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

利用废水灌溉农田，可以在气候变化条件下优化稀缺水资源的有效利用和粮食生产。越来越多的废水在用于农业之前进行处理，以尽量减少健康风险，墨西哥的梅兹奎塔尔河谷是一个重要的示范案例。FOR 5095研究单位的中心假设是，在历史悠久的废水灌溉系统中实施废水处理会产生迄今为止无法预见的风险，因为i)释放以前积累在土壤中的污染物，导致ii)浓度（共同）选择抗菌素耐药性，iii)抗生素的释放和浓度以及抗生素耐药性的选择取决于土壤类型。SP 3将通过表征单一抗生素（环丙沙星、几种MLSB抗生素、磺胺甲新唑、甲氧苄氨嘧啶）和消毒剂（季烷基铵化合物）所施加的选择压力，以及它们在采用不同敏感性菌株对的竞争实验中的组合，有助于检验这些假设。这些完成的实验将在营养肉汤溶液以及墨西哥土壤的过滤水提取物（Leptosols、Phaeozems和Vertisols）中进行，以评估悬浮胶体和溶质对抗生素和杀菌剂选择效应的影响（与SP 1合作）。我们将使用现有的baylyi不动杆菌菌株对以及其他菌株对（大肠杆菌，屎肠球菌），这些菌株对含有从墨西哥土壤和废水中通过sp4和sp5分离的质粒进行竞争实验。利用这些菌株对，确定对耐药菌株具有选择优势的最低抗生素/杀菌剂浓度，并将其整合到SP 7开发的抗生素和消毒剂的命运和效果的数学模型中。转录组实验将提供有关细菌应激反应的信息。耐药突变体的筛选及其随后的测序将显示，提取物中抗生素的存在是否会导致携带具有耐药性突变的细菌的选择。在墨西哥土壤和废水的联合实验中，通过SP 4、SP 5和SP 6分离出的性致病菌的抗生素谱将显示，细菌是否具有临床相关的多重耐药表型，或质粒是否具有对相关抗生素(如β -内酰胺类抗生素（如碳青霉烯类抗生素）或最后手段抗生素（如粘菌素）的耐药性。富含游离抗生素的土壤，甚至存在于这些土壤上生长的新鲜农产品中。通过这种方式，SP 3有助于对污水灌溉系统中污染物、抗生素耐药性和病原体之间相互作用的机制理解。

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.