Clinical antibiotic-resistance plasmids have small effects on biofilm formation and population growth in Escherichia coli in vitro.

Antimicrobial resistance (AR) mechanisms encoded on plasmids can affect other phenotypic traits in bacteria, including biofilm formation. These effects may be important contributors to the spread of AR and the evolutionary success of plasmids, but it is not yet clear how common such effects are for clinical plasmids/bacteria, and how they vary among different plasmids and host strains. Here, we used a combinatorial approach to test the effects of clinical AR plasmids on biofilm formation and population growth in clinical and laboratory Escherichia coli strains. In most of the 25 plasmid-bacterium combinations tested, we observed no significant change in biofilm formation upon plasmid introduction, contrary to the notion that plasmids frequently alter biofilm formation. In a few cases we detected altered biofilm formation, and these effects were specific to particular plasmid-bacterium combinations. By contrast, we found a relatively strong effect of a chromosomal streptomycin-resistance mutation (in rpsL) on biofilm formation. Further supporting weak and host-strain-dependent effects of clinical plasmids on bacterial phenotypes in the combinations we tested, we found growth costs associated with plasmid carriage (measured in the absence of antibiotics) were moderate and varied among bacterial strains. These findings suggest some key clinical resistance plasmids cause only mild phenotypic disruption to their host bacteria, which may contribute to the persistence of plasmids in the absence of antibiotics.

Antibiotic-degrading resistance changes bacterial community structure via species-specific responses.

Some bacterial resistance mechanisms degrade antibiotics, potentially protecting neighbouring susceptible cells from antibiotic exposure. We do not yet understand how such effects influence bacterial communities of more than two species, which are typical in nature. Here, we used experimental multispecies communities to test the effects of clinically important pOXA-48-plasmid-encoded resistance on community-level responses to antibiotics. We found that resistance in one community member reduced antibiotic inhibition of other species, but some benefitted more than others. Further experiments with supernatants and pure-culture growth assays showed the susceptible species profiting most from detoxification were those that grew best at degraded antibiotic concentrations (greater than zero, but lower than the starting concentration). This pattern was also observed on agar surfaces, and the same species also showed relatively high survival compared to most other species during the initial high-antibiotic phase. By contrast, we found no evidence of a role for higher-order interactions or horizontal plasmid transfer in community-level responses to detoxification in our experimental communities. Our findings suggest carriage of an antibiotic-degrading resistance mechanism by one species can drastically alter community-level responses to antibiotics, and the identities of the species that profit most from antibiotic detoxification are predicted by their intrinsic ability to survive and grow at changing antibiotic concentrations.

Within-patient evolution of plasmid-mediated antimicrobial resistance.

Antimicrobial resistance (AMR) in bacteria is a major threat to public health; one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been studied extensively in vitro but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalized patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug-resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed compensatory evolution of plasmid-associated fitness cost and the evolution of enhanced plasmid-mediated AMR in bacteria evolving in the gut of hospitalized patients. Crucially, we observed that the evolution of pOXA-48-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.

Translational demand is not a major source of plasmid-associated fitness costs.

Plasmids are key drivers of bacterial evolution because they are crucial agents for the horizontal transfer of adaptive traits, such as antibiotic resistance. Most plasmids entail a metabolic burden that reduces the fitness of their host if there is no selection for plasmid-encoded genes. It has been hypothesized that the translational demand imposed by plasmid-encoded genes is a major mechanism driving the fitness cost of plasmids. Plasmid-encoded genes typically present a different codon usage from host chromosomal genes. As a consequence, the translation of plasmid-encoded genes might sequestrate ribosomes on plasmid transcripts, overwhelming the translation machinery of the cell. However, the pervasiveness and origins of the translation-derived costs of plasmids are yet to be assessed. Here, we systematically altered translation efficiency in the host cell to disentangle the fitness effects produced by six natural antibiotic resistance plasmids. We show that limiting translation efficiency either by reducing the number of available ribosomes or their processivity does not increase plasmid costs. Overall, our results suggest that ribosomal paucity is not a major contributor to plasmid fitness costs. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Variability of plasmid fitness effects contributes to plasmid persistence in bacterial communities.

Plasmid persistence in bacterial populations is strongly influenced by the fitness effects associated with plasmid carriage. However, plasmid fitness effects in wild-type bacterial hosts remain largely unexplored. In this study, we determined the fitness effects of the major antibiotic resistance plasmid pOXA-48_K8 in wild-type, ecologically compatible enterobacterial isolates from the human gut microbiota. Our results show that although pOXA-48_K8 produced an overall reduction in bacterial fitness, it produced small effects in most bacterial hosts, and even beneficial effects in several isolates. Moreover, genomic results showed a link between pOXA-48_K8 fitness effects and bacterial phylogeny, helping to explain plasmid epidemiology. Incorporating our fitness results into a simple population dynamics model revealed a new set of conditions for plasmid stability in bacterial communities, with plasmid persistence increasing with bacterial diversity and becoming less dependent on conjugation. These results help to explain the high prevalence of plasmids in the greatly diverse natural microbial communities.

Pervasive transmission of a carbapenem resistance plasmid in the gut microbiota of hospitalized patients.

Infections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterize the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a 2-yr period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonized patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE.

Beyond horizontal gene transfer: the role of plasmids in bacterial evolution.

Plasmids have a key role in bacterial ecology and evolution because they mobilize accessory genes by horizontal gene transfer. However, recent studies have revealed that the evolutionary impact of plasmids goes above and beyond their being mere gene delivery platforms. Plasmids are usually kept at multiple copies per cell, producing islands of polyploidy in the bacterial genome. As a consequence, the evolution of plasmid-encoded genes is governed by a set of rules different from those affecting chromosomal genes, and these rules are shaped by unusual concepts in bacterial genetics, such as genetic dominance, heteroplasmy or segregational drift. In this Review, we discuss recent advances that underscore the importance of plasmids in bacterial ecology and evolution beyond horizontal gene transfer. We focus on new evidence that suggests that plasmids might accelerate bacterial evolution, mainly by promoting the evolution of plasmid-encoded genes, but also by enhancing the adaptation of their host chromosome. Finally, we integrate the most relevant theoretical and empirical studies providing a global understanding of the forces that govern plasmid-mediated evolution in bacteria.

Collateral sensitivity associated with antibiotic resistance plasmids.

Collateral sensitivity (CS) is a promising alternative approach to counteract the rising problem of antibiotic resistance (ABR). CS occurs when the acquisition of resistance to one antibiotic produces increased susceptibility to a second antibiotic. Recent studies have focused on CS strategies designed against ABR mediated by chromosomal mutations. However, one of the main drivers of ABR in clinically relevant bacteria is the horizontal transfer of ABR genes mediated by plasmids. Here, we report the first analysis of CS associated with the acquisition of complete ABR plasmids, including the clinically important carbapenem-resistance conjugative plasmid pOXA-48. In addition, we describe the conservation of CS in clinical E. coli isolates and its application to selectively kill plasmid-carrying bacteria. Our results provide new insights that establish the basis for developing CS-informed treatment strategies to combat plasmid-mediated ABR.

Methods to Quantify DNA Transfer in Enterococcus.

DNA uptake in Enterococcus normally occurs by conjugation, a natural process that is replicated in biomedical research to assess the transferability of different mobile genetic elements and chromosomal regions as well as to study the host range of plasmids and other conjugative elements. More efficient artificial methods to transform cells with foreign DNA as chemotransformation and electroporation are widely used in molecular genetics. Here, we described conjugation protocols to quantify DNA transfer among Enterococcus and revise current perspectives and lab strains. Protocols of electrotransformation have been previously described in this series.

Transfer dynamics of Tn6648, a composite integrative conjugative element generated by tandem accretion of Tn5801 and Tn6647 in Enterococcus faecalis.

