PlasmidEC and gplas2: an optimized short-read approach to predict and reconstruct antibiotic resistance plasmids in Escherichia coli.

Accurate reconstruction of Escherichia coli antibiotic resistance gene (ARG) plasmids from Illumina sequencing data has proven to be a challenge with current bioinformatic tools. In this work, we present an improved method to reconstruct E. coli plasmids using short reads. We developed plasmidEC, an ensemble classifier that identifies plasmid-derived contigs by combining the output of three different binary classification tools. We showed that plasmidEC is especially suited to classify contigs derived from ARG plasmids with a high recall of 0.941. Additionally, we optimized gplas, a graph-based tool that bins plasmid-predicted contigs into distinct plasmid predictions. Gplas2 is more effective at recovering plasmids with large sequencing coverage variations and can be combined with the output of any binary classifier. The combination of plasmidEC with gplas2 showed a high completeness (median=0.818) and F1-Score (median=0.812) when reconstructing ARG plasmids and exceeded the binning capacity of the reference-based method MOB-suite. In the absence of long-read data, our method offers an excellent alternative to reconstruct ARG plasmids in E. coli.

The population-level impact of Enterococcus faecalis genetics on intestinal colonization and extraintestinal infection.

IMPORTANCE: Enterococcus faecalis causes life-threatening invasive hospital- and community-associated infections that are usually associated with multidrug resistance globally. Although E. faecalis infections cause opportunistic infections typically associated with antibiotic use, immunocompromised immune status, and other factors, they also possess an arsenal of virulence factors crucial for their pathogenicity. Despite this, the relative contribution of these virulence factors and other genetic changes to the pathogenicity of E. faecalis strains remain poorly understood. Here, we investigated whether specific genomic changes in the genome of E. faecalis isolates influence its pathogenicity-infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection and intestinal colonization. Our findings indicate that E. faecalis genetics partially influence the infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection, possibly due to gut-to-bloodstream translocation, highlighting the potential substantial role of host and environmental factors, including gut microbiota, on the opportunistic pathogenic lifestyle of this bacterium.

Gut microbiome dynamics in index patients colonized with extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales after hospital discharge and their household contacts.

IMPORTANCE: Colonization with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) often precedes infections and is therefore considered as a great threat for public health. Here, we studied the gut microbiome dynamics in eight index patients colonized with ESBL-PE after hospital discharge and the impact of exposure to this index patient on the gut microbiome dynamics of their household contacts. We showed that the microbiome composition from index patients is different from their household contacts upon hospital discharge and that, in some of the index patients, their microbiome composition over time shifted toward the composition of their household contacts. In contrast, household contacts showed a stable microbiome composition over time irrespective of low-level extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) or extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) gut colonization, suggesting that, in healthy microbiomes, colonization resistance is able to prevent ESBL-PE expansion.

Unraveling Enterococcus susceptibility to quaternary ammonium compounds: genes, phenotypes, and the impact of environmental conditions.

