Antimicrobial resistance and genetic diversity of Enterococcus and Staphylococcus species from environmental water samples from the Gauteng and Limpopo provinces

Abstract

Enterococci and staphylococci are leading causes of morbidity and mortality globally due to antimicrobial resistance (AMR) and a wide arsenal of virulence factors. Environmental and zoonotic bacteria, including enterococci and staphylococci with high AMR rates are increasingly implicated in human infections. Therefore, the World Health Organization proposed a ‘One Health’ approach to identify non-clinical AMR reservoirs.

Wastewater from agricultural, healthcare and community settings is a potential reservoir of antimicrobial resistant bacteria (ARB), which may survive killing at wastewater treatment plants (WWTPs) and spread via rivers to communities, resulting in outbreaks. This study followed a ‘One Health’ approach to investigate the prevalence, AMR characteristics, virulence profiles and genetic relatedness of Enterococcus faecalis (E. faecalis), Enterococcus faecium (E. faecium) and Staphylococcus aureus (S. aureus) in environmental water samples from rivers and wastewater from a tertiary hospital, poultry and swine abattoirs, swine farms and WWTPs in Gauteng and Limpopo provinces to assess the potential public health risk. Enterococci and staphylococci were isolated on selective media, followed by multiplex polymerase chain reaction (M-PCR) assays for speciation and characterisation of antimicrobial resistance genes (ARGs), efflux pump genes and virulence genes. Pulsed-field gel electrophoresis was performed to assess genetic relatedness. Representative isolates were selected for phenotypic antimicrobial susceptibility testing (AST) using the VITEK® 2 automated system (bioMérieux, France) and for whole genome sequencing (WGS).

A total of 182 isolates (109 E. faecalis, 52 E. faecium and 21 S. aureus) were identified using M PCR. Enterococci and staphylococci carrying tetracycline ARGs (75%), macrolide ARGs (44%), aminoglycoside ARGs (24%) and vancomycin ARGs (1%), including multidrug-resistant enterococci (24%) carrying ARGs to at least three antimicrobial classes were identified. Isolates carrying multidrug efflux pumps [E. faecalis (87%), E. faecium (19%) and S. aureus (100%)] were detected. Phenotypic AST identified resistance to erythromycin (100%), tetracycline (63%), streptomycin (25%) and ciprofloxacin (6%) among 16 representative enterococci isolates, and mupirocin (100%), streptogramins (60%), macrolides (40%), benzylpenicillin (20%), clindamycin (20%), rifampicin (20%) and tetracycline (20%) resistance among five representative S. aureus isolates. Plasmids carrying ARGs were identified among the bacterial isolates by WGS which showed that these isolates can disseminate ARGs to other bacteria. Virulence genes encoding toxins, adherence, biofilm formation and immune evasion were identified among the bacteria using M-PCR assays and WGS, indicating a capacity to cause severe infections.

The PFGE profiles showed high genetic diversity among the study isolates, which indicated that these ARB are of diverse origin. Enterococcus faecalis sequence types (STs) (ST27, ST206 and ST387), E. faecium (ST94 and ST1036) and S. aureus (ST12 and ST15) associated with human infection were identified using WGS. Emerging E. faecium ST1833 and novel E. faecalis (ST1170) and E. faecium (ST2104 and ST2105) STs were identified, which showed that new ARB strains are emerging from these agricultural and environmental settings.

The study settings were identified as reservoirs of potentially pathogenic ARB. Current wastewater treatment mechanisms are insufficient in removing ARB from wastewater resulting in WWTPs releasing potentially infectious effluent into the environment and creating a health risk to communities that use this downstream water.