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Objectives: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene., Methods: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS)., Results: Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline., Conclusions: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood., Methods: In this study, Escherichia coli with the major ESC-R genes bla CTX-M-1 , bla CTX-M-15 , bla CTX-M-14 (ESBLs) and bla CMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n = 192 ESC-R E. coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences., Findings: We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E. coli lineages and host species. The IncI1 pST3 bla CTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 bla CMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 bla CMY-2 plasmid in ST131 and ST117 E. coli harbouring this plasmid. The IncI1 pST2 bla CMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 bla CTX-M-15 and F2:A-:B- bla CTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 bla CTX-M-1 and IncF F31:A4:B1 bla CTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents., Interpretation: We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR., Funding: This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain)., Competing Interests: Declaration of interests AEM is an inventor on International Patent Application No. PCT/GB2023/050906 filed in the name of Quadram Institute Bioscience–Determination and quantification of the microbial communities and antimicrobial resistance genes on food. All other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Linezolid plays a crucial role in the treatment of infections caused by multiresistant Gram-positive bacteria. The poxtA gene not only confers oxazolidinone and phenicol resistance but also decreases susceptibility to tetracycline. In this study, we investigated structural changes in mobilizable poxtA -carrying plasmids in enterococci which occurred during conjugation experiments using S1-PFGE (pulsed-field gel electrophoresis), Southern blot hybridization, and whole-genome sequencing (WGS) analysis. Two poxtA -carrying strains were identified in Enterococcus faecalis E006 and Enterococcus lactis E843, respectively. E. faecalis E006 contains the 121,520-bp conjugative plasmid pE006-121 and the 19,832-bp mobilizable poxtA -carrying plasmid pE006-19, while E. lactis E843 contains the 171,930-bp conjugative plasmid pE843-171 and the 27,847-bp mobilizable poxtA -carrying plasmid pE843-27. Moreover, both poxtA -carrying plasmids were mobilized by their respective conjugative plasmid in enterococci by plasmid fusion; one was generated by homologous recombination in E. faecalis through an identical 864-bp homologous region in the plasmids of the parental strain, while another was generated by an IS 1216E -mediated plasmid integration in E. lactis , involving a replicative transposition. IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA -carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis . During both the fusion events, the poxtA -carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle., (© 2022. The Author(s).)

Tetracyclines are the broad-spectrum agents used in veterinary medicine and food animal production. Known mechanisms of tetracycline resistance include ribosome protection, active efflux and enzymatic inactivation. However, the presence of two different tet genes conferring different resistance mechanisms on the same plasmid has rarely been reported. In this study, we identified the tandem tetracycline resistance genes tet(61)-tet(58) on the novel plasmid pT4303. These tet genes were identified for the first time in Aerococcus urinaeequi. Reduced susceptibility to doxycycline was observed in S. aureus RN4220 harboring tet(61) when an extra tet(58) was expressed. Plasmid pT4303 was electrotransformed into S. aureus RN4220, E. faecalis JH2-2, S. suis BAA and E. coli DH5α and conferred tetracycline resistance (MIC ≥ 16) in both Gram-positive and Gram-negative bacteria, assuming that it might serve as a vehicle for the dissemination of the tetracycline resistance genes tet(61) and tet(58)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The occurrence of carbapenemase-producing Enterobacteriaceae (CPE) poses a considerable risk for public health. The gene for Klebsiella pneumoniae carbapenemase-2 (KPC-2) has been reported in many countries worldwide, and KPC-2-producing strains are mainly of human origin. In this study, we identified two novel hybrid plasmids that carry either bla KPC-2 or the fosfomycin resistance gene fosA3 in the multiresistant K. pneumoniae isolate K15 of swine origin in China. The bla KPC-2 -bearing plasmid pK15-KPC was a fusion derivative of an IncF33:A-:B- incompatibility group (Inc) plasmid and chromosomal sequences of K. pneumoniae (CSKP). A 5-bp direct target sequence duplication (GACTA) was identified at the boundaries of the CSKP, suggesting that the integration might have been due to a transposition event. The bla KPC-2 -carrying pHN7A8-like plasmid and a pKPC-LK30-like IncR plasmid. Stability tests demonstrated that both novel hybrid plasmids were stably maintained in the original host without antibiotic selection but were lost from the transformants after approximately 200 generations. This is apparently the first description of a porcine sequence type 11 (ST11) 6296-1 The multireplicon fosA3 -carrying IncN-IncR plasmid pK15-FOS also showed a mosaic structure, possibly originating from a recombination between an epidemic fosA3 -carrying pHN7A8-like plasmid and a pKPC-LK30-like IncR plasmid. Stability tests demonstrated that both novel hybrid plasmids were stably maintained in the original host without antibiotic selection but were lost from the transformants after approximately 200 generations. This is apparently the first description of a porcine sequence type 11 (ST11) K. pneumoniae isolate coproducing KPC-2 and FosA3 via pK15-KPC and pK15-FOS, respectively. The multidrug resistance (MDR) phenotype of this high-risk K. pneumoniae isolate may contribute to its spread and its persistence. IMPORTANCE ST11 isolate, strain K15, which is of pig origin and coproduces KPC-2 and FosA3 via two novel hybrid plasmids. Furthermore, our findings highlight that this ST11 Enterobacteriaceae strains of animal origin have been reported increasingly, bla -carrying plasmids among the KPC-2 -positive strains from food-producing animals are still rare. In this study, we first describe the isolation and characterization of a carbapenem-resistant Klebsiella pneumoniae ST11 isolate, strain K15, which is of pig origin and coproduces KPC-2 and FosA3 via two novel hybrid plasmids. Furthermore, our findings highlight that this ST11 Klebsiella pneumoniae strain K15 is most likely of human origin and could be easily transmitted back to humans via direct contact or food intake. In light of our findings, significant attention must be paid to monitoring the prevalence and further evolution of bla KPC-2 -carrying plasmids among the Enterobacteriaceae strains of animal origin., (Copyright © 2019 Zhang et al.)