Quaternary ammonium compounds (QACs) have been extensively used in the community, healthcare facilities, and food chain, in concentrations between 20 and 30,000 mg/L. Enterococcus faecalis and Enterococcus faecium are ubiquitous in these settings and are recognized as nosocomial pathogens worldwide, but QACs' activity against strains from diverse epidemiological and genomic backgrounds remained largely unexplored. We evaluated the role of Enterococcus isolates from different sources, years, and clonal lineages as hosts of QACs tolerance genes and their susceptibility to QACs in optimal, single-stress and cross-stress growth conditions. Only 1% of the Enterococcus isolates included in this study and 0.5% of publicly available Enterococcus genomes carried qacA/B, qacC, qacG, qacJ, qacZ, qrg, bcrABC or oqxAB genes, shared with >60 species of Bacillota, Pseudomonadota, Actinomycetota, or Spirochaetota. These genes were generally found within close proximity of antibiotics and/or metals resistance genes. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of benzalkonium chloride (BC) and didecyldimethylammonium chloride ranged between 0.5 and 4 mg/L (microdilution: 37°C/20 h/pH = 7/aerobiosis) for 210 E. faecalis and E. faecium isolates (two isolates carrying qacZ). Modified growth conditions (e.g., 22°C/pH = 5) increased MICBC/MBCBC (maximum of eightfold and MBCBC = 16 mg/L) and changed bacterial growth kinetics under BC toward later stationary phases in both species, including in isolates without QACs tolerance genes. In conclusion, Enterococcus are susceptible to in-use QACs concentrations and rarely carry QACs tolerance genes. However, their potential gene exchange with different microbiota, the decreased susceptibility to QACs under specific environmental conditions, and the presence of subinhibitory QACs concentrations in various settings may contribute to the selection of particular strains and, thus, require a One Health strategy to maintain QACs effectiveness. IMPORTANCE Despite the increasing use of quaternary ammonium compounds (QACs), the susceptibility of pathogens to these antimicrobials remains largely unknown. Enterococcus faecium and Enterococcus faecalis are susceptible to in-use QACs concentrations and are not main hosts of QACs tolerance genes but participate in gene transfer pathways with diverse bacterial taxa exposed to these biocides. Moreover, QACs tolerance genes often share the same genetic contexts with antibiotics and/or metals resistance genes, raising concerns about potential co-selection events. E. faecium and E. faecalis showed increased tolerance to benzalkonium chloride under specific environmental conditions (22°C, pH = 5), suggesting that strains might be selected in settings where they occur along with subinhibitory QACs concentrations. Transcriptomic studies investigating the cellular mechanisms of Enterococcus adaptation to QACs tolerance, along with longitudinal metadata analysis of tolerant populations dynamics under the influence of diverse environmental factors, are essential and should be prioritized within a One Health strategy.

Gut Colonization by ESBL-Producing Escherichia coli in Dogs Is Associated with a Distinct Microbiome and Resistome Composition.

The gut microbiome of humans and animals acts as a reservoir of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC). Dogs are known for having a high prevalence of ESBL-EC in their gut microbiota, although their ESBL-EC carrier status often shifts over time. We hypothesized that the gut microbiome composition of dogs is implicated in ESBL-EC colonization status. Therefore, we assessed whether ESBL-EC carriage in dogs is associated with changes in the gut microbiome and resistome. Fecal samples were collected longitudinally from 57 companion dogs in the Netherlands every 2 weeks for a total of 6 weeks (n = 4 samples/dog). Carriage of ESBL-EC was determined through selective culturing and PCR and in line with previous studies, we observed a high prevalence of ESBL-EC carriage in dogs. Using 16s rRNA gene profiling we found significant associations between detected ESBL-EC carriage and an increased abundance of Clostridium sensu stricto 1, Enterococcus, Lactococcus, and the shared genera of Escherichia-Shigella in the dog microbiome. A resistome capture sequencing approach (ResCap) furthermore, revealed associations between detected ESBL-EC carriage and the increased abundance of the antimicrobial resistance genes: cmlA, dfrA, dhfR, floR, and sul3. In summary, our study showed that ESBL-EC carriage is associated with a distinct microbiome and resistome composition. IMPORTANCE The gut microbiome of humans and animals is an important source of multidrug resistant pathogens, including beta-lactamase-producing Escherichia coli (ESBL-EC). In this study, we assessed if the carriage of ESBL-EC in dogs was associated with changes in gut composition of bacteria and antimicrobial resistant genes (ARGs). Therefore, stool samples from 57 dogs were collected every 2 weeks for a total of 6 weeks. Sixty eight percent of the dogs carried ESBL-EC during at least one of the time points analyzed. By investigating the gut microbiome and resistome composition, we observed specific changes at time points when dogs were colonized with ESBL-EC compared to time points whenESBL-EC were not detected. In conclusion, our study highlights the importance to study the microbial diversity in companion animals, as gut colonization of particular antimicrobial resistant bacteria might be an indication of a changed microbial composition that is associated with the selection of particular ARGs.

Characterization of blaKPC-2 and blaNDM-1 Plasmids of a K. pneumoniae ST11 Outbreak Clone.

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring blaKPC and blaNDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including blaKPC-2 and blaNDM-1. The blaKPC gene was present on a ~56 Kbp IncN plasmid and the blaNDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the blaNDM plasmid contained genes for conjugational transfer, only the blaKPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the blaKPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The blaKPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the blaNDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored blaKPC-2 and blaNDM-1. The blaKPC-harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the blaKPC-containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem.

