Vancomycin resistance in Enterococcus faecium isolated from Danish chicken meat is located on a pVEF4-like plasmid persisting in poultry for 18 years

The occurrence of vancomycin-resistant Enterococcus faecium (VREfm) in food has public health relevance since foodborne VREfm may colonize the gut of consumers and transfer vancomycin resistance genes to the indigenous gut microbiota. Therefore, we determined occurrence and elucidated genetic traits of VREfm in Danish retail chicken meat. Three out of 40 samples (7.5%) from two slaughterhouses yielded VREfm (vancomycin MIC > 32mg/L). This is the first report of VREfm in Danish retail poultry meat since 2010 (DANMAP). All three VREfm belonged to the sequence type ST32, cluster type CT1068. Using whole genome sequencing, we detected transposon Tn1546 harbouring the vanA operon encoding vancomycin resistance. The vanA operon was located on a 43.4kb plasmid highly similar (99.9% identity across 97.5% of the sequence) to pVEF4 which was observed in VREfm in Norwegian poultry in 1998 as well as in Danish poultry in 2010. The remarkable persistence of a pVEF4-like plasmid in enterococcal populations may be explained by the presence of two independent plasmid stability systems namely the ω/ε/ζ toxin-antitoxin system and the prgOPN gene cluster. Filter mating experiments showed that the pVEF4-like plasmid could transfer between E. faecium strains in vitro and that transfer occurred concomitantly with a larger, co-residing plasmid. The data presented here indicates that poultry meat constitutes a reservoir of VREfm and further investigations are needed to assess the risk of foodborne transmission to humans.