven Proteus mirabilis isolates recovered from one chicken farm were screened for the presence of plasmid-mediated fosfomycin resistance genes by PCR. The clonal relatedness of fosA3-positive isolates, the transferability and location of fosA3, and the genetic context of the fosA3 gene were determined. Seven P. mirabilis isolates with three different pulsed-field gel electrophoresis (PFGE) patterns and five E. coli isolates belonging to sequence type 117 (ST117) and phylogenetic group D were positive for fosA3 and all carried the blaCTX-M gene. In E. coli, the genetic structures IS26-ISEcp1-blaCTX-M-65-IS26-fosA3-1758 bp-IS26 and IS26-ISEcp1-blaCTX-M-3-blaTEM-1-IS26-fosA3-1758 bp-IS26 were present on transferable IncHI2/ST3 and F2:A-:B- plasmids, respectively. However, fosA3 was located on the chromosome of the seven P. mirabilis isolates. IS26-ISEcp1-blaCTX-M-65-IS26-fosA3-1758 bp-IS26 and IS26-blaCTX-M-14-611 bp-fosA3-1222 bp-IS26 were detected in three and four P. mirabilis isolates, respectively. Minicircles that contained both fosA3 and blaCTX-M-65 were shared between E. coli and P. mirabilis. This is the first report of the fosA3 gene integrated into the chromosome of P. mirabilis isolates with the blaCTX-M gene. The emergence and clonal spread of avian pathogenic E. coli ST117 with the feature of multidrug resistance and high virulence are a serious problem.
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84. |
Mollenkopf DF,
Stull JW,
Mathys DA,
Bowman AS,
Feicht SM,
Grooters SV,
Daniels JB,
Wittum TE,
( 2017 ) Carbapenemase-Producing Enterobacteriaceae Recovered from the Environment of a Swine Farrow-to-Finish Operation in the United States. PMID : 27919894 : DOI : 10.1128/AAC.01298-16 PMC : PMC5278694 Abstract >>
Carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to public health. While use of carbapenem antimicrobials is restricted for food-producing animals, other �]-lactams, such as ceftiofur, are used in livestock. This use may provide selection pressure favoring the amplification of carbapenem resistance, but this relationship has not been established. Previously unreported among U.S. livestock, plasmid-mediated CRE have been reported from livestock in Europe and Asia. In this study, environmental and fecal samples were collected from a 1,500-sow, U.S. farrow-to-finish operation during 4 visits over a 5-month period in 2015. Samples were screened using selective media for the presence of CRE, and the resulting carbapenemase-producing isolates were further characterized. Of 30 environmental samples collected from a nursery room on our initial visit, 2 (7%) samples yielded 3 isolates, 2 sequence type 218 (ST 218) Escherichia coli and 1 Proteus mirabilis, carrying the metallo-�]-lactamase gene blaIMP-27 on IncQ1 plasmids. We recovered on our third visit 15 IMP-27-bearing isolates of multiple Enterobacteriaceae species from 11 of 24 (46%) environmental samples from 2 farrowing rooms. These isolates each also carried blaIMP-27 on IncQ1 plasmids. No CRE isolates were recovered from fecal swabs or samples in this study. As is common in U.S. swine production, piglets on this farm receive ceftiofur at birth, with males receiving a second dose at castration (?day 6). This selection pressure may favor the dissemination of blaIMP-27-bearing Enterobacteriaceae in this farrowing barn. The absence of this selection pressure in the nursery and finisher barns likely resulted in the loss of the ecological niche needed for maintenance of this carbapenem resistance gene.
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85. |
Girlich D,
Bonnin RA,
Bogaerts P,
De Laveleye M,
Huang DT,
Dortet L,
Glaser P,
Glupczynski Y,
Naas T,
( 2017 ) Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate. PMID : 27855079 : DOI : 10.1128/AAC.01697-16 PMC : PMC5278702 Abstract >>
Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired �]-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of blaOXA-58 class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a blaAmpC-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.
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86. |
Li X,
Du Y,
Du P,
Dai H,
Fang Y,
Li Z,
Lv N,
Zhu B,
Kan B,
Wang D,
( 2016 ) SXT/R391 integrative and conjugative elements in Proteus species reveal abundant genetic diversity and multidrug resistance. PMID : 27892525 : DOI : 10.1038/srep37372 PMC : PMC5124997 Abstract >>
SXT/R391 integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements that are found in most members of Enterobacteriaceae. Here, we determined fifteen SXT/R391 ICEs carried by Proteus isolates from food (4.2%) and diarrhoea patients (17.3%). BLASTn searches against GenBank showed that the fifteen SXT/R391 ICEs were closely related to that from different Enterobacteriaceae species, including Proteus mirabilis. Using core gene phylogenetic analysis, the fifteen SXT/R391 ICEs were grouped into six distinct clusters, including a dominant cluster and three clusters that have not been previously reported in Proteus isolates. The SXT/R391 ICEs shared a common structure with a set of conserved genes, five hotspots and two variable regions, which contained more foreign genes, including drug-resistance genes. Notably, a class A �]-lactamase gene was identified in nine SXT/R391 ICEs. Collectively, the ICE-carrying isolates carried resistance genes for 20 tested drugs. Six isolates were resistant to chloramphenicol, kanamycin, streptomycin, trimethoprim-sulfamethoxazole, sulfisoxazole and tetracycline, which are drug resistances commonly encoded by ICEs. Our results demonstrate abundant genetic diversity and multidrug resistance of the SXT/R391 ICEs carried by Proteus isolates, which may have significance for public health. It is therefore necessary to continuously monitor the antimicrobial resistance and related mobile elements among Proteus isolates.
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87. |
Paiva MC,
Reis MP,
Costa PS,
Dias MF,
Bleicher L,
Scholte LLS,
Nardi RMD,
Nascimento AMA,
( 2017 ) Identification of new bacteria harboring qnrS and aac(6')-Ib/cr and mutations possibly involved in fluoroquinolone resistance in raw sewage and activated sludge samples from a full-scale WWTP. PMID : 27984803 : DOI : 10.1016/j.watres.2016.11.056 Abstract >>
Wastewater treatment plants (WWTPs) harbor bacteria and antimicrobial resistance genes, favoring gene exchange events and resistance dissemination. Here, a culture-based and metagenomic survey of qnrA, qnrB, qnrS, and aac(6')-Ib genes from raw sewage (RS) and activated sludge (AS) of a full-scale municipal WWTP was performed. A total of 96 bacterial isolates were recovered from nalidixic acid-enrichment cultures. Bacteria harboring the aac(6')-Ib gene predominated in RS, whereas qnrS-positive isolates were specific to AS. Novel qnrS- and aac(6')-Ib-cr positive species were identified: Morganella morganii, Providencia rettgeri, and Pseudomonas guangdongensis (qnrS), and Alcaligenes faecalis and P. rettgeri (aac(6')-Ib-cr). Analysis of qnrS and aac(6')-Ib sequences from isolates and clone libraries suggested that the diversity of qnrS is wider than that of aac(6')-Ib. A large number of amino acid mutations were observed in the QnrS and AAC(6')-Ib proteins at previously undetected positions, whose structural implications are not clear. An accumulation of mutations at the C72, Q73, L74, A75 and M76 positions of QnrS, and D181 of AAC(6')-Ib might be important for resistance. These findings add significant information on bacteria harboring qnrS and aac(6')-Ib genes, and the presence of novel mutations that may eventually emerge in clinical isolates.
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88. |
Dixon N,
Fowler RC,
Yoshizumi A,
Horiyama T,
Ishii Y,
Harrison L,
Geyer CN,
Moland ES,
Thomson K,
Hanson ND,
( 2016 ) IMP-27, a Unique Metallo-�]-Lactamase Identified in Geographically Distinct Isolates of Proteus mirabilis. PMID : 27503648 : DOI : 10.1128/AAC.02945-15 PMC : PMC5038328 Abstract >>
A novel metallo-�]-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.
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89. |
Paul-Satyaseela M,
Murali S,
Thirunavukkarasu B,
Naraharirao MH,
Jambulingam M,
( 2016 ) Characterization of Antibiotic Resistance Profiles of Ocular Enterobacteriaceae Isolates. PMID : 27141313 : DOI : 10.1556/1886.2015.00047 PMC : PMC4838984 Abstract >>
Emergence of extended-spectrum �]-lactamase (ESBL) and fluoroquinolone resistance among ocular Enterobacteriaceae is increasing in higher frequency. Therefore, studies are being carried out to understand their multidrug resistance pattern. A total of 101 Enterobacteriaceae isolates recovered from various ocular diseases in a tertiary eye care center at Chennai, India during the period of January 2011 to June 2014 were studied. Forty one randomly chosen isolates were subjected to antibiotic susceptibility by minimum inhibitory concentration (MIC) and genotypic analysis. Of them, 16 were ESBL producers, one was carbapenemase producer and four were resistant to ertapenem which could be due to porin loss associated with AmpC production, and 17 were resistant to fluoroquinolones. Sixteen isolates harbored ESBL genes in which 14 had more than one gene and none of them were positive for blaNDM-1 gene. QNR genes were detected in 18 isolates. ESBL producers were predominantly isolated from conjunctiva. A high degree of ESBL production and fluoroquinolone resistance is seen among the genus Klebsiella sp. Hence, monitoring the rate of ESBL prevalence plays a vital role in the administration of appropriate intravitreal antibiotics to save the vision and also to reduce the development of drug resistance in ocular pathogens.
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90. |
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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91. |
Habibi M,
Asadi Karam MR,
Bouzari S,
( 2015 ) In silico design of fusion protein of FimH from uropathogenic Escherichia coli and MrpH from Proteus mirabilis against urinary tract infections. PMID : 26605246 : DOI : 10.4103/2277-9175.166164 PMC : PMC4627185 Abstract >>
Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) and Proteus mirabilis are the most important pathogens causing UTIs. The FimH from type 1 pili of UPEC and the MrpH from P. mirabilis play critical roles in the UTI process and have presented as ideal vaccine candidates against UTIs. There is no effective vaccine against UTI and the development of an ideal UTI vaccine is required. In this study, we planned to design a novel fusion protein of FimH from UPEC and MrpH from P. mirabilis. For this purpose, we modeled fusion protein forms computationally using the Iterative Threading Assembly Refinement (I-TASSER) server and evaluated their interactions with toll-like receptor 4 (TLR4). The best fusion protein was constructed using overlap extension polymerase chain reaction (OE-PCR) and the biological activity of fusion was evaluated by the induction of interleukin-8 (IL-8) in the HT-29 cell line. Our study indicated that based on the Protein Structure Analysis (ProSA)-web and the docking results, MrpH.FimH showed better results than did FimH.MrpH, and it was selected for construction. The results of bioassay on the HT-29 showed that FimH and MrpH.FimH induced significantly higher IL-8 responses than untreated cells or MrpH alone in the cell line tested. In the present study, we designed and constructed the novel fusion protein MrpH.FimH from UPEC and P. mirabilis based on in silico methods. Our bioassay results indicate that the MrpH.FimH fusion protein is active and capable of inducing immune responses.
