| 1. |
Felske AD,
Fehr W,
Pauling BV,
von Canstein H,
Wagner-Döbler I,
( 2003 ) Functional profiling of mercuric reductase (mer A) genes in biofilm communities of a technical scale biocatalyzer. PMID : 14577839 : DOI : 10.1186/1471-2180-3-22 PMC : PMC270059 Abstract >>
Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase. We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation. Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences. The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing. The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducing Ps. aeruginosa strain was identified by its unique mercuric reductase gene.
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2. |
Généreux C,
Dehareng D,
Devreese B,
Van Beeumen J,
Frère JM,
Joris B,
( 2004 ) Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-L-alanine amidase. PMID : 14507260 : DOI : 10.1042/BJ20030862 PMC : PMC1223845 Abstract >>
Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-L-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.
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3. |
Spierings G,
Ockhuijsen C,
Hofstra H,
Tommassen J,
( 1992 ) Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides. PMID : 1337052 : DOI : 10.1016/0378-1097(92)90025-j Abstract >>
The phoE gene of Citrobacter freundii, encoding a pore-forming outer membrane protein, was cloned and its nucleotide sequence was determined. The homologies in terms of identical amino acids between the C. freundii PhoE protein and those of Escherichia coli, E. cloacae and Klebsiella pneumoniae were 90%, 86% and 84%, respectively. Two synthetic oligonucleotides, corresponding to hypervariable, cell surface-exposed regions of the protein, were tested for their specificity in polymerase chain reactions. They were specific for the species C. freundii, i.e., no reaction was detected with 35 non-C. freundii strains tested, including 17 Salmonella, two C. amalonaticus and three C. diversus strains, whereas all five C. freundii strains tested were correctly recognized.
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4. |
Siebold C,
Arnold I,
Garcia-Alles LF,
Baumann U,
Erni B,
( 2003 ) Crystal structure of the Citrobacter freundii dihydroxyacetone kinase reveals an eight-stranded alpha-helical barrel ATP-binding domain. PMID : 12966101 : DOI : 10.1074/jbc.M305942200 Abstract >>
Dihydroxyacetone kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system in bacteria. Here we report the 2.5-A crystal structure of the homodimeric Citrobacter freundii dihydroxyacetone kinase complex with an ATP analogue and dihydroxyacetone. The N-terminal domain consists of two alpha/beta-folds with a molecule of dihydroxyacetone covalently bound in hemiaminal linkage to the N epsilon 2 of His-220. The C-terminal domain consists of a regular eight-helix alpha-barrel. The eight helices form a deep pocket, which includes a tightly bound phospholipid. Only the lipid headgroup protrudes from the surface. The nucleotide is bound on the top of the barrel across from the entrance to the lipid pocket. The phosphate groups are coordinated by two Mg2+ ions to gamma-carboxyl groups of aspartyl residues. The ATP binding site does not contain positively charged or aromatic groups. Paralogues of dihydroxyacetone kinase also occur in association with transcription regulators and proteins of unknown function pointing to biological roles beyond triose metabolism.
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5. |
Pickard D,
Wain J,
Baker S,
Line A,
Chohan S,
Fookes M,
Barron A,
Gaora PO,
Chabalgoity JA,
Thanky N,
Scholes C,
Thomson N,
Quail M,
Parkhill J,
Dougan G,
( 2003 ) Composition, acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7. PMID : 12923078 : DOI : 10.1128/jb.185.17.5055-5065.2003 PMC : PMC180996 Abstract >>
Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.
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6. |
Liepinsh E,
Généreux C,
Dehareng D,
Joris B,
Otting G,
( 2003 ) NMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains. PMID : 12654266 : DOI : 10.1016/s0022-2836(03)00185-2 Abstract >>
AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.
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7. |
Karasawa T,
Ito H,
Tsukamoto T,
Yamasaki S,
Kurazono H,
Faruque SM,
Nair GB,
Nishibuchi M,
Takeda Y,
( 2002 ) Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E. coli. PMID : 12438400 : DOI : 10.1128/iai.70.12.7153-7155.2002 PMC : PMC133046 Abstract >>
We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C. freundii isolates and 152 E. coli isolates from diarrheal patients by colony blot hybridization. Two strains, C. freundii 48 and E. coli 176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the cfxA and cfxB genes from E. coli 176 and designated them ecxA and ecxB, respectively. The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.
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8. |
Pepperell C,
Kus JV,
Gardam MA,
Humar A,
Burrows LL,
( 2002 ) Low-virulence Citrobacter species encode resistance to multiple antimicrobials. PMID : 12384364 : DOI : 10.1128/aac.46.11.3555-3560.2002 PMC : PMC128719 Abstract >>
Citrobacter spp. are gram-negative commensal bacteria that infrequently cause serious nosocomial infections in compromised hosts. They are often resistant to cephalosporins due to overexpression of their chromosomal beta-lactamase. During a recent study of multidrug-resistant Enterobacteriaceae (MDRE) in solid-organ transplant patients, we found that almost half of patients colonized with MDRE carried one or more cefpodoxime-resistant Citrobacter freundii, Citrobacter braakii, or Citrobacter amalonaticus strains. Pulsed-field gel electrophoresis showed that 36 unique strains of Citrobacter were present among 32 patients. Genetic and phenotypic analysis of the resistance mechanisms of these bacteria showed that the extended-spectrum beta-lactamase (ESBL) SHV-5 or SHV-12 was encoded by 8 strains (26%) and expressed by 7 strains (19%). A number of strains were resistant to other drug classes, including aminoglycosides (28%), trimethoprim-sulfamethoxazole (31%), and fluoroquinolones (8%). PCR and DNA analysis of these multiresistant strains revealed the presence of class I integrons, including the first integrons reported for C. braakii and C. amalonaticus. The integrons encoded aminoglycoside resistance, trimethoprim resistance, or both. Despite the prevalence of MDR Citrobacter spp. in our solid-organ transplant patients, only a single infection with a colonizing strain was recorded over 18 months. Low-virulence Citrobacter spp., which can persist in the host for long periods, could influence pathogen evolution by accumulation of genes encoding resistance to multiple antimicrobial classes.
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9. |
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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10. |
Kang JW,
Jeong YJ,
Kwon AR,
Yun HJ,
Kim DH,
Choi EC,
( 2001 ) Cloning, sequence analysis, and characterization of the astA gene encoding an arylsulfate sulfotransferase from Citrobacter freundii. PMID : 11534764 : Abstract >>
Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy of Citrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homology, they were designated as astA and dsbA, respectively. The amino acid sequence of astA deduced from C. freundii was highly similar to that of the Salmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, and Campylobacter jejuni, encoded by the astA genes. However, the ASST activity assay revealed different acceptor specificities. Using p-nitrophenyl sulfate (PNS) as a donor substrate, alpha-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine and tyrosine.
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11. |
Nørskov-Lauritsen N,
Sandvang D,
Hedegaard J,
Fussing V,
Mortensen KK,
Sperling-Petersen HU,
Schønheyder HC,
( 2001 ) Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county. PMID : 11444774 : DOI : 10.1099/0022-1317-50-7-636 Abstract >>
During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacterfreundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial sequencing of the gene encoding translation initiation factor 2. Fourteen of the 15 isolates were identical, as evaluated by their antibiograms and by all these typing methods. This epidemic strain harboured the aminoglycoside resistance genes aac(3)-II and ant(3")-I, with the latter located in tandem with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission.
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12. |
Haruta S,
Nukaga M,
Sawai T,
( 2001 ) Characterization of an extended-spectrum class C beta-lactamase of Citrobacter freundii. PMID : 11386417 : DOI : 10.1111/j.1348-0421.2001.tb02619.x Abstract >>
Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino beta-lactams such as ceftazidime and aztreonam. This drug resistance was due to an extended-spectrum class C beta-lactamase encoded by chromosomal gene(s). The GC3 beta-lactamase showed high amino acid sequence homology to a known C. freundii beta-lactamase, i.e., 346 of 361 amino acids were identical with those of C. freundii GN346 beta-lactamase (Tsukamoto, K. et al, Eur. J. Biochem. 188, 15-22, 1990). Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended-spectrum resistance in class C beta-lactamases (Haruta, S. et al, Microbiol. Immunol. 42, 165-169, 1998). However, Asp198 was eliminated as a cause of the extended-spectrum resistance by the substitution of Asn for Asp198. Subsequent investigation suggested that the moderate resistance to oxyimino beta-lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active-site. Some or all of the replacements are assumed to delicately modify the active-site configuration. The GC3 beta-lactamase is the first example of an extended-spectrum class C beta-lactamase in which mutations are independent of the omega loop.
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13. |
Shimamoto T,
Shimamoto T,
Xu XJ,
Okazaki N,
Kawakami H,
Tsuchiya T,
( 2001 ) A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii. PMID : 11275561 : DOI : 10.1093/oxfordjournals.jbchem.a002897 Abstract >>
Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.
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14. |
Seifert C,
Bowien S,
Gottschalk G,
Daniel R,
( 2001 ) Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii. PMID : 11298756 : DOI : 10.1046/j.1432-1327.2001.02123.x Abstract >>
The coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis. We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase. Northern blot analyses revealed that both genes were expressed during glycerol fermentation. The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3-propanediol dehydrogenase. The dhaF and dhaG gene products were copurified to homogeneity from cell-free extracts of a recombinant E. coli strain producing both His6-tagged proteins. Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da. The subunit structure of the native complex is probably alpha2beta2. The factor rapidly reactivated glycerol- or O2-inactivated hologlycerol dehydratase and activated the enzyme-cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+. The DhaF-DhaG complex and DhaF exhibited ATP-hydrolyzing activity, which was not directly linked to the reactivation of dehydratase. The purified DhaF-DhaG complex of C. freundii efficiently cross-activated the enzyme-cyanocobalamin complex and the glycerol-inactivated glycerol dehydratase of Klebsiella pneumoniae. It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.
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15. |
Liebert CA,
Watson AL,
Summers AO,
( 2000 ) The quality of merC, a module of the mer mosaic. PMID : 11116334 : Abstract >>
We examined a region of high variability in the mosaic mercury resistance (mer) operon of natural bacterial isolates from the primate intestinal microbiota. The region between the merP and merA genes of nine mer loci was sequenced and either the merC, the merF, or no gene was present. Two novel merC genes were identified. Overall nucleotide diversity, pi (per 100 sites), of the merC gene was greater (49.63) than adjacent merP (35.82) and merA (32.58) genes. However, the consequences of this variability for the predicted structure of the MerC protein are limited and putative functional elements (metal-binding ligands and transmembrane domains) are strongly conserved. Comparison of codon usage of the merTP, merC, and merA genes suggests that several merC genes are not coeval with their flanking sequences. Although evidence of homologous recombination within the very variable merC genes is not apparent, the flanking regions have higher homologies than merC, and recombination appears to be driving their overall sequence identities higher. The synonymous codon usage bias (EN(C)) values suggest greater variability in expression of the merC gene than in flanking genes in six different bacterial hosts. We propose a model for the evolution of MerC as a host-dependent, adventitious module of the mer operon.
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16. |
Ruiz J,
Ribera A,
Navia MM,
( 1999 ) Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii. PMID : 10590274 : DOI : 10.1093/jac/44.6.743 Abstract >>
The presence of gyrA, gyrB and/or parC mutations, quinolone uptake, outer membrane protein profiles and epidemiological relationship were studied in 12 clinical isolates of Citrobacter freundii. No alterations were observed in the gyrB gene of any of the strains, or gyrA or parC of the four quinolone-susceptible strains (nalidixic acid MIC of 2-4 mg/L, and a ciprofloxacin MIC of 0.006-0.06 mg/L). The quinolone-resistant strains were classified into two groups: one group (group A) composed of strains resistant to nalidixic acid but not to ciprofloxacin and another (group B) including those resistant to both antibiotics with a mutation at codon 83 of the gyrA gene (Thr-->Ile), but no alteration in either parC or gyrB genes. In group B, three of the four resistant isolates, with a nalidixic acid MIC > 1024 mg/L and ciprofloxacin MIC of 8-32 mg/L, showed concomitant mutations at codons 83 and 87 of the gyrA gene (Thr-->Ile and Asp-->Tyr, respectively) as well as a single mutation in codon 80 of the parC gene (Ser-->Ile). The fourth isolate did not possess the mutation at codon 87 of gyrA. Two strains belong to the same clone and, although they had the same type of mutations in the gyrA and parC genes, showed different MICs of ciprofloxacin. This difference was related to an efflux pump mechanism. Mutations in the gyrA and parC genes play the main role in quinolone resistance development in Citrobacter freundii, although other factors such as overexpression of efflux pumps can play a complementary role and thus modulate the final quinolone MIC.
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17. |
Young JM,
Park DC,
( 2007 ) Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences. PMID : 17451899 : DOI : 10.1016/j.syapm.2007.03.002 Abstract >>
Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.
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18. |
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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19. |
Power P,
Di Conza J,
Rodríguez MM,
Ghiglione B,
Ayala JA,
Casellas JM,
Radice M,
Gutkind G,
( 2007 ) Biochemical characterization of PER-2 and genetic environment of blaPER-2. PMID : 17438050 : DOI : 10.1128/AAC.01395-06 PMC : PMC1913245 Abstract >>
PER-2 was the first detected and the second most prevalent extended-spectrum beta-lactamase in clinical pathogens isolated in Argentina and was also reported only in other South American countries. Citrobacter freundii 33587 was isolated in 1999 in Buenos Aires and was resistant to all tested beta-lactams except cephamycins and carbapenems. The strain produced both plasmid-borne TEM-1 and PER-2 (pI 5.4), which could be transferred by conjugation. By PCR screening, thermal asymmetric interlaced PCR, and DNA sequencing, we detected an ISPa12/IS1387a insertion sequence upstream of bla(PER-2), previously reported as also being associated with bla(PER-1). The presence of similar structures upstream of bla(PER-1) and bla(PER-2) suggests a common origin and mobilization. Compared to bla(PER-1) genes, an additional putative promoter for bla(PER-2) was found. PER-2 kinetic analysis showed its high hydrolysis efficiencies toward both CTX and CAZ (k(cat)/K(m), 0.76 and 0.43 microM(-1).s(-1), respectively).
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20. |
Garvin LD,
Hardies SC,
( 1991 ) Temporal and topological clustering of diverged residues among enterobacterial dihydrofolate reductases. PMID : 1766362 : DOI : 10.1093/oxfordjournals.molbev.a040676 Abstract >>
The complete nucleotide and encoded amino acid sequences were determined for the dihydrofolate reductase (DHFR) from the bacteria Enterobacter aerogenes and Citrobacter freundii. These were compared with the closely related Escherichia coli DHFR sequence. The ancestral DHFR sequence common to these three species was reconstructed. Since that ancestor there have been seven, nine, and one amino acid replacements in E. coli, E. aerogenes, and C. freundii, respectively. In E. coli, five of its seven replacements were located in the beta-sheet portion of the protein, and all seven were located in a single restricted region of the protein. In E. aerogenes, all nine of its replacements were located within surface residues, with five clustered in a region topologically distinct from the E. coli cluster. The replaced side chains are sometimes in direct contact but more often are separated by an intervening side chain. It is argued that the temporal clustering of replacements is typical for the evolution of most proteins and that the associated topological clustering gives a picture of how evolutionary change is accommodated by protein structure.
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21. |
Go?ebiewski M,
Kern-Zdanowicz I,
Zienkiewicz M,
Adamczyk M,
Zylinska J,
Baraniak A,
Gniadkowski M,
Bardowski J,
Ceg?owski P,
( 2007 ) Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3. PMID : 17698626 : DOI : 10.1128/AAC.00457-07 PMC : PMC2151408 Abstract >>
Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.
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22. |
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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23. |
Naas T,
Aubert D,
Ozcan A,
Nordmann P,
( 2007 ) Chromosome-encoded narrow-spectrum Ambler class A beta-lactamase GIL-1 from Citrobacter gillenii. PMID : 17242148 : DOI : 10.1128/AAC.01152-06 PMC : PMC1855525 Abstract >>
A novel beta-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum beta-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A beta-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of beta-lactamases TEM-1 and SHV-1. The blaGIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C beta-lactamases was found upstream of the blaGIL-1 gene, which fits the noninducibility of beta-lactamase expression in C. gillenii. Rapid amplification of DNA 5' ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the d-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of beta-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.
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24. |
Barlow RS,
Gobius KS,
( 2006 ) Diverse class 2 integrons in bacteria from beef cattle sources. PMID : 17065187 : DOI : 10.1093/jac/dkl423 Abstract >>
The purpose of this study was to determine the diversity of class 2 integrons in bacteria isolated from beef cattle sources. The variable regions of a subset of 11 class 2 integron-containing bacteria were analysed by PCR and DNA sequencing for the presence of novel rearrangements. A total of six different class 2 integron arrays were identified and four of these were fully characterized. Three of the four arrays characterized have been previously described; however the remaining array is unlike previously described class 2 integrons. The novel class 2 integron was found in Providencia stuartii and contains an apparently functional class 2 integrase. Examination of the variable region of the P. stuartii integron identified nine open reading frames, mostly of unknown function, and represents the first report of a class 2 integron without inserted antibiotic resistance gene cassettes. This study has identified a novel class 2 integron found in P. stuartii that contains an apparently functional naturally occurring class 2 integrase. Further investigation of this novel class 2 integron is required to determine the impact of a functional class 2 integrase upon the evolution of class 2 integrons.
