Phenotypic and Genotypic Characterization of Enterococcus spp. Isolated from Freshwater Lakes and Rivers: Antimicrobial Resistance, Virulence Determinants and Biofilm Formation

Enterococci are environmentally persistent bacteria that are relevant to both water quality and the spread of antimicrobial resistance. This study aimed to phenotypically and genotypically characterize Enterococcus spp. isolated from freshwater (lakes and rivers) in north–central Poland, with a focus on antimicrobial susceptibility, virulence genes, and biofilm-forming capacity. Surface water samples were collected during the vegetation seasons of 2022 and 2023. Enterococci were isolated by culture-based methods and confirmed by MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was performed using the disk diffusion method, virulence-associated genes were detected by multiplex PCR, and biofilm formation was evaluated using a crystal violet assay. In total, 96 Enterococcus spp. isolates representing 12 species were identified from 328 freshwater samples, with E. faecalis (24.0%) and E. hirae (21.9%) being the most frequently isolated. Thirty-one isolates (32.3%) were resistant to at least one antibiotic, and two isolates were classified as multidrug-resistant. The most prevalent virulence genes were gelE, srtA, and hyl. The ace gene was detected exclusively in E. faecalis. Most isolates were non-biofilm producers, while biofilm formation was confirmed in 8 strains (6 weak and 2 moderate producers), with no strong biofilm-forming strains detected. These findings suggest that freshwater ecosystems may constitute important reservoirs of antimicrobial-resistant and virulence-associated Enterococcus spp., underscoring the importance of continuous surveillance within the One Health framework.