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

Abstract

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

Keywords: Enterococcus; Enterococcus faecalis; Enterococcus faecium; Enterococcus lactis; antimicrobial resistance; dairy cattle; long-read WGS; minimum inhibitory concentration; whole-genome sequencing.

FIG 1

Pangenome analyses of Enterococcus genomes. (A) Distribution of the 15,766 genes that make up the pangenome across the 32 isolates. The dendrogram represents the hierarchical clustering of the genomes based on the distribution of their accessory genes (presence/absence). The cluster framed in red corresponds to isolates identified as E. faecalis and in green as E. faecium by RT-PCR1. (B) Phylogenetic tree based on the core-genome single nucleotide polymorphisms (SNPs) of E. faecium and E. lactis (size = 110,229 bases). (C) Phylogenetic tree based on the core-genome SNPs of E. faecalis (size = 50,251 bases). The phylogenetic trees (B and C) were constructed using Parsnp and RaxML, and corresponding metadata such as age group, farm, sampling time, and MLST results (ST, Sequence Type; CC, Clonal Complex) are indicated for each isolate.

FIG 2

Sampling scheme and distribution of phenotypic AMR profile per farm (F1–F5), sampling (S01–S10), and age group (C: calves; H: heifers; LC: lactating cows). Each AMR pattern is depicted in a different color in the heatmap as described in the legend, which also indicates the enterococci species where each AMR pattern is present. Antimicrobials are abbreviated as follows: CHL, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; GEN, gentamicin; LZD, linezolid; Q/D, quinupristin-dalfopristin; TET, tetracycline.

FIG 3

Heatmap of the distribution of the AMR genes detected by WGS. The presence and location of the GDRs are indicated as per the legend.