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25. |
Lee SG,
Hong SP,
Kim DY,
Song JJ,
Ro HS,
Sung MH,
( 2006 ) Inactivation of tyrosine phenol-lyase by Pictet-Spengler reaction and alleviation by T15A mutation on intertwined N-terminal arm. PMID : 17094783 : DOI : 10.1111/j.1742-4658.2006.05546.x Abstract >>
Citrobacter freundiil-tyrosine phenol-lyase (TPL) was inactivated by a Pictet-Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-L-alanine (L-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards L-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with L-DOPA was detected at 45 and 40 degrees C, respectively, which was 15 degrees C lower than when using L-tyrosine as the substrate. The half-lives at 45 degrees C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mM L-DOPA. On treatment with excess pyridoxal-5'-phosphate (PLP), the L-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet-Spengler condensation with L-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (K(m,PLP)) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 degrees C, implying that the loss of the cofactor with the Pictet-Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.
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26. |
Mamaeva DV,
Morozova EA,
Nikulin AD,
Revtovich SV,
Nikonov SV,
Garber MB,
Demidkina TV,
( 2005 ) Structure of Citrobacter freundii L-methionine gamma-lyase. PMID : 16511092 : DOI : 10.1107/S1744309105015447 PMC : PMC1952331 Abstract >>
L-Methionine gamma-lyase (MGL) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes gamma-elimination of L-methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9 A resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops.
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27. |
Manukhov IV,
Mamaeva DV,
Rastorguev SM,
Faleev NG,
Morozova EA,
Demidkina TV,
Zavilgelsky GB,
( 2005 ) A gene encoding L-methionine gamma-lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine gamma-lyase. PMID : 15901718 : DOI : 10.1128/JB.187.11.3889-3893.2005 PMC : PMC1112054 Abstract >>
Citrobacter freundii cells produce L-methionine gamma-lyase when grown on a medium containing L-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded L-methionine gamma-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3'-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.
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28. |
Gangoué-Piéboji J,
Bedenic B,
Koulla-Shiro S,
Randegger C,
Adiogo D,
Ngassam P,
Ndumbe P,
Hächler H,
( 2005 ) Extended-spectrum-beta-lactamase-producing Enterobacteriaceae in Yaounde, Cameroon. PMID : 16000447 : DOI : 10.1128/JCM.43.7.3273-3277.2005 PMC : PMC1169189 Abstract >>
Organisms producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Cameroon. A total of 259 Enterobacteriaceae strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these Enterobacteriaceae strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains-i.e., 6 Klebsiella pneumoniae, 4 Escherichia coli, 1 Citrobacter freundii, and 1 Enterobacter cloacae strain-was transferred to E. coli HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of beta-lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible E. coli strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The beta-lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.
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29. |
Handal T,
Olsen I,
Walker CB,
Caugant DA,
( 2005 ) Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis. PMID : 15621454 : DOI : 10.1016/j.femsle.2004.11.023 Abstract >>
Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.
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30. |
Antson AA,
Strokopytov BV,
Murshudov GN,
Isupov MN,
Harutyunyan EH,
Demidkina TV,
Vassylyev DG,
Dauter Z,
Terry H,
Wilson KS,
( 1992 ) The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5'-phosphate-dependent enzyme. PMID : 1601133 : DOI : 10.1016/0014-5793(92)80454-o Abstract >>
The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A and it contains two subunits of the tetrameric molecule in the asymmetric unit. Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanner. The structure has been solved at 2.7 A resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.
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31. |
Stoebel DM,
( 2005 ) Lack of evidence for horizontal transfer of the lac operon into Escherichia coli. PMID : 15563718 : DOI : 10.1093/molbev/msi056 Abstract >>
The idea that Escherichia coli gained the lac operon via horizontal transfer, allowing it to invade a new niche and form a new species, has become a paradigmatic example of bacterial nonpathogenic adaptation and speciation catalyzed by horizontal transfer. Surprisingly, empirical evidence for this event is essentially nonexistent. To see whether horizontal transfer occurred, I compared a phylogeny of 14 Enterobacteriaceae based on two housekeeping genes to a phylogeny of a part of their lac operon. Although several species in this clade appear to have acquired some or all of the operon via horizontal transfer, there is no evidence of horizontal transfer into E. coli. It is not clear whether the horizontal transfer events for which there is evidence were adaptive because those species which have acquired the operon are not thought to live in high lactose environments. I propose that vertical transmission from the common ancestor of the Enterobacteriaceae, with subsequent loss of these genes in many species can explain much of the patchy distribution of lactose use in this clade. Finally, I argue that we need new, well-supported examples of horizontal transfer spurring niche expansion and speciation, particularly in nonpathogenic cases, before we can accept claims that horizontal transfer is a hallmark of bacterial adaptation.
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32. |
Abdalhamid B,
Pitout JD,
Moland ES,
Hanson ND,
( 2004 ) Community-onset disease caused by Citrobacter freundii producing a novel CTX-M beta-lactamase, CTX-M-30, in Canada. PMID : 15504875 : DOI : 10.1128/AAC.48.11.4435-4437.2004 PMC : PMC525418 Abstract >>
Strains of Citrobacter freundii intermediate to cefotaxime but sensitive to ceftazidime were isolated from four different patients in Canada. Sequencing of PCR products by use of CTX-M-specific primers revealed a new combination of four amino acid substitutions. This new gene was designated bla(CTX-M-30) and was encoded on a 3-kb plasmid. The pI of CTX-M-30 was 8.0.
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33. |
Samuel G,
Hogbin JP,
Wang L,
Reeves PR,
( 2004 ) Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens. PMID : 15375135 : DOI : 10.1128/JB.186.19.6536-6543.2004 PMC : PMC516595 Abstract >>
Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E. coli O55 and S. enterica O50. The O-antigen gene cluster sequences for E. coli O157 and E. coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica O30 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coli O157 cluster. We also determined the sequence of the S. enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E. coli O55 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.
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34. |
Hess P,
Altenhöfer A,
Khan AS,
Daryab N,
Kim KS,
Hacker J,
Oelschlaeger TA,
( 2004 ) A Salmonella fim homologue in Citrobacter freundii mediates invasion in vitro and crossing of the blood-brain barrier in the rat pup model. PMID : 15322026 : DOI : 10.1128/IAI.72.9.5298-5307.2004 PMC : PMC517473 Abstract >>
From the invasive Citrobacter freundii strain 3009, an invasion determinant was cloned, sequenced, and expressed. Sequence analysis of the determinant showed high homology with the fim determinant from Salmonella enterica serovar Typhimurium. The genes of the invasion determinant directed invasion of recombinant Escherichia coli K-12 strains into human epithelial cell lines of the bladder and gut as well as mannose-sensitive yeast agglutination and were termed fim(Cf) genes. Expression of the Fim(Cf) proteins was shown by (35)S labeling and/or Western blotting. In the infant rat model of experimental hematogenous meningitis, C. freundii strain 3009 and the in vitro invasive recombinant E. coli K-12 strain harboring the fim(Cf) determinant reached the cerebrospinal fluid, in contrast to the case for the control strain. The fim determinant was also necessary for efficient in vitro invasion by C. freundii, because a deletion mutant was strongly reduced in its invasion efficiency. The mutation could be complemented in trans by the corresponding genes. Invasion by C. freundii could be blocked only by d-mannose, GlcNAc, and chitin hydrolysate and not by other carbohydrates tested. In contrast, yeast agglutination was not affected by GlcNAc or chitin hydrolysate. This finding indicated mannose residues to be essential for both yeast agglutination and invasion, whereas GlcNAc (oligomer) residues of host cells are involved exclusively in invasion. These results showed the fim determinant of C. freundii to be responsible for d-mannose- and GlcNAc-dependent in vitro invasion without being assembled into pili and for crossing of the blood-brain barrier in the infant rat model.
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35. |
Chen H,
Ponniah G,
Salonen N,
Blum P,
( 2004 ) Culture-independent analysis of fecal enterobacteria in environmental samples by single-cell mRNA profiling. PMID : 15294770 : DOI : 10.1128/AEM.70.8.4432-4439.2004 PMC : PMC492453 Abstract >>
A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.
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36. |
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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37. |
Sánchez-Moreno I,
García-García JF,
Bastida A,
García-Junceda E,
( 2004 ) Multienzyme system for dihydroxyacetone phosphate-dependent aldolase catalyzed C-C bond formation from dihydroxyacetone. PMID : 15263954 : DOI : 10.1039/b405220j Abstract >>
A multienzyme system composed by recombinant dihydroxyacetone kinase from Citrobacter freundii, fuculose-1-phosphate aldolase and acetate kinase, allows a practical one-pot C-C bond formation catalysed by dihydroxyacetone phosphate-dependent aldolases from dihydroxyacetone and an aldehyde.
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38. |
Son MS,
Del Castilho C,
Duncalf KA,
Carney D,
Weiner JH,
Turner RJ,
( 2003 ) Mutagenesis of SugE, a small multidrug resistance protein. PMID : 14651958 : DOI : 10.1016/j.bbrc.2003.11.018 Abstract >>
The small multidrug resistance protein family has two subclasses. In this study we used a mutation approach to see what is necessary to convert a SUG subgroup member into a quaternary ammonium compound (QAC) transporter. We chose four key residues (H24, M39, I43, and A44) conserved within SUGs but conserved differently within the QAC transporters. Altogether, seven mutants were generated in Citrobacter freundii SugE. Surprisingly, the mutated SugE demonstrated an increased sensitivity to representative QACs. Additionally, ethidium uptake is found to be more prominent in the hypersensitive mutants. We conducted orientation studies using topology reporter gene fusions which indicated that SugE and the QAC transporter EmrE both have their N- and C-termini in the cytoplasm as predicted. The results imply that SugE can be converted to a QAC transporter with only a single mutation. However, because hypersensitivity was observed, the SugE mutant proteins are behaving as importers rather than as exporters.
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39. |
Wertz JE,
Goldstone C,
Gordon DM,
Riley MA,
( 2003 ) A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. PMID : 14640415 : Abstract >>
A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.
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40. |
Arduino SM,
Catalano M,
Orman BE,
Roy PH,
Centrón D,
( 2003 ) Molecular epidemiology of orf513-bearing class 1 integrons in multiresistant clinical isolates from Argentinean hospitals. PMID : 14638506 : DOI : 10.1128/aac.47.12.3945-3949.2003 PMC : PMC296183 Abstract >>
The spread of orf513-bearing class 1 integrons is associated with bla(CTX-M-2) in gram-negative clinical isolates in Argentina, with In35 being the most frequently found integron (74%). Among 65 isolates without bla(CTX-M-2), only one harbored a novel orf513-bearing class 1 integron with the dfrA3b gene. The finding of orf513 not associated with class 1 integrons in two gram-positive strains indicates the widespread occurrence of this putative site-specific recombinase.
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41. |
Jeong HS,
Bae IK,
Shin JH,
Kim SH,
Chang CL,
Jeong J,
Kim S,
Lee CH,
Ryoo NH,
Lee JN,
( 2011 ) Fecal colonization of Enterobacteriaceae carrying plasmid-mediated quinolone resistance determinants in Korea. PMID : 21830908 : DOI : 10.1089/mdr.2011.0040 Abstract >>
The aims of the current study were to investigate the prevalence and molecular characteristics of plasmid-mediated quinolone resistance (PMQR) genes from colonizing fecal organisms and to compare the incidence and subtype of these genes according to bacterial species and hospital at five tertiary-care hospitals in Korea. A total of 500 nonduplicated clinical isolates of Enterobacteriaceae were obtained from fecal specimens at five tertiary-care hospitals between March and May 2008. The PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) were amplified by PCR and confirmed by direct sequencing of the PCR products. A total of 83 (16.6%) qnr-positive isolates were detected. The prevalence rates of qnrA, qnrB, and qnrS were 1.4%, 13.6%, and 1.6%, respectively. The species distributions of qnrB-positive isolates were Klebsiella pneumoniae (37/109; 33.9%), Citrobacter freundii (10/34; 29.4%), Citrobacter braakii (8/13; 61.5%), and Escherichia coli (8/275; 2.9%). Sixteen subtypes of qnrB were detected, including seven novel variants. The prevalences of aac(6')-Ib-cr and qepA were 15.6% (n=78) and 0.6% (n=3), respectively. The aac(6')-Ib-cr gene was detected in 39 (47.0%) of 83 qnr-positive isolates and 39 (9.4%) of 417 qnr-negative isolates There was one qepA variant containing a novel mutation (Ala231Val). The prevalence of PMQR genes was high in Enterobacteriaceae from stool specimens in Korea, and there was a close relation between qnr and aac(6')-Ib-cr.
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42. |
Jacoby GA,
Griffin CM,
Hooper DC,
( 2011 ) Citrobacter spp. as a source of qnrB Alleles. PMID : 21844311 : DOI : 10.1128/AAC.05187-11 PMC : PMC3195048 Abstract >>
qnrB is the most common of the five qnr families and has the greatest number of allelic variants. Almost two-thirds of the qnrB alleles have been reported in Citrobacter spp., and several were shown to be located on the chromosome. In this study, PCR was used to investigate the prevalence of plasmid-mediated quinolone resistance genes in 71 clinical isolates belonging to the Citrobacter freundii complex. Thirty-seven percent contained qnrB alleles, including 7 (qnrB32 to qnrB38) that were novel and 1 pseudogene, while none contained qnrA, qnrC, qnrD, qnrS, or aac(6')-Ib-cr. When the strains were arrayed by related 16S rRNA sequence and further separated into subspecies by biochemical criteria, clustering of qnrB-positive strains was evident. In only two strains with qnrB2 and qnrB4 was quinolone resistance transferable by conjugation, and only these strains contained the ISCR1 sequence that is often associated with qnrB on plasmids. Five of 26 qnrB-positive strains contained integrase genes, but these included the strains with qnrB2 and qnrB4 as well as two strains with other transmissible plasmids. In a fully sequenced genome of Citrobacter youngae, a member of the C. freundii complex, another novel qnrB allele, qnrB39, occurs in a sequence of genes that is 90% identical to sequence surrounding integron-associated qnrB4 incorporated into plasmids. The chromosome of Citrobacter is the likely source of plasmid-mediated qnrB.
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43. |
Gomez SA,
Pasteran FG,
Faccone D,
Tijet N,
Rapoport M,
Lucero C,
Lastovetska O,
Albornoz E,
Galas M,
N/A N/A,
Melano RG,
Corso A,
Petroni A,
( 2011 ) Clonal dissemination of Klebsiella pneumoniae ST258 harbouring KPC-2 in Argentina. PMID : 21851480 : DOI : 10.1111/j.1469-0691.2011.03600.x Abstract >>
The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla(KPC-2) was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla(KPC-2) in an unusual genetic environment flanked by ISKpn8-�Gbla(TEM-1) and ISKpn6-like. bla(KPC-2) was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla(KPC-2) in Tn4401a.
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44. |
Shao Y,
Xiong Z,
Li X,
Hu L,
Shen J,
Li T,
Hu F,
Chen S,
( 2011 ) Prevalence of plasmid-mediated quinolone resistance determinants in Citrobacter freundii isolates from Anhui province, PR China. PMID : 21816943 : DOI : 10.1099/jmm.0.034082-0 Abstract >>
This study was conducted to detect and analyse the presence of plasmid-mediated quinolone resistance (PMQR) determinants [qnr, aac(6')-Ib-cr and qepA] among Citrobacter freundii isolates from patients in Anhui province, PR China. During 2009-2010, 31 C. freundii strains were collected from various hospital units and patient specimens. Using PCR, qnr genes were detected in eight isolates, but aac(6')-Ib-cr and qepA genes were not found. The genes qnrA1, qnrB1, qnrB2, qnrB4, qnrB10 and qnrB24 were present in 6.5, 3.2, 6.5, 3.2, 3.2 and 3.2% of C. freundii isolates, respectively. A new subgene of qnrB variant (qnrB24) was found and identified for what we believe to be the first time. PFGE after XbaI digestion of genomic DNA indicated that qnr-positive strains were not clonally related. Conjugation experiments were conducted to determine whether the qnr-carrying plasmids were self-transferable, and plasmids of transconjugants were extracted and analysed. The qnr genes were transferred from three clinical isolates to their transconjugants. Two qnrA1 genes transferred quinolone resistance with a plasmid of ~11 kb, whilst the size of the plasmid carrying the qnrB4 gene was ~64 kb. The susceptibility of positive isolates and transconjugants was tested using an agar dilution method according to Clinical and Laboratory Standards Institute guidelines, and the MICs of ciprofloxacin and levofloxacin were determined using Etest strips. Most isolates with qnr genes were resistant to fluoroquinolones and other antimicrobial agents. The MICs of transconjugants showed reduced susceptibility to fluoroquinolones.
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45. |
Pang A,
Warren MJ,
Pickersgill RW,
( 2011 ) Structure of PduT, a trimeric bacterial microcompartment protein with a 4Fe-4S cluster-binding site. PMID : 21245529 : DOI : 10.1107/S0907444910050201 Abstract >>
Propanediol metabolism in Citrobacter freundii occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. The propanediol-utilization (Pdu) microcompartment shell is constructed from thousands of hexagonal-shaped protein complexes made from seven different types of protein subunit. Here, the structure of the bacterial microcompartment protein PduT, which has a tandem structural repeat within the subunit and forms trimers with pseudo-hexagonal symmetry, is reported. This trimeric assembly forms a flat approximately hexagonally shaped disc with a central pore that is suitable for a 4Fe-4S cluster. The essentially cubic shaped 4Fe-4S cluster conforms to the threefold symmetry of the trimer with one free iron, the role of which could be to supply electrons to an associated microcompartment enzyme, PduS.
