Investigating selection for antimicrobial resistance by non-antibiotic drugs in microbial communities

Abstract

Antimicrobial resistance (AMR) is a significant growing problem, and can be co selected for by non-antibiotic compounds. Co-selection refers to the increase in antibiotic resistance genes/phenotypes in response to non-antibiotic compounds (e.g. metals). It is crucial we understand which compounds are co-selective, since they possibly threaten human health. Recent research has found that non antibiotic drugs (NADs) can co-select for AMR in single-species populations, at drug concentrations much higher human gut and environmental contexts. The antimicrobial effects of NADs at context-relevant concentrations, and on bacterial communities, are unknown. In this thesis, culture dependent and independent methods were used to determine effects of NADs individually, and in simple mixtures with an antibiotic, at human gut and environmentally relevant concentrations on a microbial community. Seven of nine NADs significantly reduced the growth of the bacterial community, of these, six posed risks to bacterial communities according to author produced environmental risk assessments using global data from the Umweltbundesamt. Diclofenac, metformin, and 17-β-estradiol were assayed using qPCR to test co selective effects. Only 17-β-estradiol selected for one of the three genes tested, intI1, at environmentally relevant and human gut concentrations. Metagenomics and metatranscriptomics were used to determine if NADs co selected for increased abundance or expression of other antibiotic resistance genes (ARGs), and/or biocide and metal resistance genes (BMRGs). Metformin reduced expression of the TolC outer membrane protein gene, which confers resistance to multiple compounds, including antibiotics. Metformin also increased 3 expression of a mercury resistance gene. 17-β-estradiol increased abundance of two arsenic resistance genes and decreased abundance of a nickel resistance gene in dose-dependent manners. Diclofenac and metformin also altered the diversity of BMRGs and ARGs respectively. Otherwise, significant detectable effects on the metagenome and metatranscriptome were limited. Finally, mixtures were tested, since pollutants are present co-currently. Ciprofloxacin with either diclofenac, metformin, or 17-β-estradiol, significantly reduced the growth of the wastewater community, compared to growth in ciprofloxacin alone. Additionally, the mixtures reduced the ciprofloxacin concentration at which intI1 was selected for, from 40µg/L to 10µg/L. These data indicate that NADs affect bacterial communities and may select for AMR at environmentally relevant concentrations. Future work should aim to identify other NADs that could be selective, both alone and in mixtures, and use experimental conditions and relevant microbial communities to fully appreciate co-selection risk. The data presented here can direct future research into the co selective effects of NADs, and their effects on human and environmental microbiomes.