Species-Specific Patterns of Gut Metabolic Modules in Dutch Individuals with Different Dietary Habits.

Diet is an important determinant of the human gut microbiome. Here, we analyzed fecal metagenomes of Dutch adults following omnivorous, pescatarian, vegan, and vegetarian diets. We compared the taxonomic composition of individuals from our study with publicly available gut metagenomes from westernized and non-westernized societies. We observed that, despite long-term transition to diets rich in plant fibers (vegan or vegetarian), the microbiomes of these were typical of westernized populations, and similar in composition to omnivores. Although there were no major differences in metabolic modules, we identified differences in the species that contributed to particular functions, such as carbohydrate degradation and short-chain fatty acid metabolism. Overall, this study shows functional redundancy of the microbiomes among westernized populations, which is independent of long-term individual dietary habits. IMPORTANCE Diet is an important modulator of the human gut microbiome, which is susceptible to increased consumption of plant fibers in vegan or vegetarian lifestyles. To investigate this, we compared the gut microbiome of Dutch adults following omnivorous, pescatarian, vegan and vegetarian diets. We did not observe major differences in the gut microbiome composition and function between individuals with different dietary habits. However, we observed differences in the species that contribute to the core functions of the gut microbiome. Our study thus emphasizes the need to better understand the species-specific functional changes associated with dietary habits in the human gut microbiome.

The interplay between community and hospital Enterococcus faecium clones within health-care settings: a genomic analysis.

BACKGROUND: The genomic relationships among Enterococcus faecium isolates are the subject of ongoing research that seeks to clarify the origins of observed lineages and the extent of horizontal gene transfer between them, and to robustly identify links between genotypes and phenotypes. E faecium is considered to form distinct groups-A and B-corresponding to isolates derived from patients who were hospitalised (A) and isolates from humans in the community (B). The additional separation of A into the so-called clades A1 and A2 remains an area of uncertainty. We aimed to investigate the relationships between A1 and non-A1 groups and explore the potential role of non-A1 isolates in shaping the population structure of hospital E faecium. METHODS: We collected short-read sequence data from invited groups that had previously published E faecium genome data. This hospital-based isolate collection could be separated into three groups (or clades, A1, A2, and B) by augmenting the study genomes with published sequences derived from human samples representing the previously defined genomic clusters. We performed phylogenetic analyses, by constructing maximum-likelihood phylogenetic trees, and identified historical recombination events. We assessed the pan-genome, did resistome analysis, and examined the genomic data to identify mobile genetic elements. Each genome underwent chromosome painting by use of ChromoPainter within FineSTRUCTURE software to assess ancestry and identify hybrid groups. We further assessed highly admixed regions to infer recombination directionality. FINDINGS: We assembled a collection of 1095 hospital E faecium sequences from 34 countries, further augmented by 33 published sequences. 997 (88%) of 1128 genomes clustered as A1, 92 (8%) as A2, and 39 (4%) as B. We showed that A1 probably emerged as a clone from within A2 and that, because of ongoing gene flow, hospital isolates currently identified as A2 represent a genetic continuum between A1 and community E faecium. This interchange of genetic material between isolates from different groups results in the emergence of hybrid genomes between clusters. Of the 1128 genomes, 49 (4%) hybrid genomes were identified: 33 previously labelled as A2 and 16 previously labelled as A1. These interactions were fuelled by a directional pattern of recombination mediated by mobile genetic elements. By contrast, the contribution of B group genetic material to A1 was limited to a few small regions of the genome and appeared to be driven by genomic sweep events. INTERPRETATION: A2 and B isolates coming into the hospital form an important reservoir for ongoing A1 adaptation, suggesting that effective long-term control of the effect of E faecium could benefit from strategies to reduce these genomic interactions, such as a focus on reducing the acquisition of hospital A1 strains by patients entering the hospital. FUNDING: Wellcome Trust.

Effects of early-life antibiotics on the developing infant gut microbiome and resistome: a randomized trial.

Broad-spectrum antibiotics for suspected early-onset neonatal sepsis (sEONS) may have pronounced effects on gut microbiome development and selection of antimicrobial resistance when administered in the first week of life, during the assembly phase of the neonatal microbiome. Here, 147 infants born at ≥36 weeks of gestational age, requiring broad-spectrum antibiotics for treatment of sEONS in their first week of life were randomized 1:1:1 to receive three commonly prescribed intravenous antibiotic combinations, namely penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime (ZEBRA study, Trial Register NL4882). Average antibiotic treatment duration was 48 hours. A subset of 80 non-antibiotic treated infants from a healthy birth cohort served as controls (MUIS study, Trial Register NL3821). Rectal swabs and/or faeces were collected before and immediately after treatment, and at 1, 4 and 12 months of life. Microbiota were characterized by 16S rRNA-based sequencing and a panel of 31 antimicrobial resistance genes was tested using targeted qPCR. Confirmatory shotgun metagenomic sequencing was executed on a subset of samples. The overall gut microbial community composition and antimicrobial resistance gene profile majorly shift directly following treatment (R2 = 9.5%, adjusted p-value = 0.001 and R2 = 7.5%, adjusted p-value = 0.001, respectively) and normalize over 12 months (R2 = 1.1%, adjusted p-value = 0.03 and R2 = 0.6%, adjusted p-value = 0.23, respectively). We find a decreased abundance of Bifidobacterium spp. and increased abundance of Klebsiella and Enterococcus spp. in the antibiotic treated infants compared to controls. Amoxicillin + cefotaxime shows the largest effects on both microbial community composition and antimicrobial resistance gene profile, whereas penicillin + gentamicin exhibits the least effects. These data suggest that the choice of empirical antibiotics is relevant for adverse ecological side-effects.

Impact of long-term dietary habits on the human gut resistome in the Dutch population.

The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilus when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature.

Functional characterization of a gene cluster responsible for inositol catabolism associated with hospital-adapted isolates of Enterococcus faecium.

Enterococcus faecium is a nosocomial, multidrug-resistant pathogen. Whole genome sequence studies revealed that hospital-associated E. faecium isolates are clustered in a separate clade A1. Here, we investigated the distribution, integration site and function of a putative iol gene cluster that encodes for myo-inositol (MI) catabolism. This iol gene cluster was found as part of an ~20 kbp genetic element (iol element), integrated in ICEEfm1 close to its integrase gene in E. faecium isolate E1679. Among 1644 E. faecium isolates, ICEEfm1 was found in 789/1227 (64.3 %) clade A1 and 3/417 (0.7 %) non-clade A1 isolates. The iol element was present at a similar integration site in 180/792 (22.7 %) ICEEfm1-containing isolates. Examination of the phylogenetic tree revealed genetically closely related isolates that differed in presence/absence of ICEEfm1 and/or iol element, suggesting either independent acquisition or loss of both elements. E. faecium iol gene cluster containing isolates E1679 and E1504 were able to grow in minimal medium with only myo-inositol as carbon source, while the iolD-deficient mutant in E1504 (E1504∆iolD) lost this ability and an iol gene cluster negative recipient strain gained this ability after acquisition of ICEEfm1 by conjugation from donor strain E1679. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources. In an intestinal colonization mouse model the colonization ability of E1504∆iolD mutant was not affected relative to the wild-type E1504 strain. In conclusion, we describe and functionally characterise a gene cluster involved in MI catabolism that is associated with the ICEEfm1 island in hospital-associated E. faecium isolates. We were unable to show that this gene cluster provides a competitive advantage during gut colonisation in a mouse model. Therefore, to what extent this gene cluster contributes to the spread and ecological specialisation of ICEEfm1-carrying hospital-associated isolates remains to be investigated.

Recovering Escherichia coli Plasmids in the Absence of Long-Read Sequencing Data.