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46. |
Selvakumaran S,
Kapley A,
Kashyap SM,
Daginawala HF,
Kalia VC,
Purohit HJ,
( 2011 ) Diversity of aromatic ring-hydroxylating dioxygenase gene in Citrobacter. PMID : 21295975 : DOI : 10.1016/j.biortech.2011.01.011 Abstract >>
Genetic and functional diversity of Citrobacter spp. for their abilities to degrade aromatic compounds was evaluated to develop mixed cultures or a consortium for bioremediation technology. Thirty Citrobacter strains isolated from various effluent treatment plants were found to degrade a range of aromatic compounds: phenol, benzoate, hydroxy benzoic acid and biotransform mono-chlorophenols and di-chlorophenol within 24 to 48 h of incubation at 30 �XC. Sequence similarity and phylogeny of the ARHD gene transcripts (730 nucleotides) depicted their diversity within 9 Citrobacter strains: HPC255, HPC369, HPC560, HPC570, HPC784, HPC1196, HPC1216, HPC1276 and HPC1299. Here, the degree of associations varied up to 84% with (i) ARHD �\-sub unit (SU), (ii) LSU of Phenylpropionate dioxygenase (PDO), (iii) Phenol hydroxylase �\-SU, (iv) Benzoate 1,2-dioxygenase, �\-SU, (v) Naphthalene dioxygenase LSU, etc. This study has provided basic information, which can be used to develop a consortium of bacteria with mutually beneficial characteristics.
|
47. |
Ronda L,
Bazhulina NP,
Morozova EA,
Revtovich SV,
Chekhov VO,
Nikulin AD,
Demidkina TV,
Mozzarelli A,
( 2011 ) Exploring methionine �^-lyase structure-function relationship via microspectrophotometry and X-ray crystallography. PMID : 20601224 : DOI : 10.1016/j.bbapap.2010.06.017 Abstract >>
Pyridoxal 5'-phosphate (PLP) dependent methionine �^-lyase catalyzes the breakdown of L-methionine to �\-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65? resolution with R(free) 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
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48. |
Tijet N,
Andres P,
Chung C,
Lucero C,
N/A N/A,
Low DE,
Galas M,
Corso A,
Petroni A,
Melano RG,
( 2011 ) rmtD2, a new allele of a 16S rRNA methylase gene, has been present in Enterobacteriaceae isolates from Argentina for more than a decade. PMID : 21078935 : DOI : 10.1128/AAC.00962-10 PMC : PMC3028771 Abstract >>
The first allele of a 16S rRNA methyltransferase gene, rmtD2, conferring very high resistance to all clinically available aminoglycosides, was detected in 7/1,064 enterobacteria collected in 2007. rmtD2 was located on a conjugative plasmid in a Tn2670-like element inside a structure similar to that of rmtD1 but probably having an independent assembly. rmtD2 has been found since 1996 to 1998 mainly in Enterobacter and Citrobacter isolates, suggesting a possible reservoir in these genera. This presumption deserves monitoring by continuous surveillance.
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49. |
Ong CL,
Beatson SA,
Totsika M,
Forestier C,
McEwan AG,
Schembri MA,
( 2010 ) Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species. PMID : 20576143 : DOI : 10.1186/1471-2180-10-183 PMC : PMC2900259 Abstract >>
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.
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50. |
Balcewich MD,
Reeve TM,
Orlikow EA,
Donald LJ,
Vocadlo DJ,
Mark BL,
( 2010 ) Crystal structure of the AmpR effector binding domain provides insight into the molecular regulation of inducible ampc beta-lactamase. PMID : 20594961 : DOI : 10.1016/j.jmb.2010.05.040 DOI : 10.1016/j.jmb.2010.05.040 Abstract >>
Hyperproduction of AmpC beta-lactamase (AmpC) is a formidable mechanism of resistance to penicillins and cephalosporins in Gram-negative bacteria such as Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is regulated by the LysR-type transcriptional regulator AmpR. ampR and ampC genes form a divergent operon with overlapping promoters to which AmpR binds and regulates the transcription of both genes. AmpR induces ampC by binding to one member of the family of 1,6-anhydro-N-acetylmuramyl peptides, which are cytosolic catabolites of peptidoglycan that accumulate during beta-lactam challenge. To gain structural insights into AmpR regulation, we determined the crystal structure of the effector binding domain (EBD) of AmpR from Citrobacter freundii up to 1.83 A resolution. The AmpR EBD is dimeric and each monomer comprises two subdomains that adopt alpha/beta Rossmann-like folds. Located between the monomer subdomains is a pocket that was found to bind the crystallization buffer molecule 2-(N-morpholino)ethanesulfonic acid. The pocket, together with a groove along the surface of subdomain I, forms a putative effector binding site into which a molecule of 1,6-anhydro-N-acetylmuramyl pentapeptide could be modeled. Amino acid substitutions at the base of the interdomain pocket either were found to render AmpR incapable of inducing ampC (Thr103Val, Ser221Ala and Tyr264Phe) or resulted in constitutive ampC expression (Gly102Glu). While the substitutions that prevented ampC induction did not alter the overall AmpR EBD structure, circular dichroism spectroscopy revealed that the nonconservative Gly102Glu mutation affected EBD secondary structure, confirming previous work suggesting that Gly102Glu induces a conformational change to result in constitutive AmpC production.
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51. |
Ferreira S,
Paradela A,
Velez J,
Ramalheira E,
Walsh TR,
Mendo S,
( 2010 ) Carriage of qnrA1 and qnrB2, blaCTX-M15, and complex class 1 integron in a clinical multiresistant Citrobacter freundii isolate. PMID : 20338709 : DOI : 10.1016/j.diagmicrobio.2010.01.003 Abstract >>
A multiresistant Citrobacter freundii strain was recovered from a catheter from a patient hospitalized in Aveiro, Portugal. This strain harbored quinolone resistance genes, qnrA1 and qnrB2, both in a large plasmid.
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52. |
Bae IK,
Park I,
Lee JJ,
Sun HI,
Park KS,
Lee JE,
Ahn JH,
Lee SH,
Woo GJ,
( 2010 ) Novel variants of the qnrB gene, qnrB22 and qnrB23, in Citrobacter werkmanii and Citrobacter freundii. PMID : 20421404 : DOI : 10.1128/AAC.01339-09 PMC : PMC2897306 Abstract >>
N/A
|
53. |
Jansson L,
Angström J,
Lebens M,
Imberty A,
Varrot A,
Teneberg S,
( 2010 ) Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii. PMID : 20171259 : DOI : 10.1016/j.biochi.2010.02.010 Abstract >>
Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB(5) toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galbeta3GalNAcbeta4(NeuAcalpha3)Galbeta4GlcbetaCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galbeta4GlcNAcbeta-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcalpha3(Fucalpha2)Galbeta4GlcNAcbeta-)terminated glycoconjugates. A B-subunit with 73% sequence identity to the B-subunits of cholera toxin and the heat-labile toxin of E. coli is produced by certain strains of enteropathogenic E. coli and by Citrobacter freundii. This C. freundii B-subunit (CFXB) has now been expressed in V. cholerae, and isolated in high yields. Glycosphingolipid binding studies show that CFXB binds to the GM1 ganglioside with high affinity. In addition, CFXB has high affinity for both neolacto-terminated and blood group A type 2-terminated glycoconjugates. The crystal structure of the pentameric arrangement of C. freundii B-subunits display high structural similarity with related proteins from E. coli and V. cholerae and oligosaccharide binding sites can be identified on the protein surface. Small changes in the 88-95 loop connecting the GM1 and blood group A binding sites explains the minor changes in affinity seen for these two ligands. However, the enhanced affinity of CFXB for neolacto-terminated structures can be sought in the Lys34Tyr substitution affording additional hydrogen bond interactions between the tyrosyl side chain and the GlcNAcbeta3Galb4Glcbeta1 segment of neolactotetraosylceramide via bridging water molecules.
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54. |
Pan J,
Hu L,
Yu F,
Chen C,
Zhang X,
( 2010 ) Coexistence of multiple antimicrobial-resistance genes in a carbapenem-resistant Citrobacter freundii clinical isolate from China. PMID : 20110384 : DOI : 10.1099/jmm.0.016287-0 Abstract >>
N/A
|
55. |
Lawrence JG,
Ochman H,
Hartl DL,
( 1991 ) Molecular and evolutionary relationships among enteric bacteria. PMID : 1955870 : DOI : 10.1099/00221287-137-8-1911 Abstract >>
Classification of bacterial species into genera has traditionally relied upon variation in phenotypic characteristics. However, these phenotypes often have a multifactorial genetic basis, making unambiguous taxonomic placement of new species difficult. By designing evolutionarily conserved oligonucleotide primers, it is possible to amplify homologous regions of genes in diverse taxa using the polymerase chain reaction and determine their nucleotide sequences. We have constructed a phylogeny of some enteric bacteria, including five species classified as members of the genus Escherichia, based on nucleotide sequence variation at the loci encoding glyceraldehyde-3-phosphate dehydrogenase and outer membrane protein 3A, and compared this genealogy with the relationships inferred by biotyping. The DNA sequences of these genes defined congruent and robust phylogenetic trees indicating that they are an accurate reflection of the evolutionary history of the bacterial species. The five species of Escherichia were found to be distantly related and, contrary to their placement in the same genus, do not form a monophyletic group. These data provide a framework which allows the relationships of additional species of enteric bacteria to be inferred. These procedures have general applicability for analysis of the classification, evolution, and epidemiology of bacterial taxa.
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56. |
Yang Q,
Wang H,
Sun H,
Chen H,
Xu Y,
Chen M,
( 2010 ) Phenotypic and genotypic characterization of Enterobacteriaceae with decreased susceptibility to carbapenems: results from large hospital-based surveillance studies in China. PMID : 19805565 : DOI : 10.1128/AAC.01099-09 PMC : PMC2798477 Abstract >>
The resistance mechanism of 49 Enterobacteriaceae isolates with decreased susceptibility to carbapenems collected from 2004 to 2008 at 16 teaching hospitals in China was investigated. Moderate- to high-level carbapenem resistance in most isolates was more closely associated with loss or decreased expression of both major porins combined with production of AmpC or extended-spectrum beta-lactamase enzymes, while KPC-2, IMP-4, and IMP-8 carbapenemase production may lead to a low to moderate level of carbapenem resistance in Enterobacteriaceae in China.
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57. |
Bartowsky E,
Normark S,
( 1991 ) Purification and mutant analysis of Citrobacter freundii AmpR, the regulator for chromosomal AmpC beta-lactamase. PMID : 1943705 : DOI : 10.1111/j.1365-2958.1991.tb01920.x Abstract >>
AmpR, the transcriptional regulator for the Citrobacter freundii ampC beta-lactamase gene, was purified. The purified AmpR had DNA-binding activity, the same molecular mass (32 kDa) on sodium dodecyl sulphate/polyacrylamide gel electrophoresis as previously described, and N-terminal sequencing of the first 15 amino acids was in agreement with that predicted from the nucleotide sequence. Two mutants were isolated that abolish DNA-binding and beta-lactamase induction and which map in the amino- and carboxyl-terminal ends of AmpR, respectively. The mutation in the amino terminus (S35F) was located in a helix-turn-helix region showing high homology to other members of the LysR regulator family. Therefore this mutation may directly abolish the contact between AmpR and its operator sequence. It is suggested that the C-terminal mutation (Y264N) affects subunit interactions in AmpR. One constitutive mutant was isolated which mapped in the centre of the ampR gene. This G102E mutant leads to constitutive beta-lactamase expression in the absence of both beta-lactam inducer and ampG, a gene essential for induction in wild-type enterobacteria. Another mutant protein, D135Y, showed wild-type properties in an ampG+ and an ampG::kan background, but could, unlike wild-type AmpR, activate the ampC gene in an ampG1 mutant background. It is thought that ampG1 is a missense mutant. These two types of ampR mutants suggest that activation of ampC transcription is dependent on the conversion of AmpR into a transcriptional activator and that this activation may normally involve interactions with AmpG.
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58. |
Tsukamoto K,
Tachibana K,
Yamazaki N,
Ishii Y,
Ujiie K,
Nishida N,
Sawai T,
( 1990 ) Role of lysine-67 in the active site of class C beta-lactamase from Citrobacter freundii GN346. PMID : 1969344 : DOI : 10.1111/j.1432-1033.1990.tb15365.x Abstract >>
Citrobacter freundii GN346 produces a class C beta-lactamase exhibiting the substrate profile of a typical cephalosporinase. The structural and promoter regions of the cephalosporinase gene, comprising 1408 nucleotides, were completely sequenced. The amino acid sequence of the mature enzyme, comprising 361 amino acids, and its molecular mass, 39,878 Da, were determined. The active site was confirmed to be Ser-64. The amino acid sequence of the enzyme differs from that of the cephalosporinase of C. freundii OS60 by nine residues. The nucleotide sequence of the promoter region suggests a possible attenuator structure. Lys-67, one of the most conserved residues found in class A and C beta-lactamases and penicillin-binding proteins, was converted into arginine, threonine or glutamic acid through site-directed mutagenesis. The Glu-67 enzyme had lost the catalytic activity and the Thr-67 enzyme only showed a trace of activity. The Arg-67 enzyme, which retained a significant amount of the activity, was purified. The Km values of the Arg-67 enzyme for cephalothin, cephaloridine and benzylpenicillin are 13-19 times those of the wild-type enzyme; the kcat values for the three substrates are 37%, 3%, and 36% those of the wild-type enzyme, respectively.
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59. |
Kuhnert P,
Korczak BM,
Stephan R,
Joosten H,
Iversen C,
( 2009 ) Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA). PMID : 19321218 : DOI : 10.1016/j.ijfoodmicro.2009.02.022 Abstract >>
Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.
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60. |
Liu W,
Chen L,
Li H,
Duan H,
Zhang Y,
Liang X,
Li X,
Zou M,
Xu L,
Hawkey PM,
( 2009 ) Novel CTX-M {beta}-lactamase genotype distribution and spread into multiple species of Enterobacteriaceae in Changsha, Southern China. PMID : 19297379 : DOI : 10.1093/jac/dkp068 Abstract >>
The aim of this study was to undertake a survey of the occurrence of CTX-M and SHV extended-spectrum beta-lactamase (ESBL) genotypes in Enterobacteriaceae from Hunan Province, China. Clinical isolates (425) from three major hospitals in Changsha, Hunan Province, were collected between October 2004 and July 2005, and their antimicrobial susceptibilities of the genotype of bla(CTX-M) and bla(SHV) were determined. Random amplified polymorphic DNA was used to characterize the clonality of all of the isolates. The overall rate of ESBL-positive isolates was 33.4% (142/425). The dominant ESBLs were CTX-M types, and were found in 109/142 (76.8%) isolates comprising seven different genera/species, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris and Providencia stuartii. The most common bla(CTX-M) genotypes were bla(CTX-M-14) (47.7%), bla(CTX-M-3) (29.4%) and bla(CTX-M-15) (17.4%). A novel gene derived from bla(CTX-M-15), bla(CTX-M-82) (Ala-40-->Pro), was identified. The dominant ESBL genotype in Hunan Province was bla(CTX-M). The high prevalence (17.4%) of bla(CTX-M-15) has not previously been reported from China. Our results identify that an epidemic of bla(CTX-M) in Changsha, Hunan Province, has evolved with the appearance and spread of bla(CTX-M-15) against the dominant genotypes bla(CTX-M-14) and bla(CTX-M-3.) The worldwide dominance of bla(CTX-M-15) could be poised to spread to China, displacing the current prevailing genotypes.
|
61. |
Hammad AM,
Ishida Y,
Shimamoto T,
( 2009 ) Prevalence and molecular characterization of ampicillin-resistant Enterobacteriaceae isolated from traditional Egyptian Domiati cheese. PMID : 19343954 : DOI : 10.4315/0362-028x-72.3.624 Abstract >>
The aim of this study was to address the prevalence and the molecular characteristics of antibiotic-resistant enteric bacteria isolated from one of the most popular types of Egyptian cheese. A total of 215 ampicillin-resistant enterobacterial isolates were obtained from 80 samples of Domiati cheese, and they were screened by PCR for a large pool of antibiotic resistance markers, including extended-spectrum beta-lactamases (ESBLs), class 1 and class 2 integrons, and plasmid-mediated quinolone resistance genes. It was determined that the most frequent mechanism of ampicillin resistance was from a TEM-1-type beta-lactamase. As well, SHV beta-lactamases, including SHV-1, SHV-25, and SHV-26, showed a high prevalence, and two novel SHV beta-lactamases, SHV-110 and SHV-111, were identified. Type CTX-M-14, OXY-1, OXA-1, and CMY-4 beta-lactamases were also detected in a few isolates. In addition, a novel AmpC beta-lactamase was detected that was designated CMY-41. Sequencing results of class 1 integrons revealed that the uncommon aminoglycoside resistance gene cassette aadA22 was found for the first time in an Escherichia coli strain. The other class 1 integrons harbored various common gene cassettes, including aadA1, aadA1a, aadA2, aadA12, dfr5, dfr7, dfr12, and dfr15. The only isolate that carried a class 2 integron contained dfrA1, sat2, and aadA1. Plasmid-mediated quinolone resistance determinants qnrS and qnrB showed a low prevalence. This study provides meaningful data on high antimicrobial resistance contained in Domiati cheese samples and reports for the first time the presence of beta-lactamases, plasmid-mediated quinolone resistance, and integrons in isolates from food of Egyptian animal origin.
