Assessing Antimicrobial and Metal Resistance Genes in Escherichia Coli from Domestic Groundwater Supplies in Rural Ireland

Abstract

Natural ecosystems can become significant reservoirs and/or pathways for antimicrobial resistance dissemination, with prevalence of co-selective stressors (e.g., metals/metalloids) and the potential to affect nearby microbiological, plant, animal, and human communities. This is further accentuated in environments that offer direct human exposure, such as drinking water. To date, however, few studies are available which investigate co-selective pressures in groundwater environments of human health significance. Accordingly, the present study analysed samples from 132 (drinking) groundwater supplies (i.e., private wells) located in rural areas of the Republic of Ireland, where land use is dominated by livestock grazing activities. In total, 48 Escherichia coli isolates were subject to whole genome sequencing and phenotypically tested for antimicrobial susceptibility, with E.coli-positive water samples further analysed for trace metal (and metalloid) concentrations. Eight isolates (i.e., 16.7%) were phenotypically resistant to antimicrobials, which was genotypically confirmed by presence of ten antimicrobial resistance genes (ARGs); namely: aph(3’’)-lb (strA; n=7), aph(6)-Id (strA; n=6), blaTEM (n=6), sul2 (n=6), tetA (n=4), floR (n=2), dfrA5 (n=1), tetB (n=1), and tetY (n=1). Additional bioinformatic analysis revealed that all ARGs were plasmid-borne, except for two sul2 genes, and that 31.2% of all tested isolates (n=15) and 37.5% of resistant ones (n=3) carried virulence genes. Study results also showed that metal concentrations found are likely insufficient to foster a co-selective environment, with no significant relationships between metal concentrations and ARG abundance. Additionally, just one genetic linkage was identified between ARGs and a metal resistance gene (MRG), namely merA, a mercury-resistant gene found on the same plasmid as blaTEM, dfrA5, strA, strB, and sul2 in the only isolate of inferred porcine (as opposed to bovine) origin. Overall, findings indicate that ARG (and MRG) acquisition is likely occurring prior to groundwater ingress, warranting this type of contamination highly preventable by ensuring appropriate source protection measures.

Keywords: Antimicrobial Resistant Bacteria, Antimicrobial Resistance Genes, Metal Resistance Genes, Groundwater, Escherichia coli, trace metals