Unveiling the Genetic Landscape of <i>Staphylococcus aureus</i> Isolated From Hospital Wastewaters: Emergence of Hypervirulent CC8 Strains in Tehran, Iran

<b>Objective(s):</b> Multidrug-resistant bacteria and priority pathogens, including MRSA, are frequently found in hospital wastewaters. It is crucial to investigate the genetic diversity, biofilm formation, and virulence analysis of <i>Staphylococcus aureus</i> isolated from hospital wastewaters. <b>Materials and Methods:</b> In this cross-sectional study, 70 <i>S. aureus</i> isolated from hospital wastewaters were subjected to characterization through antimicrobial susceptibility tests, biofilm formation, multilocus sequence typing (MLST), and PCR analysis for detecting resistance (<i>mecA</i>, <i>mecC</i>, <i>vanA</i>, <i>vanB</i>, <i>mupB</i>, <i>mupA</i>, <i>msr(A)</i>, <i>msr(B)</i>, <i>erm</i>(A), <i>erm</i>(B), <i>erm</i>(C), <i>tet</i>(M), <i>ant</i> (4⁣')<i>-Ia</i>, <i>aac</i> (6⁣')<i>-Ie/aph</i> (2⁣^″), and <i>aph</i> (3⁣')<i>-IIIa</i>) and virulence genes (<i>eta</i>, <i>etb</i>, <i>pvl</i>, and <i>tst</i>). <b>Results:</b> Our results showed that 55.7%, 31.4%, and 12.9% of isolates were classified as strong, intermediate, and weak biofilm-forming strains, respectively. Our result revealed that about three-quarters of isolates harbored <i>mecA</i> (100%), <i>ant</i> (4⁣')<i>-Ia</i> (100%), <i>tet</i>(M) (92.9%), <i>erm</i>(B) (80%), and <i>msr</i>(A) (74.3%) resistance genes. MLST revealed that the 70 isolates belonged to five clonal complexes, including CC8 (52.9%), followed by CC30 (15.7%), CC5 (14.3%), CC1 (11.4%), and CC22 (5.7%). The vast majority of <i>S. aureus</i> isolates belonged to CC8/ST239-MRSA (21.5%). Among the 39 strong biofilm producers, the majority (25.6%) belonged to CC8/ST239-MRSA clone. Our result revealed that about one-third of Panton-Valentine leukocidin (PVL)-positive strains belonged to CC30/ST30. The high-level mupirocin-resistant (HLMUPR) isolates belonged to CC8/ST239-MRSA (36%), CC30/ST30-MRSA (16%), CC8/ST8-MRSA (12%), CC5/ST5-MRSA (12%), CC8/ST585-MRSA (8%), CC5/ST225-MRSA (8%), CC5/ST1637-MRSA (4%), and CC8/ST1465-MRSA (4%) lineages carrying <i>mupA</i>. The VRSA strain belonged to the CC8/ST239-MRSA, CC8/ST8-MRSA, and CC22/ST22-MRSA clonal lineages, carrying the <i>vanA</i> determinant. <b>Conclusion:</b> These findings highlight significant genotypic diversity and high biofilm formation among our isolates. From this study, we identified highly virulent strains of <i>S. aureus</i> associated with biofilm production and drug resistance; some of these strains were highly similar, highlighting the possibility of rapid spread. The high prevalence of CC8 and CC30 clones among <i>S. aureus</i> strains reflects the emergence of these lineages as successful clones in hospital wastewaters in Iran, which is a serious concern. The study highlights the importance of wastewater surveillance to understand genetic pattern and antimicrobial resistance profiles in surrounding communities, which can in turn support public health efforts.