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92. |
Di Ilio C,
Aceto A,
Piccolomini R,
Allocati N,
Caccuri AM,
Barra D,
Federici G,
( 1989 ) N-terminal region of Proteus mirabilis glutathione transferase is not homologous to mammalian and plant glutathione transferases. PMID : 2661269 : DOI : 10.1016/0014-5793(89)80684-2 Abstract >>
The N-terminal amino acid sequence of glutathione transferase, Pm-GST-6.0, purified from Proteus mirabilis [(1988) Biochem. J. 255, 971-975] up to residue 38 and a comparative peptide fingerprint are reported. No obvious homology with the sequences of alpha, pi and mu classes of mammalian glutathione transferases as well as with those of plant glutathione transferases has been noted. These results suggest that the classification so far adopted for glutathione transferases cannot be extended to the bacterial enzyme.
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93. |
Chen L,
Al Laham N,
Chavda KD,
Mediavilla JR,
Jacobs MR,
Bonomo RA,
Kreiswirth BN,
( 2015 ) First report of an OXA-48-producing multidrug-resistant Proteus mirabilis strain from Gaza, Palestine. PMID : 25896692 : DOI : 10.1128/AAC.00565-15 PMC : PMC4468693 Abstract >>
We report the first multidrug-resistant Proteus mirabilis strain producing the carbapenemase OXA-48 (Pm-OXA-48) isolated at Al-Shifa hospital in Gaza, Palestine. Draft genome sequencing of Pm-OXA-48 identified 16 antimicrobial resistance genes, encoding resistance to �]-lactams, aminoglycosides, fluoroquinolones, phenicols, streptothricin, tetracycline, and trimethoprim-sulfamethoxazole. Complete sequencing of the bla(OXA-48)-harboring plasmid revealed that it is a 72 kb long IncL/M plasmid, harboring carbapenemase gene bla(OXA-48), extended spectrum �]-lactamase gene bla(CTX-M-14), and aminoglycoside resistance genes strA, strB, and aph(3')-VIb.
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94. |
Yugendran T,
Harish BN,
( 2016 ) High incidence of plasmid-mediated quinolone resistance genes among ciprofloxacin-resistant clinical isolates of Enterobacteriaceae at a tertiary care hospital in Puducherry, India. PMID : 27168994 : DOI : 10.7717/peerj.1995 PMC : PMC4860338 Abstract >>
Background. Plasmid-mediated quinolone resistance (PMQR) has received considerable attention recently. Data analysis in Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER) revealed 75% of the Enterobacteriaceae isolates to be ciprofloxacin-resistant in 2012. Few reports regarding the prevalence of PMQR are available from India. Hence, the present study was carried out to ascertain the prevalence of PMQR genes among clinical isolates of ciprofloxacin-resistant Enterobacteriaceae in JIPMER. Methods. The study included 642 ciprofloxacin-resistant clinical Enterobacteriaceae isolates. JIPMER hospital's annual consumption data for fluoroquinolones were retrieved from the Department of Pharmacy. The test isolates were screened for the presence of qnr A, B, D, S and aac(6')-Ib-cr genes. PMQR-positive isolates alone were tested for the presence of class I (intI1) and class II (intI2) integrons. Randomly selected PCR amplicons were sequenced and analysed using MEGA software. A total of 30 PMQR strains chosen at random were assessed for the transferability of the PMQR genes. Results. A majority of the strains exhibited high MIC values with 106 strains exhibiting MIC values >256 ?g/mL. The aac(6')-Ib-cr gene had the highest prevalence at 64% (414) while, qnrB and qnrS genes were present in 15% (97) and 10% (64) of the isolates respectively. None of the strains were positive for qnrA and qnrD. All PMQR-positive isolates were screened for class I (intI1) and class II (intI2) integrons. Class I integron was found to be predominant among the test isolates with a few of them carrying both the classes of integrons. Transferability of PMQR genes to transconjugants was identified. Conclusion. The incidence of PMQR genes in the tertiary-care setup of the JIPMER hospital was found to be high which could be probably due to the increased prescription of fluoroquinolones. Thus, there is a need for rational usage of fluoroquinolones.
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95. |
Lei CW,
Zhang AY,
Wang HN,
Liu BH,
Yang LQ,
Yang YQ,
( 2016 ) Characterization of SXT/R391 Integrative and Conjugative Elements in Proteus mirabilis Isolates from Food-Producing Animals in China. PMID : 26824957 : DOI : 10.1128/AAC.02852-15 PMC : PMC4775944 Abstract >>
SXT/R391 integrative and conjugative elements (ICEs) were detected in 8 out of 125 Proteus mirabilis isolates from food-producing animals in China. Whole-genome sequencing revealed that seven ICEs were identical to ICEPmiJpn1, carrying the cephalosporinase gene blaCMY-2. Another one, designated ICEPmiChn1, carried five resistance genes. All eight ICEs could be transferred to Escherichia coli via conjugation. The results highlight the idea that animal farms are important reservoir of the SXT/R391 ICE-containing P. mirabilis.
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96. |
Schultz E,
Haenni M,
Mereghetti L,
Siebor E,
Neuwirth C,
Madec JY,
Cloeckaert A,
Doublet B,
( 2015 ) Survey of multidrug resistance integrative mobilizable elements SGI1 and PGI1 in Proteus mirabilis in humans and dogs in France, 2010-13. PMID : 26066582 : DOI : 10.1093/jac/dkv154 Abstract >>
To characterize MDR genomic islands related to Salmonella genomic island 1 (SGI1) and Proteus genomic island 1 (PGI1) in Proteus mirabilis from human and animal sources in France in light of the previously reported cases. A total of 52 and 46 P. mirabilis clinical strains from human and animal sources, respectively, were studied for the period 2010-13. MDR was assessed by antimicrobial susceptibility testing, PCR detection of SGI1 and PGI1 and PCR mapping of the MDR regions. The diversity of the SGI1/PGI1-positive P. mirabilis strains was assessed by PFGE. Twelve P. mirabilis strains (5 humans and 7 dogs) were found to harbour an MDR island related to SGI1 or PGI1. Among them, several SGI1 variants were identified in diverse P. mirabilis genetic backgrounds. The variant SGI1-V, which harbours the ESBL bla VEB-6 gene, was found in closely genetically related human and dog P. mirabilis strains. The recently described PGI1 element was also identified in human and dog strains. Finally, one strain harboured a novel SGI genomic island closely related to SGI1 and SGI2 without an insertion of the MDR region. This study reports for the first time, to our knowledge, SGI1-positive and PGI1-positive P. mirabilis strains from dogs in France. The genetic diversity of the strains suggests several independent horizontal acquisitions of these MDR elements. The potential transmission of SGI1/PGI1-positive P. mirabilis strains between animals and humans is of public health concern, notably with regard to the spread of ESBL and carbapenemase genes, i.e. bla VEB-6 and bla NDM-1.
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97. |
Tsai YL,
Wang MC,
Hsueh PR,
Liu MC,
Hu RM,
Wu YJ,
Liaw SJ,
( 2015 ) Overexpression of an outer membrane protein associated with decreased susceptibility to carbapenems in Proteus mirabilis. PMID : 25756370 : DOI : 10.1371/journal.pone.0120395 PMC : PMC4355480 Abstract >>
Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis.
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98. |
Qin S,
Qi H,
Zhang Q,
Zhao D,
Liu ZZ,
Tian H,
Xu L,
Xu H,
Zhou M,
Feng X,
Liu HM,
( 2015 ) Emergence of Extensively Drug-Resistant Proteus mirabilis Harboring a Conjugative NDM-1 Plasmid and a Novel Salmonella Genomic Island 1 Variant, SGI1-Z. PMID : 26195511 : DOI : 10.1128/AAC.00292-15 PMC : PMC4576069 Abstract >>
Acquisition of blaNDM-1 in bacterial species, such as Proteus mirabilis that is intrinsically resistant to tetracycline, tigecycline and colistin, will make clinical treatment extremely difficult. Here, we characterized an NDM-1-producing clinical isolate of P. mirabilis (PM58) that displayed an extensively drug-resistant (XDR) phenotype, susceptible only to aztreonam. Molecular analysis revealed that PM58 harbored both a conjugative NDM-1 plasmid and a novel Salmonella genomic island 1 variant on chromosome.
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99. |
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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100. |
Guillard T,
Grillon A,
de Champs C,
Cartier C,
Madoux J,
Berçot B,
Lebreil AL,
Lozniewski A,
Riahi J,
Vernet-Garnier V,
Cambau E,
( 2014 ) Mobile insertion cassette elements found in small non-transmissible plasmids in Proteeae may explain qnrD mobilization. PMID : 24504382 : DOI : 10.1371/journal.pone.0087801 PMC : PMC3913671 Abstract >>
qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36-60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae.
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101. |
Akaboshi E,
Yip ML,
Howard-Flanders P,
( 1989 ) Nucleotide sequence of the recA gene of Proteus mirabilis. PMID : 2544862 : DOI : 10.1093/nar/17.11.4390 PMC : PMC317964 Abstract >>
N/A
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102. |
Lei CW,
Zhang AY,
Liu BH,
Wang HN,
Yang LQ,
Guan ZB,
Xu CW,
Zhang DD,
Yang YQ,
( 2015 ) Two novel Salmonella genomic island 1 variants in Proteus mirabilis isolates from swine farms in China. PMID : 25918148 : DOI : 10.1128/AAC.00120-15 PMC : PMC4468701 Abstract >>
Four different Salmonella genomic island 1 (SGI1) variants, including two novel variants, were characterized in one Salmonella enterica serovar Rissen sequence type ST1917 isolate and three Proteus mirabilis isolates from swine farms in China. One novel variant was derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb IS26 composite transposon containing the dfrA17-aadA5 cassettes and macrolide inactivation gene cluster mphA-mrx-mphR. The other one was an integron-free SGI1 and contained a 183-bp truncated S025 next to IS6100 and S044.
