Do antibiotics excreted by humans and animals drive evolution of resistance in the environment?

We show the ability of antibiotics to drive evolution of resistance at environmentally relevant concentrations is class specific. We also demonstrate that very low levels of antibiotics can increase persistence of resistant bacteria.

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Antimicrobial resistance is predicted to be the leading cause of death globally by 2050 resulting in 10 million deaths per year, many caused by antibiotic resistant bacterial infections. Implementing mitigation strategies to limit the spread of antibiotic resistance would help limit both morbidity and mortality in the future. The environment is an underappreciated reservoir of both antibiotic resistant bacteria and antibiotic resistance genes including indigenous environmental bacteria that have evolved resistance over evolutionary time and human and animal associated bacteria that are introduced through wastewater and faecal pollution. In addition, approximately 70% of antibiotics are excreted by humans and animals in an active form and can also enter waterways. The lowest concentration at which increased resistance is observed is called the minimal selective concentration (MSC).

MSCs have been determined for a range of antibiotic compounds using single species competition experiments, however, fewer antibiotics have been tested using complex microbial communities. These are more representative of the environment and provide opportunities for horizontal transfer of resistance genes. The aim this work was to determine and compare MSC values for multiple antibiotic compounds from different classes of antibiotics, including those on the EU Water Framework Directive’s (WFD) priority hazardous substances Watch List.

We investigated the selective ability of four antibiotic compounds: azithromycin (macrolide), clarithromycin (macrolide), erythromycin (macrolide) and ciprofloxacin (fluoroquinolone) that were placed on the 2018 iteration of the WFD Watch List. In addition, we also tested a limited range of tetracycline concentrations to compare our method to a previously published approach.

We used untreated wastewater as the inoculum in experimental microcosms and evolved the bacterial populations at a range of antibiotic concentrations for seven days. We used quantitative real time PCR to determine changes in resistance gene prevalence over time for both ciprofloxacin and the macrolides. In addition, for the macrolide antibiotics, we used plating and metagenome sequencing to investigate selection.

These experiments confirmed that antibiotic compounds have different selective abilities at concentrations similar to those found in polluted aquatic environments. This means that compound specific risk assessments should be undertaken to determine the risk these compounds pose in the environment. Ciprofloxacin selected for resistance at concentrations similar to those found in the environment and therefore mitigation strategies may need to be implemented. The macrolide antibiotics, however, selected for resistance at concentrations significantly higher than current environmental concentrations.

In addition, we identified that increased persistence of antibiotic resistance occurs below the threshold where increased prevalence is observed, the MSC. We have defined the threshold at which this phenomenon is observed as the minimal increased persistence concentration (MIPC). This increased persistence may lead to higher probability of environmental human exposure to resistant bacteria and opportunities for bacterial evolution, even though numbers of resistant bacteria will still decrease over time.

This information can be used by regulators to help set safe concentration limits for antibiotics in wastewater, which may help mitigate selection for antibiotic resistance in the environment. Our study also improves understanding of resistance evolution at low antibiotic concentrations that are present in the human gut during antibiotic therapy.

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Isobel Stanton

Postdoctoral Research Fellow, University of Exeter

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