The incidence of infections caused by multidrug-resistant E. coli strains has risen in the past years. Antibiotic resistance in E. coli is often mediated by acquisition and maintenance of plasmids. The study of E. coli plasmid epidemiology and genomics often requires long-read sequencing information, but recently a number of tools that allow plasmid prediction from short-read data have been developed. Here, we reviewed 25 available plasmid prediction tools and categorized them into binary plasmid/chromosome classification tools and plasmid reconstruction tools. We benchmarked six tools (MOB-suite, plasmidSPAdes, gplas, FishingForPlasmids, HyAsP and SCAPP) that aim to reliably reconstruct distinct plasmids, with a special focus on plasmids carrying antibiotic resistance genes (ARGs) such as extended-spectrum beta-lactamase genes. We found that two thirds (n = 425, 66.3%) of all plasmids were correctly reconstructed by at least one of the six tools, with a range of 92 (14.58%) to 317 (50.23%) correctly predicted plasmids. However, the majority of plasmids that carried antibiotic resistance genes (n = 85, 57.8%) could not be completely recovered as distinct plasmids by any of the tools. MOB-suite was the only tool that was able to correctly reconstruct the majority of plasmids (n = 317, 50.23%), and performed best at reconstructing large plasmids (n = 166, 46.37%) and ARG-plasmids (n = 41, 27.9%), but predictions frequently contained chromosome contamination (40%). In contrast, plasmidSPAdes reconstructed the highest fraction of plasmids smaller than 18 kbp (n = 168, 61.54%). Large ARG-plasmids, however, were frequently merged with sequences derived from distinct replicons. Available bioinformatic tools can provide valuable insight into E. coli plasmids, but also have important limitations. This work will serve as a guideline for selecting the most appropriate plasmid reconstruction tool for studies focusing on E. coli plasmids in the absence of long-read sequencing data.

Apparent nosocomial adaptation of Enterococcus faecalis predates the modern hospital era.

Enterococcus faecalis is a commensal and nosocomial pathogen, which is also ubiquitous in animals and insects, representing a classical generalist microorganism. Here, we study E. faecalis isolates ranging from the pre-antibiotic era in 1936 up to 2018, covering a large set of host species including wild birds, mammals, healthy humans, and hospitalised patients. We sequence the bacterial genomes using short- and long-read techniques, and identify multiple extant hospital-associated lineages, with last common ancestors dating back as far as the 19th century. We find a population cohesively connected through homologous recombination, a metabolic flexibility despite a small genome size, and a stable large core genome. Our findings indicate that the apparent hospital adaptations found in hospital-associated E. faecalis lineages likely predate the "modern hospital" era, suggesting selection in another niche, and underlining the generalist nature of this nosocomial pathogen.

The global dissemination of hospital clones of Enterococcus faecium.

BACKGROUND: The hospital-adapted A1 group of Enterococcus faecium remains an organism of significant concern in the context of drug-resistant hospital-associated infections. How this pathogen evolves and disseminates remains poorly understood. METHODS: A large, globally representative collection of short-read genomic data from the hospital-associated A1 group of Enterococcus faecium was assembled (n = 973). We analysed, using a novel analysis approach, global diversity in terms of both the dynamics of the accessory genome and homologous recombination among conserved genes. RESULTS: Two main modes of genomic evolution continue to shape E. faecium: the acquisition and loss of genes, including antimicrobial resistance genes, through mobile genetic elements including plasmids, and homologous recombination of the core genome. These events lead to new clones emerging at the local level, followed by the erosion of signals of clonality through recombination, and in some identifiable cases producing new clonal clusters. These patterns lead to new, emerging lineages which are able to spread globally over relatively short timeframes. CONCLUSIONS: The ability of A1 E. faecium to continually present new combinations of genes for potential selection suggests that controlling this pathogen will remain challenging but establishing a framework for understanding genomic evolution is likely to aid in tracking the threats posed by newly emerging lineages.

Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals.

BACKGROUND: Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. METHODS: We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012-2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. RESULTS: On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. CONCLUSIONS: In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.

Evolution of Colistin Resistance in the Klebsiella pneumoniae Complex Follows Multiple Evolutionary Trajectories with Variable Effects on Fitness and Virulence Characteristics.

The increasing prevalence of multidrug-resistant Klebsiella pneumoniae has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the in vitro evolutionary trajectories toward colistin resistance in four clinical K. pneumoniae complex strains and their impact on fitness and virulence characteristics. Through population sequencing during in vitro evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one K. pneumoniaesensu stricto strain, but not those of the other three K. pneumoniae complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. K. pneumoniaesensu stricto strains exhibited cross-resistance to LL-37, in contrast to the Klebsiella variicola subsp. variicola strain. Virulence, as determined in a Caenorhabditis elegans survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella.

Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogen, Enterococcus faecium.

Enterococcus faecium is a gut commensal of the gastro-digestive tract, but also known as nosocomial pathogen among hospitalized patients. Population genetics based on whole-genome sequencing has revealed that E. faecium strains from hospitalized patients form a distinct clade, designated clade A1, and that plasmids are major contributors to the emergence of nosocomial E. faecium. Here we further explored the adaptive evolution of E. faecium using a genome-wide co-evolution study (GWES) to identify co-evolving single-nucleotide polymorphisms (SNPs). We identified three genomic regions harbouring large numbers of SNPs in tight linkage that are not proximal to each other based on the completely assembled chromosome of the clade A1 reference hospital isolate AUS0004. Close examination of these regions revealed that they are located at the borders of four different types of large-scale genomic rearrangements, insertion sites of two different genomic islands and an IS30-like transposon. In non-clade A1 isolates, these regions are adjacent to each other and they lack the insertions of the genomic islands and IS30-like transposon. Additionally, among the clade A1 isolates there is one group of pet isolates lacking the genomic rearrangement and insertion of the genomic islands, suggesting a distinct evolutionary trajectory. In silico analysis of the biological functions of the genes encoded in three regions revealed a common link to a stress response. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. In conclusion, to our knowledge, this is the first study using GWES to identify genomic rearrangements, suggesting that there is considerable untapped potential to unravel hidden evolutionary signals from population genomic data.

Conditionally essential genes for survival during starvation in Enterococcus faecium E745.

BACKGROUND: The nosocomial pathogen Enterococcus faecium can survive for prolonged periods of time on surfaces in the absence of nutrients. This trait is thought to contribute to the ability of E. faecium to spread among patients in hospitals. There is currently a lack of data on the mechanisms that are responsible for the ability of E. faecium to survive in the absence of nutrients. RESULTS: We performed a high-throughput transposon mutant library screening (Tn-seq) to identify genes that have a role in long-term survival during incubation in phosphate-buffered saline (PBS) at 20 °C. A total of 24 genes were identified by Tn-seq to contribute to survival in PBS, with functions associated with the general stress response, DNA repair, metabolism, and membrane homeostasis. The gene which was quantitatively most important for survival in PBS was usp (locus tag: EfmE745_02439), which is predicted to encode a 17.4 kDa universal stress protein. After generating a targeted deletion mutant in usp, we were able to confirm that usp significantly contributes to survival in PBS and this defect was restored by in trans complementation. The usp gene is present in 99% of a set of 1644 E. faecium genomes that collectively span the diversity of the species. CONCLUSIONS: We postulate that usp is a key determinant for the remarkable environmental robustness of E. faecium. Further mechanistic studies into usp and other genes identified in this study may shed further light on the mechanisms by which E. faecium can survive in the absence of nutrients for prolonged periods of time.

Enterococcus faecium: from microbiological insights to practical recommendations for infection control and diagnostics.

Early in its evolution, Enterococcus faecium acquired traits that allowed it to become a successful nosocomial pathogen. E. faecium inherent tenacity to build resistance to antibiotics and environmental stressors that allows the species to thrive in hospital environments. The continual wide use of antibiotics in medicine has been an important driver in the evolution of E. faecium becoming a highly proficient hospital pathogen.For successful prevention and reduction of nosocomial infections with vancomycin resistant E. faecium (VREfm), it is essential to focus on reducing VREfm carriage and spread. The aim of this review is to incorporate microbiological insights of E. faecium into practical infection control recommendations, to reduce the spread of hospital-acquired VREfm (carriage and infections). The spread of VREfm can be controlled by intensified cleaning procedures, antibiotic stewardship, rapid screening of VREfm carriage focused on high-risk populations, and identification of transmission routes through accurate detection and typing methods in outbreak situations. Further, for successful management of E. faecium, continual innovation in the fields of diagnostics, treatment, and eradication is necessary.