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103. |
Siebor E,
Neuwirth C,
( 2014 ) Proteus genomic island 1 (PGI1), a new resistance genomic island from two Proteus mirabilis French clinical isolates. PMID : 25114166 : DOI : 10.1093/jac/dku314 Abstract >>
To analyse the genetic environment of the antibiotic resistance genes in two clinical Proteus mirabilis isolates resistant to multiple antibiotics. PCR, gene walking and whole-genome sequencing were used to determine the sequence of the resistance regions, the surrounding genetic structure and the flanking chromosomal regions. A genomic island of 81.1 kb named Proteus genomic island 1 (PGI1) located at the 3'-end of trmE (formerly known as thdF) was characterized. The large MDR region of PGI1 (55.4 kb) included a class 1 integron (aadB and aadA2) and regions deriving from several transposons: Tn2 (blaTEM-135), Tn21, Tn6020-like transposon (aphA1b), a hybrid Tn502/Tn5053 transposon, Tn501, a hybrid Tn1696/Tn1721 transposon [tetA(A)] carrying a class 1 integron (aadA1) and Tn5393 (strA and strB). Several ISs were also present (IS4321, IS1R and IS26). The PGI1 backbone (25.7 kb) was identical to that identified in Salmonella Heidelberg SL476 and shared some identity with the Salmonella genomic island 1 (SGI1) backbone. An IS26-mediated recombination event caused the division of the MDR region into two parts separated by a large chromosomal DNA fragment of 197 kb, the right end of PGI1 and this chromosomal sequence being in inverse orientation. PGI1 is a new resistance genomic island from P. mirabilis belonging to the same island family as SGI1. The role of PGI1 in the spread of antimicrobial resistance genes among Enterobacteriaceae of medical importance needs to be evaluated.
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104. |
Rodrigues C,
Machado E,
Ramos H,
Peixe L,
Novais ?,
( 2014 ) Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized patients: a successful story of international clones (ST15, ST147, ST336) and epidemic plasmids (IncR, IncFIIK). PMID : 25190354 : DOI : 10.1016/j.ijmm.2014.08.003 Abstract >>
The aim of this study was to characterize by a multi-level approach extended-spectrum �]-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006-7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations. The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with the amplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12 and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species in Hospitals B and C in 2006-7. Particular plasmid types were linked to blaCTX-M-15 (IncR or non-typeable plasmids), blaSHV-12 (IncR or IncHI2), blaSHV-28/-55/-106 (IncFIIK1 or IncFIIK5), blaTEM-10 (IncL/M) or blaTEM-24 (IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-type ESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15, ST147, ST336) and plasmid types (IncR, IncFIIK). Furthermore, we highlight the potential of IncFIIK plasmids (here firstly associated with blaSHV-2/-28/-55/-106) to disseminate as antibiotic resistance plasmids.
|
105. |
Girlich D,
Dortet L,
Poirel L,
Nordmann P,
( 2015 ) Integration of the blaNDM-1 carbapenemase gene into Proteus genomic island 1 (PGI1-PmPEL) in a Proteus mirabilis clinical isolate. PMID : 25239462 : DOI : 10.1093/jac/dku371 Abstract >>
To decipher the mechanisms and their associated genetic determinants responsible for �]-lactam resistance in a Proteus mirabilis clinical isolate. The entire genetic structure surrounding the �]-lactam resistance genes was characterized by PCR, gene walking and DNA sequencing. Genes encoding the carbapenemase NDM-1 and the ESBL VEB-6 were located in a 38.5 kb MDR structure, which itself was inserted into a new variant of the Proteus genomic island 1 (PGI1). This new PGI1-PmPEL variant of 64.4 kb was chromosomally located, as an external circular form in the P. mirabilis isolate, suggesting potential mobility. This is the first known description of the bla(NDM-1) gene in a genomic island structure, which might further enhance the spread of the bla(NDM-1) carbapenemase gene among enteric pathogens.
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106. |
Lei CW,
Zhang AY,
Liu BH,
Wang HN,
Guan ZB,
Xu CW,
Xia QQ,
Cheng H,
Zhang DD,
( 2014 ) Molecular characteristics of Salmonella genomic island 1 in Proteus mirabilis isolates from poultry farms in China. PMID : 25267683 : DOI : 10.1128/AAC.03992-14 PMC : PMC4249554 Abstract >>
Six out of the 64 studied Proteus mirabilis isolates from 11 poultry farms in China contained Salmonella genomic island 1 (SGI1). PCR mapping showed that the complete nucleotide sequences of SGI1s ranged from 33.2 to 42.5 kb. Three novel variants, SGI1-W, SGI1-X, and SGI1-Y, have been characterized. Resistance genes lnuF, dfrA25, and qnrB2 were identified in SGI1 for the first time.
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107. |
Wei Q,
Hu Q,
Li S,
Lu H,
Chen G,
Shen B,
Zhang P,
Zhou Y,
( 2014 ) A novel functional class 2 integron in clinical Proteus mirabilis isolates. PMID : 24235093 : DOI : 10.1093/jac/dkt456 Abstract >>
To describe a novel functional class 2 integron that was found in clinical Proteus mirabilis isolates. Class 1 and 2 integrons were screened by PCR in 153 clinical Proteus isolates. The variable regions of class 1 and 2 integrons were determined by restriction analysis and sequencing. The mutations of internal stop codons in class 2 integrons and their common promoters were also determined by sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyse the phylogenetic relations of class 2 integron-positive P. mirabilis isolates. Class 1 integrons were detected in 96 (63%) of 153 Proteus isolates: eight different gene cassette arrays were detected, including dfrA32-ereA1-aadA2, which was detected for the first time in P. mirabilis. Class 2 integrons were detected in 101 (66%) of 153 Proteus isolates: four different gene cassette arrays were detected, including dfrA1-catB2-sat2-aadA1, which was detected for the first time in a class 2 integron. A novel functional class 2 integron was detected in 38 P. mirabilis isolates with a common promoter (-35 TTTAAT|16 bp|-10 TAAAGT). The variable region of this functional class 2 integron contained dfrA14 and three novel open reading frames with unknown functions. Very similar ERIC-PCR fingerprinting patterns were detected in these 38 P. mirabilis isolates and were different from other class 2 integron-positive isolates. A novel functional class 2 integron was found for the first time in P. mirabilis. These functional class 2 integron-harbouring P. mirabilis isolates were likely to be clonally spread in our hospital.
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108. |
Premkumar L,
Kurth F,
Neyer S,
Schembri MA,
Martin JL,
( 2014 ) The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase. PMID : 24311786 : DOI : 10.1074/jbc.M113.516898 PMC : PMC3908391 Abstract >>
The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.
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109. |
Uphoff TS,
Welch RA,
( 1990 ) Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB). PMID : 2407716 : DOI : 10.1128/jb.172.3.1206-1216.1990 PMC : PMC208585 Abstract >>
We cloned a 13.5-kilobase EcoRI fragment containing the calcium-independent hemolysin determinant (pWPM110) from a clinical isolate of Proteus mirabilis (477-12). The DNA sequence of a 7,191-base-pair region of pWPM110 was determined. Two polypeptides are encoded in this region, HpmB and HpmA (in that transcriptional order), with predicted molecular masses of 63,204 and 165,868 daltons, respectively. A putative Fur-binding site was identified upstream of hpmB overlapping the -35 region of the proposed hpm promoter. In vitro transcription-translation of pWPM110 DNA and other subclones confirmed the assignment of molecular masses for the predicted polypeptides. These polypeptides are predicted to have NH2-terminal leader peptides of 17 and 29 amino acids, respectively. NH2-terminal amino acid sequence analysis of purified extracellular hemolysin (HpmA) confirmed the cleavage of the 29-amino-acid leader peptide in the secreted form of HpmA. Hemolysis assays and immunoblot analysis of Escherichia coli containing subclones expressing hpmA, hpmB, or both indicated that HpmB is necessary for the extracellular secretion and activation of HpmA. Significant nucleotide identity (52.1%) was seen between hpm and the shl hemolysin gene sequences of Serratia marcescens despite differences in the G+C contents of these genes (hpm, 38%; shl, 65%). The predicted amino acid sequences of HpmB and HpmA are also similar to those of ShlB and ShlA, the respective sequence identities being 55.4 and 46.7%. Predicted cysteine residues and major hydrophobic and amphipathic domains have been strongly conserved in both proteins. Thus, we have identified a new hemolysin gene family among gram-negative opportunistic pathogens.
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110. |
Schneider I,
Markovska R,
Marteva-Proevska Y,
Mitov I,
Markova B,
Bauernfeind A,
( 2014 ) Detection of CMY-99, a novel acquired AmpC-Type �]-lactamase, and VIM-1 in Proteus mirabilis isolates in Bulgaria. PMID : 24165184 : DOI : 10.1128/AAC.01450-13 PMC : PMC3910747 Abstract >>
N/A
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111. |
Mazzariol A,
Kocsis B,
Koncan R,
Kocsis E,
Lanzafame P,
Cornaglia G,
( 2012 ) Description and plasmid characterization of qnrD determinants in Proteus mirabilis and Morganella morganii. PMID : 22192340 : DOI : 10.1111/j.1469-0691.2011.03728.x Abstract >>
We investigated the presence of qnrC and qnrD among 756 non-replicate Enterobacteriaceae isolated in Italy, selected for being non-susceptible to fluoroquinolones and/or resistant to third-generation cephalosporins. Four Proteus mirabilis and one Morganella morganii (0.66% of the total) presented a qnrD gene, located in a 2687-base-pair plasmid that was entirely sequenced. The plasmid is un-typable, and contains no known coding region other than qnrD. That the qnrD gene was found in four unrelated P. mirabilis and in one M. morganii isolate might suggest a frequent association of this gene with the tribe Proteeae.
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112. |
( 1997 ) A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator. PMID : 9244260 : DOI : 10.1128/jb.179.15.4741-4746.1997 PMC : PMC179319 Abstract >>
Proteus swarming is the rapid cyclical population migration across surfaces by elongated cells that hyperexpress flagellar and virulence genes. The mini-Tn5 transposon mutant mns2 was isolated as a tight nonswarming mutant that did not elongate or upregulate flagellar and hemolysin genes. Individual cell motility was retained but was reduced. The transposon had inserted in the gene encoding the global transcriptional regulator Lrp (leucine-responsive regulatory protein), expression of which was upregulated in differentiating swarm cells. Swarming was restored to the lrp mutant by artificial overexpression of the flhDC flagellar regulatory master operon. Lrp may be a key component in generating or relaying signals that are required for flagellation and swarming, possibly acting through the flhDC operon.
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113. |
( 1997 ) Negative feedback from a Proteus class II flagellum export defect to the flhDC master operon controlling cell division and flagellum assembly. PMID : 9287017 : DOI : 10.1128/jb.179.17.5585-5588.1997 PMC : PMC179433 Abstract >>
The Proteus mirabilis flagellum class I flhDC operon was isolated, and its transcript was shown to originate from a sigma70 promoter 244 bp 5' of flhD and 29 bp 3' of a putative cyclic AMP receptor protein-binding site. Expression of this regulatory master operon increased strongly as cells differentiated into elongated hyperflagellated swarm filaments, and cell populations artificially overexpressing flhDC migrated sooner and faster. A class II flhA transposon mutant was reduced in flagellum class III gene expression, as would be expected from the FlgM anti-sigma28 accumulation demonstrated in Salmonella typhimurium, but was unexpectedly also reduced in cell elongation. Here, we show that levels of flhDC transcript were ca. 10-fold lower in this flagellum export mutant, indicating that in cells defective in flagellum assembly, there is additional negative feedback via flhDC. In support of this view, artificial overexpression of flhDC in the flhA mutant restored elongation but not class III flagellum gene transcription.
