Detecting co-selection through excess linkage disequilibrium in bacterial genomes.

Population genomics has revolutionized our ability to study bacterial evolution by enabling data-driven discovery of the genetic architecture of trait variation. Genome-wide association studies (GWAS) have more recently become accompanied by genome-wide epistasis and co-selection (GWES) analysis, which offers a phenotype-free approach to generating hypotheses about selective processes that simultaneously impact multiple loci across the genome. However, existing GWES methods only consider associations between distant pairs of loci within the genome due to the strong impact of linkage-disequilibrium (LD) over short distances. Based on the general functional organisation of genomes it is nevertheless expected that majority of co-selection and epistasis will act within relatively short genomic proximity, on co-variation occurring within genes and their promoter regions, and within operons. Here, we introduce LDWeaver, which enables an exhaustive GWES across both short- and long-range LD, to disentangle likely neutral co-variation from selection. We demonstrate the ability of LDWeaver to efficiently generate hypotheses about co-selection using large genomic surveys of multiple major human bacterial pathogen species and validate several findings using functional annotation and phenotypic measurements. Our approach will facilitate the study of bacterial evolution in the light of rapidly expanding population genomic data.

YajC, a predicted membrane protein, promotes Enterococcus faecium biofilm formation in vitro and in a rat endocarditis model.

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed ΔyajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium, we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane.

Dietary cystine restriction increases the proliferative capacity of the small intestine of mice.

Currently, over 88 million people are estimated to have adopted a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and can be converted to taurine and glutathione. We found earlier that intermolecular cystine bridges are essential for the barrier function of the intestinal mucus layer. Therefore, we now investigate the effect of low dietary cystine on the intestine. Mice (8/group) received a high fat diet with a normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels or bile acid conjugation after 2 weeks of low cystine feeding. In the colon, dietary cystine restriction results in an increase in goblet cell numbers, and a borderline significant increase mucus layer thickness. Gut microbiome composition and expression of stem cell markers did not change on the low cystine diet. Remarkably, stem cell markers, as well as the proliferation marker Ki67, were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of increased epithelial proliferation. In conclusion, 2 weeks of cystine restriction did not result in apparent systemic effects, but the low cystine diet increased the proliferative capacity specifically of the small intestine and induced the number of goblet cells in the colon.

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.

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.

Cohort profile of PLUTO: a perioperative biobank focusing on prediction and early diagnosis of postoperative complications.

PURPOSE: Although elective surgery is generally safe, some procedures remain associated with an increased risk of complications. Improved preoperative risk stratification and earlier recognition of these complications may ameliorate postoperative recovery and improve long-term outcomes. The perioperative longitudinal study of complications and long-term outcomes (PLUTO) cohort aims to establish a comprehensive biorepository that will facilitate research in this field. In this profile paper, we will discuss its design rationale and opportunities for future studies. PARTICIPANTS: Patients undergoing elective intermediate to high-risk non-cardiac surgery are eligible for enrolment. For the first seven postoperative days, participants are subjected to daily bedside visits by dedicated observers, who adjudicate clinical events and perform non-invasive physiological measurements (including handheld spirometry and single-channel electroencephalography). Blood samples and microbiome specimens are collected at preselected time points. Primary study outcomes are the postoperative occurrence of nosocomial infections, major adverse cardiac events, pulmonary complications, acute kidney injury and delirium/acute encephalopathy. Secondary outcomes include mortality and quality of life, as well as the long-term occurrence of psychopathology, cognitive dysfunction and chronic pain. FINDINGS TO DATE: Enrolment of the first participant occurred early 2020. During the inception phase of the project (first 2 years), 431 patients were eligible of whom 297 patients consented to participate (69%). Observed event rate was 42% overall, with the most frequent complication being infection. FUTURE PLANS: The main purpose of the PLUTO biorepository is to provide a framework for research in the field of perioperative medicine and anaesthesiology, by storing high-quality clinical data and biomaterials for future studies. In addition, PLUTO aims to establish a logistical platform for conducting embedded clinical trials. TRIAL REGISTRATION NUMBER: NCT05331118.

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.

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.

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.

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.

Whole Genome Sequence Analysis of the First Vancomycin-Resistant Enterococcus faecium Isolates from a Libyan Hospital in Tripoli.

The purpose of the study was to investigate the molecular characteristics and genetic relatedness of the first reported cases of vancomycin-resistant enterococci (VRE) from the Tripoli Medical Center, Libya. In total, 43 VRE isolates were obtained from various clinical sites throughout the years 2013-2014, including 40 vanA-type and 2 vanB-type vancomycin-resistant Enterococcus faecium isolates and 1 vanC1-type Enterococcus gallinarum. Of the 42 E. faecium, 19 isolates were subjected to whole genome sequencing. Core genome multilocus sequence typing (cgMLST) analysis revealed three sequence clusters (SCs) of clonally related isolates, which were linked to different hospital wards. The first two VRE isolates, isolated early 2013 from patients in the medical intensive care unit, were grouped in SC1 (MLST [ST] 78, vanB) and differed in only 3 of 1423 cgMLST alleles. The SC2 (n = 16, special care baby unit, neonatal intensive care unit, pediatric surgery ward, and oncology ward) and SC3 (n = 1, antenatal ward) were all ST80 vanA-VRE, but the single SC3 isolate differed in 233 alleles compared with SC2. Within SC2, isolates differed in 1-23 alleles. Comparison with a larger database of E. faecium strains indicated that all isolates clustered within the previously defined hospital clade A1. A combination of Resfinder and mlplasmid analysis identified the presence of resistance genes on different plasmid predicted genetic elements among different SCs. In conclusion, this study documents the first isolates causing outbreaks with VRE in the Libyan health care system. Further surveillance efforts using molecular typing methods to monitor spread of multidrug-resistant bacteria in the Libyan health care system are urgently needed.

