Genomic analysis of Enterococcus faecium from non-clinical settings: antimicrobial resistance, virulence, and clonal population in livestock and the urban environment

Abstract

Introduction: Enterococci are commensals of the gastrointestinal tract of humans and animals that evolved into opportunistic pathogens with high antimicrobial resistance and virulence. Multidrug-resistant Enterococcus is a major cause of hospital-acquired infections worldwide. For this reason, the characterization of non-clinical reservoirs of Enterococci and their epidemiological link to resistant hospital isolates is crucial for controlling their spread.

Methods: A total of 295 samples collected from livestock (pigs and cows, n = 135) and environment (public buses, passengers hands, and urban environments, n = 160) were screened for Enterococcus spp. E. faecium antimicrobial resistance profiles, virulence potential, and clonal population were further characterized.

Results: Enterococci were detected in 90.5% (n = 267) of the samples, with a higher prevalence in livestock (100%) than the environment (82.5%, p < 0.0001), but none of the isolates exhibited vancomycin resistance. E. faecalis was the most prevalent species (51.7%), predominantly found in livestock (62.2%), while E. faecium was more common in the environment. Of the 59 E. faecium isolates, 78% showed resistance to ≥3 antibiotic classes and contained associated resistance genes, namely tetracyclines (tetM and tetL), beta-lactams (mutations in pbp5), and high-level resistance to aminoglycosides (ant(6)-Ia and aac(6')-aph(2″)). A wide array of virulence factors was detected among E. faecium, associated with adherence, biofilm formation, and adaptation to host response, while hospital-associated virulence markers, such as IS16, were less frequent, probably due to the non-clinical nature of the isolates. Clonal population analysis revealed a diverse E. faecium population. Although no direct epidemiological link could be traced between our isolates and specific clinical isolates, infection-associated genetic backgrounds were identified in non-clinical isolates: one isolate from pigs belonged to CC17 (ST32), while four isolates belonged to CC94, including one recovered from pigs (ST296), one from cows (ST2206), one from the urban environment (ST1205), and other from buses (ST800).

Discussion: This study underscores a high prevalence of clinically relevant Enterococcus species among healthy livestock and the environment. Despite the absence of vancomycin resistance and limited hospital infection-associated clonal lineages, the presence of E. faecium with significant virulence potential and resistance to critical antibiotics in human and veterinary medicine highlights the need for continuing surveillance of non-clinical reservoirs.

Keywords: Enterococcus faecium; Enterococcus spp.; aminoglycosides high-level resistance; ampicillin resistant E. faecium; antimicrobial resistance; environment; livestock; non-clinical enterococcus reservoirs.

Figure 1

Distribution of enterococcus species by sample collection individually and regarding livestock and environment. Percentages referred to the total of positive samples in each collection.

Figure 2

Phenotypic antibiotic resistance profiles of the 59 E. faecium isolates. CN, gentamicin; S, streptomycin; HLAR, high-level aminoglycosides resistance; CIP, ciprofloxacin; LEV, levofloxacin; VAN, vancomycin; TC, teicoplanin; ERY, erythromycin; F, nitrofurantoin; LNZ, linezolid; AMP, ampicillin; C, chloramphenicol; QD, quinupristin–dalfopristin; W, trimethoprim; SXT, trimethoprim–sulfamethoxazole; TE, tetracycline; TGC, tigecycline.

Figure 3

Genomic data analysis regarding cgMLST distance-based matrix phylogeny, antimicrobial resistance, virulence, and plasmids of the 23 E. faecium selected for WGS. Antimicrobial resistance genes and mutations (vanA, vanB, cfr, optrA, or poxtA and 23S rRNA mutations) or virulence determinants (esp., ptsD, hylfm, cylA, or gelE) not detected in any isolate were not included. Dark full squares indicate the presence of the gene or mutation; ST, sequence type; CC, clonal complex.

Figure 4

Minimum spanning tree based on MLST analysis of 23 E. faecium isolates from livestock and environment and E. faecium isolated from infections in Portuguese hospitals between 2019 and 2020. AMB, Urban environment; representative isolates of E. faecium derived from this study are labeled in boldface.