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114. |
( 1997 ) A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly. PMID : 9302021 : DOI : 10.1046/j.1365-2958.1997.5021862.x Abstract >>
A TnphoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN, a flagella gene encoding a 147-amino-acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti-sigma28 gene, flgM, flanked by the class II genes, flgA, flgBCD and flhBA, and a novel putative virulence-related gene. The flgN mutation caused a substantial reduction in cell surface-associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse-labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non-oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper-like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella-related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.
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115. |
( 1997 ) Carbenicillin-hydrolysing penicillinase mediated by a plasmid of Proteus mirabilis and its relationship to the PSE-type enzymes of Pseudomonas aeruginosa. PMID : 9281821 : Abstract >>
The nucleotide sequence of a carbenicillin-hydrolysing (carbenicillinase) gene occurring in an endogenous plasmid, pCS229, of Proteus mirabilis was determined. The amino acid sequence of the mature enzyme, comprising 271 amino acids with a molecular mass of 29,506 Da, was deduced. The pCS229 carbenicillinase showed only 46.4% similarity, in the overall amino acid sequence, to the chromosomal carbenicillinase of Pr. mirabilis GN79; however, the enzyme showed about 98% similarity to a Pseudomonas-specific plasmid-encoded carbenicillinase, PSE-4, that was isolated from Pseudomonas aeruginosa. Only five of 271 amino acids differed from those of PSE-4. This study proved the close relationship between the carbenicillinase genes distributed in Pr. mirabilis and Ps. aeruginosa.
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( 1996 ) Comparative analysis of the cya locus in enterobacteria and related gram-negative facultative anaerobes. PMID : 8874804 : DOI : 10.1016/0300-9084(96)82192-4 Abstract >>
Comparison of the cya loci (cya codes for adenylyl cyclase (AC)) from a variety of phylogenetically divergent facultative anaerobic Gram-negative bacteria reveals conserved sequence features. The entire locus structure in enterobacteria is preserved, including two major promoters (a conserved cya strong promoter, P2, and a divergent promoter for a heme biosynthetic operon, hemCD) present in the upstream region of the cya gene. The region between hemC and cya is much longer in Proteus mirabilis than in other enterobacteria, and lacks the P1 upstream cya promoter. In Aeromonas hydrophila the cya promoter (the strong P2 promoter in E coli) is preserved, including a putative GATC methylation site situated immediately downstream from the -10 box. Each cya frame analyzed uses TTG as the translation start codon and is preceded by an unusual ribosome binding site. This suggests that a lower translation efficiency of the cya transcript could be the result of some selection pressure. This has been substantiated by in vitro mutagenesis and by selection of up mutations which all map at the cya ribosome binding site. In enterobacteria the cyaY frame is the only conserved reading frame downstream of cya, with the orientation opposite to that of cya. This organization is not preserved in Aeromonas. Experiments involving fusions with the lacZ gene demonstrated that cyaY is expressed. Finally, comparison of the different polypeptide sequences of ACs permits discussion of important features of the catalytic and regulatory centers of the protein.
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( 1996 ) Proteus mirabilis ambient-temperature fimbriae: cloning and nucleotide sequence of the aft gene cluster. PMID : 8926119 : PMC : PMC174387 Abstract >>
Uropathogenic Proteus mirabilis produces at least four types of fimbriae. Amino acid sequences from two peptides, derived by tryptic digestion of the structural subunit of one type of these fimbriae, the ambient-temperature fimbriae, were determined: NVVPGQPSSTQ and LIEGENQLNYNA. PCR primers, based on these sequences and that of the N terminus, were used to amplify a 359-bp fragment. A cosmid clone, isolated from a P. mirabilis genomic library by hybridization with the 359-bp PCR product, was used to determine the nucleotide sequence of the atf gene cluster. A 3,903-bp region encodes three polypeptides: AtfA, the structural subunit; AtfB, the chaperone; and AtfC, the outer membrane molecular usher. No fimbria-related genes are evident either 5' or 3' to the three contiguous genes. AtfA demonstrates significant amino acid sequence identity with type 1 major fimbrial subunits of several enteric species. The 359-bp PCR product hybridized strongly with all Proteus isolates (n = 9) and 25% of 355 Escherichia coli isolates but failed to hybridize with any of 26 isolates among nine other uropathogenic species. Ambient-temperature fimbriae of P. mirabilis may represent a novel type of fimbriae of enteric species.
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( 1996 ) Ferryl intermediates of catalase captured by time-resolved Weissenberg crystallography and UV-VIS spectroscopy. PMID : 8901874 : Abstract >>
Various enzymes use semi-stable ferryl intermediates and free radicals during their catalytic cycle, amongst them haem catalases. Structures for two transient intermediates (compounds I and II) of the NADPH-dependent catalase from Proteus mirabilis (PMC) have been determined by time-resolved X-ray crystallography and single crystal microspectrophotometry. The results show the formation and transformation of the ferryl group in the haem, and the unexpected binding of an anion during this reaction at a site distant from the haem.
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119. |
( 1993 ) The 39-kilodalton outer membrane protein of Proteus mirabilis is an OmpA protein and mitogen for murine B lymphocytes. PMID : 8406896 : PMC : PMC281256 Abstract >>
Partial amino acid sequence analysis of a major outer membrane protein of Proteus mirabilis (39-kDa protein) indicates that it is an OmpA protein. The mitogenic activities of the 39-kDa protein for murine lymphocytes were also investigated with T lymphocytes isolated by passing spleen cells over columns of nylon wool fiber and B lymphocytes obtained by treating spleen cells with monoclonal antibodies to Thy1 plus complement. The 39-kDa protein showed little activity in stimulating T cells to proliferate but was strongly mitogenic for B cells.
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( 1993 ) The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes. PMID : 8436105 : DOI : 10.1111/j.1432-1033.1993.tb17566.x Abstract >>
The complete amino acid sequence of glutathione transferase from Proteus mirabilis was determined. The sequence was reconstructed by analysis of peptides obtained after cleavage by trypsin, Glu-C and Asp-N endoproteinases. The enzyme subunit is composed of 203 amino acid residues corresponding to a molecular mass of 22856 Da. Comparison of this sequence with other known primary structures of the corresponding enzyme from different sources shows a low level of identity (17-26%) with only seven conserved residues in all the sequences considered. This novel glutathione transferase could represent the prototype of a new class, possibly including other bacterial enzymes.
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121. |
( 1996 ) Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis. PMID : 8761466 : DOI : 10.1042/bj3180157 PMC : PMC1217602 Abstract >>
The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.
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( 1995 ) A cell-surface polysaccharide that facilitates rapid population migration by differentiated swarm cells of Proteus mirabilis. PMID : 8594335 : DOI : 10.1111/j.1365-2958.1995.mmi_17061167.x Abstract >>
Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical vegetative rods into long, hyperflagellated, virulent swarm cells. A swarm-defective TnphoA insertion mutant was isolated that was not defective in cell motility, differentiation or control of the migration cycle, but was specifically impaired in the ability to undergo surface translocation as a multicellular mass. The mutation, previously shown to compromise urinary tract virulence, was located within a 1112 bp gene that restored normal swarming of the mutant when expressed in trans. The gene encoded a 40.6 kDa protein that is related to putative sugar transferases required for lipopolysaccharide (LPS) core modification in Shigella and Salmonella. The immediately distal open reading frame encoded a protein that is related to dehydrogenases involved in the synthesis of LPS O-side-chains, enterobacterial common antigen and extracellular polysaccharide (PS). Gel electrophoresis and electron microscopy showed that the mutant still made LPS but it had lost the ability to assemble a surface (capsular) PS, which gas-liquid chromatography and mass spectrometry indicated to be an acidic type II molecule rich in galacturonic acid and galactosamine. We suggest that this surface PS facilitates translocation of differentiated cell populations by reducing surface friction.
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( 1996 ) Purification and characterization of a heterodimeric 23/20-kDa proteolytic fragment of bacterial glutathione transferase B1-1. PMID : 8645008 : DOI : 10.1006/abbi.1996.0177 Abstract >>
The proteolytic attack of bacterial glutathione S-transferase (GSTB1-1) by trypsin cleaves and inactivates the enzyme. The polypeptide portion of GSTB1-1 encompassing the cleavage site (Lys35-Lys36) constitutes an exposed and flexible region of the GSTB1-1 G-site. By sequentially using a benzamidine-affinity chromatography and GSH-affinity column, a proteolyzed form of GSTB1-1 (23/20 kDa), in which only one subunit has been cleaved has been purified and characterized. Gel filtration, sequence analysis of subunits separated by HPLC, and CD experiments indicate that the 23/20-kDa GSTB1-1 form is a dimer and maintains its secondary structure. In addition, kinetic determinations reveal that the proteolytic cleavage of one polypeptide chain inactivates one active site but does not influence the catalytic efficiency of the second one. Previous refolding studies on GSTB1-1 have shown that the formation of a correct dimer precedes the recovery of the full activity of the enzyme, indicating that the dimeric structure is essential for catalytic activity of GSTB1-1. Thus, although GSTB1-1 active sites are catalytically independent and, probably, mainly located on each monomer, interactions deriving from the dimeric arrangement of the molecule appear essential for maintaining each active site in a fully active conformation. The catalytic independence of the two active sites, as well as the importance of dimeric structure for catalytic activity, has already been established for other GSTs. Thus, despite the very low sequence identity and kinetic differences between bacterial and other distant members of the GST superfamily, the results reported here indicate that important properties of the GST active site are conserved.
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124. |
( 1994 ) Genetic organization and complete sequence of the Proteus mirabilis pmf fimbrial operon. PMID : 7959033 : DOI : 10.1016/0378-1119(94)90866-4 Abstract >>
Proteus mirabilis, commonly associated with urinary tract infection, pyelonephritis and bacteremia, produces a number of fimbriae, including PMF (P. mirabilis fimbriae). Genes encoding PMF were isolated and the complete nucleotide (nt) sequence was determined. The pmf gene cluster, encoded by 5655 bp, predicts five polypeptides: PmfA (18,921 Da), PmfC (93,107 Da), PmfD (28,208 Da), PmfE (38,875 Da) and PmfF (19,661 Da). PmfA, PmfC, PmfD and PmfF share > 25% amino acid (aa) sequence identity with gene products of the pap, mrp and sfa fimbrial gene clusters. PmfE shares no similarity with any polypeptide in the SwissProt database. No regulatory gene(s) or regulatory elements were evident in the sequence. The pmf cluster shares common features with other enteric fimbrial gene clusters, but also displays features that are unique.