Objectives: To characterize an MDR blaIMP-4-harbouring plasmid from Enterobacter cloacae EC62 of swine origin in China., Methods: Plasmid pIMP-4-EC62 from E. cloacae EC62 was transferred by conjugation via filter mating into Escherichia coli J53. Plasmid DNA was extracted from an E. coli J53 transconjugant and sequenced using single-molecule real-time (SMRT) technology. MIC values for both the isolate EC62 and the transconjugant were determined using the broth microdilution and agar dilution methods. Plasmid stability in both the isolate EC62 and the transconjugant was assessed through a series of passages on antibiotic-free media., Results: Plasmid pIMP-4-EC62 is 314351 bp in length, encodes 369 predicted proteins and harbours a novel class 1 integron carrying blaIMP-4 and a group II intron. The blaIMP-4-bearing plasmid belongs to the IncHI2/ST1 incompatibility group. Sequence analysis showed that pIMP-4-EC62 carries four MDR regions and several gene clusters encoding heavy metal resistance. Plasmid pIMP-4-EC62 was stably maintained in both the E. cloacae EC62 isolate and the transconjugant E. coli J53-pIMP-4-EC62 in the absence of selective pressure. Analysis of the evolutionary relatedness of selected IncHI2 plasmids indicates that ST1-type plasmids are key carriers of carbapenemase genes among IncHI2 plasmids., Conclusions: pIMP-4-EC62 represents the first fully sequenced IncHI2-type blaIMP-4-harbouring plasmid from E. cloacae in China. Co-location of blaIMP-4 with other resistance genes on an MDR plasmid is likely to further accelerate the dissemination of blaIMP-4 by co-selection among bacteria from humans, animals and the environment under the selective pressure of other antimicrobial agents, heavy metals and disinfectants., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To investigate the presence and transferability of the poxtA gene and identify the genetic context of poxtA in two enterococcal plasmids from swine., Methods: MICs were determined by broth microdilution. A total of 114 porcine enterococci with florfenicol MICs of ≥16 mg/L were screened for the presence of the poxtA gene by PCR. Transferability of poxtA was investigated by conjugation and transformation. The poxtA-carrying plasmids were completely sequenced using the Illumina Miseq and PacBio RSII platform. The presence of circular intermediates was examined by inverse PCR., Results: The poxtA gene was present in 57.9% (66/114) of the florfenicol-resistant porcine enterococci. Two poxtA-carrying plasmids, pE035 and pE076, were identified. The conjugative 121524 bp plasmid pE035 carried poxtA and optrA along with the resistance genes erm(A), erm(B), aac(A)-aph(D), lnu(G), fexB, dfrG and bcrABDR. Three mobile elements, comprising a mobile dfrG locus, a mobile bcrABDR locus and an unconventional circularizable structure containing aac(A)-aph(D), were located on this plasmid and all proved to be active by inverse PCR. The non-conjugative 19832 bp plasmid pE076 only carried poxtA and fexB. After transfer, both the transconjugant and the transformant displayed elevated MICs of the respective antimicrobial agents., Conclusions: To the best of our knowledge, this is the first report of the co-location of the oxazolidinone resistance genes poxtA and optrA on a conjugative multiresistance plasmid from a porcine enterococcal strain. In addition, the presence of three mobile elements in such a plasmid will aid in the persistence and dissemination of poxtA and optrA among enterococci., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To identify and analyse the first ESBL gene from Mannheimia haemolytica., Methods: Susceptibility testing was performed according to CLSI. Plasmids were extracted via alkaline lysis and transferred by electrotransformation. The sequence was determined by WGS and confirmed by Sanger sequencing., Results: The M. haemolytica strain 48 showed high cephalosporin MICs. A single plasmid, designated pKKM48, with a size of 4323 bp, was isolated. Plasmid pKKM48 harboured a novel blaROB gene, tentatively designated blaROB-2, and was transferred to Pasteurella multocida B130 and to Escherichia coli JM107. PCR assays and susceptibility testing confirmed the presence and activity of the blaROB-2 gene in the P. multocida and in the E. coli recipient carrying plasmid pKKM48. The transformants had high MICs of all β-lactam antibiotics. An ESBL phenotype was seen in the E. coli transformant when applying the CLSI double-disc confirmatory test for E. coli. The blaROB-2 gene from plasmid pKKM48 differed in three positions from blaROB-1, resulting in two amino acid exchanges and one additional amino acid in the deduced β-lactamase protein. In addition to blaROB-2, pKKM48 harboured mob genes and showed high similarity to other plasmids from Pasteurellaceae., Conclusions: This study described the first ESBL gene in Pasteurellaceae, which may limit the therapeutic options for veterinarians. The transferability to Enterobacteriaceae with the functional activity of the gene in the new host underlines the possibility of the spread of this gene across species or genus boundaries., (© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Macrolide resistance in staphylococci is based on the expression of a number of genes which specify four major resistance mechanisms: (i) target site modification by methylation of the ribosomal target site in the 23S rRNA, (ii) ribosome protection via ABC-F proteins, (iii) active efflux via Major Facilitator Superfamily (MFS) transporters, and (iv) enzymatic inactivation by phosphotransferases or esterases. So far, 14 different classes of erm genes, which code for 23S rRNA methylases, have been reported to occur in staphylococci from humans, animals and environmental sources. Inducible or constitutive expression of the erm genes depends on the presence and intactness of a regulatory region known as translational attenuator. The erm genes commonly confer resistance not only to macrolides, but also to lincosamides and streptogramin B compounds. In contrast, the msr(A) gene codes for an ABC-F protein which confers macrolide and streptogramin B resistance whereas the mef(A) gene codes for a Major Facilitator Superfamily protein that can export only macrolides. Enzymatic inactivation of macrolides may be due to the macrolide phosphotransferase gene mph(C) or the macrolide esterase genes ere(A) or ere(B). Many of these macrolide resistance genes are part of either plasmids, transposons, genomic islands or prophages and as such, can easily be transferred across strain, species and genus boundaries. The co-location of other antimicrobial or metal resistance genes on the same mobile genetic element facilitates co-selection and persistence of macrolide resistance genes under the selective pressure of metals or other antimicrobial agents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lincosamide resistance in staphylococci is based on the expression of a number of genes which specify three major resistance mechanisms: (i) enzymatic inactivation by lincosamide nucleotidyltransferases, (ii) ribosome protection by ABC-F proteins, and (iii) methylation of the ribosomal target sites in the 23S rRNA by Cfr or Erm methylases. So far, only two lnu genes, lnu(A) and lnu(B), which code for different types of lincosamide nucleotidyltransferases, have been found in staphylococci. The ABC-F proteins are encoded by genes of the vga, lsa and sal classes. The corresponding proteins exhibit ATP-binding domains, but lack transmembrane regions. So far, vga(A) genes - including the variant genes vga(A) V and vga(A) LC -, vga(C) genes and vga(E) genes - including the variant gene vga(E) V -, the lsa genes lsa(B) and lsa(E), as well as the sal(A) gene have been identified in staphylococci. The aforementioned genes, except lsa(B), confer resistance not only to lincosamides, but also to pleuromutilins and streptogramin A. The cfr and erm genes code for methylases which target the adenine residues at positions 2503 and 2048 in the 23S rRNA, respectively. While the cfr gene confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A, the erm genes mediate resistance to macrolides, lincosamides and streptogramin B. Many of the aforementioned lincosamide resistance genes are located on either plasmids or transposons and as such, can easily be disseminated across strain, species, and genus boundaries. The co-location of other antimicrobial resistance genes on the same mobile genetic element facilitates co-selection and persistence of the lincosamide resistance genes under the selective pressure imposed by other antimicrobial agents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates have been increasingly reported in different reservoirs. The aims of this study were to investigate the presence of ESBL-producing E. coli in fresh vegetables and to characterise their ESBL gene-carrying plasmids. Among the 245 samples from vegetables investigated during 2011-2013, seven putative ESBL-producing E. coli (salad n = 2, sprouts n = 5) were found. They were subjected to ESBL phenotypic confirmatory tests, detection/sequencing of ESBL genes, antimicrobial susceptibility testing (AST), phylotyping, XbaI-macrorestriction analysis, multilocus sequence typing and transformation. Transformants were characterised by AST, S1-nuclease PFGE, replicon typing, conjugation and investigated for co-located antimicrobial resistance genes. Two ESBL gene-carrying plasmids were sequenced using a HiSeq 2500 system. The seven isolates were confirmed as ESBL producers, displayed unrelated XbaI-patterns and unique sequence types (STs) and belonged to the phylogroups A, B1 or D. The ESBL genes were located on plasmids. Two plasmids carrying bla CTX-M-14 genes (incompatibility group IncK or IncHI2) were seen in isolates from salad (ST973) and sprout (ST527). Two bla CTX-M-15 - (IncFIB; non-typeable) and the IncN bla CTX-M-65 - and IncHI2 bla CTX-M-125 -carrying plasmids were found in isolates from sprouts (ST410, ST847, ST10, ST542). All plasmids were conjugative, except for the IncFIA-FIB bla CTX-M-2 -carrying plasmid. Sequence analysis of two plasmids identified the ESBL genes in close location to other resistance genes: sulfonamide resistance gene sul2, streptomycin resistance genes strA and strB, the plasmid-mediated quinolone resistance gene qnrS1 and bla TEM-1 (sul2-strA-strB-IS66-bla TEM-1 -tnpR-ΔtnpA-ISEcp1-bla CTX-M-15 -Δorf477-ΔtnpA-qnrS1) or the fosfomycin resistance gene fosA3 (ΔISEcp1-bla CTX-M-125 -ΔIS903B-fosA3). These observations underline the importance of vegetables as reservoirs for multidrug resistant ESBL-producing E. coli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thirty-nine fosfomycin-resistant Escherichia coli isolates carrying fosA3 were obtained from pigs, chickens, dairy cows, and staff in four northeastern provinces of China between June 2015 and April 2016. The fosA3 gene was colocated with bla genes on conjugative plasmids of the incompatibility groups IncN ( CTX-M genes on conjugative plasmids of the incompatibility groups IncN ( n = 12), IncN-F33:A - :B - ( n = 2), IncF33:A - :B - ( n = 14), IncF14:A - :B - ( n = 2), and IncI1/sequence type 136 (ST136) ( n = 9). Four different genetic contexts of fosA3 isolates. Three potential epidemic plasmids circulated among E. coli isolates. Three potential epidemic plasmids circulated among E. coli strains from this region., (Copyright © 2017 American Society for Microbiology.)

Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely., Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing., Results: Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 ( n  = 17), IncK ( n  = 3), IncF ( n  = 1), IncX3 ( n  = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to β-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -ΔTn 1721 resistance complex., Conclusions: Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Extended-spectrum β-lactamase (ESBL)-producing isolates have been increasingly reported during recent years. The aims of this study were to characterize ESBL-producing Escherichia coli from bovine mastitis as well as their ESBL gene-carrying plasmids. A culture collection of E. coli isolated from bovine quarter milk samples (2009-2013), was screened for ESBL production using ESBL selective agar plates. Putative ESBL producers (n=16) were investigated by phenotypic confirmatory tests and were characterized by the detection/sequencing of ESBL genes, XbaI macrorestriction analysis, multilocus sequence typing (MLST), phylotyping and antimicrobial susceptibility testing. ESBL gene-carrying plasmids were investigated by transfer experiments, PCRs for the detection of co-located antimicrobial resistance genes, PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis. Twelve ESBL-producing isolates were found. They showed eleven different XbaI patterns and were distributed among eight MLST types [ST10 (n=3), ST117 (n=2), ST361 (n=1), ST362 (n=1), ST540 (n=1), ST1431 (n=2), ST1508 (n=1), and the novel ST5447 (n=1)] and the phylogenetic groups A (n=6), B1 (n=2), B2 (n=1) and D (n=3). ESBL genes bla CTX-M-1 (n=5), bla CTX-M-2 (n=2), bla CTX-M-14 and bla CTX-M-15 (n=4) were found on conjugative plasmids (35-225kb) of diverse incompatibility groups (e.g. IncF, IncI1 or HI2+P). Co-located resistance to sulfonamides, tetracycline, trimethoprim, and chloramphenicol/florfenicol was detected on five ESBL gene-carrying plasmids, but seven plasmids conferred solely resistance to β-lactam antibiotics. The presence of additional resistance genes on the ESBL gene-carrying plasmids suggests that co-selection of ESBL genes may occur even in the absence of β-lactam antibiotics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The multiresistance plasmid pAFS11, obtained from a bovine methicillin-resistant Staphylococcus aureus (MRSA) isolate, was completely sequenced and analysed for its structure and organisation. Moreover, the susceptibility to the heavy metals cadmium and copper was determined by broth macrodilution. The 49,189-bp plasmid harboured the apramycin resistance gene apmA, two copies of the macrolide/lincosamide/streptogramin B resistance gene erm(B) (both located on remnants of a truncated transposon Tn917), the kanamycin/neomycin resistance gene aadD, the tetracycline resistance gene tet(L) and the trimethoprim resistance gene dfrK. The latter three genes were part of a 7,284-bp segment which was bracketed by two copies of IS431. In addition, the cadmium resistance operon cadDX as well as the copper resistance genes copA and mco were located on the plasmid and mediated a reduced susceptibility to cadmium and copper. Moreover, a complete novel ica-like gene cluster of so far unknown genetic origin was detected on this plasmid. The ica-like gene cluster comprised four different genes whose products showed 64.4-76.9% homology to the Ica proteins known to be involved in biofilm formation of the S. aureus strains Mu50, Mu3 and N315. However, 96.2-99.4% homology was seen to proteins from S. sciuri NS1 indicating an S. sciuri origin. The finding of five different antibiotic resistance genes co-located on a plasmid with heavy metal resistance genes and an ica-like gene cluster is alarming. With the acquisition of this plasmid, antimicrobial multiresistance, heavy metal resistances and potential virulence properties may be co-selected and spread via a single horizontal gene transfer event., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plasmids play a key role in the genetic plasticity and survival of Staphylococcus aureus in challenging environments. Although many S. aureus plasmids have been described, still few studies portray the plasmid content of a given S. aureus population. The aim of this work was to characterize the plasmids carried by a collection of 53 S. aureus isolates collected in a large hospital in Lisbon, Portugal, and investigate their role in conferring resistance to several antimicrobial agents. Plasmids were present in 44 out of the 53 isolates and were grouped into eleven AccI restriction profiles. Plasmid curing of representative strains and comparison of antimicrobial susceptibility profiles between pairs of isogenic strains proved to be a valuable guidance tool in the identification of plasmid-located resistance genes. The plasmids harbored several resistance genes, namely blaZ (resistance to β-lactams), erm(C) (resistance to macrolides, lincosamides, and streptogramin B), cadA (resistance to cadmium and zinc), cadD (resistance to cadmium), and qacA and smr (resistance to biocides and dyes). This study demonstrates the impact of plasmids on the resistance properties of S. aureus, highlighting their role in the dissemination of antibiotic, heavy metal, and biocide resistance genes, and survival of this major pathogen in the hospital environment.

