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

Abstract

Objective(s): 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 Staphylococcus aureus isolated from hospital wastewaters. Materials and Methods: In this cross-sectional study, 70 S. aureus isolated from hospital wastewaters were subjected to characterization through antimicrobial susceptibility tests, biofilm formation, multilocus sequence typing (MLST), and PCR analysis for detecting resistance (mecA, mecC, vanA, vanB, mupB, mupA, msr(A), msr(B), erm(A), erm(B), erm(C), tet(M), ant (4⁣')-Ia, aac (6⁣')-Ie/aph (2⁣^″), and aph (3⁣')-IIIa) and virulence genes (eta, etb, pvl, and tst). Results: 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 mecA (100%), ant (4⁣')-Ia (100%), tet(M) (92.9%), erm(B) (80%), and msr(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 S. aureus 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 mupA. The VRSA strain belonged to the CC8/ST239-MRSA, CC8/ST8-MRSA, and CC22/ST22-MRSA clonal lineages, carrying the vanA determinant. Conclusion: These findings highlight significant genotypic diversity and high biofilm formation among our isolates. From this study, we identified highly virulent strains of S. aureus 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 S. aureus 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.

Keywords: Staphylococcus aureus; hospitals; methicillin-resistant Staphylococcus aureus; vancomycin-resistant Staphylococcus aureus; wastewaters.

Figure 1

Antibiotic resistance patterns of 70 S. aureus isolated from hospital wastewater. ERY, erythromycin; VAN, vancomycin; MUP, mupirocin; GEN, gentamicin; NIT, nitrofurantoin; TET, tetracycline; CLI, clindamycin; PEN, penicillin; RIF, rifampicin.

Figure 2

Results of microtiter plate (MTP) assay. Column A 1–4 represent positive control (S. epidermidis ATCC 35984 strain), B 1–4 represent moderate biofilm formation, C 1–4 represent weak biofilm formation, D 1–4 represent negative control (sterile tryptic soy broth medium), and E 1–4 represent the strong biofilm formation.

Figure 3

Lane M, 100-bp DNA ladder (Fermentas, London, United Kingdom); Lane 1 vanA; Lane 2 mecC; Lane 3 mupA; Lane 4 msrA; Lane 5 ermB; Lane 6 aac (6⁣′)-Ie/aph(2⁣^″); Lane 7 ermA; Lane 8 ermC; Lane 9 msrB.

Figure 4

Distribution of antibiotic resistance genes in molecular types of S. aureus isolates.

Figure 5

The maximum likelihood phylogenetic tree of combined seven loci (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) sequences was constructed using RaxML through the CIPRES science gateway. Values at the nodes represent bootstrap percentages based on 1000 replicates, and branches with bootstrap values exceeding 90% are displayed.