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

Abstract

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.

Human and Swine Hosts Share Vancomycin-Resistant Enterococcus faecium CC17 and CC5 and Enterococcus faecalis CC2 Clonal Clusters Harboring Tn 1546 on Indistinguishable Plasmids

“…Plasmid analysis included determination of size and content by PFGE of S1 nuclease-digested genomic DNA as previously described (16). Also, the identification of replication initiator proteins (rep) and maintenance systems (the toxin-antitoxin systems Axe-Txe and -ε-and the partition module parpAD1 ) was performed using recently developed PCR plasmid-typing methods, sequencing, and hybridization (25,44). Restriction fragment length polymorphism (RFLP) analysis using EcoRI or ClaI enzyme was performed for plasmids of Ͻ150 kb (13).…”

The Plasmidome of Firmicutes: Impact on the Emergence and the Spread of Resistance to Antimicrobials

“…Two types of pRUM plasmids are currently widespread among VRE and vancomycin-susceptible E. faecium isolates from different hosts. One contains RepA and Axe-Txe from pRUM and, eventually, the mobilization system of pC223 from S. aureus (70,71,(142)(143)(144). The other type is characterized by a RepA protein that is 95% identical to RepA-pRUM, lacking postsegregational killing Axe-Txe and the presence of a MOB P7 relaxase originally detected in pEF1, a plasmid with an environmental origin.…”

“…Inc18 streptococcal plasmids greatly influenced the worldwide increase of aminoglycoside-macrolide resistance among E. faecalis isolates from humans and animals during the 1970s (199). They also contributed to the spread of vancomycin resistance among E. faecium of animal origin in Europe and E. faecalis from hospitalized patients in the United States (7,70,71). Diverse narrow host range plasmids have been involved in local expansions of enterococci conferring resistance to first-line antibiotics such as gentamicin (Tn4001) or beta-lactams (ΔTn552 blaZ) (152) and beta-lactamaseproducing E. faecalis and E. faecium (152) (200)(201)(202), which highlights the role of endogenous plasmids and recombination in the adaptation of particular lineages FIGURE 16 Similarity of rep-like sequences encoding RIPs with the PriCT_1 domain.…”

“…Because many open reading frames of pHT and pMG1 plasmids do not show significant homology with any reported proteins, they used to be categorized as a new type of highly efficient conjugative plasmids with a MOB P family REL. This plasmid group is represented by relatives of pHT (pHTα, pHTβ, and pHTγ) and pMG1, which have greatly facilitated the dissemination of resistance to glycopeptides (Tn1546_vanA) and high-level resistance to aminoglycosides (Tn4001-like elements) among human E. faecium and E. avium isolates from the United States and Japan (123,124) and, to a lesser extent, European countries (7,70,71,122).…”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…Exchange of resistant strains among different ecosystems is less probable due to the supposed ecovar association, especially among hospital-associated E. faecium strains (see chapter 5), although dissemination across host barriers of vancomycin-and multi-resistant enterococci was described anecdotally, especially for the less strongly host-adapted E. faecalis strains (Manson et al, 2003a;Manson et al, 2003b;Manson et al, 2004;Agerso et al, 2008;Larsen et al, 2010;Hammerum et al, 2010;Freitas et al, 2011a). Vancomycin resistance among enterococci most probably spreads via a dissemination of mobile genetic elements of variants of the vanA-type element Tn1546 mostly located on mobilizable or conjugative plasmids (Sletvold et al, 2007;Novais et al, 2008;Sletvold et al, 2008;Freitas et al, 2009;Rosvoll et al, 2009;Sletvold et al, 2010;Laverde Gomez et al, 2011;Werner et al, 2011b;Freitas et al, 2011b). In vitro transfer of vanA plasmids has been determined in a number of studies (Werner et al, 1997;van den Braak et al, 1998;Werner et al, 2010b) and transfer in vivo in digestive tracts of animals and human volunteers was also shown (Moubareck et al, 2003;Lester et al, 2006;Lester and Hammerum 2010).…”