Objectives: This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes., Methods: A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST., Results: The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons., Conclusions: ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The multiresistance gene cfr was found in two porcine Escherichia coli isolates, one harboring it on the conjugative 33,885-bp plasmid pFSEC-01, the other harboring it in the chromosomal DNA. Sequence analysis of pFSEC-01 revealed that a 6,769-bp fragment containing the cfr gene bracketed by two IS26 elements was inserted into a plasmid closely related to pEA3 from the plant pathogen Erwinia amylovora, suggesting that pFSEC-01 may be transferred between different bacterial genera of both animal and plant origin., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traY and excA genes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The multiresistance gene cfr was found in 8/231 porcine methicillin-resistant Staphylococcus aureus isolates. They were characterized by multilocus sequence typing, spa typing, dru typing, and staphylococcal cassette chromosome mec (SCCmec) typing as ST627-t002-dt12w-IVb, ST6-t304-dt12w-IVb, ST9-t899-dt12w-IVb, ST9-t899-dt12ae-IVb, or ST63-t899-dt12v-IVb. Different cfr gene regions were detected on plasmids of ca. 35 kb in seven isolates. For the first time, an ISEnfa4-cfr-IS256 fragment was found to be inserted upstream of the ccr genes in a chromosomal SCCmec IVb element of the remaining isolate., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

