Alessandra Carattoli

Resistance Plasmid Families in Enterobacteriaceae

Bacteria carry extrachromosomal, self-replicating genetic elements called plasmids. A plasmid is defined as a double-stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under nonstressed conditions ([109

Dissemination of Clonally Related Escherichia coli Strains Expressing Extended-Spectrum β-Lactamase CTX-M-15

E. coli ST131 and ST405 and multidrug-resistant IncFII plasmids may determine spread of this lactamase.

In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

ABSTRACT In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49 S. Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.

Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants

OBJECTIVES IncF plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance and virulence genes. These plasmids are usually heterogeneous in size and carry multiple replicons, and technical difficulties can impair the comparison and detection of related plasmids by restriction fragment length polymorphism analysis. We devised a rapid sequence-based typing scheme to categorize the members of this plasmid family into homogeneous groups. METHODS We compared the available IncF replicon sequences, identifying the combination of the different IncF replicon alleles as the discriminating characteristic of these plasmid scaffolds. An IncF typing method based on PCR amplification and sequence typing of the IncF replicons was devised. A collection of IncF plasmids carrying resistance and/or virulence genes, identified in strains from different sources and geographical origins, was tested with this typing system. RESULTS We devised a replicon sequence typing (RST) scheme discriminating IncF plasmid variants. This system was tested on the collection of IncF plasmids, demonstrating that it was useful for the discrimination of plasmids carrying the same resistance gene (i.e. the bla(CTX-M-15) gene), but also recognized strictly related virulence plasmids (i.e. IncFIme plasmids). The PCR-based replicon typing (PBRT) system was also updated by including new primer pairs to allow the identification of the Salmonella, Klebsiella and Yersinia IncF plasmids. CONCLUSIONS The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.

Plasmids and the spread of resistance.

Plasmids represent one of the most difficult challenge for counteracting the dissemination of antimicrobial resistance. They contribute to the spread of relevant resistance determinants, promoting horizontal gene transfer among unrelated bacteria. Undistinguishable plasmids were identified in unrelated bacterial strains isolated at huge geographically distant area, with no apparent epidemiological links. These plasmids belong to families that are largely prevalent in naturally occurring bacteria, usually carry multiple physically linked genetic determinants, conferring resistance to different classes of antibiotics simultaneously. Plasmids also harbour virulence factors and addiction systems, promoting their stability and maintenance in the bacterial host, in different environmental conditions. The characteristics of the most successful plasmids that were at the origin of the spread of carbapenemase, expanded-spectrum β-lactamase, and plasmid-mediated quinolone resistance genes are discussed in this review.

Complete Nucleotide Sequences of Plasmids pEK204, pEK499, and pEK516, Encoding CTX-M Enzymes in Three Major Escherichia coli Lineages from the United Kingdom, All Belonging to the International O25:H4-ST131 Clone

ABSTRACT We determined the complete nucleotide sequences of three plasmids that encode CTX-M extended-spectrum β-lactamases (ESBLs) in pulsed-field gel electrophoresis-defined United Kingdom variants (strains A, C, and D) of the internationally prevalent Escherichia coli O25:H4-ST131 clone. Plasmid pEK499 (strain A; 117,536 bp) was a fusion of type FII and FIA replicons and harbored the following 10 antibiotic resistance genes conferring resistance to eight antibiotic classes: blaCTX-M-15, blaOXA-1, blaTEM-1,aac6′-Ib-cr, mph(A), catB4, tet(A), and the integron-borne dfrA7, aadA5, and sulI genes. pEK516 (strain D; 64,471 bp) belonged to incompatibility group IncFII and carried seven antibiotic resistance genes: blaCTX-M-15, blaOXA-1, blaTEM-1, aac6′-Ib-cr, catB4, and tet(A), all as in pEK499. It also carried aac3-IIa, conferring gentamicin resistance, and was highly related to pC15-1a, a plasmid encoding the CTX-M-15 enzyme in Canada. By contrast, pEK204 (strain C; 93,732 bp) belonged to incompatibility group IncI1 and carried only two resistance genes, blaCTX-M-3 and blaTEM-1. It probably arose by the transposition of Tn3 and ISEcp1-blaCTX-M-3 elements into a pCOLIb-P9-like plasmid. We conclude that (i) United Kingdom variants of the successful E. coli ST131 clone have acquired different plasmids encoding CTX-M ESBLs on separate occasions, (ii) the blaCTX-M-3 and blaCTX-M-15 genes on pEK204 and pEK499/pEK516 represent separate escape events, and (iii) IncFII plasmids harboring blaCTX-M-15 have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli.

