Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector

Wyckoff, H.A.; Barnes, Mary; Gillies, Kevin; Sandine, W. E.

doi:10.1128/aem.62.4.1481-1486.1996

Cited by 18 publications

(10 citation statements)

References 27 publications

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“…Rep_3 family includes several plasmids previously described in enterococci [ 72 - 75 ]. In our strain collection, a significant higher prevalence of Rep_3 family replicons were detected in E. faecium (n = 28) compared to E. faecalis (n = 6; p < 0.0001).…”

Section: Resultsmentioning

confidence: 99%

Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis

et al. 2015

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BackgroundThe 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 antimicrobial resistance genes encoded by mobile genetic elements (MGEs). Here, we investigate this mobilome in successful hospital associated genetic lineages, E. faecium sequence type (ST)17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) by DNA microarray analyses.ResultsThe hybridization patterns of 272 representative targets including plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29) and clustered regularly interspaced short palindromic repeats (CRISPR)-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. RCR-, Rep_3-, RepA_N- and Inc18-family plasmids were highly prevalent and with the exception of Rep_3, evenly distributed between the species. There was a considerable difference in the replicon profile, with rep17/pRUM, rep2/pRE25, rep14/EFNP1 and rep20/pLG1 dominating in E. faecium and rep9/pCF10, rep2/pRE25 and rep7 in E. faecalis strains. We observed an overall high correlation between the presence and absence of genes coding for resistance towards antibiotics, metals, biocides and their corresponding MGEs as well as their phenotypic antimicrobial susceptibility pattern. Although most IS families were represented in both E. faecalis and E. faecium, specific IS elements within these families were distributed in only one species. The prevalence of IS256-, IS3-, ISL3-, IS200/IS605-, IS110-, IS982- and IS4-transposases was significantly higher in E. faecium than E. faecalis, and that of IS110-, IS982- and IS1182-transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS981, ISEfm1 and IS1678 that 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.ConclusionsThe targeted MGEs were highly prevalent among the selected STs, underlining their potential importance in the evolution of hospital-adapted lineages of enterococci. Although the propensity of inter-species horizontal gene transfer (HGT) must be emphasized, the considerable species-specificity of these MGEs indicates a separate vertical evolution of MGEs within each species, and for E. faecalis within each ST.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1407-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis

et al. 2015

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“…PCR assays (Table 3) were optimized to amplify nine characterized enterococcal plasmid replicon groups (Garcia-Migura et al, 2007). The replicon grouping (pS86, pRUM, pMBB1, pCF10, pAM373, pRE25, pEF418, pIP501 and pHTb) is based on phylogenetic relationships between the replication genes (repA) of enterococcal plasmids (Wyckoff et al, 1996;De Boever et al, 2000;Martinez-Bueno et al, 2000;Schwarz et al, 2001;Grady & Hayes, 2003;Thompson & Collins, 2003;Hirt et al, 2005;Tomita & Ike, 2005). PCR assays targeting the TA systems o-e-z and axe-txe (Table 3) were run on the E. faecium strain collection (Ceglowski et al, 1993;Grady & Hayes, 2003;Sletvold et al, 2007).…”

Section: Dna Extraction Pcr Assays and Dna Sequencingmentioning

confidence: 99%

PCR-based plasmid typing inEnterococcus faeciumstrains reveals widely distributed pRE25-, pRUM-, pIP501- and pHTβ-related replicons associated with glycopeptide resistance and stabilizing toxin–antitoxin systems

Rosvoll

Pedersen

Sletvold

et al. 2010

FEMS Immunol Med Microbiol

104

119

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A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHTbeta (n=14) were observed in 83% of the strains, while pS86, pCF10, pAM373, pMBB1 or pEF418 were not detected. Furthermore, 61% of the strains contained the axe-txe (n=42) or/and the omega-epsilon-zeta (n=18) plasmid stabilization loci. Sequence analyses divided the omega-epsilon-zeta operon into two distinct phylogenetic groups. The present typing scheme accounted for about 60% of the total number of plasmids detected by S1 nuclease analyses, which revealed zero to seven plasmids (10 kb to >200 kb) per isolate. Interestingly, strains belonging to the clinically important clonal complex 17 (CC17) yielded a significantly higher number of plasmids (3.1) and pRUM replicons (74%) than non-CC17 strains (2.2% and 35%, respectively). A prevalent genetic linkage between the pRUM-replicon type and axe-txe was demonstrated by cohybridization analyses. The vanA resistance determinant was associated with all four replicon types, but we also confirmed the genetic linkage of vanA to unknown transferable replicons. PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments.

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“…pAHG0090b has a G+C content of 37% and is predicted to encode 6 proteins. pAHG0090a is a 1,925 bp plasmid that exhibits extensive sequence similarity to a cryptic plasmid of similar size from Enterococcus faecium 226 (pMBB1 [1,932 bp], 96% identity and 67% coverage) (Wyckoff et al, 1996), and larger cryptic plasmids from Lactococcus fermentum KC5b (pKC5b [4,392 bp], 99% identity and 75% coverge) (Pavlova et al, 2002) and Lactococcus lactis (pCRL291.1 [4,640 bp], 89% identity and 70% coverage). pAHG0090a has a G+C content of 33% and is predicted to encode a single protein predicted to function in plasmid replication.…”

Section: Resultsmentioning

confidence: 99%

Enterococcus faecalisAHG0090 is a genetically tractable bacterium and produces a secreted peptidic bioactive that suppresses NF-kB activation in human gut epithelial cells

Cuív

Giria

Hoedt

et al. 2018

Preprint

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16Enterococcus faecalis is an early coloniser of the human infant gut and contributes to the development 17 of intestinal immunity. To better understand the functional capacity of E. faecalis we constructed a 18 broad host range RP4 mobilisable vector, pEHR513112, that confers chloramphenicol resistance and 19 used a metaparental mating approach to isolate E. faecalis AHG0090 from a faecal sample collected 20 from a healthy human infant. We demonstrated that E. faecalis AHG0090 is genetically tractable and 21 could be manipulated using traditional molecular microbiology approaches. E. faecalis AHG0090 was 22 comparable to the gold-standard anti-inflammatory bacterium Faecalibacterium prausnitzii A2-165 in 23 its ability to suppress cytokine mediated NF-B activation in human gut derived LS174T goblet cell-24 like and Caco-2 enterocyte-like cell lines. E. faecalis AHG0090 and F. prausnitzii A2-165 produced 25 secreted low molecular weight NF-B suppressive peptidic bioactives. Both bioactives were sensitive 26 to heat and proteinase K treatments although the E. faecalis AHG0090 bioactive was more resilient to 27 both forms of treatment. As expected, E. faecalis AHG0090 suppressed IL-1 induced NF-B-p65 28 subunit nuclear translocation and expression of the NF-B regulated genes IL-6, IL-8 and CXCL-10. 29Finally, we determined that E. faecalis AHG0090 is distantly related to other commensal strains and 30 likely encodes niche factors that support effective colonisation of the infant gut. 31

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Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector

Cited by 18 publications

References 27 publications

Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis

Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis

PCR-based plasmid typing inEnterococcus faeciumstrains reveals widely distributed pRE25-, pRUM-, pIP501- and pHTβ-related replicons associated with glycopeptide resistance and stabilizing toxin–antitoxin systems

Enterococcus faecalisAHG0090 is a genetically tractable bacterium and produces a secreted peptidic bioactive that suppresses NF-kB activation in human gut epithelial cells

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