Investigating the presence of virulence determinants and vancomycin-resistant genes in <i>enterococci</i> isolated from the Anzali Lagoon and recreational waters in Guilan Province, Iran

This study characterizes vancomycin-resistant <i>Enterococci</i> (VRE) in Iran's Anzali Lagoon recreational waters through analysis of 268 samples collected seasonally (2019). Culture and PCR methods identified Enterococcus in 57.5% (154/268) of samples, with 40.3% (62/154) showing vancomycin resistance. Resistance gene profiling revealed <i>vanA</i> and <i>vanH</i> predominance (56.5% each, 35/62), demonstrating complete co-occurrence, while vanB was rare (3.2%, 2/62) and vanC absent. Virulence factors displayed divergent prevalence: esp (62.9%, 39/62) exceeded clinical isolate rates, whereas hyl (3.2%, 2/62) was uncommon. Biofilm assays showed VRE isolates formed significantly denser biofilms than susceptible strains (2.3-fold higher OD570, <i>p</i> < 0.001), with esp-positive isolates exhibiting the strongest biofilm production. These findings demonstrate that recreational waters harbor VRE populations with: (1) elevated esp prevalence suggesting environmental selection pressures, and (2) enhanced biofilm capacity promoting persistence. The 100% <i>vanA</i>-<i>vanH</i> co-occurrence and 62.9% esp frequency surpass WHO risk thresholds for recreational waters, indicating need for revised monitoring protocols. This study provides the first evidence of aquatic-specific VRE genotypes in the region and their potential public health impacts through resistance gene dissemination and waterborne exposure risks.