During a study on plasmid-borne antimicrobial resistance among methicillin-resistant Staphylococcus aureus (MRSA) isolates from broiler farms, an MRSA isolate was identified which carried multiple plasmids. This MRSA isolate belonged to CC398 and exhibited spa type t3015 and dru type dt11a. Plasmid profiling revealed the presence of one large and two small plasmids. The resistance genes tet(L) (tetracycline resistance), dfrK (trimethoprim resistance) and aadD (kanamycin/neomycin resistance) were located on the large plasmid. Both small plasmids, designated pSWS371 and pSWS372, carried only an erm(C) gene for macrolide/lincosamide resistance. Sequence analysis revealed that the 2458-bp plasmid pSWS371 carried only a repL gene for plasmid replication in addition to the erm(C) gene. In contrast, the 3882-bp plasmid pSWS372 harbored - in addition to the erm(C) gene - three more genes: a repF gene for plasmid replication, a cop-6 gene for a small protein potentially involved in copy number control of the plasmid and a novel pre/mob gene for a protein involved in plasmid recombination and mobilization. The erm(C) genes of both small plasmids exhibited constitutive erm(C) gene expression and analysis of the respective translational attenuators identified deletions of 16 bp and 74 bp which explain the constitutive expression. The simultaneous presence of two small plasmids that carry the same resistance gene in the same MRSA isolate is a rare observation. The fact that both plasmids belong to different incompatibility groups as specified by the different rep genes, repL and repF, explains why they can stably coexist in the same bacterial cell., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: To determine the complete nucleotide sequence of the multidrug resistance plasmid pSCEC2, isolated from a porcine Escherichia coli strain, and to analyse it with particular reference to the cfr gene region., Methods: Plasmid pSCEC2 was purified from its E. coli J53 transconjugant and then sequenced using the 454 GS-FLX System. After draft assembly, predicted gaps were closed by PCR with subsequent sequencing of the amplicons., Results: Plasmid pSCEC2 is 135 615 bp in size and contains 200 open reading frames for proteins of ≥100 amino acids. Analysis of the sequence of pSCEC2 revealed two resistance gene segments. The 4.4 kb cfr-containing segment is flanked by two IS256 elements in the same orientation, which are believed to be involved in the dissemination of the rRNA methylase gene cfr. The other segment harbours the resistance genes floR, tet(A)-tetR, strA/strB and sul2, which have previously been found on other IncA/C plasmids. Except for these two resistance gene regions, the pSCEC2 backbone displayed >99% nucleotide sequence identity to that of other IncA/C family plasmids isolated in France, Chile and the USA., Conclusions: The cfr gene was identified on an IncA/C plasmid, which is well known for its broad host range and transfer and maintenance properties. The location on such a plasmid will further accelerate the dissemination of cfr and co-located resistance genes among different Gram-negative bacteria. The genetic context of cfr on plasmid pSCEC2 underlines the complexity of cfr transfer events and confirms the role that insertion sequences play in the spread of cfr.

