The occurrence of vancomycin-resistant Enterococcus faecium (VREfm) in food is relevant to public health as 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 > 32 mg/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 43.4 kb plasmid highly similar (99.9% identity across 97.5% of the sequence) to pVEF4, which was observed in VREfm in Norwegian poultry in 1998 and 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, 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 indicate that poultry meat constitutes a reservoir of VREfm and further investigations are needed to assess the risk of foodborne transmission to humans.

A growing number of humans are being infected by antibiotic resistant Staphylococcus aureus originating from livestock. The preference of S. aureus for humans or animals is in part determined by factors encoded by viruses (phages) that reside in the bacterial genome.

Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage‐encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus . ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high‐affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis . Analysis of β‐galactosidase expression indicated that decision frequency is independent of host factors. The white “lysogenic” phenotype, which relies on the expression of cI , could be switched to a stable blue “lytic” phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus .

Prophages of the ΦSa3int family are commonly found in human-associated strains of Staphylococcus aureus where they encode factors for evading the human innate immune system. In contrast, they are usually absent in livestock-associated methicillin-resistant S. aureus (LA-MRSA) strains where the phage attachment site is mutated compared to the human strains. However, ΦSa3int phages have been found in a small subset of LA-MRSA strains belonging to clonal complex 398 (CC398), including a lineage that is widespread in pig farms in Northern Jutland, Denmark. This lineage contains amino acid changes in the DNA topoisomerase V and the DNA gyrase encoded by grlA and gyrA, respecively, which have been associated with fluoroquinolone (FQ) resistance. As both of these enzymes are involved in DNA supercoiling, we speculated that the mutations might impact recombination between the ΦSa3int phage and the bacterial chromosome to enhance phage integration. To examine this, we introduced the FQ resistance mutations into S. aureus 8325-4attBLA that carry the mutated CC398-like bacterial attachment site for ΦSa3int phages. Our results failed to reveal significant differences between the FQ-resistant mutant and the wildtype strain with respect to phage integration or phage release. Thus, we conclude that mutations in grlA and gyrA do not contribute to the presence of the ΦSa3int phage in LA-MRSA CC398.

In recent years there has been an increase in human infections with methicillin-resistant Staphylococcus aureus (MRSA) originating from livestock and strains carrying bacterial viruses of the Sa3int-family have disseminated into the community. Sa3int phages express immune evasion factors and are common in human staphylococcal strains. As the bacterial attachment site (attB) for Sa3int phages is mutated in livestock-associated strains, the integration frequency is low and a key question is how the phages are established. Here we show that Sa3int phages adapt to alternative bacterial integration sites by mutating the phage attachment sequence, attP, leading to enhanced integration at these sites. Using a model strain carrying the mutated attBLA of livestock-associated strains we find that once established, the Sa3int phage, Φ13 is inducible with release of heterogenous phage populations carrying mutations in attP that in part increase homology to alternative integration sites or attBLA. Compared to the original phage, the adaptive mutations increase phage integration in new rounds of infection. Also, Sa3int phages induced from livestock-associated outbreak strains reveal mutated attP sequences. We suspect that promiscuity of the phage-encoded recombinase allows this adaptation and propose it may explain how phages mediate "host jumps" that are regularly observed for staphylococcal lineages.