Theoretical considerations and empirical predictions of the pharmaco- and population dynamics of heteroresistance.

Antibiotics are considered one of the most important contributions to clinical medicine in the last century. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulation models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend that the definition of heteroresistance should include a consideration of the rate of return to susceptibility.

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

Imagining Kant's theory of scientific knowledge: philosophy and education in microbiology.

In the field of observational and experimental natural sciences (as is the case for microbiology), recent decades have been overinfluenced by overwhelming technological advances, and the space of abstraction has been frequently disdained. However, the predictable future of biological sciences should necessarily recover the synthetic dimension of "natural philosophy." We should understand the nature of Microbiology as Science, and we should educate microbiology scientists in the process of thinking. The critical process of thinking "knowing what we can know" is entirely based on Kant's Critique of Pure Reason. However, this book is extremely difficult to read (even for Kant himself) and almost inaccessible to modern experimental natural scientists. Professional philosophers might have been able to explain Kant to scientists; unfortunately, however, they do not get involved this type of education for science. The intention of this review is to introduce natural scientists, particularly microbiologists and evolutionary biologists, to the main rigorous processes (aesthetics, analytics, dialectics) that Kant identified to gain access to knowledge, always a partial knowledge, given that the correspondence between truth and reality is necessarily incomplete. This goal is attempted by producing a number of "images" (figures) to help the non-expert reader grasp the essential of Kant's message and by making final observations paralleling the theory of scientific knowledge with biological evolutionary processes and the role of evolutionary epistemology in science education. Finally, the influence of Kant's postulates in key-fields of microbiology, from taxonomy to systems biology is discussed.

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.

Threats of antibiotic resistance: an obliged reappraisal.

We are living in a society of fear, where the objectivity in estimating risks is distorted by the media and the interested parties. During more than half of a century, the feeling of antibiotic resistance as an apocalyptic phenomenon able to push our society to the high mortality rates caused by infectious diseases in the dark pre-antibiotic ages has been steadily rising. However, at the current status of modern medicine, at least in the high-medium income countries, mortality by lack of efficacy of the antibiotic armamentarium in the therapy of infections is a problem, but not a catastrophe. The threat of antibiotic resistance has many other aspects than failures of therapy in the individual patient. Among them, the increase in the frequency of severe and potentially lethal infections, as bacteremia, the population biology alterations of the healthy microbiota, the global acceleration of bacterial evolution by selecting natural genetic tools mediating microbial interactions, and, most importantly, by modifying the equilibrium and composition of environmental microbial communities. All these threats have huge implications for human health as members of a Biosphere entirely rooted in a menaced microbiosphere.

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.

Stratified reconstruction of ancestral Escherichia coli diversification.

BACKGROUND: Phylogenetic analyses of the bacterial genomes based on the simple classification in core- genes and accessory genes pools could offer an incomplete view of the evolutionary processes, of which some are still unresolved. A combined strategy based on stratified phylogeny and ancient molecular polymorphisms is proposed to infer detailed evolutionary reconstructions by using a large number of whole genomes. This strategy, based on the highest number of genomes available in public databases, was evaluated for improving knowledge of the ancient diversification of E. coli. This staggered evolutionary scenario was also used to investigate whether the diversification of the ancient E. coli lineages could be associated with particular lifestyles and adaptive strategies. RESULTS: Phylogenetic reconstructions, exploiting 6220 available genomes in Genbank, established the E. coli core genome in 1023 genes, representing about 20% of the complete genome. The combined strategy using stratified phylogeny plus molecular polymorphisms inferred three ancient lineages (D, EB1A and FGB2). Lineage D was the closest to E. coli root. A staggered diversification could also be proposed in EB1A and FGB2 lineages and the phylogroups into these lineages. Several molecular markers suggest that each lineage had different adaptive trajectories. The analysis of gained and lost genes in the main lineages showed that functions of carbohydrates utilization (uptake of and metabolism) were gained principally in EB1A lineage, whereas loss of environmental-adaptive functions in FGB2 lineage were observed, but this lineage showed higher accumulated mutations and ancient recombination events. The population structure of E. coli was re-evaluated including up to 7561 new sequenced genomes, showing a more complex population structure of E. coli, as a new phylogroup, phylogroup I, was proposed. CONCLUSIONS: A staggered reconstruction of E. coli phylogeny is proposed, indicating evolution from three ancestral lineages to reach all main known phylogroups. New phylogroups were confirmed, suggesting an increasingly complex population structure of E. coli. However these new phylogroups represent < 1% of the global E. coli population. A few key evolutionary forces have driven the diversification of the two main E. coli lineages, metabolic flexibility in one of them and colonization-virulence in the other.

Antibiotic Killing of Diversely Generated Populations of Nonreplicating Bacteria.

Nonreplicating bacteria are known to be (or at least commonly thought to be) refractory to antibiotics to which they are genetically susceptible. Here, we explore the sensitivity to killing by bactericidal antibiotics of three classes of nonreplicating populations of planktonic bacteria: (i) stationary phase, when the concentration of resources and/or nutrients are too low to allow for population growth; (ii) persisters, minority subpopulations of susceptible bacteria surviving exposure to bactericidal antibiotics; and (iii) antibiotic-static cells, bacteria exposed to antibiotics that prevent their replication but kill them slowly if at all, the so-called bacteriostatic drugs. Using experimental populations of Staphylococcus aureus Newman and Escherichia coli K-12 (MG1655) and, respectively, nine and seven different bactericidal antibiotics, we estimated the rates at which these drugs kill these different types of nonreplicating bacteria. In contrast to the common belief that bacteria that are nonreplicating are refractory to antibiotic-mediated killing, all three types of nonreplicating populations of these Gram-positive and Gram-negative bacteria are consistently killed by aminoglycosides and the peptide antibiotics daptomycin and colistin, respectively. This result indicates that nonreplicating cells, irrespectively of why they do not replicate, have an almost identical response to bactericidal antibiotics. We discuss the implications of these results to our understanding of the mechanisms of action of antibiotics and the possibility of adding a short-course of aminoglycosides or peptide antibiotics to conventional therapy of bacterial infections.

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.

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.

Basic Sciences Fertilizing Clinical Microbiology and Infection Management.

Basic sciences constitute the most abundant sources of creativity and innovation, as they are based on the passion of knowing. Basic knowledge, in close and fertile contact with medical and public health needs, produces distinct advancements in applied sciences. Basic sciences play the role of stem cells, providing material and semantics to construct differentiated tissues and organisms and enabling specialized functions and applications. However, eventually processes of "practice deconstruction" might reveal basic questions, as in de-differentiation of tissue cells. Basic sciences, microbiology, infectious diseases, and public health constitute an epistemological gradient that should also be an investigational continuum. The coexistence of all these interests and their cross-fertilization should be favored by interdisciplinary, integrative research organizations working simultaneously in the analytical and synthetic dimensions of scientific knowledge.

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.

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.