The aim of this study was to determine the complete sequence of the multi-resistance plasmid pV7037 to gain insight into the structure and organization of this plasmid. Of the four XbaI clones of pV7037, one clone of 17,577 bp has already been sequenced and shown to carry a multi-resistance gene cluster. The remaining three clones of approximately 12.5, 6.5 and 4.5 kb were sequenced, the entire plasmid sequence correctly assembled and investigated for reading frames. In addition, two reading frames one coding for an ABC transporter and the other coding for an rRNA methylase were cloned and expressed in a S. aureus host to see whether they confer antimicrobial resistance properties. Plasmid pV7037 proved to be 40,971 bp in size. Besides the previously determined resistance gene cluster, it carried a functionally active tet(L) gene for tetracycline resistance, a complete cadDX operon for cadmium resistance and also a variant of the β-lactamase transposon Tn552. Two single bp deletions, which resulted in frame shifts, functionally deleted the genes for the BlaZ β-lactamase and the signal transducer protein BlaR1 in this Tn552 variant of pV7037. Plasmid pV7037 seems to be composed of various parts previously known from plasmids and transposons of staphylococci and other Gram-positive bacteria. However, there are also parts of the plasmid which do not show any homology to so far known sequences deposited in the databases. The novel ABC transporter and rRNA methylase genes identified on pV7037 do not seem to play a role in antimicrobial resistance. The co-location of numerous antimicrobial resistance genes bears the risk of co-transfer and co-selection of resistance genes, but also persistence of resistance genes even if no direct selective pressure by the use of the respective antimicrobial agents is applied., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Nineteen linezolid-resistant Staphylococcus epidermidis and two Staphylococcus aureus isolates recovered from two medical institutions in northeast Ohio and an S. aureus cfr index strain previously collected in the same facilities during the 2007 SENTRY Antimicrobial Surveillance Program were investigated for the genetic basis of oxazolidinone resistance and the location of cfr. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST). The location of cfr was determined by Southern blotting and hybridization. Plasmid sequencing was performed using the 454 Life Sciences (Roche) GS-FLX DNA platform. The two S. aureus isolates showed unique PFGE patterns but were multilocus sequence type 5 (ST5) and spa type t002, whereas the S. aureus index strain was ST239 and t037. Southern blot and hybridization experiments showed that cfr was plasmid located and that the S. epidermidis isolates, one of the S. aureus isolates, and the S. aureus index strain shared an identical cfr-carrying plasmid (39.3 kb). Sequencing results confirmed these findings. A 10-kb fragment containing cfr showed the highest identity (99.9%) to a 9.5-kb fragment of plasmid pSCFS3 from a bovine Staphylococcus lentus isolate from Germany. In addition, these 39.3-kb plasmids from human S. epidermidis and S. aureus exhibited BglII restriction profiles very similar to that observed for plasmid pSCFS3. The cfr-carrying plasmid detected in the remaining S. aureus isolate (7.9 kb) was distinct and showed the highest identity to the chromosomal cfr integrate found in the chromosomal DNA of a Proteus vulgaris isolate from a pig in China.

This study describes three novel erm(T)-carrying multiresistance plasmids that also harbor cadmium and copper resistance determinants. The plasmids, designated pUR1902, pUR2940, and pUR2941, were obtained from porcine and human methicillin-resistant Staphylococcus aureus (MRSA) of the clonal lineage ST398. In addition to the macrolide-lincosamide-streptogramin B (MLSB) resistance gene erm(T), all three plasmids also carry the tetracycline resistance gene tet(L). Furthermore, plasmid pUR2940 harbors the trimethoprim resistance gene dfrK and the MLSB resistance gene erm(C), while plasmids pUR1902 and pUR2941 possess the kanamycin/neomycin resistance gene aadD. Sequence analysis of approximately 18.1 kb of the erm(T)-flanking region from pUR1902, 20.0 kb from pUR2940, and 20.8 kb from pUR2941 revealed the presence of several copies of the recently described insertion sequence ISSau10, which is probably involved in the evolution of the respective plasmids. All plasmids carried a functional cadmium resistance operon with the genes cadD and cadX, in addition to the multicopper oxidase gene mco and the ATPase copper transport gene copA, which are involved in copper resistance. The comparative analysis of S. aureus RN4220 and the three S. aureus RN4220 transformants carrying plasmid pUR1902, pUR2940, or pUR2941 revealed an 8-fold increase in CdSO4 and a 2-fold increase in CuSO4 MICs. The emergence of multidrug resistance plasmids that also carry heavy metal resistance genes is alarming and requires further surveillance. The colocalization of antimicrobial resistance genes and genes that confer resistance to heavy metals may facilitate their persistence, coselection, and dissemination.