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62. |
Pallecchi L,
Riccobono E,
Mantella A,
Bartalesi F,
Sennati S,
Gamboa H,
Gotuzzo E,
Bartoloni A,
Rossolini GM,
( 2009 ) High prevalence of qnr genes in commensal enterobacteria from healthy children in Peru and Bolivia. PMID : 19364872 : DOI : 10.1128/AAC.01722-08 PMC : PMC2687207 Abstract >>
A remarkable prevalence of qnrB (54%) and, at a lower level, of qnrS (14%) was discovered in pools of commensal enterobacteria from 310 healthy children living in Peru and Bolivia, using a metagenomic approach. Analysis of randomly selected enterobacterial pools revealed that qnrB was mainly carried by Escherichia coli and qnrS by Klebsiella pneumoniae. Investigation of 11 qnrB-positive isolates and 9 qnrS-positive isolates revealed the presence of plasmid-borne qnrB19 (n = 8), qnrB2 (n = 2), qnrB10 (n = 1), and qnrS1 (n = 9) genes.
|
63. |
Ahmed AM,
Shimamoto T,
( 2008 ) Emergence of a cefepime- and cefpirome-resistant Citrobacter freundii clinical isolate harbouring a novel chromosomally encoded AmpC beta-lactamase, CMY-37. PMID : 18619820 : DOI : 10.1016/j.ijantimicag.2008.04.019 Abstract >>
Citrobacter freundii strain 4306 was isolated from a urine specimen of a patient in March 2006 in Palestine. This strain showed a unique multidrug resistance phenotype, as it was resistant both to 7-alpha-methoxy- and oxyimino-cephalosporins, including cefepime, cefpirome and monobactams, in addition to quinolones, streptomycin and trimethoprim/sulfamethoxazole. Clavulanic acid did not act synergistically with cephalosporins by the double-disk synergy test. Molecular characterisation showed that the resistance to 7-alpha-methoxy- and oxyimino-cephalosporins was due to a novel AmpC beta-lactamase, designated CMY-37, with an isoelectric point of approximately 9.0. CMY-37 is a variant of C. freundii chromosomal AmpC enzymes with at least seven amino acid substitutions. One of these substitutions, L316I, is located within the R2 loop that is considered the hotspot region responsible for the extended substrate spectrum in class C beta-lactamases. The blaCMY-37 gene was cloned and expressed in Escherichia coli TG1. CMY-37 is chromosomally encoded and is not associated with ISEcp1-like element. Phylogenetic analysis suggested that CMY-37 is the origin of many plasmid-mediated AmpC beta-lactamases. This study highlights the emergence of cefepime and cefpirome resistance in C. freundii owing to a new type of AmpC beta-lactamase.
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64. |
Pasteran FG,
Otaegui L,
Guerriero L,
Radice G,
Maggiora R,
Rapoport M,
Faccone D,
Di Martino A,
Galas M,
( 2008 ) Klebsiella pneumoniae Carbapenemase-2, Buenos Aires, Argentina. PMID : 18598660 : DOI : 10.3201/eid1407.070826 PMC : PMC2600346 Abstract >>
N/A
|
65. |
Tamang MD,
Seol SY,
Oh JY,
Kang HY,
Lee JC,
Lee YC,
Cho DT,
Kim J,
( 2008 ) Plasmid-mediated quinolone resistance determinants qnrA, qnrB, and qnrS among clinical isolates of Enterobacteriaceae in a Korean hospital. PMID : 18725444 : DOI : 10.1128/AAC.01633-07 PMC : PMC2573118 Abstract >>
Screening of 368 consecutive nonreplicate clinical isolates of Enterobacteriaceae resistant to nalidixic acid and at least one extended-spectrum beta-lactam revealed the presence of qnrA, qnrB, and qnrS determinants, and identified novel qnrB variants, in Citrobacter freundii isolates. This study also revealed, for the first time, the linkage of qnrB, armA, and extended-spectrum and/or AmpC-type beta-lactamase genes on large conjugative plasmids.
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66. |
Sekiguchi J,
Morita K,
Kitao T,
Watanabe N,
Okazaki M,
Miyoshi-Akiyama T,
Kanamori M,
Kirikae T,
( 2008 ) KHM-1, a novel plasmid-mediated metallo-beta-lactamase from a Citrobacter freundii clinical isolate. PMID : 18765691 : DOI : 10.1128/AAC.01337-07 PMC : PMC2573105 Abstract >>
A novel gene, bla(KHM-1), encoding a metallo-beta-lactamase, KHM-1, was cloned from a clinical isolate of Citrobacter freundii resistant to most beta-lactam antibiotics. Escherichia coli expressing bla(KHM-1) was resistant to all broad-spectrum beta-lactams except for monobactams and showed reduced susceptibility to carbapenems. Recombinant KHM-1 exhibited EDTA-inhibitable hydrolytic activity against most beta-lactams, with an overall preference for cephalosporins.
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67. |
Parsons JB,
Dinesh SD,
Deery E,
Leech HK,
Brindley AA,
Heldt D,
Frank S,
Smales CM,
Lünsdorf H,
Rambach A,
Gass MH,
Bleloch A,
McClean KJ,
Munro AW,
Rigby SE,
Warren MJ,
Prentice MB,
( 2008 ) Biochemical and structural insights into bacterial organelle form and biogenesis. PMID : 18332146 : DOI : 10.1074/jbc.M709214200 Abstract >>
Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.
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68. |
Zhang R,
Yang L,
Cai JC,
Zhou HW,
Chen GX,
( 2008 ) High-level carbapenem resistance in a Citrobacter freundii clinical isolate is due to a combination of KPC-2 production and decreased porin expression. PMID : 18287296 : DOI : 10.1099/jmm.0.47576-0 Abstract >>
An imipenem-resistant isolate of Citrobacter freundii ZJ163 (MIC 256 microg ml(-1)) isolated from a Chinese hospital was investigated. The C. freundii ZJ163 isolate exhibited high-level resistance to carbapenems, penicillins, cephalosporins, cefoxitin, aztreonam, quinolones and aminoglycosides. Isoelectric focusing (IEF) demonstrated three beta-lactamases with pIs of 5.4 (TEM-1), 6.7 (KPC-2) and 7.9 (CTX-M-14). Two different transconjugants (types A and B) were obtained by conjugation studies. The type A transconjugant exhibited reduced susceptibility or resistance to penicillins, cephalosporins and aztreonam, but was susceptible to carbapenems, quinolones and aminoglycosides. The antimicrobial susceptibility patterns of the type B transconjugant were similar to that of type A, except for its significantly reduced carbapenem susceptibility (imipenem MIC 2 microg ml(-1)). IEF, specific PCRs and DNA sequence analysis indicated that the type A transconjugant produced CTX-M-14 beta-lactamase with a pI of 7.9, that the type B transconjugant produced KPC-2 beta-lactamase with a pI of 6.7 and that the beta-lactamase with a pI of 5.4 was TEM-1. PCR analysis and sequencing confirmed the presence of the ampC gene in the chromosomal DNA from C. freundii ZJ163, although no activity of AmpC beta-lactamase was detected by IEF. Urea/SDS-PAGE analysis of outer-membrane proteins revealed that the levels of the 41 and 38 kDa porins were decreased in C. freundii ZJ163. It was concluded that production of KPC-2 combined with decreased expression of porins contributes to high-level resistance to carbapenems in C. freundii ZJ163.
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69. |
Nikulin A,
Revtovich S,
Morozova E,
Nevskaya N,
Nikonov S,
Garber M,
Demidkina T,
( 2008 ) High-resolution structure of methionine gamma-lyase from Citrobacter freundii. PMID : 18219122 : DOI : 10.1107/S0907444907065390 Abstract >>
Pyridoxal 5'-phosphate-dependent methionine gamma-lyase (MGL) is involved in the metabolism of sulfur-containing amino acids. The enzyme is a promising target in some anaerobic pathogens and is effective in cancer-cell treatment. The structure of the MGL holoenzyme from Citrobacter freundii has previously been determined at 1.9 A resolution. By modification of the crystallization procedure, the previously determined structure of C. freundii MGL has been improved to 1.35 A resolution with R and R(free) values of 0.152 and 0.177, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers in detail and to reveal the structurally invariant regions that are responsible for monomer-monomer recognition during the formation of the active enzyme. Details of the mode of cofactor binding and of the flexible regions that may be involved in substrate recognition and binding are also described.
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70. |
Gasiunas G,
Sasnauskas G,
Tamulaitis G,
Urbanke C,
Razaniene D,
Siksnys V,
( 2008 ) Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family. PMID : 18086711 : DOI : 10.1093/nar/gkm1090 PMC : PMC2241918 Abstract >>
The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5'-CCGC/GG-3' ('/' indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg2+, Mn2+, Co2+, Zn2+, Ni2+, Cu2+ and Ca2+. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.
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71. |
Ong CL,
Ulett GC,
Mabbett AN,
Beatson SA,
Webb RI,
Monaghan W,
Nimmo GR,
Looke DF,
McEwan AG,
Schembri MA,
( 2008 ) Identification of type 3 fimbriae in uropathogenic Escherichia coli reveals a role in biofilm formation. PMID : 18055599 : DOI : 10.1128/JB.01523-07 PMC : PMC2223576 Abstract >>
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens.
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72. |
Sánchez-Céspedes J,
Vila J,
( 2007 ) Partial characterisation of the acrAB locus in two Citrobacter freundii clinical isolates. PMID : 17631985 : DOI : 10.1016/j.ijantimicag.2007.05.010 Abstract >>
We studied the mechanisms of resistance to fluoroquinolones in two Citrobacter freundii strains (1.44 and 1.38) isolated from the same patient and belonging to the same clone by pulsed-field gel electrophoresis. This study allowed partial characterisation of the acrA and acrB genes of this microorganism. As previously reported, the two strains showed the same substitutions in the GyrA and ParC proteins (Thr-83-->Ile and Asp-87-->Tyr in GyrA and Ser-83-->Ile in ParC). However, differences were observed in the amount of ciprofloxacin accumulated, with strain 1.38 showing less accumulation. Expression of genes in both strains was analysed using DNA microarrays for Escherichia coli. Ten genes were overexpressed in strain 1.38 compared with strain 1.44, including genes acrA and acrB. Nucleotide similarity between the partially sequenced acrA and acrB genes of C. freundii and E. coli was 80.7% and 85%, respectively. The acrA and acrB genes of C. freundii are similar to those described in E. coli and their overexpression may play an important role in modulating the final minimum inhibitory concentration of fluoroquinolones in collaboration with mutations in the gyrA and parC genes.
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73. |
Quiroga MP,
Andres P,
Petroni A,
Soler Bistué AJ,
Guerriero L,
Vargas LJ,
Zorreguieta A,
Tokumoto M,
Quiroga C,
Tolmasky ME,
Galas M,
Centrón D,
( 2007 ) Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina. PMID : 17938184 : DOI : 10.1128/AAC.00726-07 PMC : PMC2167984 Abstract >>
Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6')-Ib-cr within the variable region of integrons. The three arrays differed from bla(CTX-M-2)-bearing integrons, which are broadly distributed in Argentina.
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74. |
Cattoir V,
Poirel L,
Rotimi V,
Soussy CJ,
Nordmann P,
( 2007 ) Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. PMID : 17561500 : DOI : 10.1093/jac/dkm204 Abstract >>
To develop a rapid and reliable single-tube-based PCR technique for detecting simultaneously the plasmid-mediated quinolone resistance qnrA, qnrB and qnrS genes. After multiple alignments, primers were designed to detect known qnr variants (six for qnrA-, six for qnrB- and two for qnrS-like genes). They were used for screening a collection of 64 expanded-spectrum beta-lactamase (ESBL)-producing enterobacterial isolates from Kuwait, collected from 2002 to 2004, as ESBL genes have been often associated with qnr genes. Sequencing was performed to identify qnr and associated ESBL genes. In optimized conditions, all positive controls (used separately or mixed) confirmed the specificity of the PCR primers. Out of 64 isolates, only 3 isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like and qnrS-like gene was detected. A qnrB2 gene was detected in an Enterobacter cloacae K34 (SHV-12+) isolate, whereas qnrB1-like (termed qnrB7) and qnrB6-like (termed qnrB8) genes were identified from E. cloacae K37 (SHV-12+) and Citrobacter freundii K70 (VEB-1b+) isolates, respectively. We report here a fast and reliable technique for rapid screening of qnr-positive strains to be used for epidemiological surveys. A low prevalence of Qnr determinants among ESBL-producing Enterobacteriaceae was identified in the study with Kuwaiti isolates.
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75. |
Jiang X,
Yin Z,
Yin X,
Fang H,
Sun Q,
Tong Y,
Xu Y,
Zhang D,
Feng J,
Chen W,
Song Y,
Wang J,
Chen S,
Zhou D,
( 2017 ) Sequencing of blaIMP-Carrying IncN2 Plasmids, and Comparative Genomics of IncN2 Plasmids Harboring Class 1 Integrons. PMID : 28424761 : DOI : 10.3389/fcimb.2017.00102 PMC : PMC5371602 Abstract >>
This work presents the complete nucleotide sequences of p0801-IMP from Klebsiella pneumoniae, p7121-IMP from K. oxytoca, and p17285-IMP from Citrobacter freundii, which are recovered from three different cases of nosocomial infection. These three plasmids represent the first fully sequenced blaIMP-carrying IncN2 plasmids. Further comparative genomics analysis of all the five integron-carrying IncN2 plasmids p0801-IMP, p7121-IMP, p17285-IMP, pJIE137, and p34983-59.134kb indicates that they possess conserved IncN2 backbones with limited genetic variations with respect to gene content and organization. Four class 1 integrons (blaIMP-1-carrying In1223 in p0801-IMP/p7121-IMP, blaIMP-8-carrying In655 in p17285-IMP, In27 in pJIE137, and In1130 in p34983-59.134kb), two insertion sequence-based transposition units (ISEcp1-orfRA1-14 in p17285-IMP, and ISEcp1-blaCTX-M-62-�Gorf477-orfRA1-14 in pJIE137), and a novel Tn1696-related transposon Tn6325 carrying In1130 in p34983-59.134kb are indentified in the plasmid accessory regions. In1223 and In655 represent ancestral Tn402-associated integrons, while In27 and In1130 belong to complex class 1 integrons. The relatively small IncN2 backbones are able to integrate different mobile elements which carry various resistance markers, promoting the accumulation and spread of antimicrobial resistance genes among enterobacterial species.
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76. |
Liang Q,
Yin Z,
Zhao Y,
Liang L,
Feng J,
Zhan Z,
Wang H,
Song Y,
Tong Y,
Wu W,
Chen W,
Wang J,
Jiang L,
Zhou D,
( 2017 ) Sequencing and comparative genomics analysis of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA. PMID : 28390961 : DOI : 10.1016/j.ijantimicag.2017.01.021 Abstract >>
Incompatibility group IncHI plasmids are important vectors of antibiotic resistance in Enterobacteriaceae. In this study, a scheme for typing IncHI into five separately clustering subgroups, including previously designated IncHI1-3 as well as IncHI4-5, was proposed based on sequenced IncHI plasmids. The complete nucleotide sequences of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA from clinical Enterobacter cloacae, Citrobacter freundii and Raoultella ornithinolytica isolates, respectively, were determined and were compared with IncHI2 and IncHI5 reference plasmids. Considerable genetic conservation was observed within the backbone sequences of each of the IncHI2 and IncHI5 subgroups, but the backbone sequences of the two subgroups were dramatically different from each other. However, the conjugal transfer regions tra1 and tra2 as well as the tellurium resistance gene cluster ter were present in all five plasmids. A number of accessory regions associated with integrons, transposons and insertion sequence-based mobile elements have been inserted at various sites of the plasmid backbones, among which were several large regions harbouring genes conferring resistance to multiple classes of antibiotics. Data generated from this study provide us with a deeper understanding of the diversification of IncHI-type resistance plasmids.
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77. |
Cookson AL,
Biggs PJ,
Marshall JC,
Reynolds A,
Collis RM,
French NP,
Brightwell G,
( 2017 ) Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure. PMID : 28404985 : DOI : 10.1038/s41598-017-00890-6 PMC : PMC5429811 Abstract >>
Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. 'Animal' was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.
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78. |
Morlon J,
Chartier M,
Bidaud M,
Lazdunski C,
( 1988 ) The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1. PMID : 2832701 : DOI : 10.1007/bf00330599 Abstract >>
The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48 X 10(6]. Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 and Clo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.
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79. |
Lindquist S,
Lindberg F,
Normark S,
( 1989 ) Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC beta-lactamase gene. PMID : 2786868 : DOI : 10.1128/jb.171.7.3746-3753.1989 PMC : PMC210120 Abstract >>
Citrobacter freundii encodes an inducible chromosomal beta-lactamase. Induction requires the product of the ampR gene, which is transcribed in the opposite orientation from the ampC beta-lactamase gene. We show here that the AmpR protein acts as a transcriptional activator by binding to a DNA region immediately upstream of the ampC promoter. The DNase I footprint pattern was not affected by growth in the presence of beta-lactam inducer or by the use of extracts prepared from cells carrying the ampD2 allele leading to semiconstitutive production of beta-lactamase. It is suggested that activation of AmpR facilitates binding or open complex formation for RNA polymerase at the ampC promoter. The AmpR-binding site overlaps the ampR promoter, and beta-galactosidase activity was decreased from an ampR-lacZ transcriptional fusion when AmpR was expressed from a coresident plasmid, suggesting that ampR is autogenously controlled. The AmpR protein belongs to a family of highly homologous transcriptional activators that includes LysR, which regulates the E. coli lysine synthetase gene, and the NodD protein, which regulates expression of a number of genes involved in nodulation in Rhizobium. The lack of sequence homology to any known beta-lactam-binding protein suggests that AmpR does not bind directly to the beta-lactam inducer but interacts with a second messenger of unknown nature.