BACKGROUND: Tn5801 [tet(M)], a Tn916-like element with site-specific affinity for the 3' end of the housekeeping gene guaA, may integrate at different chromosomal sites. OBJECTIVES: To characterize the genetic context of Tn5801 to define its transfer dynamics and impact on the evolution of Enterococcus faecalis (Efs). METHODS: WGS (Illumina HiSeq 2500) was performed on the Efs clinical strain Ef1 and primary and secondary transconjugants of Efs strains JH2-2 [which naturally contains Tn5801.B23, an unusual variant that lacks tet(M)], OG1RF and OG1SS carrying different copies of Tn5801-like elements. The transposon structures were analysed using a range of bioinformatics tools allowing us to identify the context of Tn5801-like elements. Growth rates at different tetracycline concentrations (0.5-20 mg/L) were estimated using a Synergy HTX plate reader. RESULTS: Tn5801.B15 [tet(M), 20.3 kb] exists and can be transferred either singly or within Tn6648 (53.2 kb), a composite element that comprises Tn5801.B15 and Tn6647, a newly identified 32.8 kb transposon that contains the prgABCT operon of pheromone-responsive plasmids. These transposons are able to integrate at specific 11 nt sequences at the 3' end of guaA and at other chromosomal sites in Efs genomes, thus being able to generate tandem accretions. These events may increase the number of tet(M) copies, enhancing tetracycline resistance in the recipient strain. CONCLUSIONS: This study describes Tn6647 and Tn6648 (comprising Tn6647 and Tn5801.B15) and highlights the diversity of mechanisms for conjugative mobilization and chromosomal insertion of these elements, which can result in tandem accretion. This strategy would facilitate the adaptation of Efs clones to environmental challenges.

Outbreak of NDM-1+CTX-M-15+DHA-1-producing Klebsiella pneumoniae high-risk clone in Spain owing to an undetectable colonised patient from Pakistan.

Here we describe an outbreak due to NDM-1+CTX-M-15+DHA-1-producing Klebsiella pneumoniae (NDM-1-Kp) in Spain related to a patient previously admitted to a healthcare centre in an endemic area (Pakistan). Nine colonised patients were detected in the Neurosurgery ward between September 2015 and February 2016 during the R-GNOSIS European Project. NDM-1-Kp isolates from clinical samples were also recovered in three of these patients. Surveillance culture at admission was negative in the index case, but NDM-1-Kp colonisation was detected 27 days later after receiving antibiotic treatment. Co-colonisation with a second NDM-1-Kp isolate was identified in this patient 61 days post-admission. Overall length of stay (LOS = 75 days) (P < 0.01) and LOS until carbapenemase detection (LOS-1 = 36 days) was longer in NDM-1-Kp carriers than in patients with other carbapenemase-producing Enterobacterales. Intervention strategies were implemented after the outbreak declaration and NDM-1-Kp transmission was contained. Among the NDM-1-Kp isolates, two clones [ST437 (index case and Patient 2) and ST101 (index case and Patients 3-9)] with different IncFIB NDM-1-containing plasmids were identified. Whole-genome sequencing revealed a high content of antimicrobial resistance genes in both isolates in addition to a large number of virulence factors. Colonisation with other epidemic (OXA-48-ST11-K. pneumoniae and VIM-1-ST54-K. pneumoniae) and non-epidemic (VIM-1-ST908-K. pneumoniae and VIM-ST431-Escherichia coli) clones was also detected in two NDM-1 carriers. Implementation of adequate infection control measures and uninterrupted active surveillance programmes for detecting patients with a low colonisation status are crucial to prevent the introduction and dissemination of NDM-type enzymes in our region.

Phylogenomics of Enterococcus faecalis from wild birds: new insights into host-associated differences in core and accessory genomes of the species.

