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.

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.

The endometrial microbiota of women with or without a live birth within 12 months after a first failed IVF/ICSI cycle.

The endometrial microbiota composition may be associated with implantation success. However, a 'core' composition has not yet been defined. This exploratory study analysed the endometrial microbiota by 16S rRNA sequencing (V1-V2 region) of 141 infertile women whose first IVF/ICSI cycle failed and compared the microbiota profiles of women with and without a live birth within 12 months of follow-up, and by infertility cause and type. Lactobacillus was the most abundant genus in the majority of samples. Women with a live birth compared to those without had significantly higher Lactobacillus crispatus relative abundance (RA) (p = 0.029), and a smaller proportion of them had ≤ 10% L. crispatus RA (42.1% and 70.4%, respectively; p = 0.015). A smaller proportion of women in the male factor infertility group had ≤ 10% L. crispatus RA compared to women in the unexplained and other infertility causes groups combined (p = 0.030). Women with primary infertility compared to secondary infertility had significantly higher L. crispatus RA (p = 0.004); lower proportions of them had ≤ 10% L. crispatus RA (p = 0.009) and > 10% Gardnerella vaginalis RA (p = 0.019). In conclusion, IVF/ICSI success may be associated with L. crispatus RA and secondary infertility with endometrial dysbiosis, more often than primary infertility. These hypotheses should be tested in rigorous well-powered longitudinal studies.

BCG Vaccination of Health Care Workers Does Not Reduce SARS-CoV-2 Infections nor Infection Severity or Duration: a Randomized Placebo-Controlled Trial.

Bacillus Calmette-Guerin (BCG) vaccination has been hypothesized to reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severity, and/or duration via trained immunity induction. Health care workers (HCWs) in nine Dutch hospitals were randomized to BCG or placebo vaccination (1:1) in March and April 2020 and followed for 1 year. They reported daily symptoms, SARS-CoV-2 test results, and health care-seeking behavior via a smartphone application, and they donated blood for SARS-CoV-2 serology at two time points. A total of 1,511 HCWs were randomized and 1,309 analyzed (665 BCG and 644 placebo). Of the 298 infections detected during the trial, 74 were detected by serology only. The SARS-CoV-2 incidence rates were 0.25 and 0.26 per person-year in the BCG and placebo groups, respectively (incidence rate ratio, 0.95; 95% confidence interval, 0.76 to 1.21; P = 0.732). Only three participants required hospitalization for SARS-CoV-2. The proportions of participants with asymptomatic, mild, or moderate infections and the mean infection durations did not differ between randomization groups. In addition, unadjusted and adjusted logistic regression and Cox proportional hazards models showed no differences between BCG and placebo vaccination for any of these outcomes. The percentage of participants with seroconversion (7.8% versus 2.8%; P = 0.006) and mean SARS-CoV-2 anti-S1 antibody concentration (13.1 versus 4.3 IU/mL; P = 0.023) were higher in the BCG than placebo group at 3 months but not at 6 or 12 months postvaccination. BCG vaccination of HCWs did not reduce SARS-CoV-2 infections nor infection duration or severity (ranging from asymptomatic to moderate). In the first 3 months after vaccination, BCG vaccination may enhance SARS-CoV-2 antibody production during SARS-CoV-2 infection. IMPORTANCE While several BCG trials in adults were conducted during the 2019 coronavirus disease epidemic, our data set is the most comprehensive to date, because we included serologically confirmed infections in addition to self-reported positive SARS-CoV-2 test results. We also collected data on symptoms for every day during the 1-year follow-up period, which enabled us to characterize infections in detail. We found that BCG vaccination did not reduce SARS-CoV-2 infections nor infection duration or severity but may have enhanced SARS-CoV-2 antibody production during SARS-CoV-2 infection in the first 3 months after vaccination. These results are in agreement with other BCG trials that reported negative results (but did not use serological endpoints), except for two trials in Greece and India that reported positive results but had few endpoints and included endpoints that were not laboratory confirmed. The enhanced antibody production is in agreement with prior mechanistic studies but did not translate into protection from SARS-CoV-2 infection.

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.

Air pollution from livestock farms and the oropharyngeal microbiome of COPD patients and controls.

