Dynamics of drug resistance in environmental bacteria within an aquatic ecosystem.

Recently there has been a rapid increase in the incidence and prevalence of drug-resistant bacteria and antimicrobial resistance genes in the environment, largely attributed to selection pressure from the environmental presence of antimicrobials such as antibiotics, biocides, and heavy metals, as well as other physicochemical stressors. such as Poly aromatic hydrocarbons, pH, temperature, and reactive oxygen. However, the concentrations at which these antimicrobials could elicit resistance are poorly understood. Such lack of information could hamper the development of standards for the environmental surveillance of antmicrobials with potential adverse effects on human, animal and environmental health. In this study, Water samples were collected from all the points that impact the environment directly around the Darvill wastewater treatment plant, namely the treatment plant effluent discharge point, the upstream and downstream from the effluent discharge point. Antibiotics, heavy metals, and biocides were identified and quantified from the water samples, and we ascertained the effect of environmental concentrations of some of these selected stressors on the antibiotic resistance in previously susceptible Escherichia coli. Heavy metals concentrations were determined using the United States Environmental Protection Agency (US EPA) method 200.7. Biocide and antibiotic residue concentrations were determined using validated ultra-high-performance liquid chromatography with tandem mass spectrometry-based methods. E. coli was identified and quantified using the Colilert-18TM system from IDEXX, while antimicrobial susceptibility was performed using the disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. The concentration of antibiotics observed ranged from sulfamethoxazole (286.180 μg/L) to penicillin (2.2 μg/L); for metals, sodium (27.734 mg/L) to iron (0.001 mg/L); and for biocides, benzalkonium chloride (BAC) 12 (7.805 μg/L) to BenthEZ (0.035 μg/L). There was observed increase in the pollutant concentrations in the effluent and downstream samples compared to the upstream samples, suggesting that the WWTP might be a potential source of interest, indicating that these pollutants, were not completely removed at the WWTP. Thirty days' exposure of wholly susceptible E. coli ATCC 25922 strains, to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC 12 and DADMAC 10 was conducted but could not trigger phenotypic resistance. Genotypic analysis of the WGS on the exposed isolates, found only the macrolide resistance mdf (A) gene (which was also present in the control) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes compared to the control. Only one resistance gene was detected, robA, a member of the ArcC/XylS family, that regulates the ArcAB-TolC multi-drug efflux, that contributes to multi-drug resistance. The other 18 genes we detected were tolerance conferring genes, acnB, cusA, degQ, epmA, hsmP, mlc, purH, queG, srlE, tsaB, yddh and yqhH genes, in all the exposed isolates. filA genes in only the oxytetracycline and BAC 12-exposed isolates, mutM gene in zinc exposed isolates, nudK gene in all the exposed isolates except the DADMAC 10 exposed isolates, ptsG gene in only the oxytetracycline-exposed isolates, and ompD in only DADMAC 12-exposed isolates. All the genes detected in the exposed isolates were also detected in the environmental isolates, except the robA gene. These genes detected encode for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations. In addition, we observed that the 30-days exposed isolates developed increased tolerance to high (25 x MIC) concentrations of ampicillin by 30 to 50% when compared to unexposed control. BAC 12-exposed isolates had the highest tolerance increase. The increased tolerance seems to emanate from multi gene induced persister cells formations, as well as tolerance gene expressions. The MSW of the exposed isolates to ampicillin and amoxicillin, also slightly increased compared to the control indicating the amplification of persister cells during the 30-day exposure but the MSW remained same to oxytetracycline. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues, as observed in the aquatic environment, cannot induce phenotypic resistance but can encode for genes responsible for the development of persistence and tolerance in bacteria, which seems to be the pathway towards eventual antimicrobial resistance in environmental bacteria.