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80. |
Hammerum AM,
Hansen F,
Nielsen HL,
Jakobsen L,
Stegger M,
Andersen PS,
Jensen P,
Nielsen TK,
Hansen LH,
Hasman H,
Fuglsang-Damgaard D,
( 2016 ) Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of blaNDM-1 to Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca. PMID : 27494919 : DOI : 10.1093/jac/dkw289 Abstract >>
An outbreak of NDM-1-producing Citrobacter freundii and possible secondary in vivo spread of blaNDM-1 to other Enterobacteriaceae were investigated. From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. In silico resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 C. freundii, 6 Klebsiella pneumoniae, 4 Escherichia coli and 1 Klebsiella oxytoca) and 1 meropenem-susceptible K. oxytoca. The sequences of the meropenem-resistant C. freundii isolates were compared by phylogenetic analyses. In vitro susceptibility to 21 antimicrobial agents was tested. Furthermore, in vitro conjugation and plasmid characterization was performed. From the seven patients, 13 highly clonal ST18 NDM-1-producing C. freundii were isolated. The ST18 NDM-1-producing C. freundii isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the C. freundii isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The E. coli and K. pneumoniae from different patients belonged to different STs, indicating in vivo transfer of blaNDM-1 in the individual patients. This was further supported by in vitro conjugation and detection of a 154 kb IncA/C2 plasmid with blaNDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the blaNDM-1 plasmid was unknown. To our knowledge, this is the first report of an NDM-1-producing C. freundii outbreak and secondary in vivo spread of an IncA/C2 plasmid with blaNDM-1 to other Enterobacteriaceae.
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81. |
Farkas A,
Cr?ciuna? C,
Chiriac C,
Szekeres E,
Coman C,
Butiuc-Keul A,
( 2016 ) Exploring the Role of Coliform Bacteria in Class 1 Integron Carriage and Biofilm Formation During Drinking Water Treatment. PMID : 27079455 : DOI : 10.1007/s00248-016-0758-0 Abstract >>
This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacE�G1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.
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82. |
Kannan P,
Cowan GM,
Daniel AS,
Gann AA,
Murray NE,
( 1989 ) Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes. PMID : 2585490 : DOI : 10.1016/0022-2836(89)90001-6 Abstract >>
We have identified the recognition sequence for the Citrobacter freundii restriction endonuclease CfrA, a member of the A-family of type I R-M enzymes. This bipartite target sequence differs in both its components from those of other type I enzymes. We determined the nucleotide sequence of its specificity gene (hsdS) and a comparison of this with its relative EcoA identifies two extensive variable regions, an organization analogous to that found in the K-family of type I R-M enzymes. The specificity polypeptides of the A-family, unlike those of K, have an N-terminal conserved region, and this includes a sequence repeated within the central conserved region. A second repeat sequence, identified at the amino acid level, coincides with the only sequence similarity common to all type I S polypeptides. Sequences immediately downstream from the hsdS genes of EcoA, CfrA, EcoK, B and D are almost identical, consistent with an allelic chromosomal location.
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83. |
Klimasauskas S,
Timinskas A,
Menkevicius S,
Butkienè D,
Butkus V,
Janulaitis A,
( 1989 ) Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases. PMID : 2690010 : DOI : 10.1093/nar/17.23.9823 PMC : PMC335216 Abstract >>
The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.
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84. |
Garvin LD,
Hardies SC,
( 1989 ) Nucleotide sequence for the htpR gene from Citrobacter freundii. PMID : 2664713 : DOI : 10.1093/nar/17.12.4889 PMC : PMC318053 Abstract >>
N/A
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85. |
Jan AT,
Azam M,
Choi I,
Ali A,
Haq QM,
( N/A ) Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India. PMID : 26887227 : DOI : 10.1016/j.bjm.2015.11.023 PMC : PMC4827696 Abstract >>
Mercury, which is ubiquitous and recalcitrant to biodegradation processes, threatens human health by escaping to the environment via various natural and anthropogenic activities. Non-biodegradability of mercury pollutants has necessitated the development and implementation of economic alternatives with promising potential to remove metals from the environment. Enhancement of microbial based remediation strategies through genetic engineering approaches provides one such alternative with a promising future. In this study, bacterial isolates inhabiting polluted sites were screened for tolerance to varying concentrations of mercuric chloride. Following identification, several Pseudomonas and Klebsiella species were found to exhibit the highest tolerance to both organic and inorganic mercury. Screened bacterial isolates were examined for their genetic make-up in terms of the presence of genes (merP and merT) involved in the transport of mercury across the membrane either alone or in combination to deal with the toxic mercury. Gene sequence analysis revealed that the merP gene showed 86-99% homology, while the merT gene showed >98% homology with previously reported sequences. By exploring the genes involved in imparting metal resistance to bacteria, this study will serve to highlight the credentials that are particularly advantageous for their practical application to remediation of mercury from the environment.
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86. |
Geli V,
Baty D,
Pattus F,
Lazdunski C,
( 1989 ) Topology and function of the integral membrane protein conferring immunity to colicin A. PMID : 2668695 : DOI : 10.1111/j.1365-2958.1989.tb00216.x Abstract >>
The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N- and C-terminal regions were found to be directed towards the cytoplasm. Oligonucleotide-directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.
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87. |
Feng J,
Qiu Y,
Yin Z,
Chen W,
Yang H,
Yang W,
Wang J,
Gao Y,
Zhou D,
( 2015 ) Coexistence of a novel KPC-2-encoding MDR plasmid and an NDM-1-encoding pNDM-HN380-like plasmid in a clinical isolate of Citrobacter freundii. PMID : 26260129 : DOI : 10.1093/jac/dkv232 Abstract >>
The objective of this study was to characterize the molecular mechanism of coproduction of KPC-2 and NDM-1 in Citrobacter freundii. C. freundii strain 112298 was isolated from a human case of septic shock in a Chinese teaching hospital. The major carbapenemase and ESBL genes were detected by PCR. The MIC values were determined by using VITEK 2 and antimicrobial susceptibility was judged by CLSI standards. The resistance plasmid was transferred into Escherichia coli by electroporation, followed by plasmid DNA isolation from the electroporant, and then fully sequenced and compared with closely related plasmids. Strain 112298 produces KPC-2 and NDM-1, encoded by the novel non-typeable plasmid p112298-KPC and an IncX3-type plasmid p112298-NDM, respectively. In p112298-KPC, a Tn1722-based blaKPC-2-carrying transposon is associated with several additional resistance modules, constituting a single MDR region. Assembly of these resistance modules is likely mediated by homologous recombination between five copies of IS26 elements at different sites within the MDR region. p112298-NDM is a very close relation of pNDM-HN380. blaNDM-1 in p112298-NDM is carried by a Tn125 variant, which differs from the prototype Tn125 as observed in pNDM-BJ01 by disruption of an upstream copy of ISAba125 by IS5 and absence of a downstream copy of ISAba125. Production of KPC-2 and NDM-1 by p112298-KPC and p112298-NDM, respectively, makes C. freundii 112298 highly resistant to carbapenems and, moreover, these two plasmids still harbour genes for resistance to cephalosporins, chloramphenicol, chromate, fosfomycin, quaternary ammonium, rifampicin and sulphonamides.
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88. |
Huang YM,
Zhong LL,
Zhang XF,
Hu HT,
Li YQ,
Yang XR,
Feng LQ,
Huang X,
Tian GB,
( 2015 ) NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China. PMID : 26055374 : DOI : 10.1128/AAC.04682-14 PMC : PMC4505197 Abstract >>
We identified New Delhi metallo-�]-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.
|
89. |
Campos MJ,
Palomo G,
Hormeño L,
Patrocínio Rodrigues A,
Sánchez-Benito R,
Píriz S,
Quesada A,
( 2015 ) Detection of QnrB54 and its novel genetic context in Citrobacter freundii isolated from a clinical case. PMID : 25512409 : DOI : 10.1128/AAC.03895-14 PMC : PMC4335882 Abstract >>
N/A
|
90. |
Porres-Osante N,
Sáenz Y,
Somalo S,
Torres C,
( 2015 ) Characterization of Beta-lactamases in Faecal Enterobacteriaceae Recovered from Healthy Humans in Spain: Focusing on AmpC Polymorphisms. PMID : 25501887 : DOI : 10.1007/s00248-014-0544-9 Abstract >>
The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.
|
91. |
Antonelli A,
D'Andrea MM,
Vaggelli G,
Docquier JD,
Rossolini GM,
( 2015 ) OXA-372, a novel carbapenem-hydrolysing class D �]-lactamase from a Citrobacter freundii isolated from a hospital wastewater plant. PMID : 26126492 : DOI : 10.1093/jac/dkv181 Abstract >>
The objective of this study was to characterize a novel class D carbapenemase, named OXA-372, identified in a carbapenem-resistant Citrobacter freundii strain (Cfr-FI-07) isolated from a hospital wastewater plant in central Italy. Cfr-FI-07 was isolated using a selective chromogenic medium for carbapenem-resistant Enterobacteriaceae. Carbapenemase production was confirmed by spectrophotometric assay. WGS was carried out using an Illumina MiSeq platform. The functional profile of OXA-372 was investigated by expression of the cloned gene in Escherichia coli and by analysis of kinetic parameters of the purified enzyme. C. freundii Cfr-FI-07 produced carbapenemase activity, but tested negative for common carbapenemase genes. WGS confirmed the absence of known carbapenemase genes and revealed the presence of a novel class D �]-lactamase (DBL) determinant, named blaOXA-372, encoding a protein distantly related to other DBLs. In E. coli, production of OXA-372 conferred resistance to penicillins, including temocillin, and reduced susceptibility to carbapenems, while susceptibility to expanded-spectrum cephalosporins was virtually unaffected. This substrate specificity was confirmed by kinetic characterization of the purified enzyme, which exhibited high catalytic efficiencies for carbapenems (kcat/KM values ? 0.22 s(-1) �P �gM(-1)). The blaOXA-372 gene was associated with a genetic platform of original structure consisting of a Tn402/Tn5053 hybrid transposon derivative, named Tn6255, inserted into a TnPa38-like transposon, named Tn6256, located on an IncA/C-IncN plasmid of approximately 140 kb. OXA-372 is a novel class D carbapenemase, belonging to a new lineage of DBLs, encoded by a gene associated with mobile elements. Functional properties revealed similarities, but also some differences, compared with other class D carbapenemases.
|
92. |
Morozova EA,
Revtovich SV,
Anufrieva NV,
Kulikova VV,
Nikulin AD,
Demidkina TV,
( 2014 ) Alliin is a suicide substrate of Citrobacter freundii methionine �^-lyase: structural bases of inactivation of the enzyme. PMID : 25372692 : DOI : 10.1107/S1399004714020938 Abstract >>
The interaction of Citrobacter freundii methionine �^-lyase (MGL) and the mutant form in which Cys115 is replaced by Ala (MGL C115A) with the nonprotein amino acid (2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) was investigated. It was found that MGL catalyzes the �]-elimination reaction of alliin to form 2-propenethiosulfinate (allicin), pyruvate and ammonia. The �]-elimination reaction of alliin is followed by the inactivation and modification of SH groups of the wild-type and mutant enzymes. Three-dimensional structures of inactivated wild-type MGL (iMGL wild type) and a C115A mutant form (iMGL C115A) were determined at 1.85 and 1.45 ? resolution and allowed the identification of the SH groups that were oxidized by allicin. On this basis, the mechanism of the inactivation of MGL by alliin, a new suicide substrate of MGL, is proposed.
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93. |
Kuznetsov NA,
Faleev NG,
Kuznetsova AA,
Morozova EA,
Revtovich SV,
Anufrieva NV,
Nikulin AD,
Fedorova OS,
Demidkina TV,
( 2015 ) Pre-steady-state kinetic and structural analysis of interaction of methionine �^-lyase from Citrobacter freundii with inhibitors. PMID : 25398880 : DOI : 10.1074/jbc.M114.586511 PMC : PMC4281767 Abstract >>
Methionine �^-lyase (MGL) catalyzes the �^-elimination of l-methionine and its derivatives as well as the �]-elimination of l-cysteine and its analogs. These reactions yield �\-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL�Pl-cycloserine complex has been solved at 1.6 ? resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.
|
94. |
Amos GC,
Hawkey PM,
Gaze WH,
Wellington EM,
( 2014 ) Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment. PMID : 24797064 : DOI : 10.1093/jac/dku079 PMC : PMC4054988 Abstract >>
Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river. River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate bla(CTX-M-15) mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology. We report the first examples of bla(CTX-M-15) in UK river sediment; the prevalence of bla(CTX-M-15) was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The bla(CTX-M-15) -gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river. The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health.
|
95. |
Revtovich SV,
Faleev NG,
Morozova EA,
Anufrieva NV,
Nikulin AD,
Demidkina TV,
( 2014 ) Crystal structure of the external aldimine of Citrobacter freundii methionine �^-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of �\- and �]-protons of competitive inhibitors. PMID : 24463191 : DOI : 10.1016/j.biochi.2014.01.007 Abstract >>
The three-dimensional structure of the external aldimine of Citrobacter freundii methionine �^-lyase with competitive inhibitor glycine has been determined at 2.45 ? resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of C�\-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of C�\-proton to C4'-atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic C�\-protons of glycine may be explained on the basis of external aldimine structure. The exchange of C�\-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from C�\-atom of glycine to C4'-atom of the cofactor. The exchange of C�\-pro-(S)-proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of �]-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid.
|
96. |
Halová D,
Papousek I,
Jamborova I,
Masarikova M,
Cizek A,
Janecko N,
Oravcova V,
Zurek L,
Clark AB,
Townsend A,
Ellis JC,
Literak I,
( 2014 ) Plasmid-mediated quinolone resistance genes in Enterobacteriaceae from American crows: high prevalence of bacteria with variable qnrB genes. PMID : 24247140 : DOI : 10.1128/AAC.01849-13 PMC : PMC3910892 Abstract >>
N/A
|
97. |
Pang A,
Frank S,
Brown I,
Warren MJ,
Pickersgill RW,
( 2014 ) Structural insights into higher order assembly and function of the bacterial microcompartment protein PduA. PMID : 24873823 : DOI : 10.1074/jbc.M114.569285 PMC : PMC4139245 Abstract >>
Bacterial microcompartments are large proteinaceous assemblies that are found in the cytoplasm of some bacteria. These structures consist of proteins constituting a shell that houses a number of enzymes involved in specific metabolic processes. The 1,2-propanediol-utilizing microcompartment is assembled from seven different types of shell proteins, one of which is PduA. It is one of the more abundant components of the shell and intriguingly can form nanotubule-like structures when expressed on its own in the cytoplasm of Escherichia coli. We propose a model that accounts for the size and appearance of these PduA structures and underpin our model using a combinatorial approach. Making strategic mutations at Lys-26, Val-51, and Arg-79, we targeted residues predicted to be important for PduA assembly. We present the effect of the amino acid residue substitution on the phenotype of the PduA higher order assemblies (transmission electron microscopy) and the crystal structure of the K26D mutant with one glycerol molecule bound to the central pore. Our results support the view that the hexamer-hexamer interactions seen in PduA crystals persist in the cytoplasmic structures and reveal the profound influence of the two key amino acids, Lys-26 and Arg-79, on tiling, not only in the crystal lattice but also in the bacterial cytoplasm. Understanding and controlling PduA assemblies is valuable in order to inform manipulation for synthetic biology and biotechnological applications.
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98. |
Smet A,
Vaes R,
Praud K,
Doublet B,
Daminet S,
Cloeckaert A,
Haesebrouck F,
( 2014 ) New broad-spectrum �]-lactamases emerging among Enterobacteriaceae from healthy cats and dogs: a public health concern? PMID : 24916079 : DOI : 10.1016/j.ijantimicag.2014.03.006 Abstract >>
N/A
|
99. |
Manageiro V,
Ferreira E,
Caniça M,
Manaia CM,
( 2014 ) GES-5 among the �]-lactamases detected in ubiquitous bacteria isolated from aquatic environment samples. PMID : 24267783 : DOI : 10.1111/1574-6968.12340 Abstract >>
In this study, we investigated the �]-lactamase-encoding genes responsible for �]-lactam resistance phenotypes detected among 56 Gram-negative isolates (Gamma- and Alpha-proteobacteria) recovered from wastewater, urban streams, and drinking water. The �]-lactam resistance mechanisms detected in 36 isolates comprised the presence of class A (blaTEM-1 , blaSHV-1 , blaSHV-11 , blaGES-5), class B (ImiS, L1), class C (blaCMY-2 , blaCMY-34 , blaCMY-65 , blaCMY-89 , blaCMY-90 , blaACC-5 , blaACT-13), and class D (blaOXA-309)�]-lactamase-encoding genes, some variants described for the first time here. Notably, the results showed antimicrobial resistance genes related not only to commonly used antibiotics, but also to carbapenems, providing the first description of a GES-5-producing Enterobacteriaceae. The importance of ubiquitous bacteria thriving in aquatic environments as reservoirs or carriers of clinically relevant resistance determinants was confirmed, and the need to monitor water habitats as potential sources for the emergence and/or spread of antibiotic resistance in the environment was highlighted.