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125. |
( 1993 ) Proteus mirabilis fimbriae: N-terminal amino acid sequence of a major fimbrial subunit and nucleotide sequences of the genes from two strains. PMID : 8094384 : PMC : PMC302815 Abstract >>
Proteus mirabilis, a common cause of urinary tract infection in hospitalized and catheterized patients, produces mannose-resistant/klebsiella-like (MR/K) and mannose-resistant/proteus-like (MR/P) hemagglutinins. The gene encoding the major structural subunit of a fimbria, possibly MR/K, was identified in two strains. A degenerate oligonucleotide probe based on the N terminus of the Proteus uroepithelial cell adhesin and antiserum raised against the denatured polypeptide were used to screen a cosmid gene bank of strain HU1069. A cosmid clone that reacted with the probe and antiserum was identified, and a fimbria-like open reading frame was determined by nucleotide sequencing. The predicted N-terminal amino acid sequence of the processed polypeptide, ENETPAPKVSSTKGEIQLKG (residues 23 to 42), did not match the uroepithelial cell adhesin N terminus but, rather, matched exactly the N-terminal amino acid sequence of a polypeptide with an apparent molecular size of 19.5 kDa isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a fimbrial preparation from strain HI4320 expressing MR/K hemagglutinin. By using an oligonucleotide from the HU1069 open reading frame, the fimbrial gene was isolated and sequenced from a cosmid gene bank clone of strain HI4320. A 552-bp open reading frame predicts a 184-amino-acid polypeptide including a 22-amino-acid hydrophobic leader sequence. The unprocessed polypeptide is predicted to be 18,921 Da; the processed polypeptide is predicted to be 16,749 Da. The predicted amino acid sequence of the polypeptide encoded by the gene, designated pmfA, displayed 36% exact matches with the mannose-resistant fimbrial subunit encoded by smfA of Serratia marcescens but only 15% exact matches with the predicted sequence encoded by mrkA of Klebsiella pneumoniae.
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126. |
Nemec A,
Haywood-Farmer A,
Mackie GA,
( 1995 ) Conserved amino acid residues in the primary structure of ribosomal protein S20 from selected gram-negative bacteria. PMID : 7640306 : DOI : 10.1016/0167-4781(95)00100-u Abstract >>
Partial DNA sequences encoding ribosomal protein S20 have been determined for a number of Gram-negative bacteria including three species of Aeromonas, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella typhimurium and compared to the known sequence from Escherichia coli. The derived amino acid sequence is well conserved, particularly in the C-terminal one-third of S20. In contrast, there is much less conservation of the nucleotide sequence or potential secondary structure in the corresponding mRNA.
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127. |
Gygi D,
Bailey MJ,
Allison C,
Hughes C,
( 1995 ) Requirement for FlhA in flagella assembly and swarm-cell differentiation by Proteus mirabilis. PMID : 7783646 : DOI : 10.1111/j.1365-2958.1995.tb02383.x Abstract >>
Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical rods into long, aseptate swarm cells that overexpress flagella and virulence factors, particularly haemolysin. A non-swarming Tn5phoA mutant was unable to synthesize flagella, to fully elongate or to induce high levels of the toxin. The mutation lay within a 2091 bp gene encoding a homologue of the Escherichia coli FlhA belonging to a family of proteins that are required for assembly of flagella or virulence proteins and that are suggested to act either directly in membrane translocation and/or in regulating synthesis of the export apparatus. In trans expression of multicopy flhA restored cell elongation and migration and generated differentiation-specific hyperexpression of flagellin and toxin genes to levels above those seen in the wild-type strain. Transcription of flhA was strongly induced during differentiation, from its own putative sigma 28 promoter. The results suggest a mechanistic coupling of flagella assembly and swarm-cell differentiation.
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128. |
( 1994 ) The single-stranded-DNA-binding proteins (SSB) of Proteus mirabilis and Serratia marcescens. PMID : 7925378 : DOI : 10.1111/j.1432-1033.1994.00613.x Abstract >>
The single-stranded-DNA-binding (SSB) proteins from Proteus mirabilis and Serratia marcescens were purified from overproducing Escherichia coli strains, which were devoid of their own ssb gene. The strains harboured an endA insertion mutation and a xonA mutation resulting in the absence of endonuclease I and exonuclease I activities from the preparations. The amino acid sequences of the SSB of all three species are nearly identical in the N-terminal parts of the proteins that contain the DNA-binding domain, but differ in the C-terminal parts. Both proteins have an apparent binding-site size of 65 and 35 nucleotides at high and low salt concentrations, respectively. The association-rate constant for binding to poly(dT) is 3.2 x 10(8) M-1 s-1 for P. mirabilis SSB (PmiSSB) and 3.4 x 10(8) M-1 s-1 for S. marcescens SSB (SmaSSB). These binding parameters are very similar to those of E. coli SSB (EcoSSB). The structural similarity of the proteins is also documented by the finding that they can exchange subunits among each other to form mixed tetramers. The transcriptional regulation of the ssb and uvrA genes from P. mirabilis and S. marcescens in SOS-induced E. coli cells was studied using lacZ fusions. While the uvrA genes were inducible, there was no induction of the ssb genes transcribed divergently from the uvrA genes. Apparently, regions with nucleotide sequence similarity to the E. coli SOS-box preceding the ssb genes of P. mirabilis and S. marcescens had no gross effect on the transcription. Studies on growth of the cells and recovery from ultraviolet damage indicate that the heterologous SSB proteins support DNA replication and recombinational DNA repair of E. coli with the same efficiency as the E. coli SSB protein. Interactions with other E. coli proteins involved in these processes either do not occur, or are not impeded.
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129. |
( 1994 ) Cloning and sequencing of the Proteus mirabilis gene for a single-stranded DNA-binding protein (SSB) and complementation of Escherichia coli ssb point and deletion mutations. PMID : 8012606 : DOI : 10.1099/00221287-140-4-889 Abstract >>
The gene of Proteus mirabilis coding for a single-stranded DNA-binding protein (SSB) was cloned in Escherichia coli from a genomic library. It restored the UV resistance and the rate of cell division of an E. coli ssb-113 mutant to the same extent as the cloned E. coli ssb+ gene did. An E. coli mutant with deleted ssb was viable with the P. mirabilis ssb+ gene provided on a single-copy-number plasmid and had the same cell division rate as with the E. coli ssb+ gene on the same vector plasmid. The recovery from UV damage of an excision repair deficient (uvrA) mutant deleted for the ssb gene was identical with the ssb+ gene from P. mirabilis or E. coli, suggesting full substitution in recombinational DNA repair of the homologous by the heterologous SSB protein. The nucleotide sequence of the gene revealed that the SSB has 81% amino acid sequence homology with the E. coli SSB and only 58-63% with various plasmid SSBs. The data provide evidence that the bacterial chromosomally coded SSBs and the plasmid encoded SSBs constitute separate groups.
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130. |
( 1994 ) Sequence and genetic analysis of multiple flagellin-encoding genes from Proteus mirabilis. PMID : 7926835 : DOI : 10.1016/0378-1119(94)90230-5 Abstract >>
Surface-induced overproduction of flagellin is one of the hallmarks of Proteus mirabilis swarmer cell differentiation. In this study, we analyzed the nucleotide (nt) and amino acid (aa) sequences, and expression of the P. mirabilis flagellin-encoding gene (fliC) region. The nt sequence analysis of a 3567-bp region reveals three ORFs, each with homology to known Escherichia coli flagellar genes. The first ORF corresponds to fliD, the gene encoding the flagellar filament capping protein, FliD (HAP2). The second and third ORFs are highly homologous to each other and to fliC genes from many other Gram- bacteria. To distinguish between the two alleles, we have designated these genes fliC1 and fliC2. Sequences highly homologous to promoter sites for the alternate sigma factor of RNA polymerase, sigma 28, are found 5' to the start of each gene. Additionally, both fliC1 and fliC2 have a conserved direct tandem repeat (DTR) sequence upstream from the sigma 28 promoter that may have functional significance in the transcriptional control of fliC expression during swarmer cell differentiation. Both FliC1 and FliC2 were produced in E. coli, but only FliC1 could complement FliC- mutants of E. coli. Southern hybridization data indicate the presence of fliC1 and fliC2 in six distinct P. mirabilis strains, indicating that multiple flagellin-encoding genes are common in P. mirabilis. Hybridization data also suggest the presence of a third flagellin-encoding gene, fliC3, in all isolates. The possible significance of multiple fliC in swarmer cell differentiation is discussed.
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131. |
Gouet P,
Jouve HM,
Dideberg O,
( 1995 ) Crystal structure of Proteus mirabilis PR catalase with and without bound NADPH. PMID : 7791219 : DOI : 10.1006/jmbi.1995.0350 Abstract >>
A catalase from a peroxide resistant mutant of Proteus mirabilis binds NADPH tightly. Interestingly, this enzyme can be stripped of NADPH without loss of the catalatic activity. It is the only known non-mammalian catalase able to bind NADPH. The structure without cofactor was solved by molecular replacement using the structure of beef liver catalase as a model. The structure was refined to an R-factor of 19.3% in the range 8 to 2.2 A resolution. According to the sequence, a methionine sulphone was positioned in the haem active site. This oxidized form of methionine is particular to Proteus mirabilis catalase and likely to produce some steric hindrance in the active site. Two important water molecules are positioned in the haem distal site. These two water molecules are not located in the structure of beef liver catalase, but are supposed to account for the catalytic mechanism. The liganded form was obtained by soaking crystals of the unliganded form into an NADPH solution. The structure was refined to an R-factor of 15.9% in the range of 8 to 3.1 A resolution using the unliganded structure as a model. The NADPH was clearly located in the electron density map with the same conformation as in beef liver catalase. The NADPH binding induces slight structural changes. However, the imidazole ring of a histidine residue (His284) rotates about 50 degrees to accommodate the cofactor. The electron transfer from NADPH to the haem molecule was examined and several pathways are proposed.
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132. |
Thaller MC,
Lombardi G,
Berlutti F,
Schippa S,
Rossolini GM,
( 1995 ) Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes. PMID : 7894706 : DOI : 10.1099/00221287-141-1-147 Abstract >>
The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.