Whole-Genome Pyrosequencing of an Epidemic Multidrug-Resistant Acinetobacter baumannii Strain Belonging to the European Clone II Group

ABSTRACT The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated blaOXA-58 carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes of A. baumannii ATCC 17978 and Acinetobacter baylyi ADP1, with the aim of identifying novel genes related to virulence and drug resistance. A. baumannii ACICU has a single chromosome of 3,904,116 bp (which is predicted to contain 3,758 genes) and two plasmids, pACICU1 and pACICU2, of 28,279 and 64,366 bp, respectively. Genome comparison showed 86.4% synteny with A. baumannii ATCC 17978 and 14.8% synteny with A. baylyi ADP1. A conspicuous number of transporters belonging to different superfamilies was predicted for A. baumannii ACICU. The relative number of transporters was much higher in ACICU than in ATCC 17978 and ADP1 (76.2, 57.2, and 62.5 transporters per Mb of genome, respectively). An antibiotic resistance island, AbaR2, was identified in ACICU and had plausibly evolved by reductive evolution from the AbaR1 island previously described in multiresistant strain A. baumannii AYE. Moreover, 36 putative alien islands (pAs) were detected in the ACICU genome; 24 of these had previously been described in the ATCC 17978 genome, 4 are proposed here for the first time and are present in both ATCC 17978 and ACICU, and 8 are unique to the ACICU genome. Fifteen of the pAs in the ACICU genome encode genes related to drug resistance, including membrane transporters and ex novo acquired resistance genes. These findings provide novel insight into the genetic basis of A. baumannii resistance.

Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella

OBJECTIVES The aim of this study was to identify and characterize plasmids carrying qnrS1, qnrB2 and qnrB19 genes identified in Salmonella strains from The Netherlands. The identification of plasmids may help to follow the dissemination of these resistance genes in different countries and environments. METHODS Plasmids from 33 qnr-positive Salmonella strains were transferred to Escherichia coli and analysed by restriction, Southern blot hybridization, PCR and sequencing of resistance determinants. They were also assigned to incompatibility groups by PCR-based replicon typing, including three additional PCR assays for the IncU, IncR and ColE groups. The collection included isolates from humans and one from chicken meat. RESULTS Five IncN plasmids carrying qnrS1, qnrB2 and qnrB19 genes were identified in Salmonella enterica Bredeney, Typhimurium PT507, Kentucky and Saintpaul. qnrS1 genes were also located on three further plasmid types, belonging to the ColE (in Salmonella Corvallis and Anatum), IncR (in Salmonella Montevideo) and IncHI2 (in Salmonella Stanley) groups. CONCLUSIONS Multiple events of mobilization, transposition and replicon fusion generate the complexity observed in qnr-positive isolates that are emerging worldwide. Despite the fact that the occurrence of qnr genes in bacteria from animals is scarcely reported, these genes are associated with genetic elements and located on plasmids that are recurrent in animal isolates.

Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum β-lactamases in Escherichia coli and Salmonella of human and animal origin

OBJECTIVES Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of beta-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment. METHODS pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for beta-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST. RESULTS Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried bla(CMY-2), bla(CTX-M-15), bla(CTX-M-1), bla(CTX-M-14), bla(TEM-52), bla(SHV-12) or bla(TEM-1) beta-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST. CONCLUSIONS The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among beta-lactamase producers isolated from animals and humans.

Replicon Typing of Plasmids Carrying CTX-M or CMY β-Lactamases Circulating among Salmonella and Escherichia coli Isolates

ABSTRACT Replicon typing of plasmids carrying blaCTX-M or blaCMY β-lactamase genes indicates a predominance of I1 and A/C replicons among blaCMY-carrying plasmids and five different plasmid scaffolds associated with the different types of blaCTX-M genes (I1, FII, HI2, K, and N). These results demonstrate the association of certain β-lactamase genes with specific plasmid backbones.