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100. |
Ruggiero M,
Kerff F,
Herman R,
Sapunaric F,
Galleni M,
Gutkind G,
Charlier P,
Sauvage E,
Power P,
( 2014 ) Crystal structure of the extended-spectrum �]-lactamase PER-2 and insights into the role of specific residues in the interaction with �]-lactams and �]-lactamase inhibitors. PMID : 25070104 : DOI : 10.1128/AAC.00089-14 PMC : PMC4187900 Abstract >>
PER-2 belongs to a small (7 members to date) group of extended-spectrum �]-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most �]-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 ? and evaluated the possible role of several residues in the structure and activity toward �]-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted �[ loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A �]-lactamases. PER �]-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A ("A" indicates an insertion according to Ambler's scheme for residue numbering in PER �]-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different �]-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.
|
101. |
Wendel AF,
Brodner AH,
Wydra S,
Ressina S,
Henrich B,
Pfeffer K,
Toleman MA,
Mackenzie CR,
( 2013 ) Genetic characterization and emergence of the metallo-�]-lactamase GIM-1 in Pseudomonas spp. and Enterobacteriaceae during a long-term outbreak. PMID : 23877696 : DOI : 10.1128/AAC.00118-13 PMC : PMC3811479 Abstract >>
Since the first isolation in 2002, the metallo-�]-lactamase GIM-1 has not been detected outside Germany. The data presented here, for 50 clinical blaGIM-1-positive isolates, including Pseudomonas spp. and Enterobacteriaceae (Enterobacter cloacae, Klebsiella oxytoca, Serratia marcescens, Escherichia coli, and Citrobacter freundii), collected between 2007 and 2012 at the original site in an ongoing outbreak, demonstrate a diverse genetic background and dissemination of the gene conferring resistance to enteric bacteria.
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102. |
Delgado G,
Souza V,
Morales R,
Cerritos R,
González-González A,
Méndez JL,
Vázquez V,
Cravioto A,
( 2013 ) Genetic characterization of atypical Citrobacter freundii. PMID : 24069274 : DOI : 10.1371/journal.pone.0074120 PMC : PMC3771896 Abstract >>
The ability of a bacterial population to survive in different niches, as well as in stressful and rapidly changing environmental conditions, depends greatly on its genetic content. To survive such fluctuating conditions, bacteria have evolved different mechanisms to modulate phenotypic variations and related strategies to produce high levels of genetic diversity. Laboratories working in microbiological diagnosis have shown that Citrobacter freundii is very versatile in its colony morphology, as well as in its biochemical, antigenic and pathogenic behaviours. This phenotypic versatility has made C. freundii difficult to identify and it is frequently confused with both Salmonella enterica and Escherichia coli. In order to determine the genomic events and to explain the mechanisms involved in this plasticity, six C. freundii isolates were selected from a phenotypic variation study. An I-CeuI genomic cleavage map was created and eight housekeeping genes, including 16S rRNA, were sequenced. In general, the results showed a range of both phenotypes and genotypes among the isolates with some revealing a greater similarity to C. freundii and some to S. enterica, while others were identified as phenotypic and genotypic intermediary states between the two species. The occurrence of these events in natural populations may have important implications for genomic diversification in bacterial evolution, especially when considering bacterial species boundaries. In addition, such events may have a profound impact on medical science in terms of treatment, course and outcomes of infectious diseases, evading the immune response, and understanding host-pathogen interactions.
|
103. |
Campos J,
Mourão J,
Pestana N,
Peixe L,
Novais C,
Antunes P,
( 2013 ) Microbiological quality of ready-to-eat salads: an underestimated vehicle of bacteria and clinically relevant antibiotic resistance genes. PMID : 24036261 : DOI : 10.1016/j.ijfoodmicro.2013.08.005 Abstract >>
The increase demand for fresh vegetables is causing an expansion of the market for minimally processed vegetables along with new recognized food safety problems. To gain further insight on this topic we analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying acquired antibiotic resistance genes, food products scarcely considered in surveillance studies. A total of 50 RTS (7 brands; split or mixed leaves, carrot, corn) were collected in 5 national supermarket chains in Porto region (2010). They were tested for aerobic mesophilic counts, coliforms and Escherichia coli counts as well as for the presence of Salmonella and Listeria monocytogenes. Samples were also plated in different selective media with/without antibiotics before and after enrichment. The E. coli, other coliforms and Enterococcus recovered were characterized for antibiotic resistance profiles and clonality with phenotypic and genetic approaches. A high number of RTS presented poor microbiological quality (86%--aerobic mesophilic counts, 74%--coliforms, 4%--E. coli), despite the absence of screened pathogens. In addition, a high diversity of bacteria (species and clones) and antibiotic resistance backgrounds (phenotypes and genotypes) were observed, mostly with enrichment and antibiotic selective media. E. coli was detected in 13 samples (n=78; all types and 4 brands; phylogenetic groups A, B1 and D; none STEC) with resistance to tetracycline [72%; tet(A) and/or tet(B)], streptomycin (58%; aadA and/or strA-strB), sulfamethoxazole (50%; sul1 and/or sul2), trimethoprim (50%; dfrA1 or dfrA12), ampicillin (49%; blaTEM), nalidixic acid (36%), ciprofloxacin (5%) or chloramphenicol (3%; catA). E. coli clones, including the widespread group D/ST69, were detected in different samples from the same brand or different brands pointing out to a potential cross-contamination. Other clinically relevant resistance genes were detected in 2 Raoultella terrigena carrying a bla(SHV-2) and 1 Citrobacter freundii isolate with a qnrB9 gene. Among Enterococcus (n=108; 35 samples; Enterococcus casseliflavus--40, Enterococcus faecalis--20, Enterococcus faecium--18, Enterococcus hirae--9, Enterococcus gallinarum--5, and Enterococcus spp.--16) resistance was detected for tetracyclines [6%; tet(M) and/or tet(L)], erythromycin [3%; erm(B)], nitrofurantoin (1%) or ciprofloxacin (1%). The present study places ready-to-eat salads within the spectrum of ecological niches that may be vehicles for antibiotic resistance bacteria/genes with clinical interest (e.g. E. coli-D-ST69; bla(SHV-2)) and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.
|
104. |
Cambau E,
Scheftel JM,
Bertrand X,
Guillard T,
Moret H,
( 2012 ) High-resolution melting analysis for rapid characterization of qnr alleles in clinical isolates and detection of two novel alleles, qnrB25 and qnrB42. PMID : 22850691 : DOI : 10.1093/jac/dks292 Abstract >>
qnr genes are plasmid-mediated quinolone resistance genes. Five qnr families have been described with several alleles (7 alleles of qnrA, 53 alleles of qnrB, 1 allele of qnrC, 1 allele of qnrD and 6 alleles of qnrS). Their detection requires a PCR specific for each qnr family and further sequencing for allele characterization. High-resolution melt curve analysis (HRMA) was coupled to multiplex and simplex real-time PCR assays for detection and characterization of qnrA, qnrB and qnrS alleles. The protocol was set using 27 reference strains harbouring the most frequent alleles and was applied to 55 clinical isolates unknown for qnr positivity. Out of the 27 reference strains tested, 21 alleles showed distinct profiles using HRMA: 6 qnrA, 12 qnrB and 3 qnrS. For the qnrB alleles showing similar profiles, we gathered them into four groups that were easily distinguished. For the alleles that we could not test, in silico analysis showed that they would be identified using the HRMA protocol set. Among the clinical isolates, 28 qnr-positive isolates were detected and the qnr allele was characterized as 8 qnrA1, 4 qnrB1, 5 qnrB2, 3 qnrB4, 1 qnrB8, 1 qnrB5, 3 qnrS1 and 1 qnrS2, with concordant results with PCR sequencing. Two new qnrB alleles were detected and distinguished using HMRA. They were further designated as qnrB25 and qnrB42. We developed an HRMA assay for characterizing the qnr alleles in clinical isolates. This high-throughput method can be used to screen a large number of isolates. This method allowed the detection of new qnrB alleles.
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105. |
Bai L,
Xia S,
Lan R,
Liu L,
Ye C,
Wang Y,
Jin D,
Cui Z,
Jing H,
Xiong Y,
Bai X,
Sun H,
Zhang J,
Wang L,
Xu J,
( 2012 ) Isolation and characterization of cytotoxic, aggregative Citrobacter freundii. PMID : 22470435 : DOI : 10.1371/journal.pone.0033054 PMC : PMC3310003 Abstract >>
Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 "cytotoxic and aggregative C. freundii." Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii.
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106. |
Revtovich SV,
Morozova EA,
Khurs EN,
Zakomirdina LN,
Nikulin AD,
Demidkina TV,
Khomutov RM,
( 2011 ) Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine �^-lyase with substrates. PMID : 21639836 : DOI : 10.1134/S0006297911050063 Abstract >>
Crystal structures of Citrobacter freundii methionine �^-lyase complexes with the substrates of �^- (L-1-amino-3-methylthiopropylphosphinic acid) and �]- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 ? resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.
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107. |
Dahyot S,
Mammeri H,
( 2012 ) Hydrolysis spectrum extension of CMY-2-like �]-lactamases resulting from structural alteration in the Y-X-N loop. PMID : 22232281 : DOI : 10.1128/AAC.05630-11 PMC : PMC3294937 Abstract >>
The Citrobacter freundii isolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) �]-lactamase. The chromosome-borne bla(AmpC-CHA) gene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded the Escherichia coli TOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resulting E. coli TOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 �]-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficient E. coli strain. Moreover, it exhibited increased catalytic efficiency (k(cat)/K(m)) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (k(cat)), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A �]-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.
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108. |
( 1997 ) Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates. PMID : 9055422 : PMC : PMC168397 Abstract >>
Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and delta 8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5' side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3' side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus delta 8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5' and 3' proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.
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109. |
( 1997 ) The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity. PMID : 9174368 : DOI : 10.1021/bi962917+ Abstract >>
The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.
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110. |
( 1997 ) Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii. PMID : 9311142 : DOI : 10.1111/j.1574-6968.1997.tb12675.x Abstract >>
We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.
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111. |
( 1997 ) Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates. PMID : 9361435 : PMC : PMC168768 Abstract >>
Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components.
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112. |
( 1997 ) Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron. PMID : 9021185 : PMC : PMC163707 Abstract >>
The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.
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113. |
( 1996 ) Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii. PMID : 8824629 : DOI : 10.1128/jb.178.19.5793-5796.1996 PMC : PMC178423 Abstract >>
The genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B12-free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively. The enzyme complex has the composition alpha2beta2gamma2. Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T. Tobimatsu, T. Hara, M. Sakaguchi, Y. Kishimoto, Y. Wada, M. Isoda, T. Sakai, and T. Toraya, J. Biol. Chem. 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.
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114. |
( 1993 ) Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples. PMID : 8423084 : PMC : PMC302761 Abstract >>
By hybridizing colonies grown from 928 individual stool samples of patients suffering from diarrhea with oligonucleotide probes 772 and 849 complementary to Shiga-like toxin I (SLT-I) and SLT-II gene sequences, respectively, we identified two strains that hybridized with probe 849, which biochemical identification revealed as Citrobacter freundii. An additional five slt-II probe-positive isolates were screened from 81 beef samples. Polymerase chain reaction analysis and restriction of amplified products provided evidence for slt-II-related genes in all seven strains. From C. freundii LM 76, the genes encoding the A and B subunits were cloned in pUC 18 vectors and sequenced. The gene encoding the A subunit differed from that of Escherichia coli slt-IIvhc in 4 bases, resulting in two amino acid residue differences. In 11, 13, and 11 nucleotides, differentiation of slt-IIA, slt-IIcA, and vtx2haA, respectively, was found. These differences affected the predicted amino acid sequence as follows: there were six amino acid differences with SLT-IIA, five with SLT-IIcA, and four with VTx2haA. The nucleotide sequence of the gene encoding the B subunit is identical to slt-IIvhcB and differed from slt-IIcB and vtx2haB by only a single nucleotide base, but this resulted in a predicted amino acid sequence identical to that reported for these toxins. We therefore termed the toxin genes C. freundii slt-IIcA and slt-IIcB. Culture filtrates inoculated with material from the colonies from primary cultures were cytotoxic to Vero cells. Neutralization assays with antisera to E. coli SLT-I, SLT-II, and SLT-IIvhc revealed that antibodies against SLT-IIvhc reduced the C. freundii cytotoxic activity specifically and to the same degree as with the E. coli SLT-IIvhc control strain. In five of the seven strains tested, subcultivation on both a liquid or solid medium resulted in loss of cytotoxic activity. With polymerase chain reaction, we demonstrated that loss of cytotoxic activity ran parallel with the loss of slt genes. These data demonstrate the intergeneric occurrence of SLT-II-related toxins, which may well be a new marker of enteropathogenicity in C. freundii. Our findings that the toxin genes belong to the slt-II family plus their evident instability in the majority of strains should help pave the way to a better understanding of their role in diarrhea or food poisoning.
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115. |
( 1996 ) Crystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 A resolution. PMID : 8568865 : DOI : 10.1006/jmbi.1996.0015 Abstract >>
The X-ray crystal structure of Citrobacter freundii restriction endonuclease Cfr10I has been determined at a resolution of 2.15 A by multiple isomorphous replacement methods and refined to an R-factor of 19.64%. The structure of Cfr10I represents the first structure of a restriction endonuclease recognizing a degenerated nucleotide sequence. Structural comparison of Cfr10I with previously solved structures of other restriction enzymes suggests that recognition of specific sequence occurs through contacts in the major and the minor grooves of DNA. The arrangement of the putative active site residues shows some striking differences from previously described restriction endonucleases and supports a two-metal-ion mechanism of catalysis.
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116. |
( 1993 ) Cloning and sequences of the genes encoding the CfrBI restriction-modification system from Citrobacter freundii. PMID : 8335262 : DOI : 10.1016/0378-1119(93)90698-3 Abstract >>
The genes encoding the CfrBI restriction and modification (R-M) systems from Citrobacter freundii and recognizing the sequence 5'-CCWWGG-3' (W = A or T) were cloned in Escherichia coli McrBC- cells. The nucleotide (nt) sequences of the genes were determined. Two large open reading frames were found. Deletion analysis showed that one of them [1128 nt coding for 376 amino acids (aa)] corresponds to a methyltransferase (MTase)-encoding gene and the other (1065 nt coding for 355 aa) to a restriction endonuclease-encoding gene. The genes are oriented divergently and separated by 76 bp. A CfrBI site (5'-m4CCATGG) was found in the intergenic region of the cfrBIRM genes. Analysis of the deduced aa sequence of M.CfrBI made it possible to determine the typical features of a m4C-specific MTase. Limited homology between the M.CfrBI and R.CfrBI proteins was also found.
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117. |
( 1993 ) Sequences of wild-type and mutant ampD genes of Citrobacter freundii and Enterobacter cloacae. PMID : 8383940 : DOI : 10.1128/aac.37.2.224 PMC : PMC187643 Abstract >>
The ampD gene product regulates the expression of AmpC beta-lactamase in gram-negative bacteria and is proposed to be involved in peptidoglycan metabolism. In this study, we sequenced the ampD wild type and three mutant genes of Enterobacter cloacae and Citrobacter freundii. They exhibited a high degree of homology with the corresponding gene of Escherichia coli except in the carboxy termini, where, in the wild-type genes of E. cloacae and C. freundii, four additional amino acids yielding the Ser-X-X-Lys motif were found. Evidence that this C-terminal region of the ampD gene product is necessary for activity was shown by constructing a deletion of the last 16 amino acids. The spontaneous mutation of ampD02 is an out-of-frame insertion and yields an inactive AmpD protein. The single-base-pair substitution of Gly for Asp-121 in ampD05 is responsible for a hyperinducible phenotype. These results demonstrate regions of the ampD gene and the corresponding protein which have functional importance for the induction of AmpC beta-lactamase in E. cloacae.
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118. |
( 1993 ) Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia. PMID : 8500884 : PMC : PMC280873 Abstract >>
Citrobacter freundii biotype 4280 produces attaching and effacing (AE) lesions in the large intestine of laboratory mice and is the causative agent of transmissible murine colonic hyperplasia. AE lesions are also produced by enteropathogenic Escherichia coli in humans. Southern analysis revealed that biotype 4280, but not 20 other strains of C. freundii, contained DNA homologous to the eae (E. coli attaching and effacing) gene which is necessary for AE activity by enteropathogenic E. coli in vitro. We have cloned and determined the nucleotide sequence of the C. freundii eae homolog. Our findings suggest that the eae locus of C. freundii biotype 4280 is necessary for AE activity and has a role in the pathogenesis of transmissible murine colonic hyperplasia.
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119. |
( 1994 ) Cloning and sequencing of the gene for the lactose carrier of Citrobacter freundii. PMID : 7945341 : DOI : 10.1006/bbrc.1994.2407 Abstract >>
The gene coding for the lactose carrier of Citrobacter freundii was cloned into the plasmid pBR322. The gene was sequenced and the amino acid sequence was found to be 70% identical to the lactose carrier of E. coli. All of the charged residues in the membrane spanning region were conserved. The sugar specificity is somewhat different from that of E. coli. The C. freundii carrier has less activity for lactose and more activity for o-nitrophenyl-galactose (ONPG) than the carrier of E. coli.
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120. |
( 1994 ) Intergeneric transfer and recombination of the 6-phosphogluconate dehydrogenase gene (gnd) in enteric bacteria. PMID : 7937867 : DOI : 10.1073/pnas.91.21.10227 PMC : PMC44991 Abstract >>
The gnd gene, encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44), was sequenced in 87 strains of 15 species assigned to five nominal genera of the Enterobacteriaceae, including 36 isolates of Salmonella enterica and 32 strains of Escherichia coli. In S. enterica, the effective (realized) rate of recombination of horizontally transferred gnd sequences is only moderately higher than the rates for other chromosomal housekeeping genes. In contrast, recombination at gnd has occurred with such high frequency in Escherichia coli that the indicated evolutionary relationships among strains are not congruent with those estimated by sequence analysis of other genes and by multilocus enzyme electrophoresis. E. coli and S. enterica apparently have not exchanged gnd sequences, but those of several strains of E. coli have been imported from species of Citrobacter and Klebsiella. The relatively frequent exchange of gnd within and among taxonomic groups of the Enterobacteriaceae, compared with other housekeeping genes, apparently results from its close linkage with genes that are subject to diversifying selection, including those of the rfb region determining the structure of the O antigen polysaccharide.
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121. |
( 1994 ) Cloning and analysis of translational control for genes encoding the Cfr9I restriction-modification system. PMID : 8163180 : DOI : 10.1016/0378-1119(94)90132-5 Abstract >>
The complete type-II Cfr9I restriction-modification (R-M) system of Citrobacter freundii strain RFL9, recognizing the DNA sequence CCCGGG, has been cloned and expressed, and functionally active enzymes have been produced in Escherichia coli. Both the methyltransferase (MTase; M.Cfr9I) and restriction endonuclease (ENase; R.Cfr9I) were found to be encoded on a 2.3-kb cloned fragment in the same transcriptional orientation, but differing in translational phases. The last codon (underlined) (ATGA) of the MTase-encoding gene (Cfr9IM) overlaps with the start codon for the ENase-encoding gene (overlined) (cfr9IR). A nucleotide sequence complementary to a predicted Shine-Dalgarno sequence preceding cfr9IR is within this gene. Predicted free energy (delta G) for formation of the mRNA secondary structure involving these complementary sequences was found to be -16.1 kcal/mol. Amino-acid sequence homology of 80% was found between R.Cfr9I and R.XcyI.
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122. |
Thampapillai G,
Lan R,
Reeves PR,
( 1994 ) Molecular evolution in the gnd locus of Salmonella enterica. PMID : 7815922 : DOI : 10.1093/oxfordjournals.molbev.a040165 Abstract >>
The gnd gene, the structural gene for 6-phosphogluconate dehydrogenase, was sequenced and analyzed in 34 isolates from different serovars of the seven subspecies of Salmonella enterica to provide comparative information on the evolution in this gene, which has been studied extensively in Escherichia coli. The gene tree obtained by the neighbor-joining method in general gave separate branches for each subspecies, with the few exceptions readily explained by recombination. There is evidence of recombination involving transfer of long (more than 400 bp) and short (30-150 bp) segments of DNA. Four of the six long-segment transfers detected are at the 5' end of the gene, and in all four cases a variant of the chi sequence is located close to the recombination junction and appears to have mediated the recombination events. We suggest that in these four cases and in a fifth case with intersubspecies transfer of the whole gnd gene, the adjacent rfb (O antigen) locus may have been transferred in the same event. The estimates of the number of synonymous substitutions per synonymous site, KS, and the number of nonsynonymous substitutions per nonsynonymous site, KA, within the E. coli and S. enterica gnd genes, and also between the two species show an interesting distribution, with KS being lower toward the ends of the gene and KA in particular being lower in the first than in the second domain. In S. enterica, synonymous sites also seem to be subjected to negative selection. The ratio of KA to KS was higher within S. enterica and E. coli than between them, which may indicate that intraspecies variation is essentially between clones and that mildly deleterious mutations can be fixed within clones, which would thus raise KA within species.
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123. |
( 1993 ) Three-dimensional structure of tyrosine phenol-lyase. PMID : 7916622 : DOI : 10.1021/bi00067a006 Abstract >>
Tyrosine phenol-lyase (EC 4.1.99.2) from Citrobacter freundii has been cloned and the primary sequence deduced from the DNA sequence. From the BrCN digest of the NaBH4-reduced holoenzyme, five peptides were purified and sequenced. The amino acid sequences of the peptides agreed with the corresponding parts of the tyrosine phenol-lyase sequence obtained from the gene structure. K257 is the pyridoxal 5'-phosphate binding residue. Assisted by the sequence data, the crystal structure of apotyrosine phenol-lyase, a pyridoxal 5'-phosphate-dependent enzyme, has been refined to an R-factor of 16.2% at 2.3-A resolution using synchrotron radiation diffraction data. The tetrameric molecule has 222 symmetry, with one of the axes coincident with the crystallographic 2-fold symmetry axis of the crystal which belongs to the space group P2(1)2(1)2 with a = 76.0 A, b = 138.3 A, and c = 93.5 A. Each subunit comprises 14 alpha-helices and 16 beta-strands, which fold into a small and a large domain. The coenzyme-binding lysine residue is located at the interface between the large and small domains of one subunit and the large domain of a crystallographically related subunit. The fold of the large, pyridoxal 5'-phosphate binding domain and the location of the active site are similar to that found in aminotransferases. Most of the residues which participate in binding of pyridoxal 5'-phosphate in aminotransferases are conserved in the structure of tyrosine phenol-lyase. Two dimers of tyrosine phenol-lyase, each of which has a domain architecture similar to that found in aspartate aminotransferases, are bound together through a hydrophobic cluster in the center of the molecule and intertwined N-terminal arms.
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124. |
Daniel R,
Boenigk R,
Gottschalk G,
( 1995 ) Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli. PMID : 7721705 : DOI : 10.1128/jb.177.8.2151-2156.1995 PMC : PMC176860 Abstract >>
1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent Km values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 microM, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system.
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125. |
Bishop RE,
Penfold SS,
Frost LS,
Höltje JV,
Weiner JH,
( 1995 ) Stationary phase expression of a novel Escherichia coli outer membrane lipoprotein and its relationship with mammalian apolipoprotein D. Implications for the origin of lipocalins. PMID : 7559452 : DOI : 10.1074/jbc.270.39.23097 Abstract >>
We report a novel outer membrane lipoprotein of Escherichia coli. DNA sequencing between ampC and sugE at the 94.5 min region of the E. coli chromosome revealed an open reading frame specifying 177 amino acid residues. Primer extension analysis demonstrated that the promoter is activated at the transition between exponential and stationary growth phases under control of the rpoS sigma factor gene, and this was confirmed in vivo by monitoring expression of beta-galactosidase activity from a lacZ translational fusion. The amino acid sequence exhibited 31% identity with human apolipoprotein D (apoD), which is a component of plasma high density lipoprotein and belongs to the eukaryotic family of lipocalins. The bacterial lipocalin (Blc) contained a short deletion of 7 amino acid residues corresponding to a hydrophobic surface loop that is thought to facilitate the physical interaction between apoD and high density lipoprotein. However, Blc exhibited a typical prokaryotic lipoprotein signal peptide at its amino terminus. Overexpression, membrane fractionation, and metabolic labeling with [3H]palmitate demonstrated that Blc is indeed a globomycin-sensitive outer membrane lipoprotein. Blc represents the first bacterial member of the family of lipocalins and may serve a starvation response function in E. coli.
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126. |
Blumenberg M,
Yanofsky C,
( 1982 ) Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria. PMID : 6749821 : PMC : PMC221374 Abstract >>
The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.
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127. |
Lloubes RP,
Chartier MJ,
Journet AM,
Varenne SG,
Lazdunski CJ,
( 1984 ) Nucleotide sequence of the gene for the immunity protein to colicin A. Analysis of codon usage of immunity proteins as compared to colicins. PMID : 6383827 : DOI : 10.1111/j.1432-1033.1984.tb08432.x Abstract >>
The nucleotide sequence of the structural gene for the immunity protein to colicin A (cai) has been established. This sequence consists of 534 base pairs. According to the predicted amino acid sequence, the polypeptide chain of this immunity protein comprises 178 amino acids and has a relative molecular mass of 20462. As expected from its localization in the inner membrane, large hydrophobic fragments are found along the polypeptide chain that also contains clusters of mostly positively charged residues. The cai like the ceiA genes encode proteins that are weakly expressed as compared to the corresponding colicins (A and E1). Codon usage reflects this difference. In contrast, the four genes for immunity to cloacin DF13 and to colicin E3 and for these bacteriocins, all of which are highly expressed and are organized in operon, display similar codon usage. These results are discussed with regards to the possible relationship between expressivity and codon usage.
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128. |
Morlon J,
Lloubès R,
Varenne S,
Chartier M,
Lazdunski C,
( 1983 ) Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate. PMID : 6313941 : DOI : 10.1016/s0022-2836(83)80148-x Abstract >>
The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.
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129. |
Morlon J,
Lloubes R,
Chartier M,
Bonicel J,
Lazdunski C,
( 1983 ) Nucleotide sequence of promoter, operator and amino-terminal region of caa, the structural gene of colicin A. PMID : 6641715 : PMC : PMC555186 Abstract >>
The nucleotide sequence of 378 bp covering the promoter-operator regions and the region coding for the N-terminal portion of the colicin A gene was determined. These assignments were made possible by the determination of the N-terminal 12 amino acids of the colicin A protein. DNA sequence homologies between operator regions of recA, lexA, uvrA, uvrB, cea and caa genes are discussed.
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130. |
Daniel R,
Stuertz K,
Gottschalk G,
( 1995 ) Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. PMID : 7635824 : DOI : 10.1128/jb.177.15.4392-4401.1995 PMC : PMC177189 Abstract >>
Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent Kms of the enzyme for glycerol and NAD+ were 1.27 mM and 57 microM, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent Kms were 30 and 70 microM, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the sigma 54 bacterial enhancer-binding protein family.
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131. |
Sawai T,
Yamaguchi A,
Tsukamoto K,
( N/A ) Amino acid sequence, active-site residue, and effect of suicide inhibitors on cephalosporinase of Citrobacter freundii GN346. PMID : 3263684 : DOI : 10.1093/clinids/10.4.721 Abstract >>
The structural gene for a cephalosporinase of Citrobacter freundii GN346 was sequenced. From the nucleotide sequence, the entire amino acid sequence of the mature enzyme with 361 amino acids and a molecular weight of 39,867 was determined. The active-site serine was directly confirmed to be serine 64 by studies in which the enzyme was labeled with dansylpenicillin. In investigations comparing the inhibitory effect of sulbactam (penicillanic acid sulfone) and cloxacillin sulfone on the cephalosporinase and on TEM-2-type penicillinase, sulbactam was found to be an effective progressive inhibitor but a poor competitive inhibitor for the cephalosporinase. The cephalosporinase and the inhibitor formed a long-lived complex with a half-life of 550 minutes. Cloxacillin sulfone could not inactivate the cephalosporinase progressively but irreversibly inactivated the penicillinase.
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132. |
Lindberg F,
Normark S,
( 1986 ) Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene. PMID : 3486121 : DOI : 10.1111/j.1432-1033.1986.tb09601.x Abstract >>
The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.
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133. |
Yeom SJ,
Kim M,
Kwon KK,
Fu Y,
Rha E,
Park SH,
Lee H,
Kim H,
Lee DH,
Kim DM,
Lee SG,
( 2018 ) A synthetic microbial biosensor for high-throughput screening of lactam biocatalysts. PMID : 30498220 : DOI : 10.1038/s41467-018-07488-0 PMC : PMC6265244 Abstract >>
Biocatalytic cyclization is highly desirable for efficient synthesis of biologically derived chemical substances, such as the commodity chemicals �`-caprolactam and �_-valerolactam. To identify biocatalysts in lactam biosynthesis, we develop a caprolactam-detecting genetic enzyme screening system (CL-GESS). The Alcaligenes faecalis regulatory protein NitR is adopted for the highly specific detection of lactam compounds against lactam biosynthetic intermediates. We further systematically optimize the genetic components of the CL-GESS to enhance sensitivity, achieving 10-fold improvement. Using this highly sensitive GESS, we screen marine metagenomes and find an enzyme that cyclizes �s-amino fatty acids to lactam. Moreover, we determine the X-ray crystal structure and catalytic residues based on mutational analysis of the cyclase. The cyclase is also used as a helper enzyme to sense intracellular �s-amino fatty acids. We expect this simple and accurate biosensor to have wide-ranging applications in rapid screening of new lactam-synthesizing enzymes and metabolic engineering for lactam bio-production.
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134. |
Yamaguchi A,
Adachi H,
Sawai T,
( 1987 ) Identification of the active site of Citrobacter freundii beta-lactamase using dansyl-penicillin. PMID : 3496243 : DOI : 10.1016/0014-5793(87)81031-1 Abstract >>
The active site sequence of a beta-lactamase encoded by chromosomal gene(s) in Citrobacter freundii GN346 was determined using dansyl-penicillin as a fluorescent probe. The tryptic digest of the labelled enzyme gave a fluorescent peptide containing 22 amino acids. The sequence of this peptide was identical to the consensus sequence of class C beta-lactamases, Gly-Ser-X-Ser-Lys. The residue labelled was the serine adjacent to the glycine. The active site sequence corresponded to positions 46-67 of the entire sequence of the Citrobacter freundii beta-lactamase determined on the basis of the DNA sequence of the structural gene [(1986) Eur. J. Biochem. 156, 441-445]. The labelled serine corresponded to Ser-64.
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135. |
Cavard D,
Lloubès R,
Morlon J,
Chartier M,
Lazdunski C,
( 1985 ) Lysis protein encoded by plasmid ColA-CA31. Gene sequence and export. PMID : 3889552 : DOI : 10.1007/bf00327516 Abstract >>
A gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion. The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A. Transcription of cal is in the same direction as caa, that is in the opposite direction to cai. Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins. The C-terminal part also contains a core of hydrophobic residues. The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13. The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A. The cal protein undergoes slow processing with loss of the N-terminal "signal" region and the mature form is released into the medium together with colicin A.
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136. |
Amos GCA,
Ploumakis S,
Zhang L,
Hawkey PM,
Gaze WH,
Wellington EMH,
( 2018 ) The widespread dissemination of integrons throughout bacterial communities in a riverine system. PMID : 29374269 : DOI : 10.1038/s41396-017-0030-8 PMC : PMC5864220 Abstract >>
Anthropogenic inputs increase levels of antimicrobial resistance (AMR) in the environment, however, it is unknown how these inputs create this observed increase, and if anthropogenic sources impact AMR in environmental bacteria. The aim of this study was to characterise the role of waste water treatment plants (WWTPs) in the dissemination of class 1 integrons (CL1s) in the riverine environment. Using sample sites from upstream and downstream of a WWTP, we demonstrate through isolation and culture-independent analysis that WWTP effluent significantly increases both CL1 abundance and antibiotic resistance in the riverine environment. Characterisation of CL1-bearing isolates revealed that CL1s were distributed across a diverse range of bacteria, with identical complex genetic resistance determinants isolated from both human-associated and common environmental bacteria across connected sites. Over half of sequenced CL1s lacked the 3'-conserved sequence ('atypical' CL1s); surprisingly, bacteria carrying atypical CL1s were on average resistant to more antibiotics than bacteria carrying 3'-CS CL1s. Quaternary ammonium compound (QAC) resistance genes were observed across 75% of sequenced CL1 gene cassette arrays. Chemical data analysis indicated high levels of boron (a detergent marker) downstream of the WWTP. Subsequent phenotypic screening of CL1-bearing isolates demonstrated that ~90% were resistant to QAC detergents, with in vitro experiments demonstrating that QACs could solely select for the transfer of clinical antibiotic resistance genes to a naive Escherichia coli recipient. In conclusion, this study highlights the significant impact of WWTPs on environmental AMR, and demonstrates the widespread carriage of clinically important resistance determinants by environmentally associated bacteria.
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137. |
Anderson MT,
Mitchell LA,
Zhao L,
Mobley HLT,
( 2018 ) Citrobacter freundii fitness during bloodstream infection. PMID : 30087402 : DOI : 10.1038/s41598-018-30196-0 PMC : PMC6081441 Abstract >>
Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.
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138. |
Shiuan D,
Campbell A,
( 1988 ) Transcriptional regulation and gene arrangement of Escherichia coli, Citrobacter freundii and Salmonella typhimurium biotin operons. PMID : 2971595 : DOI : 10.1016/0378-1119(88)90397-6 Abstract >>
The bio operons of Citrobacter freundii and Escherichia coli K-12 (strain C600) were isolated by screening lambda banks for complementation of E. coli bio mutants. These were compared with the previously isolated bio operon of Salmonella typhimurium and previous data on E. coli K-12. The restriction maps of the operon are very different in the three species, but no difference in gene order was found. Operator-promoter DNA, identified by repressible titration and by biotin-repressible transcription in E. coli, was sequenced and compared to the published E. coli K-12 sequence. In the segment previously identified as operator/bioB promoter, C. freundii and S. typhimurium DNA are identical and differ from E. coli only by 2 bp. The DNA to the right of this segment (indicated by previous data to be the bioA promoter of E. coli) has diverged in all three species, and only E. coli has a sequence resembling a consensus promoter.
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139. |
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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140. |
Burke KA,
Wilcox G,
( 1987 ) The araC gene of Citrobacter freundii. PMID : 2965663 : DOI : 10.1016/0378-1119(87)90188-0 Abstract >>
The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.
