Project description:Enterococcus faecium has emerged as a major opportunistic pathogen for two decades, with the spread of hospital-adapted multidrug-resistant clones. Members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of the study was to evaluate globally the impact of subinhibitory concentrations (SICs) of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium. Transcriptomic analysis was performed by RNA-seq (HiSeq 2500, Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/L, i.e. MIC 1/8). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced whereas 286 were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Amongst upregulated genes, EFAU004_02294 (fold change of 14.3) encoded a protein (EfmQnr) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive FQ resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292 coding for the collagen adhesin Acm was also induced by SIC of ciprofloxacin (fold change of 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both Efmqnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving a fluoroquinolone therapy.
Project description:The enterococci comprise a genus of 49 low-GC content Gram-positive commensal species within the Firmicutes phylum that are known to occupy diverse habitats, notably the gastrointestinal core microbiota of nearly every phylum, including human. Of particular clinical relevance are two rogue species of enterococci, Enterococcus faecalis and the distantly related Enterococcus faecium, standing among the nefarious multi-drug resistant and hospital-acquired pathogens. Despite increasing evidence for RNA-based regulation in the enterococci, including regulation of virulence factors, their transcriptome structure and arsenal of regulatory small sRNAs (sRNAs) are not thoroughly understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptomes of E. faecalis V583 and E. faecium AUS0004. We identified 2517 and 2771 transcription start sites (TSS) in E. faecalis and E. faecium, respectively. Based on the identified TSS, we created a global map of s70 promoter motifs. We also revealed features of 5’ and 3’UTRs across the genomes. The transcriptome maps also predicted 150 and 128 sRNA candidates in E. faecalis and E. faecium, respectively, some of which have been identified in previous studies and many of which are new. Finally, we validated several of the predicted sRNAs by Northern Blot in biologically relevant conditions. Comprehensive TSS mapping of two representative strains will provide a valuable resource for the continued development of RNA biology in the Enterococci.
Project description:Enterococcus faecium has become a major opportunistic pathogen with the emergence of multidrug-resistant clones that are well-adapted to the hospital environment. As part of the vast diversity of gut microbiota, they are faced with different environmental stress, including antimicrobial pressure. By contrast, little is known about the effect of non-antibiotic molecules on bacterial physiology while numerous drugs are used in inpatients, especially those hospitalized in intensive care units (ICUs). The aim of this study was to investigate the impact of the most prescribed xenobiotics in ICUs on fitness, pathogenicity and antimicrobial resistance of E. faecium. Several phenotypic analysis was carried out and we rapidly brought to light that caspofungin, an antifungal agent belonging to the echinocandin family, seemed to have an important impact on E. faecium growth. Since the fungal target of caspofungin [beta-(1,3)-glucan synthase] is absent in enterococci, the mechanism of caspofungin action was investigated by several approaches. First, we decided to confirm this result by electronic microscopy and a peptidoglycan analysis by Ultra Performance Liquid Chromatography coupled with mass spectrometry (UPLC-MS/MS). Again, we highlighted that caspofungin even at subinhibitory concentrations (SICs) seemed to have an impact on cell wall organization especially in muropeptide precursors abundance. Then, a transcriptomic analysis was performed by RNA-seq (HiSeq 2500, Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the presence or absence of caspofungin SIC (8 mg/L i.e., ¼ of the MIC). Transcriptomic analysis showed that the expression of 568 genes (19.9% of the genome) was significantly altered in the presence of caspofungin SIC, with 323 genes induced (fold change >2, p-value <0.1) and 245 genes repressed (fold change <-2, p-value <0.1). Regarding the repressed genes, the pdhABCD operon is largely downregulated (fold changes -4.3, -9.7, -6.9 and -6.4, respectively). This operon encoded components of the pyruvate deshydrogenase multienzyme complex involved in bacterial energetic pathway by the citrate cycle (i.e., TCA cycle). Moreover, it seemed that the glycerol metabolism pathway and in particular the glpOKF operon is downregulated too. The dramatic alteration of TCA seemed to have an drastic impact on bacterial cells viability indeed decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan.
Project description:Regulatory RNAs (sRNAs) are now considered as major players in many physiological and adaptive responses in pathogenic bacteria. sRNAs have been extensively studied in Gram-negative bacteria, but less information is available in Gram-positive pathogens. There is a spread of multidrug-resistant (MDR) opportunistic organisms, grouped as “ESKAPE” pathogens, which comprise enterococci, a leading cause of hospital-acquired infections and outbreaks with emergence of MDR isolates, especially vancomycin-resistant Enterococcus faecium (VREF). Note that no information about sRNA expression is known in this major opportunistic pathogen. By transcriptomic and genomic analyses using E. faecium Aus0004 reference strain, 249 transcribed IGRs, including sRNA candidates, were detected and, using a series of cut-offs, this set was lowered down to 54 sRNAs while 7 that were predicted based on comparative sequence analysis. RNA-seq was performed with and without subinhibitory concentrations (SIC) of daptomycin, a cyclic lipopeptide antibiotic used for VREF infections. Under daptomycin SIC exposure, 260 genes (9.1% of the genome) had a significant alteration of expression including 80 upregulated genes and 180 downregulated genes. Among the repressed genes, a large proportion (55%) coded for proteins involved in carbohydrate and transport metabolism. Also, we focused on the 9 sRNAs exhibiting the highest expression, and all of them were confirmed as expressed along bacterial growth by Northern blots and qPCR. Out of these 9 sRNAs, four had significantly lower or higher expression in the presence of daptomycin SIC, and therefore responded to antibiotic exposure. Finally, we also tested the expression of these 9 sRNAs in a collection of isogenic Aus0004 mutants with increasing levels of daptomycin resistance, and we observed by qPCR that some sRNAs had a significantly modified expression in daptomycin resistance mutants. It highlights the significant implication of some of the E. faecium sRNAs in the early steps of the development of daptomycin resistance. This is the first experimental genome-wide sRNA identification in Gram-positive E. faecium, a leading cause of hospital acquired infections.
Project description:The transcriptome of Enterococcus faecium E1162 growing in Brain heart Infusion Broth was compared in the mid-exponential growth phase (A660 = 0.3) at 25 C and 37 C.
Project description:Enterococcus faecium is a member of the human gut microbiota that has evolved to be a problematic nosocomial pathogen and a leading cause of bloodstream infections in hospitalized patients. Treatment of E. faecium infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here we explored the collateral effects of rifampin resistance-associated mutations in the E. faecium RNA polymerase β-subunit (RpoB). Of 14,384 publicly available E. faecium genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 E. faecium isolates collected from patients at a single medical center, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin class antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on E. faecium gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth, but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival in the hospital environment. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping E. faecium physiology and antibiotic resistance, two important features of this hospital-associated pathogen.