The prevalence of drug-resistant and virulent Staphylococcus spp. in a municipal wastewater treatment plant and their spread in the environment

Drug-resistant and pathogenic Staphylococcus spp. strains can reach surface waters and air with wastewater evacuated to the environment. These strains increase the environmental pool of genetic determinants conferring antibiotic resistance and virulence, and constitute a health risk for the employees of wastewater treatment plants (WWTP) who come into daily contact with bioaerosols. The aim of this study was to identify the genetic determinants of drug resistance and virulence in Staphylococcus spp. strains isolated from untreated (UWW) and treated wastewater (TWW), an activated sludge (AS) bioreactor, river water collected upstream and downstream from the wastewater discharge point (URW and DRW), and WWTP employees. All isolates were analysed for the presence of the rpoB gene, and were subjected to clonal analysis by ERIC fingerprinting. As a result, 249 of the 455 analysed isolates were selected for PCR. The presence of the gene encoding nuclease activity in S. aureus (nuc), the methicillin resistance gene (mecA), vancomycin resistance gene (vanA), antiseptic resistance gene (qacA/B) and virulence genes (sasX, pvl, tst1, hla, sec) was determined. The prevalence of nuc, mecA, vanA and qacA/B genes in wastewater and river water was determined by quantitative PCR (qPCR). In the group of strains isolated from wastewater and water samples, 63% were identified as S. aureus, and 20% of the strains carried the vanA gene. The hla virulence gene was present in 80% of the isolates, and the pvl gene was detected in 27% of the isolates. In the group of strains isolated from the employees, 82% were identified as S. aureus, and the presence of vanA and mecA genes was confirmed in 14% and 16% of the isolates, respectively. The most prevalent virulence gene was hla (74%), whereas pvl was observed in 43% of the isolates. The quantitative analysis revealed the highest concentrations of the studied genes in UWW samples, at 2.56x104 gene copies/ml for nuc, 1.18x103 gene copies /ml for mecA, 8.28x105 gene copies /ml for vanA and 3.83x105 gene copies /ml for qacA/B. Some of analysed genes were identified in the isolates from both URW and DRW samples, as well as in genomic DNA of these samples. These results indicate that wastewater is not effectively treated in the analysed WWTP, which could contribute to the dissemination of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) to the environment. An analysis of the genetic relatedness of selected isolates revealed clusters of strains originating from UWW samples, AS samples and the employees. These observations suggest that ARGs and ARB are transmitted by wastewater bioaerosols to the upper respiratory tract mucosa of the plant’s employees, thus increasing their exposure to infectious factors.