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.

mlplasmids: a user-friendly tool to predict plasmid- and chromosome-derived sequences for single species.

Assembly of bacterial short-read whole-genome sequencing data frequently results in hundreds of contigs for which the origin, plasmid or chromosome, is unclear. Complete genomes resolved by long-read sequencing can be used to generate and label short-read contigs. These were used to train several popular machine learning methods to classify the origin of contigs from Enterococcus faecium, Klebsiella pneumoniae and Escherichia coli using pentamer frequencies. We selected support-vector machine (SVM) models as the best classifier for all three bacterial species (F1-score E. faecium=0.92, F1-score K. pneumoniae=0.90, F1-score E. coli=0.76), which outperformed other existing plasmid prediction tools using a benchmarking set of isolates. We demonstrated the scalability of our models by accurately predicting the plasmidome of a large collection of 1644 E. faecium isolates and illustrate its applicability by predicting the location of antibiotic-resistance genes in all three species. The SVM classifiers are publicly available as an R package and graphical-user interface called 'mlplasmids'. We anticipate that this tool may significantly facilitate research on the dissemination of plasmids encoding antibiotic resistance and/or contributing to host adaptation.

Identification of a Novel Genomic Island Associated with vanD-Type Vancomycin Resistance in Six Dutch Vancomycin-Resistant Enterococcus faecium Isolates.

Genomic comparison of the first six Dutch vanD-type vancomycin-resistant Enterococcus faecium (VRE) isolates with four vanD gene clusters from other enterococcal species and anaerobic gut commensals revealed that the vanD gene cluster was located on a genomic island of variable size. Phylogenetic inferences revealed that the Dutch VRE isolates were genetically not closely related and that genetic variation of the vanD-containing genomic island was not species specific, suggesting that this island is transferred horizontally between enterococci and anaerobic gut commensals.

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.

Characterization of Enterococcus Isolates Colonizing the Intestinal Tract of Intensive Care Unit Patients Receiving Selective Digestive Decontamination.

Enterococci have emerged as important opportunistic pathogens in intensive care units (ICUs). In this study, enterococcal population size and Enterococcus isolates colonizing the intestinal tract of ICU patients receiving Selective Digestive Decontamination (SDD) were investigated. All nine patients included in the study showed substantial shifts in the enterococcal 16S rRNA gene copy number in the gut microbiota during the hospitalization period. Furthermore, 41 Enterococcus spp. strains were isolated and characterized from these patients at different time points during and after ICU hospitalization, including E. faecalis (n = 13), E. faecium (n = 23), and five isolates that could not unequivocally assigned to a specific species (E. sp. n = 5) Multi locus sequence typing revealed a high prevalence of ST 6 in E. faecalis isolates (46%) and ST 117 in E. faecium (52%). Furthermore, antibiotic resistance phenotypes, including macrolide and vancomycin resistance, as well as virulence factor-encoding genes [asa1, esp-fm, esp-fs, hyl, and cyl (B)] were investigated in all isolates. Resistance to ampicillin and tetracycline was observed in 25 (61%) and 19 (46%) isolates, respectively. Furthermore, 30 out of 41 isolates harbored the erm (B) gene, mainly present in E. faecium isolates (78%). The most prevalent virulence genes were asa1 in E. faecalis (54%) and esp (esp-fm, 74%; esp-fs, 39%). Six out of nine patients developed nosocomial enterococcal infections, however, corresponding clinical isolates were unfortunately not available for further analysis. Our results show that multiple Enterococcus species, carrying several antibiotic resistance and virulence genes, occurred simultaneously in patients receiving SDD therapy, with varying prevalence dynamics over time. Furthermore, simultaneous presence and/or replacement of E. faecium STs was observed-, reinforcing the importance of screening multiple isolates to comprehensively characterize enterococcal diversity in ICU patients.

Antibiotic-Driven Dysbiosis Mediates Intraluminal Agglutination and Alternative Segregation of Enterococcus faecium from the Intestinal Epithelium.

UNLABELLED: The microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faecium agglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faecium from the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitro mimics agglutination of E. faecium in vivo. Also, the Ca(2+) levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faecium or other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks. IMPORTANCE: Infections with antibiotic-resistant enterococci are an emerging worldwide problem because enterococci are resistant to most of the antibiotics used in hospitals. During antibiotic treatment, the normal bacteria are replaced by resistant enterococci within the gut, from which they can spread and cause infections. We studied antibiotic-mediated intestinal proliferation of multidrug-resistant Enterococcus faecium and the effects on intestinal architecture. We demonstrated that antibiotics allow proliferation of E. faecium in the gut, alter the mucus-associated gut bacterial layer, and reduce the colon wall, mucus thickness, and amount of Muc-2 protein. E. faecium is agglutinated in the intestine in a matrix consisting of host molecules. We hypothesize that this matrix maintains a segregation of E. faecium from the epithelium. Understanding the processes that occur in the gut during antibiotic treatment may provide clues for future mucosal vaccination strategies to control E. faecium or other multidrug-resistant opportunistic pathogens, thereby preventing infections, hospital transmission, and outbreaks.