Air pollution from livestock farms is known to affect respiratory health of patients with chronic obstructive pulmonary disease (COPD). The mechanisms behind this relationship, however, remain poorly understood. We hypothesise that air pollutants could influence respiratory health through modulation of the airway microbiome. Therefore, we studied associations between air pollution exposure and the oropharyngeal microbiota (OPM) composition of COPD patients and controls in a livestock-dense area. Oropharyngeal swabs were collected from 99 community-based (mostly mild) COPD cases and 184 controls (baseline), and after 6 and 12 weeks. Participants were non-smokers or former smokers. Annual average livestock-related outdoor air pollution at the home address was predicted using dispersion modelling. OPM composition was analysed using 16S rRNA-based sequencing in all baseline samples and 6-week and 12-week repeated samples of 20 randomly selected subjects (n = 323 samples). A random selection of negative control swabs, taken every sampling day, were also included in the downstream analysis. Both farm-emitted endotoxin and PM10 levels were associated with increased OPM richness in COPD patients (p < 0.05) but not in controls. COPD case-control status was not associated with community structure, while correcting for known confounders (multivariate PERMANOVA p > 0.05). However, members of the genus Streptococcus were more abundant in COPD patients (Benjamini-Hochberg adjusted p < 0.01). Moderate correlation was found between ordinations of 20 subjects analysed at 0, 6, and 12 weeks (Procrustes r = 0.52 to 0.66; p < 0.05; Principal coordinate analysis of Bray-Curtis dissimilarity), indicating that the OPM is relatively stable over a 12 week period and that a single sample sufficiently represents the OPM. Air pollution from livestock farms is associated with OPM richness of COPD patients, suggesting that the OPM of COPD patients is susceptible to alterations induced by exposure to air pollutants.

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.

CRISPR-Cas is associated with fewer antibiotic resistance genes in bacterial pathogens.

The acquisition of antibiotic resistance (ABR) genes via horizontal gene transfer (HGT) is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of ABR. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of ABR. We analysed approximately 40 000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens, including Enterococcus, Staphylococcus, Acinetobacter and Pseudomonas. We modelled the association between CRISPR-Cas and indicators of HGT, and found that pathogens with a CRISPR-Cas system were less likely to carry ABR genes than those lacking this defence system. Analysis of the mobile genetic elements (MGEs) targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of ABR. These results suggest a potential 'immunocompromised' state for multidrug-resistant strains that may be exploited in tailored interventions that rely on MGEs, such as phages or phagemids, to treat infections caused by bacterial pathogens. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Low IgA Associated With Oropharyngeal Microbiota Changes and Lung Disease in Primary Antibody Deficiency.

Common Variable Immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) are primary antibody deficiencies characterized by hypogammaglobulinemia and recurrent infections, which can lead to structural airway disease (AD) and interstitial lung disease (ILD). We investigated associations between serum IgA, oropharyngeal microbiota composition and severity of lung disease in these patients. In this cross-sectional multicentre study we analyzed oropharyngeal microbiota composition of 86 CVID patients, 12 XLA patients and 49 healthy controls (HC) using next-generation sequencing of the 16S rRNA gene. qPCR was used to estimate bacterial load. IgA was measured in serum. High resolution CT scans were scored for severity of AD and ILD. Oropharyngeal bacterial load was increased in CVID patients with low IgA (p = 0.013) and XLA (p = 0.029) compared to HC. IgA status was associated with distinct beta (between-sample) diversity (p = 0.039), enrichment of (Allo)prevotella, and more severe radiographic lung disease (p = 0.003), independently of recent antibiotic use. AD scores were positively associated with Prevotella, Alloprevotella, and Selenomonas, and ILD scores with Streptococcus and negatively with Rothia. In clinically stable patients with CVID and XLA, radiographic lung disease was associated with IgA deficiency and expansion of distinct oropharyngeal bacterial taxa. Our findings highlight IgA as a potential driver of upper respiratory tract microbiota homeostasis.

Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli.

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

Low-calcium diet in mice leads to reduced gut colonization by Enterococcus faecium.

The aim of this study was to determine whether dietary intervention influenced luminal Ca2+ levels and Enterococcus faecium gut colonization in mice. For this purpose, mice fed semi-synthetic food AIN93 were compared to mice fed AIN93-low calcium (LC). Administration of AIN93-LC resulted in lower luminal Ca2+ levels independent of the presence of E. faecium. Furthermore, E. faecium gut colonization was reduced in mice fed AIN93-LC based on culture, and which was in concordance with a reduction of Enterococcaceae in microbiota analysis. In conclusion, diet intervention might be a strategy for controlling gut colonization of E. faecium, an important opportunistic nosocomial pathogen.

Roux-Y Gastric Bypass and Sleeve Gastrectomy directly change gut microbiota composition independent of surgery type.

