Simple SummaryEnterococci are among the most responsible agents for nosocomial infections and are globally prevalent antibiotic-resistant microorganisms. The risk of calves being fed colostrum contaminated with these bacteria or antimicrobial-resistant bacteria, leading to the colonization of their gastrointestinal tract, is a concern of public health. The objective of this study was to investigate whether bovine colostrum can act as a reservoir and vehicle for the dissemination of antibiotic-resistantEnterococcusspp. via the food chain. The sensitivity to 14 antibiotics using the disk diffusion method, as well as the presence of antibiotic-resistant genes and virulence genes, were analyzed in calf colostrum samples.E. faecalis,E. faeciumandE. gallinarumwere identified in the colostrum samples. The results demonstrated a very high percentage (92.1%) of isolates classified as multidrug-resistant (≥3 antimicrobial classes). Additionally, 52% of the isolates showed the presence of ≥4 virulence genes.E. faeciumwas most likely to carry erythromycin and tetracycline resistance genes, as well as virulence genes. This study revealed that colostrum serves as a reservoir and/or vehicle for the spread of antibiotic resistance and virulence genes. These results have to be analyzed in a One Health perspective to better combat this spread to humans, other animals and the environment.AbstractEnterococciare considered among the most prevalent global multidrug-resistant microorganisms globally. Their dissemination is a global concern, particularly by food-producing animals for both animals and humans. The aim of this study was to identify the species and investigate the antibiotic resistance and virulence profile ofEnterococcusin bovine colostrum. Out of 88 presumptiveEnterococcusisolates, species identification and susceptibility to 14 antimicrobials were tested using the disk diffusion method. An analysis of the antibiotic resistance and virulence genes was performed on the most prevalent species, using specific PCR assays.Enterococcus faecalis(54.5%),E. faecium(14.8%) andE. gallinarum(6.8%) were the identified species. To the best of our knowledge, this is the first report ofE. gallinarumin bovine colostrum. The majority of the isolates showed resistance to quinupristin-dalfopristin (95.9%), erythromycin (80.7%), tetracycline (80.7%) and streptomycin (58%). Ninety-two percent of isolates were classified as multidrug-resistant. The most frequently detected resistance genes weretet(K) (61.1%),tet(M) (75.9%),tet(L) (90.7%),erm(B) (55.6%) andant(6)-Ia (46.3%). The most prevalent virulence factors werecpd,esp,aggandcylLL.Enterococcus faeciumshowed a higher probability of carrying theerm(C),tet(M),aceandgel(E) genes (p< 0.05). These results demonstrated that colostrum can constitute an important reservoir and vehicle for the dissemination of antibiotic resistance and virulence genes to the three niches included in a One Health perspective (humans, animals and the environment), highlighting the importance of hygiene sanitary measures to mitigate colostrum microbial contamination.