Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

Abstract

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n ‫؍‬ 136) and glycopeptide-susceptible (n ‫؍‬ 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmidmediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that "plasmid addiction systems" may contribute to the persistence of GREF in nonselective environments.

Diversity of Tn 1546 and Its Role in the Dissemination of Vancomycin-Resistant Enterococci in Portugal

“…Difficulties also arise from the different methods used for this purpose, which complicates comparison among studies (14,44,45). Tn1546 may be located in conjugative plasmids (14,20,25,43,47) or in larger composite transposons, as documented for isolates with chromosomally located vanA (15). Horizontal transfer of conjugative plasmids containing Tn1546 seems to have played a relevant role in the recent increase of VRE in Portugal, as vanA was plasmid located in most VRE isolates studied and specific plasmids containing the most prevalent Tn1546 variants were identified in representative distinct clones collected from different cities and years (13).…”

“…Nevertheless, the role of prevalent and persistent E. faecalis and E. faecium strains in the maintenance of the resistance by acquiring different genetic elements must not be underestimated, highlighting the complex epidemiology associated with successful spread of multidrug-resistant enterococci, as illustrated in Poland and the United States, where both successful plasmids and prevalent clones contributed to endemicity (11,21,22). Plasmid persistence has been described in areas with both high and low antibiotic selective pressure, as in the United States versus Norway or Denmark, suggesting that factors other than antibiotic pressure might be important in VRE maintenance and epidemiology (17,20).…”

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

“…Based on the sequence alignment of the axe-txe, par, parDE, ccd, mazEF, relBE, higBA, vagCD, and phd-doc TA genes in the National Center for Biotechnology Information database, pairs of fixed primers specific to each TA system were designed to amplify internal fragments with sizes ranging from 456 bp to 1.04 kb. The sequence of primers to amplify the --gene system was identical to those reported (44). The sequences of all primers are listed in SI Table 4. PCR amplification was performed in a final volume of 100 l containing 1-3 l of purified total DNA or plasmid DNA, 1ϫ PCR buffer (20 mM Tris⅐HCl/50 mM KCl, pH 8.4), 1.5 mM MgCl 2 , 0.2 mM each deoxynucleoside triphosphate, 0.5 M each primer, and 2.5 units of Taq polymerase (Invitrogen, Carlsbad, CA).…”

Prevalence, Persistence, and Molecular Characterization of Glycopeptide-Resistant Enterococci in Norwegian Poultry and Poultry Farmers 3 to 8 Years after the Ban on Avoparcin

“…Our recent study of GRE persistence on two Norwegian farms previously exposed to avoparcin concluded that the GRE population in poultry is heterogeneous with a common plasmid-borne Tn1546 element shared by distinct genetic lineages (21). It was suggested that Tn1546 might be stably maintained through physical linkage to a putative pRE25-related postsegregational killing (PSK) system (21).…”