Bariatric surgery in morbid obesity, either through sleeve gastrectomy (SG) or Roux-Y gastric bypass (RYGB), leads to sustainable weight loss, improvement of metabolic disorders and changes in intestinal microbiota. Yet, the relationship between changes in gut microbiota, weight loss and surgical procedure remains incompletely understood. We determined temporal changes in microbiota composition in 45 obese patients undergoing crash diet followed by SG (n = 22) or RYGB (n = 23). Intestinal microbiota composition was determined before intervention (baseline, S1), 2 weeks after crash diet (S2), and 1 week (S3), 3 months (S4) and 6 months (S5) after surgery. Relative to S1, the microbial diversity index declined at S2 and S3 (p < 0.05), and gradually returned to baseline levels at S5. Rikenellaceae relative abundance increased and Ruminococcaceae and Streptococcaceae abundance decreased at S2 (p < 0.05). At S3, Bifidobacteriaceae abundance decreased, whereas those of Streptococcaceae and Enterobacteriaceae increased (p < 0.05). Increased weight loss between S3-S5 was not associated with major changes in microbiota composition. No significant differences appeared between both surgical procedures. In conclusion, undergoing a crash diet and bariatric surgery were associated with an immediate but temporary decline in microbial diversity, with immediate and permanent changes in microbiota composition, independent of surgery type.

Group IIA-Secreted Phospholipase A2 in Human Serum Kills Commensal but Not Clinical Enterococcus faecium Isolates.

Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to killing by serum, we uncover here that normal human serum effectively kills Enterococcus faecium Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in "noninflamed" serum was considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal concentration of hGIIA in serum effectively kills commensal E. faecium and that resistance of clinical E. faecium to hGIIA could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients.

Enterococcus faecium TIR-Domain Genes Are Part of a Gene Cluster Which Promotes Bacterial Survival in Blood.

Enterococcus faecium has undergone a transition to a multidrug-resistant nosocomial pathogen. The population structure of E. faecium is characterized by a sharp distinction of clades, where the hospital-adapted lineage is primarily responsible for bacteremia. So far, factors that were identified in hospital-adapted strains and that promoted pathogenesis of nosocomial E. faecium mainly play a role in adherence and biofilm production, while less is known about factors contributing to survival in blood. This study identified a gene cluster, which includes genes encoding bacterial Toll/interleukin-1 receptor- (TIR-) domain-containing proteins (TirEs). The cluster was found to be unique to nosocomial strains and to be located on a putative mobile genetic element of phage origin. The three genes within the cluster appeared to be expressed as an operon. Expression was detected in bacterial culture media and in the presence of human blood. TirEs are released into the bacterial supernatant, and TirE2 is associated with membrane vesicles. Furthermore, the tirE-gene cluster promotes bacterial proliferation in human blood, indicating that TirE may contribute to the pathogenesis of bacteremia.

Lipoteichoic acid synthesis inhibition in combination with antibiotics abrogates growth of multidrug-resistant Enterococcus faecium.

Enterococcus faecium is a multidrug-resistant (MDR) nosocomial pathogen causing significant morbidity in debilitated patients. New antimicrobials are needed to treat antibiotic-resistant E. faecium infections in hospitalised patients. E. faecium incorporates lipoteichoic acid (LTA) (1,3-polyglycerol-phosphate linked to glycolipid) in its cell wall. The small-molecule inhibitor 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] specifically blocks the activity of Staphylococcus aureus LtaS synthase, which polymerises 1,3-glycerolphosphate into LTA polymers. Here we characterised the effects of the small-molecule inhibitor 1771 on the growth of E. faecium isolates, alone (28 strains) or in combination with the antibiotics vancomycin, daptomycin, ampicillin, gentamicin or linezolid (15 strains), and on biofilm formation (16 strains). Inhibition of LTA synthesis at the surface of the cell by compound 1771 in combination with current antibiotic therapy abrogates enterococcal growth in vitro but does not affect mature E. faecium biofilms. Targeting LTA synthesis may provide new possibilities to treat MDR E. faecium infections.

Expression of the Gene for Autotransporter AutB of Neisseria meningitidis Affects Biofilm Formation and Epithelial Transmigration.

Neisseria meningitidis is a Gram-negative bacterium that resides as a commensal in the upper respiratory tract of humans, but occasionally, it invades the host and causes sepsis and/or meningitis. The bacterium can produce eight autotransporters, seven of which have been studied to some detail. The remaining one, AutB, has not been characterized yet. Here, we show that the autB gene is broadly distributed among pathogenic Neisseria spp. The gene is intact in most meningococcal strains. However, its expression is prone to phase variation due to slipped-strand mispairing at AAGC repeats located within the DNA encoding the signal sequence and is switched off in the vast majority of these strains. Moreover, various genetic disruptions prevent autB expression in most of the strains in which the gene is in phase indicating a strong selection against AutB synthesis. We observed that autB is expressed in two of the strains examined and that AutB is secreted and exposed at the cell surface. Functionality assays revealed that AutB synthesis promotes biofilm formation and delays the passage of epithelial cell layers in vitro. We hypothesize that this autotransporter is produced during the colonization process only in specific niches to facilitate microcolony formation, but its synthesis is switched off probably to evade the immune system and facilitate human tissue invasion.

Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation.

Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of ß-methyl-D-glucoside. ß-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis.