CMY-31 and CMY-36 cephalosporinases encoded by ColE1-like plasmids.
ABSTRACT: Two CMY-2 derivatives, CMY-31 (Gln(215)-->Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala(77)-->Cys and Gln(193)-->Glu) from Klebsiella pneumoniae, were characterized. Both cephalosporinases functionally resembled CMY-2. bla(CMY) alleles occurred as parts of a putative transposon comprising ISEcp1B and a Citrobacter freundii-derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca.
Project description:This study exploited the possibility to detect Citrobacter freundii-derived CMY-2-like cephalosporinases in Enterobacteriaceae clinical isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Periplasmic proteins were prepared using a modified sucrose method and analyzed by MALDI-TOF MS. A ca. 39,850-m/z peak, confirmed to represent a C. freundii-like ?-lactamase by in-gel tryptic digestion followed by MALDI-TOF/TOF MS, was observed only in CMY-producing isolates. We have also shown the potential of the assay to detect ACC- and DHA-like AmpC-type ?-lactamases.
Project description:Twenty-nine Proteus mirabilis isolates from 17 Polish hospitals were analyzed. The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to beta-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers. Indeed, beta-lactamases with a pI of approximately 9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants. The enzymes were of Citrobacter freundii origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones. The bla(CMY) genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of approximately 10 kb. In the CMY-12- and -15-producing isolates, an additional fragment of approximately 4.5 kb hybridized with the bla(CMY) probe as well, which could have arisen from a duplication event during the evolution of the genes. In all of the isolates, the ISEcp1 mobile element, which most probably is involved in mobilization of the C. freundii ampC gene, was placed at the same distance from the 5' ends of the bla(CMY) genes, and sequences located between them were identical in isolates carrying each of the four genes. These data suggested that a single chromosome-to-chromosome transfer of the ampC gene from C. freundii to P. mirabilis could have initiated the spread and evolution of the AmpC-producing P. mirabilis in Poland. The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the P. mirabilis isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates.
Project description:Plasmid pTKH11, originally obtained by electroporation of a Klebsiella oxytoca plasmid preparation into Escherichia coli XAC, expressed a high level of an AmpC-like beta-lactamase. The enzyme, designated CMY-5, conferred resistance to extended-spectrum beta-lactams in E. coli; nevertheless, the phenotype was cryptic in the K. oxytoca donor. Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 beta-lactamase (blaCMY-5). The blaCMY-5 product was similar to the plasmidic CMY-2 beta-lactamase of K. pneumoniae and the chromosomal AmpC of Citrobacter freundii, with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2. blaCMY-5 was followed by the Blc and SugE genes of C. freundii, and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome of C. freundii; these results confirmed that C. freundii AmpC was the evolutionary origin of the plasmidic cephamycinases. In the K. oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63. Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible. Incompatibility of the two plasmids might explain the cryptic phenotype of blaCMY-5 in K. oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.
Project description:CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-30) and Ser (CMY-42) for Val211 in the ?-loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino-?-lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oximino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the ?-loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site (?-loop-Q120 loop-R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the covalently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adaptation of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).
Project description:Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.
Project description:In the last decade, detection of antibiotic resistant bacteria from wildlife has received increasing interest, due to the potential risk posed by those bacteria to wild animals, livestock or humans at the interface with wildlife, and due to the ensuing contamination of the environment. According to World Health Organization, cephalosporins are critically important antibiotics to human health. However, acquired resistance to β-lactams is widely distributed and is mainly mediated by extended-spectrum beta-lactamase and AmpC beta-lactamases, such as cephalosporinases. This work thus aimed to compile and analyse the information available on the emergence and dissemination of cephalosporinases in wildlife worldwide. Results suggest a serious scenario, with reporting of cephalosporinases in 46 countries from all continents (52% in Europe), across 188 host species, mainly birds and mammals, especially gulls and ungulates. The most widely reported cephalosporinases, CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2, were also the most common in wild animals, in agreement with their ubiquity in human settings, including their association to high-risk clones of <i>Escherichia coli</i> (<i>E. coli</i>), such as the worldwide distributed CTX-M-15/ST131 <i>E. coli</i>. Altogether, our findings show that anthropogenic activities affect the whole ecosystem and that public policies promoting animal and environmental surveillance, as well as mitigation measures to avoid antimicrobial misuse and AMR spread, are urgently needed to be out in practise.
Project description:The aim of the present study was to investigate the epidemiological link of multidrug-resistant Klebsiella oxytoca isolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification of bla gene types and the mapping of the integron-containing metallo-?-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistant K. oxytoca clinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the same K. oxytoca clonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 ?-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifying bla(OXY) gene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistant K. oxytoca isolates causing extrahospital infections.
Project description:While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.
Project description:We report multifocal detection (four different cities in northern Italy) of Proteus mirabilis isolates resistant to both oxyimino- and 7-alpha-methoxy-cephalosporins and producing a novel acquired AmpC-like beta-lactamase. The enzyme, named CMY-16, is a variant of the CMY/LAT lineage, which differs from the closest homologues, CMY-4 and CMY-12, by a single amino acid substitution (A171S or N363S, respectively) and from CMY-2 by two substitutions (A171S and W221R). Expression of the cloned bla(CMY-16) gene in Escherichia coli decreased susceptibility to penicillins, cephalosporins, and aztreonam. Tazobactam was more effective than clavulanate at antagonizing the enzyme activity. Genotyping, by random amplification of polymorphic DNA and pulsed-field gel electrophoresis of genomic DNA digested with SfiI, showed that isolates were clonally related to each other, although not identical. The bla(CMY-16) gene was not transferable to E. coli by conjugation or transformation. In all isolates, it was chromosomally located and inserted in a conserved genetic environment. PCR mapping experiments revealed that the bla(CMY-16) was flanked by ISEcp1 and the blc gene, similar to other genes of this lineage from plasmids of Salmonella enterica, Klebsiella spp., and E. coli. Overall, these results revealed multifocal spreading of a CMY-16-producing P. mirabilis clone in northern Italy. This finding represents the first report of an acquired AmpC-like beta-lactamase in Proteus mirabilis from Italy and underscores the emergence of similar resistance determinants in the European setting.
Project description:Background:CMY-2 is the most prevalent pAmpC ?-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. Methods:Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1-PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. Results:The blaCMY-2-containing region (ISEcp1-blaCMY-2-?blc-?yggR-?tnp1-orf7-orf8-orf9-?tnp2-?hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10-8-10-9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. Conclusions:Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure.