Evolution and selection of resistant bacteria in the presence of subinhibitory concentrations of micropollutants

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The use of wastewater for irrigation of agricultural land optimizes the efficient use of scarce water resources and the production of food under conditions of climate change. Wastewater is increasingly treated prior to its use in agriculture in order to minimize health risks, with the Mezquital Valley, Mexico, representing an important model case. The central hypothesis of the research unit FOR 5095 is that hitherto unforeseen risks arise from the implementation of wastewater treatment in long-established wastewater irrigation systems, due to i) the release of pollutants that have formerly been accumulated in soils, leading to ii) concentrations that (co-)select for antimicrobial resistance with iii) release and concentrations of antibiotics as well as selection of antibiotic resistance depending on soil type. SP 3 will contribute to testing these hypotheses by characterizing the selection pressure exerted by single antibiotics (ciprofloxacin, several MLSB antibiotics, sulfamethoxazole, trimethoprim) and disinfectants (quaternary alkylammonium compounds) as well as by their combinations in competition experiments employing strain pairs with different susceptibilities. These completion experiments will be conducted in nutrient broth solutions as well as in filtered aqueous extracts of Mexican soils (Leptosols, Phaeozems and Vertisols), in order to assess the influence of suspended colloids and solutes on the selection effects of antibiotics and biocides (collaboration with SP 1). We will use existing Acinetobacter baylyi strain pairs as well as other strain pairs (Escherichia coli, Enterococcus faecium) harboring plasmids that are isolated from Mexican soil and wastewater by SP 4 and SP 5 for the competition experiments. Employing these strain pairs, the minimum antibiotic/biocide concentrations that give selection advantages to the resistant strains will be determined and integrated into the mathematical model of the fate and effects of antibiotics and disinfectants developed by SP 7. Transcriptome experiments will give information about the stress responses of the bacteria. Screening for resistant mutants and their subsequent sequencing will show whether the presence of antibiotics in the extracts leads to selection of bacteria harboring mutations that confer resistance. The antibiograms of facultative pathogenic bacteria, that are isolated during the joint experiments from Mexican soil and wastewater by SP 4, SP 5, and SP 6, will show whether bacteria with clinically relevant multi-resistance phenotypes or plasmids that confer resistance to relevant antibiotics like beta-lactams (e.g., carbapenems) or antibiotics of last resort (e.g., colistin), are enriched in soils with a higher availability of free antibiotics or are even present on fresh produce grown on these soils. In this way, SP 3 contributes to a mechanistic understanding of interactions between pollutants, antibiotic resistance and pathogens in changing wastewater irrigation systems.