The clinical Staphylococcus epidermidis isolate 426-3147L exhibits an unusually high resistance to linezolid that exceeds 256 μg/ml. The presence of the cfr gene, encoding the RNA methyltransferase targeting an rRNA nucleotide located in the linezolid binding site, accounts for a significant fraction of resistance. The association of cfr with a multicopy plasmid is one of the factors that contribute to its elevated expression. Mapping of the cfr transcription start sites identified the native cfr promoter. Furthermore, analysis of the cfr transcripts in Staphylococcus epidermidis 426-3147L showed that some of them originate from the upstream plasmid-derived promoters whose activity contributes to efficient cfr transcription. The genetic environment of the cfr gene and its idiosyncratic transcription pattern result in increased activity of Cfr methyltransferase, leading to a high fraction of the ribosomes being methylated at A2503 of the 23S rRNA. Curing of the Staphylococcus epidermidis 426-3147L isolate from the cfr-containing plasmid reduced the linezolid MIC to 64 μg/ml, indicating that other determinants contribute to resistance. Nucleotide sequence analysis revealed the presence of the C2534T mutation in two of the six 23S rRNA gene alleles as well as the presence of mutations in the genes of ribosomal proteins L3 and L4, which were previously implicated in linezolid resistance. Thus, the combination of resistance mechanisms operating through alteration of the drug target site appears to cause an unusually high level of linezolid resistance in the isolate.

Seventy-seven porcine Enterococcus isolates with florfenicol MICs of ≥16 μg of were/ml screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. Three isolates-Enterococcus thailandicus 3-38 (from a porcine rectal swab collected at a pig farm), Enterococcus thailandicus W3, and Enterococcus faecalis W9-2 (the latter two from sewage at a different farm), carried the cfr gene. The SmaI pulsed-field gel electrophoresis patterns of the three isolates differed distinctly. In addition, E. faecalis W9-2 was assigned to a new multilocus sequence type ST469. Mating experiments and Southern blot analysis indicated that cfr is located on conjugative plasmids pW3 (∼75 kb) from E. thailandicus W3, p3-38 (∼72 kb) from E. thailandicus 3-38, and pW9-2 (∼55 kb) from E. faecalis W9-2; these plasmids differed in their sizes, additional resistance genes, and the analysis of the segments encompassing the cfr gene. Sequence analysis revealed that all plasmids harbored a 4,447-bp central region, in which cfr was bracketed by two copies of the novel insertion sequence ISEnfa4 located in the same orientation. The sequences flanking the central regions of these plasmids, including the partial tra gene regions and a ω-ε-ζ toxin-antitoxin module, exhibited >95% nucleotide sequence identity to the conjugative plasmid pAMβ1 from E. faecalis. Conjugative plasmids carrying cfr appear to play an important role in the dissemination and maintenance of the multiresistance gene cfr among enterococcal isolates and possibly other species of Gram-positive bacteria.

Objectives: The aims of this study were to identify small staphylococcal plasmids that carry either the trimethoprim resistance gene dfrK or the apramycin resistance gene apmA and analyse them for their structure and organization with regard to their potential role as precursors of large multiresistance plasmids that carry these genes., Methods: Trimethoprim- or apramycin-resistant staphylococci from the strain collections of the two participating institutions were investigated for the presence of plasmid-borne dfrK or apmA genes. The dfrK- or apmA-carrying plasmids were sequenced completely and compared with sequences deposited in the databases., Results: Two small plasmids, the 4957 bp dfrK-carrying plasmid pKKS966 from porcine Staphylococcus hyicus subsp. hyicus and the 4809 bp apmA-carrying plasmid pKKS49 from porcine methicillin-resistant Staphylococcus aureus were identified. Structural analysis revealed that both plasmids had a similar organization, comprising a single resistance gene (dfrK or apmA), a plasmid replication gene (rep) and three partly overlapping genes for mobilization proteins (mobA, mobB and mobC). Comparisons showed 71%-82% amino acid identity between the Rep and Mob proteins of these two plasmids; however, distinctly lesser percentages of identity to Rep and Mob proteins of staphylococci and other bacteria deposited in the databases were detected., Conclusions: Both plasmids, pKKS966 and pKKS49, appeared not to be typical staphylococcal plasmids. The homology to larger plasmids that harbour the genes apmA and/or dfrK was limited to these resistance genes and their immediate upstream and downstream regions and thus suggested that these small plasmids were not integrated into larger plasmids.

