Seasonal and spatial dynamics of Enterococcus and antimicrobial resistance in a one health dairy ecosystem in Western Canada.

Antimicrobial resistance (AMR) in Enterococcus poses an emerging One Health concern due to its capacity for environmental persistence and cross-sector transmission. This two-year longitudinal study investigated the spatial and seasonal distribution of Enterococcus across interconnected animal, environmental, and human wastewater compartments in a peri-urban region of western Canada. We aimed to i.) characterize Enterococcus species across varying stages of dairy cow maturity, farm environments and nearby municipal wastewater systems and ii.) determine the AMR profiles and genomic relationships of E. faecalis and E. faecium. Among the 1133 Enterococcus isolates collected, over 24 species were identified, with E. hirae (n = 319) being the most prevalent followed by E. faecium (n = 256) and E. faecalis (n = 223). Species distributions varied significantly by season across most sample types. Antimicrobial susceptibility testing of E. faecalis (n = 58) and E. faecium (n = 61) isolates revealed four, multi-drug resistance E. faecalis isolates, with resistance to tetracycline (18%) and quinupristin-dalfopristin (18%) most common. Genomic analysis of E. faecalis (n = 158) and E. faecium (n = 171) isolates revealed 36 resistance determinants, with aac(6')-Ii, lsa(A), msr(C), tet(M) and tet(L) being the predominant resistance genes. Phylogenomic analysis revealed high diversity within both species, comprising 88 sequence types (STs) for E. faecalis and 100 for E. faecium, including 46 novel STs. Most of the closely related isolates (≤10 SNPs) originated from the same sample type and site (>74%), reflecting localized transmission dynamics. Evidence of cross-domain exchange and seasonal persistence was confined to organic dairy farms, while no genomic relatedness was observed between livestock-associated isolates and those from wastewater. These findings show that dairy-associated Enterococcus populations are diverse yet spatially contained, with limited connectivity to the human wastewater domain. Targeted One Health surveillance can help identify localized points to guide AMR mitigation in regions of intensive dairy production.