Wild birds have been suggested to be reservoirs of antimicrobial resistant and/or pathogenic Enterococcus faecalis (Efs) strains, but the scarcity of studies and available sequences limit our understanding of the population structure of the species in these hosts. Here, we analysed the clonal and plasmid diversity of 97 Efs isolates from wild migratory birds. We found a high diversity, with most sequence types (STs) being firstly described here, while others were found in other hosts including some predominant in poultry. We found that pheromone-responsive plasmids predominate in wild bird Efs while 35% of the isolates entirely lack plasmids. Then, to better understand the ecology of the species, the whole genome of fivestrains with known STs (ST82, ST170, ST16 and ST55) were sequenced and compared with all the Efs genomes available in public databases. Using several methods to analyse core and accessory genomes (AccNET, PLACNET, hierBAPS and PANINI), we detected differences in the accessory genome of some lineages (e.g. ST82) demonstrating specific associations with birds. Conversely, the genomes of other Efs lineages exhibited divergence in core and accessory genomes, reflecting different adaptive trajectories in various hosts. This pangenome divergence, horizontal gene transfer events and occasional epidemic peaks could explain the population structure of the species.

Carbapenemases on the move: it's good to be on ICEs.

BACKGROUND: The evolution and spread of antibiotic resistance is often mediated by mobile genetic elements. Integrative and conjugative elements (ICEs) are the most abundant conjugative elements among prokaryotes. However, the contribution of ICEs to horizontal gene transfer of antibiotic resistance has been largely unexplored. RESULTS: Here we report that ICEs belonging to mating-pair formation (MPF) classes G and T are highly prevalent among the opportunistic pathogen Pseudomonas aeruginosa, contributing to the spread of carbapenemase-encoding genes (CEGs). Most CEGs of the MPFG class were encoded within class I integrons, which co-harbour genes conferring resistance to other antibiotics. The majority of the integrons were located within Tn3-like and composite transposons. Conserved attachment site could be predicted for the MPFG class ICEs. MPFT class ICEs carried the CEGs within composite transposons which were not associated with integrons. CONCLUSIONS: The data presented here provides a global snapshot of the different CEG-harbouring ICEs and sheds light on the underappreciated contribution of these elements to the evolution and dissemination of antibiotic resistance on P. aeruginosa.

First Report of an OXA-48- and CTX-M-213-Producing Kluyvera Species Clone Recovered from Patients Admitted in a University Hospital in Madrid, Spain.

Enterobacterales species other than Klebsiella pneumoniae also contribute to OXA-48 carbapenemase endemicity. We studied the emergence of an OXA-48-producing Kluyvera species clone, which expresses the novel CTX-M-213 enzyme, colonizing patients in our hospital. Rectal swabs from patients admitted in four wards (March 2014 to March 2016; R-GNOSIS project) were seeded onto Chromo ID-ESBL) and Chrom-CARB/OXA-48 chromogenic agar plates. Carbapenemases and extended-spectrum ß-lactamases (ESBLs) were characterized (PCR, sequencing, cloning, and site-directed mutagenesis), and antibiotic susceptibility was determined. Clonal relatedness was established (XbaI pulsed-field gel electrophoresis [XbaI-PFGE]), and plasmid content was studied (transformation, S1 nuclease digestion-PFGE, SB-hybridization, restriction fragment length polymorphism [RFLP] analysis [DraI and HpaI], and PCR [incompatibility group and repA, traU, and parA genes]). Whole-genome sequencing (WGS) (Illumina HiSeq-2500) and further bioinformatics analysis of plasmids (PLACNET and plasmidSPAdes) were performed. Patients' charts were reviewed. Six unrelated patients (median age, 75 years [range, 59 to 81 years]; 4/6 male patients) colonized with OXA-48-producing Kluyvera species isolates (>95% similarity of the PFGE pattern) were identified. Nosocomial acquisition was demonstrated. In two patients, OXA-48-producing Kluyvera species isolates coexisted with OXA-48-producing Raoultella ornithinolytica, K. pneumoniae, and Escherichia coli The blaOXA-48 gene was located on an ∼60-kb IncL plasmid related to IncL/M-pOXA-48a and the novel blaCTX-M-213 gene in a conserved chromosomal region of Kluyvera species isolates. CTX-M-213, different from CTX-M-13 (K56E) but conferring a similar ß-lactam resistance profile, was identified. Genomic analysis also revealed a 177-kb IncF plasmid (class I integron harboring sul1 and aadA2) and an 8-kb IncQ plasmid (IS4-blaFOX-8). We describe the first blaOXA-48 plasmid in Kluyvera spp. and the novel chromosomal CTX-M-213 enzyme and highlight further nosocomial dissemination of blaOXA-48 through clonal lineages or plasmids related to IncL/M-pOXA-48a.

Antimicrobial Resistance in Enterococcus spp. of animal origin.

Enterococci are natural inhabitants of the intestinal tract in humans and many animals, including food-producing and companion animals. They can easily contaminate the food and the environment, entering the food chain. Moreover, Enterococcus is an important opportunistic pathogen, especially the species E. faecalis and E. faecium, causing a wide variety of infections. This microorganism not only contains intrinsic resistance mechanisms to several antimicrobial agents, but also has the capacity to acquire new mechanisms of antimicrobial resistance. In this review we analyze the diversity of enterococcal species and their distribution in the intestinal tract of animals. Moreover, resistance mechanisms for different classes of antimicrobials of clinical relevance are reviewed, as well as the epidemiology of multidrug-resistant enterococci of animal origin, with special attention given to beta-lactams, glycopeptides, and linezolid. The emergence of new antimicrobial resistance genes in enterococci of animal origin, such as optrA and cfr, is highlighted. The molecular epidemiology and the population structure of E. faecalis and E. faecium isolates in farm and companion animals is presented. Moreover, the types of plasmids that carry the antimicrobial resistance genes in enterococci of animal origin are reviewed.

Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters.

OBJECTIVES: Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment. METHODS: Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996-2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates. RESULTS: Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine). CONCLUSIONS: To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA.

Multiple adaptive routes of Salmonella enterica Typhimurium to biocide and antibiotic exposure.

BACKGROUND: Biocides and antibiotics are used to eradicate or prevent the growth of microbial species on surfaces (occasionally on catheters), or infected sites, either in combination or sequentially, raising concerns about the development of co-resistance to both antimicrobial types. The effect of such compounds on Salmonella enterica, a major food-borne and zoonotic pathogen, has been analysed in different studies, but only few works evaluated its biological cost, and the overall effects at the genomic and transcriptomic levels associated with diverse phenotypes resulting from biocide exposure, which was the aim of this work. RESULTS: Exposure to triclosan, clorhexidine, benzalkonium, (but not to hypochlorite) resulted in mutants with different phenotypes to a wide range of antimicrobials even unrelated to the selective agent. Most biocide-resistant mutants showed increased susceptibility to compounds acting on the cell wall (ß-lactams) or the cell membranes (poly-L-lysine, polymyxin B, colistin or toxic anions). Mutations (SNPs) were found in three intergenic regions and nine genes, which have a role in energy production, amino acids, carbohydrates or lipids metabolism, some of them involved in membrane transport and pathogenicity. Comparative transcriptomics of biocide-resistant mutants showed over-expression of genes encoding efflux pumps (sugE), ribosomal and transcription-related proteins, cold-shock response (cpeE) and enzymes of microaerobic metabolism including those of the phosphotransferase system. Mainly ribosomal, metabolic and pathogenicity-related genes had affected expression in both in vitro-selected biocide mutants and field Salmonella isolates with reduced biocide susceptibility. CONCLUSIONS: Multiple pathways can be involved in the adaptation of Salmonella to biocides, mainly related with global stress, or involving metabolic and membrane alterations, and eventually causing "collateral sensitivity" to other antimicrobials. These changes might impact the bacterial-environment interaction, imposing significant bacterial fitness costs which may reduce the chances of fixation and spread of biocide resistant mutants.