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133. |
Bijlsma IG,
van Dijk L,
Kusters JG,
Gaastra W,
( 1995 ) Nucleotide sequences of two fimbrial major subunit genes, pmpA and ucaA, from canine-uropathogenic Proteus mirabilis strains. PMID : 7670636 : DOI : 10.1099/13500872-141-6-1349 Abstract >>
Proteus mirabilis strains were isolated from dogs with urinary tract infection (UTI) and fimbriae were prepared from two strains. The N-terminal amino acid sequences of the major fimbrial subunits were determined and both sequences appeared identical to the N-terminal amino acid sequence of a urinary cell adhesin (UCA) (Wray, S. K., Hull, S. I., Cook, R. G., Barrish, J. & Hull, R. A., 1986, Infect Immun 54, 43-49). The genes of two different major fimbrial subunits were cloned using oligonucleotide probes that were designed on the basis of the N-terminal UCA sequence. Nucleotide sequencing revealed the complete ucaA gene of 540 bp (from strain IVB247) encoding a polypeptide of 180 amino acids, including a 22 amino acid signal sequence peptide, and the pmpA (P. mirabilis P-like pili) gene of 549 bp (from strain IVB219) encoding a polypeptide of 183 amino acids, including a 23 amino acid signal sequence. Hybridization experiments gave clear indications of the presence of both kinds of fimbriae in many UTI-related canine P. mirabilis isolates. However, the presence of these fimbriae could not be demonstrated in P. vulgaris or other Proteus-related species. Database analysis of amino acid sequences of major subunit proteins revealed that the UcaA protein shares about 56% amino acid identity with the F17A and F111A major fimbrial subunits from bovine enterotoxigenic Escherichia coli. In turn, the PmpA protein more closely resembled the pyelonephritis-associated pili (Pap)-like major subunit protein from UTI-related E. coli. The evolutionary relationship of UcaA, PmpA and various other fimbrial subunit proteins is presented in a phylogenetic tree.
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134. |
( 1994 ) Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression. PMID : 7910820 : DOI : 10.1128/jb.176.11.3412-3419.1994 PMC : PMC205519 Abstract >>
Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.
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135. |
Buzy A,
Bracchi V,
Sterjiades R,
Chroboczek J,
Thibault P,
Gagnon J,
Jouve HM,
Hudry-Clergeon G,
( 1995 ) Complete amino acid sequence of Proteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site. PMID : 7786407 : Abstract >>
The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.
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136. |
Cook SW,
Mody N,
Valle J,
Hull R,
( 1995 ) Molecular cloning of Proteus mirabilis uroepithelial cell adherence (uca) genes. PMID : 7729924 : PMC : PMC173269 Abstract >>
Proteus mirabilis bacteria are a common cause of hospital-acquired urinary tract infection. In a previous study, we described a P. mirabilis fimbrial protein, UCA, that adhered to human uroepithelial cells. Genes sufficient for expression of UCA adherence were cloned into Escherichia coli K-12. E. coli bacteria that contained the uca recombinant plasmid adhered to human uroepithelial cells. In addition, the ucaA gene encoding the structural component of UCA pili was subcloned, and its DNA sequence was determined. Amino acid sequence homology (30 to 50%) was found between mature UcaA protein and pilins from pathogenic bacteria representing several genera, including E. coli F17, G, and type 1C pilins, Haemophilus M43 pilin, and a Bordetella pilin.
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137. |
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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138. |
Massad G,
Zhao H,
Mobley HL,
( 1995 ) Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad. PMID : 7592338 : DOI : 10.1128/jb.177.20.5878-5883.1995 PMC : PMC177413 Abstract >>
Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.
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139. |
Wassif C,
Cheek D,
Belas R,
( 1995 ) Molecular analysis of a metalloprotease from Proteus mirabilis. PMID : 7592325 : DOI : 10.1128/jb.177.20.5790-5798.1995 PMC : PMC177400 Abstract >>
Proteus mirabilis is known for its ability to differentiate from swimmer to swarmer cells, a process crucial for the pathogenesis of these bacteria during urinary tract infections. Among the many virulence factors produced during swarmer cell differentiation is an extracellular metalloprotease. A cosmid containing a large fragment of P. mirabilis chromosomal DNA was obtained by measuring protease expression in recombinant Escherichia coli. The recombinant and native enzymes were purified to over 95% homogeneity from culture supernatants by use of phenyl-Sepharose affinity chromatography and found to be identical. The activity of the 55-kDa enzyme was stimulated by divalent cations (Ca2+ > Mg2+) and inhibited by a chelator of these cations. The enzyme possesses substrate specificity for both serum and secretory forms of immunoglobulin A1 (IgA1) and IgA2 as well as IgG and, unlike classic IgA proteases, digested to completion both human and mouse IgA. Following subcloning, a 5-kb DNA fragment encoding recombinant protease activity was identified by insertional mutagenesis with Tn5. Four open reading frames were identified within this 5-kb region by limited nucleotide sequence analysis of DNA flanking the transposon. The nucleotide and deduced amino acid sequences of the metalloprotease structural gene (zapA) were obtained. Computerized homology studies revealed that the P. mirabilis metalloprotein is a member of the serralysin family of proteases and may be part of an operon comprising genes encoding an ATP-dependent ABC transporter in addition to the metalloprotease. The relevance of the metalloprotease to swarmer cell differentiation and pathogenicity is discussed.
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140. |
Ching G,
Inouye M,
( 1985 ) Evolution of the lipoprotein gene in the enterobacteriaceae. Cloning and DNA sequence of the lpp gene from Proteus mirabilis. PMID : 3903165 : DOI : 10.1016/0022-2836(85)90066-x Abstract >>
We cloned the lipoprotein gene from Proteus mirabilis and determined its DNA sequence. Comparison with the lpp genes from Escherichia coli, Serratia marcescens, Erwinia amylovora and Morganella morganii revealed several unique features of the evolution of the lpp gene in the Enterobacteriaceae and enabled us to establish phylogenetic relationships between these bacteria.
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141. |
Charles IG,
Keyte JW,
Shaw WV,
( 1985 ) Nucleotide sequence analysis of the cat gene of Proteus mirabilis: comparison with the type I (Tn9) cat gene. PMID : 3900035 : PMC : PMC214219 Abstract >>
In Proteus mirabilis PM13 chloramphenicol resistance is mediated by the cat gene, a single copy of which is present in both resistant and sensitive isolates and which reverts at a high frequency. RNA measurements show an about 8.5-fold increase in cat-specific mRNA in cells expressing the resistance phenotype as compared with those which are sensitive to chloramphenicol. DNA sequence analysis has revealed a high degree of homology between the P. mirabilis cat gene and the type I cat variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. Sequence homology between the strain PM13 cat variant and Tn9 cat was not apparent however in the 5' and 3' flanking regions. Segments of near identity were seen when the upstream sequence of the cat of P. mirabilis was compared with the 5' regions of the Salmonella typhimurium flagellin genes H1 and H2, which are alternately expressed by a flip-flop control mechanism involving an invertible promoter and a trans-acting product.
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142. |
Bie L,
Fang M,
Li Z,
Wang M,
Xu H,
( 2018 ) Identification and Characterization of New Resistance-Conferring SGI1s (Salmonella Genomic Island 1) in Proteus mirabilis. PMID : 30619228 : DOI : 10.3389/fmicb.2018.03172 PMC : PMC6305713 Abstract >>
Salmonella genomic island 1 (SGI1) is a resistance-conferring chromosomal genomic island that contains an antibiotic resistance gene cluster. The international spread of SGI1-containing strains drew attention to the role of genomic islands in the dissemination of antibiotic resistance genes in Salmonella and other Gram-negative bacteria. In this study, five SGI1 variants conferring multidrug and heavy metal resistance were identified and characterized in Proteus mirabilis strains: SGI1-PmCAU, SGI1-PmABB, SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48. The genetic structures of SGI1-PmCAU and SGI1-PmABB were identical to previously reported SGI1s, while structural analysis showed that SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48 are new SGI1 variants. SGI1-PmJN16 is derived from SGI1-Z with the MDR region containing a new gene cassette array dfrA12-orfF-aadA2-qacE�G1-sul1-chrA-orf1. SGI1-PmJN40 has an unprecedented structure that contains two right direct repeat sequences separated by a transcriptional regulator-rich DNA fragment, and is predicted to form two different extrachromosomal mobilizable DNA circles for dissemination. SGI1-PmJN48 lacks a common ORF S044, and its right junction region exhibits a unique genetic organization due to the reverse integration of a P. mirabilis chromosomal gene cluster and the insertion of part of a P. mirabilis plasmid, making it the largest known SGI1 to date (189.1 kb). Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria, which promotes the horizontal transfer of antibiotic and heavy metal resistance genes. The identification and characterization of the new SGI1 variants in this work suggested the diversity of SGI1 structures and their significant roles in the evolution of bacteria.
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143. |
Ching G,
Inouye M,
( 1986 ) Expression of the Proteus mirabilis lipoprotein gene in Escherichia coli. Existence of tandem promoters. PMID : 3007466 : Abstract >>
The Ipp gene from Proteus mirabilis was cloned onto pBR322 and expressed in Escherichia coli. The P. mirabilis lpp gene is unique in that it has two tandem promoters transcribing two mRNAs that differ in length by approximately 70 nucleotides at their 5'-ends. The two mRNAs thus encode the identical lipoprotein. The P. mirabilis prolipoprotein has a 19-amino acid signal peptide and a 59-amino acid lipoprotein sequence. In spite of the substantial differences in the amino acid sequence from the E. coli prolipoprotein, the P. mirabilis prolipoprotein is normally modified and processed in E. coli, and the resultant lipoprotein is assembled in the E. coli outer membrane as is the E. coli lipoprotein.
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144. |
Jiang X,
Yu T,
Liu L,
Li Y,
Zhang K,
Wang H,
Shi L,
( 2017 ) Examination of Quaternary Ammonium Compound Resistance in Proteus mirabilis Isolated from Cooked Meat Products in China. PMID : 29312157 : DOI : 10.3389/fmicb.2017.02417 PMC : PMC5732425 Abstract >>
The aim of this study was to examine the presence of genes responsible for resistance to quaternary ammonium compounds (QACs) and the association of qac genes with class 1 integrons in Proteus mirabilis isolated from cooked meat products. A total of 52 P. mirabilis isolates (29.2%) were detected from 178 samples, and their minimum inhibitory concentrations (MICs) of benzalkonium chloride (BC) ranged from 4 to >32 �gg/mL. The isolates with BC MICs of 24 �gg/mL were observed most frequently. PCR assays indicated that mdfA, ydgE/ydgF, qacE, qacE�G1, emrE, sugE(c), and sugE(p) were commonly present (32.7%-100%) in these isolates, but qacH was less prevalent (3.8%). Five groups of resistance gene cassettes were identified in 10 intI1-positive isolates. An unusual gene cassette array dfrA32-ereA-aadA2 was found in one foodborne isolate of P. mirabilis. Two isolates harbored qacH- and sul3- associated non-classic integrons: aadA2-cmlA1-aadA1-qacH-IS440-sul3 and a new arrangement dfrA32-ereA1-aadA2-cmlA1-aadA1-qacH-IS440-sul3, which is first reported in P. mirabilis. Non-classic class 1 integrons were located on conjugative plasmids of 100 kb in two tested isolates. Our data showed that the QAC resistance genes were commonly present among P. mirabilis isolates from cooked meats and qacH was associated with non-classic class 1 integrons. The creation of transconjugants demonstrated that qacH-associated non-classic class 1 integrons were located on conjugative plasmids and therefore could facilitate the co-dissemination of disinfectant and antimicrobial resistance genes among bacteria, an increasing area of concern.
