Methicillin Resistant Staphylococcus aureus from Wastewater in Finland

Population-level data on multidrug-resistant bacteria could complement clinical data and be used as an early warning tool. This study assessed the reliability of wastewater surveillance as a pandemic preparedness tool and used a culture-based approach to isolate methicillin-resistant Staphylococcus aureus (MRSA). Wastewater samples (n=80) were collected from ten cities across Finland between February 2021 and January 2022 and cultural isolation was done using CHROMagar MRSA. Phenotypic antimicrobial susceptibility testing (AST) was done by broth microdilution assay using EUSTAPF sensititre plates. Whole genome sequencing was done to determine the spa types, antibiotic resistance genes (ARGs), virulence-associated genes, plasmids, and the genomic relatedness of isolates. The prevalence of MRSA in wastewater samples was 27.5% (n=22/80). There were seasonal and temporal variations in the occurrence of MRSA. Phenotypic AST showed that MRSA isolates were extensively drug-resistant with >80.0% of isolates being resistant to sulfamethoxazole/trimethoprim, tetracycline, fusidic acid, and erythromycin. Four isolates (18.2%) were vancomycin resistant. Molecular typing of isolates revealed that 36.4% (n=8) and 31.8% (n=7) of the isolates belonged to the t304 and t008 spa types respectively. In addition, two spa types (t011 and t034) belonging to the CC398 complex were detected in wastewater samples. There was high genotypic concordance with the national clinical bacterial resistance data. The mecA gene was found in all isolates (n=22) and three tetracycline resistance determinants (tet38, tetK, and tetM) were detected with tet38 being the most abundant (81.8%, n=18/22). Three isolates harboured the plasmid-mediated sat4 gene that confers resistance to Streptothricin. In addition, resistance determinants to macrolide antibiotics (mph(C)/msr(A) and fosfomycin (fosB) were detected in the seven isolates that belonged to spa type t008. All isolates except one harboured the SCCmec_type_IVa(2B). Six ST8 isolates were PVL (Panton–Valentine leucocidin) and ACME (Arginine Catabolic Mobile Element) positive and belong to the globally disseminated USA300 clone. The Inc18 plasmid was the most abundant as it was detected in 72.7% (n=16/22) of the isolates. Other plasmid replicons detected were the rep_trans and repA_N which were detected in 45.4% (n=10/22) and 40.9% (n=9/22) of the isolates respectively. Ten isolates harboured at least three plasmid replicons and no plasmid replicons were detected in four isolates (ST6/t304). The core genome MLST of the isolates revealed that isolates aggregated into several clusters. Two genomically indistinguishable clusters were identified: ST6/t304 belonging to cluster type CT12405 (=20 allelic differences) and ST8/t008 belonging to cluster type CT1925 (<8 allelic differences). Culture-based wastewater surveillance for MRSA using clinical media after a pre-enrichment step was sensitive and could reliably predict the occurrence of this pathogen at the population level. More genomic studies and insights are needed to ascertain the source of molecular features such as ARGs and plasmids.