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 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: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:Teixobactin is the first novel antimicrobial to be discovered in decades and represents a new class of antimicrobials. Teixobactin shows great promise with proven efficacy against multi-drug resistant organisms such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococci (VRE), and Mycobacterium tuberculosis. VRE infections are notoriously difficult to treat with complex and adaptable cell wall stress response systems, which confer intrinsic resistance to a wide variety of antimicrobials. The aim of this study was to isolate the teixobactin-induced transcriptional response by challenging lab strain Enterococcus faecalis JH2-2 with sub-MIC levels of teixobactin using RNA sequencing. Two cultures of E. faecalis were grown to an OD600 of 0.2 and subsequently split into three to form a total of six cultures (two samples, with three technical replicates each), and grown to an OD600 of 0.5. One set of three cultures were treated with 0.5 ug/ml (0.25 x MIC), and all six cultures were grown for a further 1h. Cells were harvested by centrifugation and stored at -80 degrees C. RNA was extracted using TRIzol-chloroform and RNA samples were run through the RNAeasy Minikit (Qiagen). The Agilent RNA 6000 Nano kit and the Agilent 2100 Bioanalyzer (RIN >8), was used to verify RNA quality as per the manufacturer’s instructions, and RNA concentration was determined using a NanoDrop ND-100 spectrophotometer. Ribosomal RNA was removed from total RNA using Ribo-Zero and cDNA libraries were created using the Illumina TruSeq™ stranded total RNA library prep kit. Sequencing was completed using Illumina MiSeq_v3 generating 150 bp single end reads. Adapter sequences were removed from raw fastq files using Flexbar and reads shorter than 50bp were discarded. Sequence reads from each sample were mapped against the E. faecalis JH2-2 genome (NZ_KI518257.1) using Bowtie to produce a table of raw read counts for JH2-2 genes for each of the replicates. Statistical and principal component analysis were performed using the Bioconductor DESeq package.

Dataset Information

Grad-seq Enterococcus faecalis and faecium

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