Industrial dog food is a vehicle of multidrug-resistant enterococci carrying virulence genes often linked to human infections.

The increase in the number of pets in recent years has been followed by an exponential growth of the industrial pet food sector, which has been accompanied by new food safety risks, namely antibiotic resistance. The aim of this study was to investigate whether dog food commercially available in Portugal is a reservoir of clinically-relevant antibiotic resistant Enterococcus. Fifty-five samples (25 brands; 22 wet, 14 raw frozen, 8 dry, 7 treats and 4 semi-wet) were collected on 9 commercial surfaces in the Porto region (September 2019 to January 2020). Most samples were obtained from brands that are commercialized worldwide (n = 21/25). Sample (25 g) processing included pre-enrichment and enrichment steps in culture media without/with 3 antibiotics, and then plating into selective media without/with the same antibiotics. Susceptibility was studied for 13 antibiotics (disk diffusion; Etest; microdilution) according to EUCAST/CLSI. Clinically-relevant species (E. faecium and E. faecalis), antibiotic resistance (vanA, vanB, optrA, poxtA) and virulence (e.g. ptsD, esp, sgrA) genes were identified by PCR. Other species of Enterococcus were identified by MALDI-TOF MS. Clonality was established by MLST in selected isolates. Enterococcus (n = 184; 7 species; >85% E. faecium and E. faecalis) were detected in 30 samples (54%) of different types (14 raw, 16 heat treated-7 dry, 6 wet, 3 treats). E. faecium and E. faecalis were more frequent in dry and wet samples, respectively. More than 40% of enterococci recovered were resistant to erythromycin, tetracycline, quinupristin-dalfopristin, streptomycin, gentamicin, chloramphenicol, ampicillin or ciprofloxacin, and to a lesser extent to linezolid (23%; optrA, poxtA) or vancomycin and teicoplanin (2% each; vanA). Multidrug-resistant isolates (31%), including to vancomycin and linezolid, were obtained mostly from raw foods, although also detected in wet samples or treats, and mainly from culture media supplemented with antibiotics. Samples subjected to thermal treatment mostly carried non-MDR isolates. The variety of clones observed included strains previously identified in hospitalized patients (E. faecium ST17/ST80; E. faecalis ST40), farm animals, pets and environmental strains. This study shows that dog food from international brands is a vehicle of clinically-relevant enterococci carrying resistance to last resort antibiotics and relevant virulence genes, thus positioning pet food as an important source of antibiotic resistance spread within the One Health context. The high incidence of Enterococcus in a variety of dog food samples indicates the need to review selection of raw materials, manufacturing and hygiene practices in an emerging food sector growing worldwide.