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Johnson T.J.,University of Minnesota | Shepard S.M.,University of Minnesota | Rivet B.,University of Minnesota | Danzeisen J.L.,University of Minnesota | Carattoli A.,Parasitic and Immuno Mediated Diseases
Plasmid | Year: 2011

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the bla TEM and bla CMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of bla TEM and bla CMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC. © 2011 Elsevier Inc.

Villa L.,Parasitic and Immuno Mediated Diseases | Poirel L.,University Paris - Sud | Nordmann P.,University Paris - Sud | Carta C.,Oncology and Molecular Medicine | Carattoli A.,Parasitic and Immuno Mediated Diseases
Journal of Antimicrobial Chemotherapy | Year: 2012

Objectives: To characterize plasmid pNDM-MAR recovered from a Klebsiella pneumoniae isolate of sequence type (ST) 15 from Morocco, carrying the genes encoding the metallo-β-lactamase NDM-1, the extended-spectrum β-lactamase (ESBL) CTX-M-15 and the qnrB1 quinolone resistance determinant. Methods: The plasmid DNA sequence was obtained by using the 454-Genome Sequencer FLX procedure on a library constructed from total plasmid DNA obtained from an Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Results: Plasmid pNDM-MAR was 267 242 bp long and encoded 177 predicted proteins. It harboured novel replicons and transfer loci, defining a novel plasmid type within the IncH plasmid family. The bla NDM-1 gene was flanked by genetic elements that are distinct from those observed in other bla NDM-1-positive plasmids, including the groES and groEL chaperonin genes. This plasmid harboured the ESBL gene bla CTX-M-15 together with the quinolone resistance gene qnrB1. In addition, it harboured genes encoding resistance to tellurite, mercury, chloramphenicol and aminoglycosides. Interestingly, pNDM-MAR did not carry any 16S rRNA methylase gene, in contrast to other bla NDM-1-positive plasmids. Conclusions: This study underlines the diversity of genetic vehicles involved in the spread of the bla NDM-1 gene. Plasmid pNDM-MAR differed significantly from all known bla NDM-1-bearing plasmids. Comparative analysis of the pNDM-MAR sequence identified a novel type of IncH plasmid. © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Garcia-Fernandez A.,Parasitic and Immuno Mediated Diseases | Miriagou V.,Hellenic Pasteur Institute | Papagiannitsis C.C.,Hellenic Pasteur Institute | Giordano A.,University of Rome La Sapienza | And 3 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2010

Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and β-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem- and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Towner K.J.,University of Nottingham | Evans B.,University of Edinburgh | Evans B.,University of Manchester | Villa L.,Parasitic and Immuno Mediated Diseases | And 4 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2011

Ninety-six genetically diverse multidrug-resistant clinical isolates of Acinetobacter baumannii from 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D β-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase (rep) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups of rep genes. Groups detected were GR6 (repAci6; 93 isolates), GR2 (including repAci1 [67 isolates] and repAci2 [3 isolates]), GR16 (repApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 (repAci3; 4 isolates), GR4 (repAci4; 3 isolates), GR10 (repAciX; 1 isolate), and GR14 (repp4AYE; 1 isolate). Variations in rep gene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of the repAci1, repAci3, repAci4, and repAciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated with repAci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated with repAci1. Most intrinsic Acinetobacter plasmids are non-self-transferable, but the almost ubiquitous repAci6 gene was strongly associated with a potential tra locus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains of A. baumannii. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Carattoli A.,Parasitic and Immuno Mediated Diseases | Villa L.,Parasitic and Immuno Mediated Diseases | Poirel L.,University Paris - Sud | Bonnin R.A.,University Paris - Sud | Nordmann P.,University Paris - Sud
Antimicrobial Agents and Chemotherapy | Year: 2012

The blaNDM-1 gene has been reported to be often located on broad-host-range plasmids of the IncA/C type in clinical but also environmental bacteria recovered from the New Delhi, India, area. IncA/C-type plasmids are the main vehicles for the spread of the cephalosporinase gene blaCMY-2, frequently identified in the United States, Canada, and Europe. In this study, we completed the sequence of IncA/C plasmid pNDM-KN carrying the bla NDM-1 gene, recovered from a Klebsiella pneumoniae isolate from Kenya. This sequence was compared with those of three IncA/C-type reference plasmids from Escherichia coli, Yersinia ruckeri, and Photobacterium damselae. Comparative analysis showed that the blaNDM-1 gene was located on a widely diffused plasmid scaffold known to be responsible for the spread of blaCMY-2-like genes and consequently for resistance to broad-spectrum cephalosporins. Considering that IncA/C plasmids possess a broad host range, this scaffold might support a large-scale diffusion of the blaNDM-1 gene among Gram-negative rods. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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