Cross-Sector Antimicrobial Resistance and Virulence in Enterococcus spp. from Humans, Animals and the Environment

Background/Objectives: Antimicrobial resistance is a major public health concern requiring integrated surveillance across human, animal, and environmental sectors. Enterococcus spp. are widely distributed opportunistic bacteria with the capacity to acquire and disseminate resistance and virulence determinants. This study aimed to characterize species distribution, phenotypic antimicrobial resistance, resistance genes, and virulence-associated traits in Enterococcus spp. from One Health sources. Methods: Enterococci were recovered from 66 samples collected in Lisbon, Portugal, between late 2022 and early 2024, including healthy and sick humans, healthy and sick animals, canteen food, surface water, and public transport surfaces. RAPD-PCR was used to assess genetic diversity among 90 isolates and select 74 representative enterococci. Species identification, resistance gene screening, and virulence gene detection were performed by PCR. Antimicrobial susceptibility was assessed by disk diffusion against 12 antibiotics using CLSI criteria. Vancomycin resistance was further evaluated by agar dilution and Etest when applicable. Results: Enterococcus faecalis predominated, representing 63/74 isolates (85.1%), followed by Enterococcus faecium (5/74, 6.8%) and other Enterococcus spp. (6/74, 8.1%). Antibiotic resistance was detected across all One Health sectors. Sick Human isolates showed higher resistance than Healthy Human isolates. E. faecium showed higher resistance than E. faecalis, and all E. faecium isolates across sectors were multidrug-resistant. Rifampicin and quinupristin-dalfopristin resistance were frequent (except in E. faecalis), while linezolid resistance was not detected. Resistance and virulence determinants were distributed across sectors. Conclusions: Enterococci from human, animal, and environmental sources carried antimicrobial resistance and virulence-associated traits. These findings support integrated One Health surveillance to monitor resistant enterococci across interconnected reservoirs.