Dissemination of Novel Antimicrobial Resistance Mechanisms through the Insertion Sequence Mediated Spread of Metabolic Genes.

The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein (ACP) reductase, which is also an important target for the development of narrow spectrum antibiotics. The analysis of triclosan resistant Staphylococcus aureus isolates had previously shown that in about half of the strains, the mechanism of triclosan resistance consists on the heterologous duplication of the triclosan target gene due to the acquisition of an additional fabI allele derived from Staphylococcus haemolyticus (sh-fabI). In the current work, the genomic sequencing of 10 of these strains allowed the characterization of two novel composite transposons TnSha1 and TnSha2 involved in the spread of sh-fabI. TnSha1 harbors one copy of IS1272, whereas TnSha2 is a 11.7 kb plasmid carrying TnSha1 present either as plasmid or in an integrated form generally flanked by two IS1272 elements. The target and mechanism of integration for IS1272 and TnSha1 are novel and include targeting of DNA secondary structures, generation of blunt-end deletions of the stem-loop and absence of target duplication. Database analyses showed widespread occurrence of these two elements in chromosomes and plasmids, with TnSha1 mainly in S. aureus and with TnSha2 mainly in S. haemolyticus and S. epidermidis. The acquisition of resistance by means of an insertion sequence-based mobilization and consequent duplication of drug-target metabolic genes, as observed here for sh-fabI, is highly reminiscent of the situation with the ileS2 gene conferring mupirocin resistance, and the dfrA and dfrG genes conferring trimethoprim resistance both of which are mobilized by IS257. These three examples, which show similar mechanisms and levels of spread of metabolic genes linked to IS elements, highlight the importance of this genetic strategy for recruitment and rapid distribution of novel resistance mechanisms in staphylococci.

Diversity and Evolution of the Tn5801-tet(M)-Like Integrative and Conjugative Elements among Enterococcus, Streptococcus, and Staphylococcus.

This work describes the diversity and evolution of Tn5801 among enterococci, staphylococci, and streptococci based on analysis of the 5,073 genomes of these bacterial groups available in gene databases. We also examined 610 isolates of Enterococcus (from 10 countries, 1987 to 2010) for the presence of this and other known CTn-tet(M) elements due to the scarcity of data about Tn5801 among enterococci. Genome location (by ICeu-I-pulsed-field gel electrophoresis [PFGE] hybridization/integration site identification), conjugation and fitness (by standard methods), Tn5801 characterization (by long-PCR mapping/sequencing), and clonality (by PFGE/multilocus sequence typing [MLST]) were studied. Twenty-three Tn5801 variants (17 unpublished) clustered in two groups, designated "A" (25 kb; n = 14; predominant in Staphylococcus aureus) and "B" (20 kb; n = 9; predominant in Streptococcus agalactiae). The percent GC content of the common backbone suggests a streptococcal origin of Tn5801 group B, with further acquisition of a 5-kb fragment that resulted in group A. Deep sequence analysis allowed identification of variants associated with clonal lineages of S. aureus (clonal complex 8 [CC8], sequence type 239 [ST239]), S. agalactiae (CC17), Enterococcus faecium (ST17/ST18), or Enterococcus faecalis (ST8), local variants, or variants located in different species and geographical areas. All Tn5801 elements were chromosomally located upstream of the guaA gene, which serves as an integration hot spot. Transferability was demonstrated only for Tn5801 type B among E. faecalis clonal backgrounds, which eventually harbored another Tn5801 copy. The study documents early acquisition of Tn5801 by Enterococcus, Staphylococcus, and Streptococcus. Clonal waves of these pathogens seem to have contributed to the geographical spread and local evolution of the transposon. Horizontal transfer, also demonstrated, could explain the variability observed, with the isolates often containing sequences of different origins.