Jumamurat R. Bayjanov

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

ABSTRACT 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 Ca2+ 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. 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.

Comparative gut microbiota and resistome profiling of intensive care patients receiving selective digestive tract decontamination and healthy subjects

BackgroundThe gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed “selective decontamination of the digestive tract” (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes (“the resistome”) of ICU patients during SDD and of healthy subjects.ResultsFrom ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs.The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6′)-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects.ConclusionsThe gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

ABSTRACT Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.

RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum

BackgroundThe Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq).ResultsWe first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010xa0nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745.ConclusionsGenes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.

The two component system ChtRS contributes to chlorhexidine tolerance in Enterococcus faecium

ABSTRACT Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrug-resistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNA-binding response regulator (ChtR), which contributed to CHX tolerance in E. faecium. Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX- and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR. Transmission electron microscopy showed that upon challenge with CHX, the ΔchtR and ΔchtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans. Morphological abnormalities were also observed upon exposure of the ΔchtR and ΔchtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials.

Limited influence of hospital wastewater on the microbiome and resistome of wastewater in a community sewerage system

ABSTRACT Effluents from wastewater treatment plants (WWTPs) have been proposed to act as point sources of antibiotic‐resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in the environment. Hospital sewage may contribute to the spread of ARB and ARGs as it contains the feces and urine of hospitalized patients, who are more frequently colonized with multi‐drug resistant bacteria than the general population. However, whether hospital sewage noticeably contributes to the quantity and diversity of ARGs in the general sewerage system has not yet been determined. Here, we employed culture‐independent techniques, namely 16S rRNA gene sequencing and nanolitre‐scale quantitative PCRs, to assess the role of hospital effluent as a point source of ARGs in the sewerage system, through comparing microbiota composition and levels of ARGs in hospital sewage with WWTP influent with and without hospital sewage. Compared to other sites, hospital sewage was richest in human‐associated bacteria and contained the highest relative levels of ARGs. Yet, the relative abundance of ARGs was comparable in the influent of WWTPs with and without hospital sewage, suggesting that hospitals do not contribute importantly to the quantity and diversity of ARGs in the investigated sewerage system.

Therapy and Outcome of Staphylococcus aureus Infections of Intracorporeal Ventricular Assist Devices: Staphylococcus aureus VAD INFECTIONS

Abstract Infection of the driveline or pump pocket is a common complication in patients with ventricular assist devices (VADs) and Staphylococcus aureus is the main pathogen causing such infections. Limited evidence is currently available to guide the choice of antibiotic therapy and the duration of treatment in these patients. Patients at the University Medical Center Utrecht who developed a VAD‐related S. aureus infection between 2007 and 2016 were retrospectively assessed. Blood culture isolates were typed by whole genome sequencing to differentiate between relapses and reinfections, and to determine whether antibiotic therapy had led to acquisition of resistance mutations. Twenty‐eight patients had S. aureus VAD infections. Ten of these patients also suffered S. aureus bacteremia. Discontinuation of antibiotic therapy was followed by relapse in 50% of the patients without prior S. aureus bacteremia and in 80% of patients with bacteremia. Oral cephalexin could ultimately suppress the infection for the duration of follow‐up in 8/8 patients without S. aureus bacteremia and in 3/6 patients with S. aureus bacteremia. Clindamycin failed as suppressive therapy in 4/4 patients. Cephalexin appears an adequate choice for antibiotic suppression of VAD infections with methicillin‐susceptible S. aureus. In patients without systemic symptoms, it may be justified to attempt to stop therapy after treatment of the acute infection, but antibiotic suppression until heart transplant seems indicated in patients with S. aureus bacteremia.

Fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum

Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. Here, we identify genes that contribute to growth of E. faecium in human serum. We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, to completion and then compared its transcriptome during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. A high-throughput transposon sequencing (Tn-seq) approach was used to identify conditionally essential genes in E. faecium E745 during growth in serum. Genes involved in de novo nucleotide biosysnthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants also exhibited significantly attenuated virulence in an intravenous zebrafish infection model. We conclude that genes involved in carbohydrate and nucleotide metabolism of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism.

The microbiome and resistome of hospital sewage during passage through the community sewer system

Effluents from wastewater treatment plants (WWTPs) have been proposed to act as point sources of antibiotic-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in the environment. Hospital sewage may contribute to the spread of ARB and ARGs as it contains the feces and urine of hospitalized patients, who are more frequently colonized with multi-drug resistant bacteria than the general population. However, whether hospital sewage noticeably contributes to the quantity and diversity of ARGs in the general sewerage system has not yet been determined. Here, we employed culture-independent techniques, namely 16S rRNA and nanolitre-scale quantitative PCRs, to describe the role of hospital effluent as a point source of ARGs in the sewer system, through comparing microbiota composition and levels of ARGs in hospital sewage with WWTP influent, WWTP effluent and the surface water in which the effluent is released. Compared to other sample sites, hospital sewage was richest in human-associated bacteria and contained the highest relative levels of ARGs. Yet, the abundance of ARGs was comparable in WWTPs with and without hospital wastewater, suggesting that hospitals do not contribute to the spread of ARGs in countries with a functioning sewerage system.

Gut microbiota and resistome dynamics in intensive care patients receiving selective digestive tract decontamination

Background Critically ill patients hospitalized in an Intensive Care Unit (ICU) are at increased risk of acquiring potentially life-threatening infections with opportunistic pathogens. The gut microbiota of ICU patients forms an important reservoir for these infectious agents. To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed ‘Selective decontamination of the digestive tract’ (SDD), may be used. SDD has previously been shown to improve clinical outcome in ICU patients, but the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes (‘the resistome’) of ICU patients during SDD. Results During ICU-stay, 30 fecal samples of ten patients were collected. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. As a control group, feces from ten healthy subjects were collected twice, with a one-year interval. Gut microbiota and resistome composition were determined using 16S rRNA phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by low microbial diversity, decreased levels of E. coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6′)-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants and tetracyclines respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects. Conclusions The microbiota and resistome of ICU patients and healthy subjects were noticeably different, but importantly, levels of E. coli remained low during ICU hospitalization, presumably due to SDD therapy. Selection for four antibiotic resistance genes was observed, but none of these are of particular concern as they do not contribute to clinically relevant resistance. Our data support the ecological safety of SDD, at least in settings with low levels of circulating antibiotic resistance.