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145. |
Yu X,
Torzewska A,
Zhang X,
Yin Z,
Drzewiecka D,
Cao H,
Liu B,
Knirel YA,
Rozalski A,
Wang L,
( 2017 ) Genetic diversity of the O antigens of Proteus species and the development of a suspension array for molecular serotyping. PMID : 28817637 : DOI : 10.1371/journal.pone.0183267 PMC : PMC5560731 Abstract >>
Proteus species are well-known opportunistic pathogens frequently associated with skin wound and urinary tract infections in humans and animals. O antigen diversity is important for bacteria to adapt to different hosts and environments, and has been used to identify serotypes of Proteus isolates. At present, 80 Proteus O-serotypes have been reported. Although the O antigen structures of most Proteus serotypes have been identified, the genetic features of these O antigens have not been well characterized. The O antigen gene clusters of Proteus species are located between the cpxA and secB genes. In this study, we identified 55 O antigen gene clusters of different Proteus serotypes. All clusters contain both the wzx and wzy genes and exhibit a high degree of heterogeneity. Potential functions of O antigen-related genes were proposed based on their similarity to genes in available databases. The O antigen gene clusters and structures were compared, and a number of glycosyltransferases were assigned to glycosidic linkages. In addition, an O serotype-specific suspension array was developed for detecting 31 Proteus serotypes frequently isolated from clinical specimens. To our knowledge, this is the first comprehensive report to describe the genetic features of Proteus O antigens and to develop a molecular technique to identify different Proteus serotypes.
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146. |
Helmy OM,
Kashef MT,
( 2017 ) Different phenotypic and molecular mechanisms associated with multidrug resistance in Gram-negative clinical isolates from Egypt. PMID : 29263684 : DOI : 10.2147/IDR.S147192 PMC : PMC5726372 Abstract >>
We set out to investigate the prevalence, different mechanisms, and clonal relatedness of multidrug resistance (MDR) among third-generation cephalosporin-resistant Gram-negative clinical isolates from Egypt. A total of 118 third-generation cephalosporin-resistant Gram-negative clinical isolates were included in this study. Their antimicrobial susceptibility pattern was determined using Kirby-Bauer disk diffusion method. Efflux pump-mediated resistance was tested by the efflux-pump inhibitor-based microplate assay using chlorpromazine. Detection of different aminoglycoside-, �]-lactam-, and quinolone-resistance genes was done using polymerase chain reaction. The genetic diversity of MDR isolates was investigated using random amplification of polymorphic DNA. Most of the tested isolates exhibited MDR phenotypes (84.75%). The occurrence of efflux pump-mediated resistance in the different MDR species tested was 40%-66%. Acinetobacter baumannii isolates showed resistance to most of the tested antibiotics, including imipenem. The blaOXA-23-like gene was detected in 69% of the MDR A. baumannii isolates. The MDR phenotype was detected in 65% of Pseudomonas aeruginosa isolates, of which only 23% exhibited efflux pump-mediated resistance. On the contrary, efflux-mediated resistance to piperacillin and gentamicin was recorded in 47.5% of piperacillin-resistant and 25% of gentamicin-resistant MDR Enterobacteriaceae. Moreover, the plasmid-mediated quinolone-resistance genes (aac(6')-Ib-cr, qnrB, and qnrS) were detected in 57.6% and 83.33% of quinolone-resistant MDR Escherichia coli and Klebsiella pneumoniae isolates, respectively. The �]-lactamase-resistance gene blaSHV-31 was detected for the first time in one MDR K. pneumoniae isolate from an endotracheal tube specimen in Egypt, accompanied by blaTEM-1, blaCTX-M-15, blaCTX-M-14, aac(6')-Ib-cr, qnrS, and multidrug efflux-mediated resistance. MDR phenotypes are predominant among third-generation cephalosporin-resistant Gram-negative bacteria in Egypt and mediated by different mechanisms, with an increased role of efflux pumps in Enterobacteriaceae.
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147. |
Siebor E,
de Curraize C,
Neuwirth C,
( 2018 ) Genomic context of resistance genes within a French clinical MDR Proteus mirabilis: identification of the novel genomic resistance island GIPmi1. PMID : 29669108 : DOI : 10.1093/jac/dky126 Abstract >>
To determine the location of the antibiotic resistance genes in the MDR Proteus mirabilis PmPHI clinical isolate. WGS and de novo assembly were performed. BLAST searches were used to identify relevant contigs. PCR and Sanger sequencing were used to link the fragments of interest and fill the gaps. P. mirabilis PmPHI was resistant to six classes of antibiotics: penicillins, aminoglycosides, phenicols, tetracyclines, folate inhibitors and fluoroquinolones. A novel genomic resistance island (GIPmi1) of 55.8 kb was located at the 3' end of trmE. The backbone shared 93% identity with a genomic sequence of Enterobacter cloacae DSM 16690. The MDR region was composed of two class 1 integrons [one Tn402-type (estX-qacE) and one In5-type (aadB-aadA2)], separated by a region containing many parts of transposons. An external circular form of GIPmi1 was detected; however, mobilization by an A/C plasmid failed. In addition, an SXT/R391 integrative and conjugative element (ICEPmiFra1) was inserted into prfC. It carried floR, the sul2-strA-strB cluster and a composite transposon flanked by two copies of a tISPpu12 element that contains a class 1 integron (dfrA32-ereA1-aadA2), Tn4352 (aphA1a) and tetA(C). A class 2 integron (dfrA1-sat2-aadA1) was also identified on Tn7 as well as point mutations in gyrA and parC accounting for quinolone resistance. The finding of the new genomic island GIPmi1 belonging to the same superfamily of genomic islands as SGI1/SGI2/PGI1/AGI1 and of the integrative conjugative element ICEPmiFra1 (SXT/R391 family) suggested that these genetic elements might be key mediators of resistance gene acquisition in P. mirabilis.
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148. |
( 2013 ) Prevalence and plasmid characterization of the qnrD determinant in Enterobacteriaceae isolated from animals, retail meat products, and humans. PMID : 23557071 : DOI : 10.1089/mdr.2012.0146 Abstract >>
qnrD, unlike other qnr genes, is mainly located on small nonconjugative plasmids. We investigated the presence of qnrD among 1,373 Enterobacteriaceae isolates in China. Twelve qnrD-positive strains were detected, and all were nonsusceptible to fluoroquinolones. The complete sequence of plasmids showed that the qnrD determinants were located on two plasmids with a respective size of ~4.2 and 2.7 k-bp. Interestingly, the identification of qnrD in this study revealed the highest prevalence of Proteeae among Enterobacteriaceae identified.
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149. |
( 2013 ) Emergence of Salmonella genomic island 1 (SGI1) among Proteus mirabilis clinical isolates in Dijon, France. PMID : 23580563 : DOI : 10.1093/jac/dkt100 Abstract >>
Salmonella genomic island 1 (SGI1) is often encountered in antibiotic-resistant Salmonella enterica and exceptionally in Proteus mirabilis. We investigated the prevalence of SGI1-producing clinical isolates of P. mirabilis in our hospital (Dijon, France). A total of 57 strains of P. mirabilis resistant to amoxicillin and/or gentamicin and/or trimethoprim/sulfamethoxazole isolated from August 2011 to February 2012 as well as 9 extended-spectrum �]-lactamase (ESBL)-producing P. mirabilis from our collection were tested for the presence of SGI1 by PCR. The complete SGI1 structure from positive isolates [backbone and multidrug resistance (MDR) region] was sequenced. SGI1 was detected in 7 isolates; 5 out of the 57 isolates collected during the study period (9%) and 2 out of the 9 ESBL-producing strains of our collection. The structures of the seven SGI1s were distinct. Three different backbones were identified: one identical to the SGI1 backbone from the epidemic Salmonella Typhimurium DT104, one with variations already described in SGI1-K from Salmonella Kentucky (deletion and insertion of IS1359 in the region spanning from S005 to S009) and one with a variation never detected before (deletion from S005 to S009). Six different MDR regions were identified: four simple variants containing resistance genes already described and two variants harbouring a very complex structure including regions derived from several transposons and IS26 elements with aphA1a never reported to date in SGI1. SGI1 variants are widely distributed among P. mirabilis clinical strains and might spread to other commensal Enterobacteriaceae. This would become a serious public health problem.
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150. |
Wang M,
Xu H,
Bie L,
Wu H,
Wang XH,
( 2017 ) Identification and characterization of new members of the SXT/R391 family of integrative and conjugative elements (ICEs) in Proteus mirabilis. PMID : 28602701 : DOI : 10.1016/j.ijantimicag.2017.01.045 Abstract >>
Integrative and conjugative elements (ICEs) are self-transmissible chromosomal mobile elements that play significant roles in the dissemination of antimicrobial resistance genes. Identification of the structures and functions of ICEs, particularly those in pathogens, improves understanding of the dissemination of antimicrobial resistance. This study identified new members of the sulfamethoxazole-trimethoprim (SXT)/R391 family of ICEs that could confer multi-drug resistance in the opportunistic pathogen Proteus mirabilis, characterized their genetic structures, and explored their evolutionary connection with other members of this family of ICEs. Three new members of the SXT/R391 family of ICEs were detected in six of 77 P. mirabilis strains isolated in China: ICEPmiChn2 (one strain), ICEPmiChn3 (one strain) and ICEPmiChn4 (three strains). All three new ICEs harbour antimicrobial resistance genes from diverse origins, suggesting their capability in acquiring foreign genes and serving as important carriers for antimicrobial resistance genes. Structural analysis showed that ICEPmiChn3 is a particularly interesting and unique ICE that has lost core genes involved in conjugation, and could not transfer to other cells via conjugation. This finding confirmed the key roles of these missing genes in conjugation. Further phylogenetic analysis suggested that ICEs in geographically close strains are also connected evolutionarily, and ICEPmiChn3 lost its conjugation cassette from a former mobile ICE. The identification and characterization of the three new members of the SXT/R391 family of ICEs in this work leads to suggestions of core ICE genes essential for conjugation, and extends understanding on the structures of ICEs, evolutionary relationships between ICEs, and the antimicrobial resistance mechanisms of P. mirabilis.