Objectives: The aims of this study were to identify qnr genes among quinolone-resistant Escherichia coli isolates from defined disease conditions of companion and farm animals obtained in the BfT-GermVet study, and to gain insight into their localization and the organization of the qnr gene regions., Methods: The qnr genes were detected by PCR and confirmed by sequencing. qnr-positive isolates were checked for mutations in DNA gyrase and topoisomerase IV genes by PCR and sequencing of the quinolone resistance-determining regions. Multilocus sequence typing (MLST) was performed for the qnr-positive E. coli isolates. Plasmids harbouring qnr genes were transferred by conjugation into E. coli recipients, subjected to PCR-based replicon typing and plasmid-MLST, and one qnrB19-carrying plasmid was sequenced completely., Results: Only 2 of 417 E. coli isolates investigated carried qnr genes. Both isolates originated from horses and showed MLST type ST86. They harboured conjugative qnrB19-carrying plasmids, which proved to be indistinguishable by restriction analysis, belonged to incompatibility group IncN, showed plasmid-MLST type ST8 and did not carry other resistance genes. The qnrB19 gene was flanked by copies of the insertion sequence IS26. One of these plasmids, pQNR2078, was sequenced completely and had a size of 42 379 bp. Except for the resistance gene region, plasmid pQNR2078 closely resembled the bla(CTX-M-65)-carrying plasmid pKC396 from E. coli., Conclusions: qnr genes were rarely detected among E. coli from animals in the BfT-GermVet study. The qnrB19 gene was detected on conjugative plasmids, with IS26 being likely involved in the mobility of qnrB19.

A novel plasmid-borne resistance gene cluster comprising the genes erm(T) for macrolide-lincosamide-streptogramin B resistance, dfrK for trimethoprim resistance, and tet(L) for tetracycline resistance was identified in a porcine methicillin-resistant Staphylococcus aureus sequence type 398 (ST398) strain. This erm(T)-dfrK-tet(L) region was flanked by copies of the novel IS element ISSau10. The erm(T) region resembled that of Streptococcus pyogenes plasmid pRW35. The erm(T) gene of pKKS25 was expressed constitutively due to a 57-bp deletion in the erm(T) translational attenuator.

A novel ABC transporter gene, vga(C), was identified on the 14,365-bp multiresistance plasmid pKKS825 in a porcine methicillin (meticillin)-resistant Staphylococcus aureus isolate of sequence type 398. The vga(C) gene encodes a 523-amino-acid protein which confers resistance not only to streptogramin A antibiotics but also to lincosamides and pleuromutilins. Plasmid pKKS825 also carries the resistance genes aadD, tet(L), and dfrK, which may enable the coselection of vga(C) under selective pressure by kanamycin/neomycin, tetracyclines, and trimethoprim.

Background: Salmonella enterica serovar Enteritidis is an important frequently reported zoonotic pathogen and a common cause of human gastroenteritis worldwide. The highly conserved Sero-specific plasmids (SSPs) and Salmonella plasmid virulence (Spy) genes have been shown to mediate extra-intestinal colonization and systemic infection. The objective of current study was to document the presence of SSPs and SpvB/SpvC genes prevailing in the indigenous population of serovar Enteritidis., Methods: A total of 48 epidemiologically unrelated strains of Salmonella enteritidis were included in the study. Preparation of plasmids DNA suitable for endonuclease digestion and separation of respective fragments by agarose gel electrophoresis followed previously described protocols. The plasmids of Escherichia coli V517, 1-kbp ladder, and lamda DNA HindIII fragments served as DNA size standards. Transfer of DNA fragments from agarose gels to nitrocellulose membranes was achieved by capillary blot procedure. An ECL labeled 3.6 kbp HindIII fragment of plasmid PRQ 51 was used as probe for SpvB/SpvC gene detection., Results: Plasmid DNA fingerprinting revealed the presence of two different profiles of approximately 55 kbp and 90 kbp and were identified as virulence plasmids by DNA hybridization. The SpvB/SpvC genes were located on HindIII fragments of 3.6 kbp in each of the two types of virulence plasmids., Conclusion: The study confirms the presence of SSPs and SpvB/SpvC genes in indigenous strains of S. enteritidis isolated from Northern Punjab area of Pakistan and substantiate the previous data on such findings from other parts of the world.

Objectives: To analyse the sequence and transfer properties of two tetracycline resistance plasmids found in clinical isolates of Actinobacillus pleuropneumoniae in order to assess their role in the spread of tetracycline resistance., Methods: The plasmids designated p9956 and p12494 were purified from A. pleuropneumoniae and completely sequenced. The transfer properties of both plasmids were evaluated by electroporation and/or conjugation into Pasteurella multocida and Escherichia coli., Results: Both plasmids showed a modular structure consisting of three regions involved in mobilization, tetracycline resistance or replication. The mobilization regions included the mobA gene, encoding a relaxase, a protein involved in plasmid transfer. The tetracycline resistance regions were closely related and consisted of the tet(H) gene and its repressor gene tetR. The tetracycline resistance phenotype was transferred successfully to P. multocida and in the case of p9956 also to E. coli by electroporation of the plasmids. Moreover, plasmid p9956 could be mobilized in E. coli with the assistance of RP4 conjugal transfer functions., Conclusions: For the first time, the complete sequences of two tet(H)-carrying plasmids from A. pleuropneumoniae were determined. These two plasmids differed from one another and from known tet(H)-carrying plasmids from Pasteurella or Mannheimia spp. Structural analysis confirmed that these plasmids consisted of segments that have been previously detected in members of the families Pasteurellaceae and Enterobacteriaceae.

Objectives: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR., Methods: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization., Results: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events., Conclusions: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae.