PATO: Pangenome Analysis Toolkit.

MOTIVATION: We present the Pangenome Analysis Toolkit (PATO) designed to simultaneously analyze thousands of genomes using a desktop computer. The tool performs common tasks of pangenome analysis such as core-genome definition and accessory genome properties and includes new features that help characterize population structure, annotate pathogenic features and create gene sharedness networks. PATO has been developed in R to integrate with the large set of tools available for genetic, phylogenetic and statistical analysis in this environment. RESULTS: PATO can perform the most demanding bioinformatic analyses in minutes with an accuracy comparable to state-of-the-art software but 20-30× times faster. PATO also integrates all the necessary functions for the complete analysis of the most common objectives in microbiology studies. Finally, PATO includes the necessary tools for visualizing the results and can be integrated with other analytical packages available in R. AVAILABILITYAND IMPLEMENTATION: The source code for PATO is freely available at https://github.com/irycisBioinfo/PATO under the GPLv3 license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ecogenetics of antibiotic resistance in Listeria monocytogenes.

The acquisition process of antibiotic resistance in an otherwise susceptible organism is shaped by the ecology of the species. Unlike other relevant human pathogens, Listeria monocytogenes has maintained a high rate of susceptibility to the antibiotics used for decades to treat human and animal infections. However, L. monocytogenes can acquire antibiotic resistance genes from other organisms' plasmids and conjugative transposons. Ecological factors could account for its susceptibility. L. monocytogenes is ubiquitous in nature, most frequently including reservoirs unexposed to antibiotics, including intracellular sanctuaries. L. monocytogenes has a remarkably closed genome, reflecting limited community interactions, small population sizes and high niche specialization. The L. monocytogenes species is divided into variants that are specialized in small specific niches, which reduces the possibility of coexistence with potential donors of antibiotic resistance. Interactions with potential donors are also hampered by interspecies antagonism. However, occasional increases in population sizes (and thus the possibility of acquiring antibiotic resistance) can derive from selection of the species based on intrinsic or acquired resistance to antibiotics, biocides, heavy metals or by a natural tolerance to extreme conditions. High-quality surveillance of the emergence of resistance to the key drugs used in primary therapy is mandatory.

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks.

BACKGROUND: Vancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. This study's aim was to analyse the effects of acquiring Tn1546-carrying plasmids with proven epidemicity in different bacterial host backgrounds. METHODS: Widespread Tn1546-carrying plasmids of different families RepA_N (n = 5), Inc18 (n = 4) and/or pHTß (n = 1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in the presence/absence of vancomycin. Plasmid stability was analysed (300 generations). WGS (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n = 49) was performed. SNP calling (Breseq software) of non-evolved strains was used for comparison. RESULTS: All plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-carrying plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2% to 18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9%-49%). Stability of Tn1546-carrying plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. SNPs and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. CONCLUSIONS: The stability of E. faecium Tn1546-carrying plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

Evaluation of CHROMagar™-Serratia agar, a new chromogenic medium for the detection and isolation of Serratia marcescens.

A comparative analysis of the performance of the new selective chromogenic CHROMagar™-Serratia culture medium for detection and isolation of Serratia marcescens was undertaken. A total of 134 clinical isolates (95 S. marcescens with and without carbapenemase production and 39 non-S. marcescens isolates) and 96 epidemiological samples (46 rectal swabs and 50 from environmental surfaces) were studied. Diagnostic values when compared with CHROMagar™-Orientation medium were 96.8% sensitivity, 100% specificity, 100% positive predictive value and 88.5% negative predictive value. In conclusion, CHROMagar™-Serratia shows an excellent ability for differentiation of S. marcescens among clinical isolates and in environmental samples.

Simulating the Influence of Conjugative-Plasmid Kinetic Values on the Multilevel Dynamics of Antimicrobial Resistance in a Membrane Computing Model.

Bacterial plasmids harboring antibiotic resistance genes are critical in the spread of antibiotic resistance. It is known that plasmids differ in their kinetic values, i.e., conjugation rate, segregation rate by copy number incompatibility with related plasmids, and rate of stochastic loss during replication. They also differ in cost to the cell in terms of reducing fitness and in the frequency of compensatory mutations compensating plasmid cost. However, we do not know how variation in these values influences the success of a plasmid and its resistance genes in complex ecosystems, such as the microbiota. Genes are in plasmids, plasmids are in cells, and cells are in bacterial populations and microbiotas, which are inside hosts, and hosts are in human communities at the hospital or the community under various levels of cross-colonization and antibiotic exposure. Differences in plasmid kinetics might have consequences on the global spread of antibiotic resistance. New membrane computing methods help to predict these consequences. In our simulation, conjugation frequency of at least 10-3 influences the dominance of a strain with a resistance plasmid. Coexistence of different antibiotic resistances occurs if host strains can maintain two copies of similar plasmids. Plasmid loss rates of 10-4 or 10-5 or plasmid fitness costs of ≥0.06 favor plasmids located in the most abundant species. The beneficial effect of compensatory mutations for plasmid fitness cost is proportional to this cost at high mutation frequencies (10-3 to 10-5). The results of this computational model clearly show how changes in plasmid kinetics can modify the entire population ecology of antibiotic resistance in the hospital setting.

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.

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.

2CS-CHXT Operon Signature of Chlorhexidine Tolerance among Enterococcus faecium Isolates.

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years that has recently acquired higher relevance in nosocomial infection control worldwide. Despite this, CHX tolerance among Enterococcus faecium bacteria, representing one of the leading agents causing nosocomial infections, has been poorly understood. This study provides new phenotypic and molecular data for better identification of CHX-tolerant E. faecium subpopulations in community and clinical contexts. The chlorhexidine MIC (MICCHX) distribution of 106 E. faecium isolates suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of the 2CS-CHXT operon, identified here. It encodes glucose and amino acid-polyamine-organocation family transporters, besides the DNA-binding response regulator ChtR, with a P102H mutation previously described only in CHX-tolerant clade A1 E. faecium, and the ChtS sensor. 2CS-CHXT seems to be associated with three regulons modulating diverse bacterial biological functions. Combined data from normal MIC distribution and 2CS-CHXT operon characterization support a tentative epidemiological cutoff (ECOFF) of 8 mg/liter to CHX, which is useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds calls for the prudent use of CHX in multiple contexts.IMPORTANCE Chlorhexidine is one of the substances included in the World Health Organization's list of essential medicines, which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic in health care (skin, hands, mouthwashes, eye drops) since the 1950s, its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here, we provide a comprehensive study on chlorhexidine tolerance among strains of Enterococcus faecium, one of the leading nosocomial agents worldwide, and identify a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Our data allowed for the proposal of a tentative epidemiological cutoff of 8 mg/liter, which is useful to detect tolerant E. faecium populations in surveillance studies in community and clinical contexts. The prediction of 2CS-CHXT regulons will also facilitate the design of future experimental studies to better uncover chlorhexidine tolerance among E. faecium bacteria.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

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.

Distribution of putative virulence markers in Enterococcus faecium: towards a safety profile review.

Objectives: The criteria for identification of Enterococcus faecium (Efm) with the ability to cause human infections are currently being debated by the European Food Safety Authority (EFSA). Strains that have an MIC of ampicillin of ≤ 2 mg/L and lack IS16/esp/hyl genes should be regarded as safe for use as feed additives in animal nutrition, despite the lack of knowledge about putative virulence marker (PVM) distribution in community Efm. We analysed the distribution of major PVM and ampicillin phenotypes in large Efm collections to investigate further the safety of strains from a public health perspective. Methods: Thirty-three PVM were assessed by PCR/sequencing among clonally disparate Efm (n = 328; 1986-2015) from different origins. We analysed ampicillin susceptibility (Etest/broth microdilution) according to EUCAST guidelines, clonal relationship (MLST) and genomic location of PVM (S1-PFGE/hybridization). Results: Infection-derived Efm were more enriched in PVM and the increase in ampicillin MIC was positively correlated with an enrichment in different PVM. PVM coding for surface (esp/sgrA/ecbA/complete acm) and pili proteins, or others enhancing colonization (hyl/ptsD/orf1481) or plasticity (IS16), were strongly associated with clinical Efm (mostly clade A1), but also observed in clades A2/B at different rates. ptsD was a good marker of ampicillin-resistant Efm. ptsD, IS16, orf1481, sgrA and hospital variants of complete pili gene clusters are proposed as markers to assess the safety of Efm strains. Conclusions: Our study expands on the distribution of PVM in diverse Efm lineages and demonstrates the enrichment in infection-derived strains of PVM not previously included in EFSA's list of Efm safety criteria. The evidence of relevant Efm infection markers can impact the risk assessment of Efm strains in different public health contexts.

AcCNET (Accessory Genome Constellation Network): comparative genomics software for accessory genome analysis using bipartite networks.

AcCNET (Accessory genome Constellation Network) is a Perl application that aims to compare accessory genomes of a large number of genomic units, both at qualitative and quantitative levels. Using the proteomes extracted from the analysed genomes, AcCNET creates a bipartite network compatible with standard network analysis platforms. AcCNET allows merging phylogenetic and functional information about the concerned genomes, thus improving the capability of current methods of network analysis. The AcCNET bipartite network opens a new perspective to explore the pangenome of bacterial species, focusing on the accessory genome behind the idiosyncrasy of a particular strain and/or population. AVAILABILITY AND IMPLEMENTATION: AcCNET is available under GNU General Public License version 3.0 (GPLv3) from http://sourceforge.net/projects/accnet CONTACT: : valfernandez.vf@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online.

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.

Long-term clonal dynamics of Enterococcus faecium strains causing bloodstream infections (1995-2015) in Spain.

OBJECTIVES: To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes. METHODS: All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hylEfm) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS). RESULTS: The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006. CONCLUSIONS: Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients.

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

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.

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.

Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

OBJECTIVES: Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units. METHODS: From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed. RESULTS: VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids). CONCLUSIONS: Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids.

The impact of host metapopulation structure on the population genetics of colonizing bacteria.

Many key bacterial pathogens are frequently carried asymptomatically, and the emergence and spread of these opportunistic pathogens can be driven, or mitigated, via demographic changes within the host population. These inter-host transmission dynamics combine with basic evolutionary parameters such as rates of mutation and recombination, population size and selection, to shape the genetic diversity within bacterial populations. Whilst many studies have focused on how molecular processes underpin bacterial population structure, the impact of host migration and the connectivity of the local populations has received far less attention. A stochastic neutral model incorporating heightened local transmission has been previously shown to fit closely with genetic data for several bacterial species. However, this model did not incorporate transmission limiting population stratification, nor the possibility of migration of strains between subpopulations, which we address here by presenting an extended model. We study the consequences of migration in terms of shared genetic variation and show by simulation that the previously used summary statistic, the allelic mismatch distribution, can be insensitive to even large changes in microepidemic and migration rates. Using likelihood-free inference with genotype network topological summaries we fit a simpler model to commensal and hospital samples from the common nosocomial pathogens Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium. Only the hospital data for E. faecium display clearly marked deviations from the model predictions which may be attributable to its adaptation to the hospital environment.