Whole-genome long-read sequencing to unveil <i>Enterococcus</i> antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of <i>Enterococcus lactis</i>

<i>Enterococcus faecalis</i> (<i>Efs</i>) and <i>Enterococcus faecium</i> (<i>Efm</i>) are major causes of multiresistant healthcare-associated or nosocomial infections. <i>Efm</i> has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to <i>Enterococcus lactis</i> (<i>Elc</i>). However, identification techniques do not routinely differentiate <i>Elc</i> from <i>Efm</i>. As part of a longitudinal study to investigate the antimicrobial resistance of <i>Enterococcus</i> in dairy cattle, isolates initially identified as <i>Efm</i> were confirmed as <i>Elc</i> after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An <i>Efm</i>-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 <i>Efs</i>, 23 <i>Efm</i>, and 59 <i>Elc</i>. Resistance, determined by broth microdilution, was more frequent in <i>Efs</i> than in <i>Efm</i> and <i>Elc</i> 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 <i>Efm</i> was encoded by the <i>optrA</i> gene, located in plasmids downstream of the <i>fexA</i> gene. Although most <i>Elc</i> lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that <i>Elc</i> is more prevalent than <i>Efm</i> in dairy cattle but carries fewer ARGs and virulence genes. However, <i>Elc</i> can carry multi-drug-resistant plasmids like those harbored by <i>Efm</i> and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCE<i>Enterococcus</i> 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 <i>Enterococcus lactis</i> (<i>Elc</i>) from <i>Enterococcus faecium</i> (<i>Efm</i>) results in a biased interpretation of <i>Efm</i> epidemiology. The <i>Efm</i> species-specific real-time PCR assay developed here will help to properly identify <i>Efm</i> (only the formerly known clade A) in future studies. Here, we showed that <i>Elc</i> is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than <i>Enterococcus faecalis</i> (<i>Efs</i>) and <i>Efm</i>, 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 (<i>Efs</i>, <i>Efm</i>, and <i>Elc</i>) 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.