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:Enterococci are normal inhabitants of the gastrointestinal tracts of humans and animals and thanks to their capability to tolerate different environmental conditions and their high rates of gene transfer, they are able to colonize various ecological niches, as food matrices. Enterococcus faecalis bacteria are defined as “border line” microorganisms. From one side they are used as food starters, bio-control agents and probiotics to improve human or animal health. From the other side, in the last 2 two decades enterococci have emerged as important nosocomial pathogens, because bearing high-level of resistance to antibiotics and several putative virulence factors. In this study the proteomic (LC-MS/MS) and the phenotypic characterization (enzymatic methods) of three strains of E. faecalis with different origin were performed in order to investigate the differences and/or similarities occurring between pathogenic and health promoting bacteria. The E. faecalis D27 isolated as cheese contaminant, E. faecalis Symbioflor 1 a probiotic strain and E. faecalis UW3114 a clinical isolate involved in urinary tract infection were the objects of the study. The comparison of cytosolic protein expression profiles of the three strains, highlighted statistically significant changes in the abundance of proteins mainly involved in specific metabolic pathways, nutrient transport, stress response and cell wall modulation. Moreover, especially in the food contaminant and the clinical isolate, several proteins with potential pathogenic implications were found. The analysis of the extracellular proteome provided interesting results concerning proteins involved in bacterial communication, such as pheromones and conjugative elements and also proteins able to interact with human components. The phenotypic assays evaluating i) biofilm formation ii) hemolytic activity on blood agar plates iii) protease activity, allowed typing of bacteria in relation to well-known pathogenic traits. The obtained results confirmed the pathogenic profile associated to the clinical isolate as compared to the food contaminant and to the probiotic and allowed to elucidate the risks associated with the poor characterized foodborne E. faecalis D27.
Project description:Enterococci are normal inhabitants of the gastrointestinal tracts of humans and animals and thanks to their capability to tolerate different environmental conditions and their high rates of gene transfer, they are able to colonize various ecological niches, as food matrices. Enterococcus faecalis bacteria are defined as “border line” microorganisms. From one side they are used as food starters, bio-control agents and probiotics to improve human or animal health. From the other side, in the last 2 two decades enterococci have emerged as important nosocomial pathogens, because bearing high-level of resistance to antibiotics and several putative virulence factors. In this study the proteomic (LC-MS/MS) and the phenotypic characterization (enzymatic methods) of three strains of E. faecalis with different origin were performed in order to investigate the differences and/or similarities occurring between pathogenic and health promoting bacteria. The E. faecalis D27 isolated as cheese contaminant, E. faecalis Symbioflor 1 a probiotic strain and E. faecalis UW3114 a clinical isolate involved in urinary tract infection were the objects of the study. The comparison of cytosolic protein expression profiles of the three strains, highlighted statistically significant changes in the abundance of proteins mainly involved in specific metabolic pathways, nutrient transport, stress response and cell wall modulation. Moreover, especially in the food contaminant and the clinical isolate, several proteins with potential pathogenic implications were found. The analysis of the extracellular proteome provided interesting results concerning proteins involved in bacterial communication, such as pheromones and conjugative elements and also proteins able to interact with human components.
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:Lactobacillus rhamnosus GG has become one of the most widely marketed and studied probiotic strains. Several genes important for probiotic function have been identified, including the spaCBA-srtC1 gene cluster encoding pili, which have been shown to be important for certain of its probiotic properties. The spaCBA-srtC1 gene cluster has been reported to be unstable in L. rhamnosus GG isolated from liquid dairy products and therefore the present study examined the L. rhamnosus GG genome stability throughout an industrial production process from the original deposit to the freeze-dried products including intermediate fermentations and single colony isolates prepared from these samples. The results showed that the original deposit was identical to the reference ATCC and that the genome sequence stayed fully intact throughout the production process. No SNPs or larger genomic changes occurred in any of the samples throughout the production process and the spaCBA-srtC1 gene locus was fully conserved and intact in all 31 samples examined. In addition, phenotypic expression of pili was demonstrated using immune-gold labelling EM. The images showed that pili production was preserved throughout the production process and that the number of pili were consistent in all batches. The present study extends the scope of previous findings to an industrial setting and shows that the region around the spaCBA-srtC1 cluster exhibits high stability in L. rhamnosus GG in an industrial production process.
Project description:Enterococcus faecalis is a major nosocomial pathogen frequently isolated from non- healing wound infections. The major factor responsible for the virulence and establishment of infection is its ability to form robust biofilm. This renders the recalcitrant nature of Enterococci towards the current treatment strategies. In the present study, the quantitative proteomic approach is carried out to elucidate the protein expression levels in E. faecalis at planktonic and biofilm stages. This will help to identify biofilm associated pathways in E. faecalis which inturn can be considered as novel targets for biofilm inhibition.