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.

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