Project description:The success of Enterococcus faecium and E. faecalis evolving as multi-resistant nosocomial pathogens is associated with their ability to acquire and share adaptive traits, including mobile genetic elements (MGE) encoding antimicrobial resistance. Here, we define the mobilome in representative successful hospital associated genetic lineages, E. faecium ST17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) using DNA microarray analyses. The hybridization patterns of 272 targets representing plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29), and CRISPR-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. Although plasmids belonging to the RCR-, Rep_3-, RepA_N- and Inc18-families were well represented with no significant differences in prevalence, the presence of specific replicon classes differed highly between the species; E. faecium was dominated by rep17/pRUM, rep2/pRE25, rep14/EFNP1 and rep20/pLG1 and E. faecalis by rep9/pCF10, rep2/pRE25 and rep7. Tn916-elements conferring tetracycline resistance (tetM) were found in all E. faecalis strains, but only in two E. faecium strains. A significant higher prevalence of IS256-, IS3-, ISL3-, IS200/IS605-, IS110-, IS982-, and IS4-transposases were detected in E. faecium, and of IS110-, IS982- and IS1182-transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS981, ISEfm1 and IS1678 which have only been reported in few enterococcal isolates, were well represented in the E. faecium strains. E. faecalis ST40 strains harboured possible functional CRISPR-Cas systems, and still resistance and prophage sequences were generally well represented. Gene targets defined as the enterococcal mobilome, including plasmids, IS elements and transposons, resistance determinants, prophage sequences and CRISPR-Cas systems were highly prevalent, underlining their potential importance in the evolution of hospital associated STs. An association between axe-txe to the RepA_N-family and ω-ε-ζ to the Inc18-family, implicates the contribution of TA-systems in stable plasmid maintenance carrying virulence and resistance determinants in enterococci. The concurrent presence of defined MGE and their associated resistance markers was generally confirmed and illustrates the importance of horizontal gene transfer in the development of multidrug resistant enterococci.
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:The success of Enterococcus faecium and E. faecalis evolving as multi-resistant nosocomial pathogens is associated with their ability to acquire and share adaptive traits, including mobile genetic elements (MGE) encoding antimicrobial resistance. Here, we define the mobilome in representative successful hospital associated genetic lineages, E. faecium ST17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) using DNA microarray analyses. The hybridization patterns of 272 targets representing plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29), and CRISPR-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. Although plasmids belonging to the RCR-, Rep_3-, RepA_N- and Inc18-families were well represented with no significant differences in prevalence, the presence of specific replicon classes differed highly between the species; E. faecium was dominated by rep17/pRUM, rep2/pRE25, rep14/EFNP1 and rep20/pLG1 and E. faecalis by rep9/pCF10, rep2/pRE25 and rep7. Tn916-elements conferring tetracycline resistance (tetM) were found in all E. faecalis strains, but only in two E. faecium strains. A significant higher prevalence of IS256-, IS3-, ISL3-, IS200/IS605-, IS110-, IS982-, and IS4-transposases were detected in E. faecium, and of IS110-, IS982- and IS1182-transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS981, ISEfm1 and IS1678 which have only been reported in few enterococcal isolates, were well represented in the E. faecium strains. E. faecalis ST40 strains harboured possible functional CRISPR-Cas systems, and still resistance and prophage sequences were generally well represented. Gene targets defined as the enterococcal mobilome, including plasmids, IS elements and transposons, resistance determinants, prophage sequences and CRISPR-Cas systems were highly prevalent, underlining their potential importance in the evolution of hospital associated STs. An association between axe-txe to the RepA_N-family and M-OM-^I-M-NM-5-M-NM-6 to the Inc18-family, implicates the contribution of TA-systems in stable plasmid maintenance carrying virulence and resistance determinants in enterococci. The concurrent presence of defined MGE and their associated resistance markers was generally confirmed and illustrates the importance of horizontal gene transfer in the development of multidrug resistant enterococci. All together 272 DNA targets representing mobile genetic elements and antimicrobial resistance determinants associated with enterococci were spotted on a CustomArray 4x2K microarray from CustomArray Inc. Each fourplex microarray slide contain four identical sectors that were stripped and re-hybridized up to six times. Each target was represented by 1-5 probes each. The total of 1250 probes were Tm balanced by altering their lenght between 35 and 40 nucleotides. Total DNA of 41 samples were hybridized and a control strain, the fully sequenced E. faecalis V585, was included in one of the four sectors on each slide in each set of hybridization to monitor the overall array and hybridization quality.
Project description:Young adult N2 Caenorhabditis elegans were infected with Enterococcus faecalis or Enterococcus faecium for 8 h to determine the transcriptional host response to each enterococcal species. Analysis of differential gene expression in C. elegans young adults exposed to four different bacteria: heat-killed Escherichia coli strain OP50 (control), wild-type E. faecalis MMH594, wild-type E. faecium E007, or Bacillus subtilis PY79 (sigF::kan). Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection. Brain-heart infusion agar plates (10 ug/ml kanamycin) were used.
Project description:E. faecium is inherantly resistant to cephalosporins. Resistance is lost in Class A penicillin binding protein PbfF PonA mutants, but is reversible by pencillin exposure. E. faecium Affymetrix GeneChips were used to compare E. faecium expression properties of pbfF ponA mutant cells in the absence or presence of penicillin exposure. Significant differences were observed between the expression properties of mock and penicillin treated E. faecium CV571 (pbfF ponA double mutant) cells.
Project description:In order to successfully survive in and to colonize the gastrointestinal tract, bacteria need to develop strategies to overcome bile acid stress. The most prominent bile acids are the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA). In this study, we investigated the stress response of E. faecalis and E. faecium to sublethal concentrations of these three bile acids on the proteome level using DIA-MS. As both species showed similar IC50 for DCA and CDCA in growth experiments and both were highly resistant towards CA, we assumed similar changes to their protein expression profiles. Moreover, we investigated proteomic differences of E. faecalis grown under aerobic or microaerophilic conditions. Our findings showed similarities, but also species-specific variations in the response to the different bile acids, which reveal potential differences in the adaptation process. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis, but to a lesser extent in E. faecium. Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a “general bile acid response”. Among these, ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Specific for all E. faecalis samples was the up-expression of several subunits of a V-type ATPase and the down-expression of proteins involved in pyruvate-, citrate- and folate metabolism. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions in comparison to aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously.
Project description:We investigated the RNA-protein interactome of Enterococcus faecalis V583 and Enterococcus faecium Aus0004 by native gradient fractionation of complexes coupled to RNA-sequencing. Whole bacterial cell lysates were analysed by size and density in a glycerol gradient. At native conditions, RNA-protein complexes stay intact and sediment as a whole. Sedimentation profiles of individual RNAs appear correlated in case of interaction in a complex. The profile of KhpB caught our attention and we determined its RNA interactome by immunoprecipitation that suggests a role at the post-transcriptional level, binding notably several tRNAs, sRNAs, and 3’UTRs.
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:Enterococcus faecalis, a member of the human gastrointestinal microbiota, is a Gram-positive, opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can suppress or evade immune-mediated clearance by macrophages to promote persistent infection, although the exact mechanisms and bacterial factor(s) involved are not well-defined. In this study, we examined E. faecalis factor(s) involved in suppressing macrophage activation, as well the macrophage pathways modulated by E. faecalis to suppress activation. We observed that E. faecalis prevents ERK and p65 phosphorylation and reduces MyD88 expression leading to a reduction in NF-κB activity. We identified E. faecalis lactate dehydrogenase, which is important for lactic acid production by E. faecalis, to be necessary for macrophage suppression and demonstrated that E. faecalis lactate dehydrogenase-mediated immune suppression promotes E. coli survival during polymicrobial wound infection. Taken together, these results suggest that that E. faecalis-derived lactic acid is involved in macrophage subversion and may help to promote the virulence of co-infecting bacteria.