Whole-genome long-read sequencing to unveil antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of .

() and () are major causes of multiresistant healthcare-associated or nosocomial infections. has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to (). However, identification techniques do not routinely differentiate from . As part of a longitudinal study to investigate the antimicrobial resistance of in dairy cattle, isolates initially identified as were confirmed as after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An -specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 , 23 , and 59 . Resistance, determined by broth microdilution, was more frequent in than in and but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in was encoded by the gene, located in plasmids downstream of the gene. Although most lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that is more prevalent than in dairy cattle but carries fewer ARGs and virulence genes. However, can carry multi-drug-resistant plasmids like those harbored by and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCE species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish () from () results in a biased interpretation of epidemiology. The species-specific real-time PCR assay developed here will help to properly identify (only the formerly known clade A) in future studies. Here, we showed that is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than () and , it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (, , and ) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.