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151. |
( 2012 ) Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants. PMID : 23118975 : DOI : 10.1371/journal.pone.0048301 PMC : PMC3485190 Abstract >>
Lysine racemase, a pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (�\/�])(8) barrel and a C-terminal �]-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in �\-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.
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152. |
( 2013 ) TEM-187, a new extended-spectrum �]-lactamase with weak activity in a Proteus mirabilis clinical strain. PMID : 23478954 : DOI : 10.1128/AAC.01761-12 PMC : PMC3632918 Abstract >>
A Proteus mirabilis clinical strain (7001324) was isolated from urine sample of a patient hospitalized in a long-term-care facility. PCR and cloning experiments performed with this strain identified a novel TEM-type �]-lactamase (TEM-187) differing by four amino acid substitutions (Leu21Phe, Arg164His, Ala184Val, and Thr265Met) from TEM-1. This characterization provides further evidence for the diversity of extended-spectrum �]-lactamases (ESBL) produced by P. mirabilis and for their potential spread to other Enterobacteriaceae due to a lack of sensitive detection methods used in daily practice.
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153. |
( 2013 ) A binational cohort study of intestinal colonization with extended-spectrum �]-lactamase-producing Proteus mirabilis in patients admitted to rehabilitation centres. PMID : 23210906 : DOI : 10.1111/1469-0691.12072 Abstract >>
The aims of our study were to analyse the risk factors for colonization by Extended-spectrum �]-lactamases (ESBL)-producing Proteus mirabilis (ESBL-PM) in rehabilitation patients and to characterize the molecular features of these strains. The study was conducted in two rehabilitation centres located in Rome, Italy (Fondazione Santa Lucia IRCCS (FSL)), and Tel-Aviv, Israel (Tel-Aviv Sourasky Medical Center (TASMC)). Carriage of ESBL-PM was surveyed by rectal swabs. Strain typing was performed by pulsed-field gel electrophoresis (PFGE). Identification of ESBL genes was done by PCR and sequencing. Patients admitted to the same institutions without ESBL carriage were included as controls. The study group included 70 and 41 patients from FSL and TASMC, respectively. In FSL, the multivariate analysis identified severe acute brain injury (OR = 15, 95% CI = 3.2-69.5, p 0.001), decubitus ulcer (OR = 3.5, 95% CI = 1.2-9.8, p 0.018) and recent treatment with quinolones (OR = 5.7, 95% CI = 1.07-30.1, p 0.042) as independent risk factors. ESBL-PM carriers stayed longer in the hospital on average and were less likely to be discharged home. No significant risk factor was identified in TASMC. There were no similarities in PFGE types or ESBL genes between the ESBL-PM isolates from the two institutions. In both hospitals, a variety of PFGE types existed but a single ESBL type predominated, namely TEM-92 in FSL (n = 64/70; 91%) and CTX-M-2 in TASMC (n = 37/41; 90%). A new TEM ESBL variant, TEM-177 was identified in FSL. The clonal diversity and the predominance of a single ESBL type suggested that horizontal gene transfer played an important role in dissemination of resistance. The development of a population analysis tool that would allow tracing deeper genetic relationships is required.
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154. |
( 2012 ) Integrons, �]-lactamase and qnr genes in multidrug resistant clinical isolates of Proteus mirabilis and P. vulgaris. PMID : 23030307 : DOI : 10.1111/j.1600-0463.2012.02923.x Abstract >>
Thirty-three isolates of Proteus mirabilis and two P. vulgaris were examined for their antimicrobial resistance, the presence of integrons with regard to gene cassette content, and genetic determinants of �]-lactam and low-level quinolone resistance. Integrons were detected in 23 (69.7%) P. mirabilis isolates; six (18.2%) of them had class 1 integrons, 11 (33.3%) possessed class 2 integrons and six (18.2%) carried integrons of both classes. One P. vulgaris strain possessed class 1 and class 2 integrons. The presence of integrons was associated with increased frequency of resistance to gentamicin, ciprofloxacin, sulfamethoxazole and co-trimoxazole. Moreover, integron presence was associated with increased resistance range in terms of both the number of antimicrobials and the number of classes of antimicrobials to which a strain was resistant. Class 1 integrons contained aadA1, aadB-aadA1, dfrA1-aadA1, bla(PSE-1) -aadA1 and aacA4-orfA-orfB-aadA1 gene cassette arrays, whereas all class 2 integrons had a dfrA1-sat2-aada1 array. �]-lactamase genes not associated with integrons comprised bla(TEM-2) , bla(DHA-1) and bla(CMY-15) . Plasmid-mediated fluoroquinolone resistance was determined by qnrD and qnrS1 genes. This is the first report of P. vulgaris strains harbouring qnrD genes in Europe.
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155. |
( 2013 ) The novel CTX-M-116 �]-lactamase gene discovered in Proteus mirabilis is composed of parts of the CTX-M-22 and CTX-M-23 genes. PMID : 23318795 : DOI : 10.1128/AAC.01471-12 PMC : PMC3591887 Abstract >>
The novel �]-lactamase gene bla(CTX-M-116) was identified in a Proteus mirabilis nosocomial isolate recovered from the urine of a patient in Moscow in 2005. DNA sequence analysis showed bla(CTX-M-116) to be a hybrid gene consisting of 5' bla(CTX-M-23) (nucleotides 1 to 278) and 3' bla(CTX-M-22) (nucleotides 286 to 876) moieties separated by an intervening putative site of recombination (GTTAAAT). A retrospective analysis of available bla(CTX-M) genes in the GenBank database revealed 19 bla(CTX-M) genes that display the same hybrid structure.
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156. |
( 1998 ) Cloning and characterisation of the Proteus mirabilis xerD gene. PMID : 9675854 : DOI : 10.1111/j.1574-6968.1998.tb13071.x Abstract >>
The Xer site-specific recombination system is involved in the stable maintenance of replicons (certain plasmids and chromosomes) in Escherichia coli and other bacteria by converting multimers into monomers. This system requires a cis-acting DNA sequence (the chromosomal dif site or the ColE1 cer site) and two trans-acting factors: the XerC and XerD recombinases, which belong to the lambda integrase family of tyrosine site-specific recombinases. In addition, in order to resolve plasmid multimers into monomers, two additional factors are required: the ArgR and PepA proteins. We have previously shown the presence of xerC and xerD genes (and their function) by Southern hybridisation and by in vivo recombination in a wide variety of Enterobacteriaceac. We have now cloned and sequenced the xerD gene of Proteus mirabilis using degenerate and inverse PCR methods. This gene encodes a tyrosine recombinase which is highly similar to the E. coli XerD recombinase, is capable of complementing an E. coli xerD mutant, and displays sequence-specific DNA binding activity.
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157. |
( 1998 ) A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications. PMID : 9655824 : Abstract >>
Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.
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158. |
( 1998 ) Characterization of Proteus mirabilis precocious swarming mutants: identification of rsbA, encoding a regulator of swarming behavior. PMID : 9829920 : PMC : PMC107696 Abstract >>
Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull's-eye colony often associated with cultures of P. mirabilis. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.
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159. |
( 1998 ) Characterisation of CMY-4, an AmpC-type plasmid-mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis. PMID : 9868767 : DOI : 10.1111/j.1574-6968.1998.tb13323.x Abstract >>
A strain of Proteus mirabilis resistant to beta-lactams, including cefoxitin, was isolated from the urine of a woman from Tunisia. Its antibiotic susceptibility pattern and that of the Escherichia coli transconjugant suggested the presence of an AmpC-type beta-lactamase. Two bands of beta-lactamase activity (pI 5.4 and 9.2) were detected by isoelectric focusing. The nucleotide sequence of the gene encoding the AmpC-type enzyme was determined. The deduced amino acid sequence was 98-99% identical to CMY-3 and to those of the plasmid-mediated AmpC-type beta-lactamases originated from Citrobacter freundii and 97% identical to the chromosome-encoded beta-lactamase of a Tunisian clinical isolate of C. freundii. This enzyme differs from CMY-2 by one substitution (Arg for Trp at position 221) and from CMY-3 by two substitutions (Glu for Gly at position 42 and Ser for Asn at position 363) and we propose the denomination CMY-4.
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160. |
( 1998 ) Novel genes that upregulate the Proteus mirabilis flhDC master operon controlling flagellar biogenesis and swarming. PMID : 9723914 : DOI : 10.1046/j.1365-2958.1998.00967.x Abstract >>
By screening for restoration of multicellular migration in a non-swarming but motile Proteus mirabilis mutant lacking the FIgN facilitator of flagella assembly, we identified four distinct genes that, in trans and multicopy, increased flagella production and cell length. Each of the genes upregulated expression of the flhDC master operon that controls flagellar biogenesis, cell division and swarming, not only in the mutant but also in the wild type. The genes were named umoA, umoB, umoC and umoD. Disruption of each of the wild-type chromosomal umo genes caused corresponding reductions in swarming and cell elongation, which correlated with decreased expression of the flhDC operon. The umoA, umoB, umoC and umoD genes are not closely linked, and only umoB is part of an operon. The sequences of the calculated gene products, UmoA (20.6 kDa), UmoB (78.0 kDa), UmoC (15.2 kDa) and UmoD (19.2 kDa), contain putative N-terminal secretion signals and predict a location in the cell membranes or periplasm. UmoB and UmoD have sequence similarity to the Escherichia coli uncharacterized open reading frames YrfF and YcfJ respectively; UmoA and UmoC have no known homologues. The umoB and umoC gene transcripts were present at very low levels, but umoA and umoD expression was similar to that of flhDC and increased in parallel with flhDC expression during differentiation into elongated hyperflagellated swarm cells. Like flhDC, umoA and umoD expression was subject to negative feedback in aflagellar assembly mutant lacking the FlhA inner membrane component of the export machinery. Assays of umo gene expression and cross-complementation indicated that the umo genes do not act in sequence within a pathway to upregulate flhDC, but revealed that umoA and umoD are reciprocally upregulated by FlhDC. Our findings strengthen the picture of the flhDC master operon as a major assimilatory checkpoint in Proteus mirabilis and other Gram-negative bacteria and expand the view of a complex regulatory network coupled to flagellar biogenesis.
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161. |
( 1998 ) A new tetracycline resistance determinant cloned from Proteus mirabilis. PMID : 9838156 : DOI : 10.1016/s0167-4781(98)00210-3 Abstract >>
The chromosomal inducible Tcr determinant from Proteus mirabilis was cloned and the nucleotide sequence of both the structural and repressor genes determined. The deduced amino acid sequence of the structural protein shows the highest similarity to TetA(H) from Pasteurella multocida (78.4%), followed by TetA(B) from Tn10 (50.9%). Based on this analysis, we suggest that this new determinant can be assigned to a new class, TetJ.
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162. |
( 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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