Whole-genome long-read sequencing to unveil
Enterococcus
antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of
Enterococcus lactis
Authors: Ocejo Medelin , Mugica Maitane , Oporto Beatriz , Lavín José Luis , Hurtado Ana 
Published: January 2024

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.

IMPORTANCE

Enterococcus
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.