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141. |
Liu L,
Lan R,
Liu L,
Wang Y,
Zhang Y,
Wang Y,
Xu J,
( 2017 ) Antimicrobial Resistance and Cytotoxicity of Citrobacter spp. in Maanshan Anhui Province, China. PMID : 28775715 : DOI : 10.3389/fmicb.2017.01357 PMC : PMC5518651 Abstract >>
Objectives:Citrobacter spp. especially Citrobacter freundii, is frequently causing nosocomial infections, and increasingly becoming multi-drug resistant (MDR). In this study, we aimed to determine the genetic diversity and relationships of Citrobacter spp. from diarrheal patients and food sources, their antimicrobial resistance profiles and in vitro virulence properties. Methods: Sixty two Citrobacter isolates, including 13 C. freundii, 41 C. youngae and eight C. braakii isolates, were obtained from human diarrheal patients and food sources. Multilocus Sequence Typing (MLST) of seven housekeeping genes and antimicrobial susceptibility testing using the broth microdilution method according to CLSI recommendations were carried out. Adhesion and cytotoxicity to HEp-2 cells were performed. PCR and sequencing were used to identify blaCTX-M, blaSHV, blaTEM and qnr genes. Results: The 62 isolates were divided into 53 sequence types (STs) with all STs being novel, displaying high genetic diversity. ST39 was a predominant ST shared by 5 C. youngae strains isolated from four foods and a diarrheal patient. All isolates were resistant to cefoxitin, and sensitive to imipenem, meropenem and amikacin. The majority of Citrobacter isolates (61.3%) were MDR of three or more antibiotics out of the 22 antibiotics tested. Two C. freundii isolates each carried the blaTEM-1 gene and a variant of qnrB77. Three Citrobacter isolates each carried qnrS1 and aac(6')-Ib-cr genes. Seven isolates that showed strong cytotoxicity to HEp-2 cells were MDR. Conclusions:Citrobacter spp. from human and food sources are diverse with variation in virulence properties and antibiotic resistance profiles. Food may be an important source of Citrobacter species in transmission to humans. C. freundii and C. youngae are potential foodborne pathogens.
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142. |
Guarino A,
Giannella R,
Thompson MR,
( 1989 ) Citrobacter freundii produces an 18-amino-acid heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heat-stable enterotoxin (ST Ia). PMID : 2912902 : PMC : PMC313149 Abstract >>
We purified and sequenced the heat-stable enterotoxin produced by Citrobacter freundii. The toxin was detected during purification by reaction with monoclonal antibody to Escherichia coli heat-stable enterotoxin. The C. freundii toxin amino acid sequence was identical to that of the 18-amino-acid heat-stable enterotoxin (STa) produced by toxigenic E. coli.
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143. |
Ma L,
Yin Z,
Zhang D,
Zhan Z,
Wang Q,
Duan X,
Gao H,
Liang Q,
Zhao Y,
Feng J,
Zhao Y,
Tong Y,
Dai E,
Zhou D,
( 2017 ) Comparative genomics of type 1 IncC plasmids from China. PMID : 29140102 : DOI : 10.2217/fmb-2017-0072 Abstract >>
This study dealt with genomic characterization of type 1 IncC resistance plasmids, capable of spreading across taxonomic borders, from China. p112298-tetA was sequenced and compared with type 1 IncC reference plasmid pR148 and two available sequenced type 1 IncC plasmids pHS36-NDM and pVAS3-1 from China. These plasmids contained one or more exogenous resistance islands, which included the ARI-A islands, the ARI-B islands, the ISEcp1-blaCMY units and the bla KPC-2 region and were inserted at various sites in the IncC backbone and thus represented three distinct lineages. Complex rearrangement and homologous recombination events have occurred during evolution of p112298-tetA, making it significantly differ modularly from the other three plasmids with respect to both plasmid backbone and exogenous resistance regions.
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144. |
( 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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145. |
Preston KE,
Tine JA,
( 2017 ) Acquisition of a second multi-drug resistance-encoding element by IncM1 plasmid pACM130 abolished conjugative transfer. PMID : 28571994 : DOI : 10.1016/j.plasmid.2017.05.003 Abstract >>
Within the IncM plasmid family there is a lineage that has a transposon Tn1721-based multiple-resistance island inserted in the backbone gene mucB. So far, this group includes R1215, p202c, pIGT15, pARM26, and pACM1, from Europe and the USA. A new member of this group, pACM130, was isolated at the same American hospital as pACM1 and has a similar resistance island, but also carries a copy of Tn1331 that interrupts the traY gene in the conjugation operon. The conjugative phenotype of this plasmid has been abolished, though pACM130 could be mobilized by an intact traY cloned into a laboratory vector and transformed into the same donor bacterium.
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146. |
( 2013 ) Complete sequence of the IncT-type plasmid pT-OXA-181 carrying the blaOXA-181 carbapenemase gene from Citrobacter freundii. PMID : 23357767 : DOI : 10.1128/AAC.01297-12 PMC : PMC3623325 Abstract >>
The gene encoding the carbapenemase OXA-181 (an OXA-48 variant) was identified from a Citrobacter freundii isolate coproducing NDM-1. The whole sequence of plasmid pT-OXA-181 bearing the blaOXA-181 gene was determined and revealed a 84-kb mobilizable but non-self-conjugative IncT-type plasmid. It totally differs from the 7.6-kb ColE-type and blaOXA-181-bearing plasmid recently identified in a Klebsiella pneumoniae isolate. However, in both plasmids, insertion sequence ISEcp1 might have played a role in acquisition of the blaOXA-181 gene.
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147. |
( 2013 ) Complex integrons containing qnrB4-ampC (bla(DHA-1)) in plasmids of multidrug-resistant Citrobacter freundii from wastewater. PMID : 23461518 : DOI : 10.1139/cjm-2012-0576 Abstract >>
Microbial populations in wastewater treatment plants (WWTPs) are increasingly being recognized as environmental reservoirs of antibiotic resistance genes. PCR amplicons for plasmid-mediated quinolone resistance determinants qnrA, qnrB, and qnrS were recorded in samples from a WWTP in Vancouver, British Columbia. Six strains of ciprofloxacin-resistant Citrobacter freundii were isolated and found to carry mutations in gyrA and parC, as well as multiple plasmid-borne resistance genes, collectively including qnrB; aac(6')-Ib-cr; �]-lactamase-encoding genes from molecular classes A (blaTEM-1), C (ampC), D (blaOXA-1, blaOXA-10); and genes for resistance to 5 other types of antibiotics. In 3 strains, large (>60 kb) plasmids carried qnrB4 and ampC as part of a complex integron in a 14 kb arrangement that has been reported worldwide but, until recently, only among pathogenic strains of Klebsiella. Analysis of single-nucleotide polymorphisms in the qnrB4-ampC regions infers 2 introductions into the WWTP environment. These results suggest recent passage of plasmid-borne fluoroquinolone and �]-lactam resistance genes from pathogens to bacteria that may be indigenous inhabitants of WWTPs, thus contributing to an environmental pool of antibiotic resistance.
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148. |
Revtovich SV,
Morozova EA,
Kulikova VV,
Anufrieva NV,
Osipova TI,
Koval VS,
Nikulin AD,
Demidkina TV,
( 2017 ) Crystal structure of mutant form Cys115His of Citrobacter freundii methionine �^-lyase complexed with l-norleucine. PMID : 28602917 : DOI : 10.1016/j.bbapap.2017.06.001 Abstract >>
The mutant form of Citrobacter freundii methionine �^-lyase with the replacement of active site Cys115 for His has been found to be inactive in the �^-elimination reaction of methionine while fully active in the �^-elimination reaction of O-acetyl-l-homoserine and in the �]-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45? resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the �^- and �]-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.
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149. |
( 2013 ) Characterization of qnrB-like genes in Citrobacter species of the American Type Culture Collection. PMID : 23529729 : DOI : 10.1128/AAC.02396-12 PMC : PMC3716155 Abstract >>
Among five American Type Culture Collection (ATCC) Citrobacter strains, qnrB60 in Citrobacter freundii ATCC 6879, an isolate from the preantibiotic era, and qnrB61 in Citrobacter braakii ATCC 51113(T), a type strain belonging to the C. freundii complex, were identified. Meanwhile, a truncated qnrB-like pseudogene was identified in C. freundii ATCC 8090(T) and ATCC 43864. No qnrB-like sequence was found in Citrobacter koseri ATCC 27028(T). These findings underscore the close relationship between this species and qnrB.
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150. |
( 1990 ) Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. PMID : 2300174 : DOI : 10.1038/343284a0 Abstract >>
Beta-Lactamases (EC 3.5.2.6, 'penicillinases') are a family of enzymes that protect bacteria against the lethal effects of cell-wall synthesis of penicillins, cephalosporins and related antibiotic agents, by hydrolysing the beta-lactam antibiotics to biologically inactive compounds. Their production can, therefore, greatly contribute to the clinical problem of antibiotic resistance. Three classes of beta-lactamases--A, B and C--have been identified on the basis of their amino-acid sequence; class B beta-lactamases are metalloenzymes, and are clearly distinct from members of class A and C beta-lactamases, which both contain an active-site serine residue involved in the formation of an acyl enzyme with beta-lactam substrates during catalysis. It has been predicted that class C beta-lactamases share common structural features with D,D-carboxypeptidases and class A beta-lactamases, and further, suggested that class A and class C beta-lactamases have the same evolutionary origin as other beta-lactam target enzymes. We report here the refined three-dimensional structure of the class C beta-lactamase from Citrobacter freundii at 2.0-A resolution and confirm the predicted structural similarity. The refined structure of the acyl-enzyme formed with the monobactam inhibitor aztreonam at 2.5-A resolution defines the enzyme's active site and, along with molecular modelling, indicates a mechanism for beta-lactam hydrolysis. This leads to the hypothesis that Tyr 150 functions as a general base during catalysis.
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151. |
( 2013 ) Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump. PMID : 22969080 : DOI : 10.1093/jac/dks357 Abstract >>
To characterize the pNDM-CIT plasmid identified in Citrobacter freundii carrying genes encoding the metallo-�]-lactamase NDM-1 and the 16S RNA methylase ArmA. The complete DNA sequence of pNDM-CIT was obtained by using the 454-Genome Sequencer FLX procedure on a library obtained using plasmid DNA purified from the pNDM-CIT Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Comparative analysis with IncHI1 incompatibility group plasmids was performed using BLASTN and BLASTP algorithms. Plasmid pNDM-CIT was 288::920 bp and revealed an IncHI1 plasmid scaffold, showing novel resistance and potential virulence determinants. The bla(NDM-1) gene was identified within a novel genetic context, flanked by a duplication of the class 1 integron on both sides. The replicase gene repAciN, originating from Acinetobacter spp. plasmids, was identified in a close association with the Tn1548::armA transposon and the macrolide resistance mel-mph2 cluster. The same structure was identified in silico from a series of enterobacterial plasmids carrying the armA gene. The repAciN gene probably represents a remnant sign of the original occurrence of the armA gene in Acinetobacter plasmids. A CP4-like prophage sequence was identified in pNDM-CIT, containing a resistance-nodulation-cell division/multidrug resistance (RND/MDR) efflux pump cluster surrounded by two IS1-like elements. This resistance determinant, associated with such a prophage sequence, has never been reported on plasmids. Plasmid pNDM-CIT differed significantly from all known bla(NDM-1)-carrying plasmids identified in Enterobacteriaceae, since it combines the metallo-�]-lactamase NDM-1, the 16S RNA methylase ArmA and a cryptic prophage carrying the RND/MDR efflux pump.
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152. |
( 1998 ) The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death. PMID : 9677290 : DOI : 10.1006/jmbi.1998.1894 Abstract >>
Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations by means of post-segregational killing. However, several chromosome-encoded addiction modules may provide an entirely distinct function in the programmed cell death of moribund subpopulations under starvation conditions. We now report a novel chromosomal bacteriolytic module of Escherichia coli called the entericidin locus, which is activated in stationary phase under high osmolarity conditions by sigmaS and simultaneously repressed by the osmoregulatory EnvZ/OmpR signal transduction pathway. The entericidin locus encodes tandem paralogous genes (ecnAB) and directs the synthesis of two small cell-envelope lipoproteins. An attenuator precedes ecnA and an ompR-sensitive sigmaS promoter governs expression of ecnB. The entericidin A lipoprotein is an antidote to the bacteriolytic lipoprotein entericidin B. The entericidins are predicted to adopt amphipathic alpha-helical structures and to reciprocally modulate membrane stability. The entericidin locus is not present on any known plasmids, but is conserved in the homologous region of the Citrobacter freundii chromosome. Although the cloned C. freundii entericidin locus is expressed in E. coli independently of ompR, it carries an additional ompR-like gene called ecnR. The organization of the entericidin locus as a chromosomal antidote/toxin gene pair, which is regulated by both positive and negative osmotic signals during starvation, is consistent with an emerging paradigm of programmed bacterial cell death.
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153. |
( 1998 ) gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. PMID : 9756773 : PMC : PMC105915 Abstract >>
Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 microg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.
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154. |
( 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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155. |
( 1997 ) Comparison of ViaB regions of Vi-positive organisms. PMID : 9418239 : DOI : 10.1111/j.1574-6968.1997.tb12752.x Abstract >>
We cloned the vipR genes from Salmonella paratyphi C, S. dublin, and Citrobacter freundii strains and compared them with the S. typhi sequence to clarify the genetic relationship of the ViaB regions of Vi-positive organisms. ViaB regions were divided into two groups based on their sequences, the Salmonella and C. freundii groups. The vipR coding sequences of the Salmonella group were identical. Southern blot hybridization results using the full-length ViaB region as a probe support these findings.
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156. |
( 1997 ) Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers. PMID : 9439003 : Abstract >>
The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.
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157. |
( 1998 ) Ceftazidime-resistant Enterobacteriaceae isolates from three Polish hospitals: identification of three novel TEM- and SHV-5-type extended-spectrum beta-lactamases. PMID : 9517925 : PMC : PMC105491 Abstract >>
Twelve ceftazidime-resistant isolates of the family Enterobacteriaceae (11 Klebsiella pneumoniae isolates and 1 Escherichia coli isolate) were collected in 1995 from three Polish hospitals located in different cities. All were identified as producers of extended-spectrum beta-lactamases (ESBLs). Detailed analysis of their beta-lactamase contents revealed that six of them expressed SHV-5-like ESBLs. The remaining six were found to produce three different TEM enzymes, each characterized by a pI value of 6.0 and specified by new combinations of amino acid substitutions. The amino acid substitutions compared to the TEM-1 beta-lactamase sequence were Gly238Ser, Glu240Lys, and Thr265Met for TEM-47; Leu21Phe, Gly238Ser, Glu240Lys, and Thr265Met for TEM-48; and Leu21Phe, Gly238Ser, Glu240Lys, Thr265Met, and Ser268Gly for TEM-49. The new TEM beta-lactamases, TEM-47, TEM-48, and TEM-49, belong to a subfamily of TEM-2-related enzymes. Genes coding for TEM-47 and TEM-49 could have originated from the TEM-48-encoding sequence by various single genetic events. The new TEM derivatives probably document the already advanced microevolution of ESBLs ongoing in Polish hospitals, in a majority of which no monitoring of ESBL producers was performed before 1996.
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158. |
( 1998 ) Phylogenetic relationships of Salmonella based on DNA sequence comparison of atpD encoding the beta subunit of ATP synthase. PMID : 9561735 : DOI : 10.1111/j.1574-6968.1998.tb12933.x Abstract >>
DNA sequences covering 57% of atpD encoding the beta subunit of ATP synthase were determined for 16 strains of Salmonella enterica, two strains of S. bongori, and one strain each of Citrobacter freundii and Yersinia enterocolitica, and comparison was made with the published Escherichia coli and Enterobacter aerogenes sequences. The phylogenetic tree based on maximum-likelihood analysis showed separation of the subspecies of S. enterica except for two serotypes of subspecies II which were unsupported by a common node. The two serotypes of S. bongori were separated from S. enterica and related to the serotypes of subspecies II. A tight relationship was found between S. enterica subspecies IIIa consisting of monophasic serotypes and subspecies IIIb consisting of diphasic serotypes. This is in conflict with results obtained for most other housekeeping genes and the 23S rRNA gene separating mono- from diphasic subspecies.
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159. |
( 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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( 1998 ) Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. PMID : 9559791 : PMC : PMC105550 Abstract >>
A group of cefotaxime-resistant Citrobacter freundii and Escherichia coli isolates were collected by a clinical laboratory in a hospital in Warsaw, Poland, in July 1996. Detailed analysis has shown that all of these produced a beta-lactamase (pI, 8.4) belonging to the CTX-M family, one of the minor extended-spectrum beta-lactamase families with a strong cefotaxime-hydrolyzing activity. Sequencing has revealed that C. freundii isolates produced a new CTX-M-3 enzyme which is very closely related to the CTX-M-1/MEN-1 beta-lactamase, sporadically identified in Europe over a period of 6 years. Amino acid sequences of these two beta-lactamases differ at four positions: Val77Ala, Asp114Asn, Ser140Ala, and Asn288Asp (the first amino acid of each pair refers to CTX-M-1/MEN-1 and second refers to CTX-M-3). The partial sequence of the E. coli CTX-M gene was identical to the corresponding region of bla(CTX-M-3), but a transconjugant of the E. coli isolate expressed higher levels of resistance to beta-lactams than did C. freundii transconjugants. These resistance differences correlated with differences in plasmid DNA restriction patterns. Our results suggest that CTX-M genes have been spread among different species of the family Enterobacteriaceae in the hospital and that the CTX-M-3-expressing C. freundii strain causing routine urinary tract infections has been maintained for a relatively long time in the hospital environment.
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