Antibiotic resistance presents a serious and still growing threat to human health. Environmental exposure levels required to select for resistance are unknown for most antibiotics. Here, we evaluated different experimental approaches and ways to interpret effect measures, in order to identify what concentration of trimethoprim that are likely to select for resistance in aquatic environments. When grown in complex biofilms, selection for resistant E. coli increased at 100 µg/L, whereas there was only a non-significant trend with regards to changes in taxonomic composition within the tested range (0–100 µg/L). Planktonic co-culturing of 149 different E. coli strains isolated from sewage again confirmed selection at 100 µg/L. Finally, pairwise competition experiments were performed with engineered E. coli strains carrying different trimethoprim resistance genes (dfr) and their sensitive counterparts. While strains with introduced resistance genes grew slower than the sensitive ones at 0 and 10 µg/L, a significant reduction in cost was found already at 10 µg/L. Defining lowest effect concentrations by comparing proportion of resistant strains to sensitive ones at the same time point, rather than to their initial ratios, will reflect the advantage a resistance factor can bring, while ignoring exposure-independent fitness costs. As costs are likely to be highly dependent on the specific environmental and genetic contexts, the former approach might be more suitable as a basis for defining exposure limits with the intention to prevent selection for resistance. Based on the present and other studies, we propose that 1 µg/L would be a reasonably protective exposure limit for trimethoprim in aquatic environments.

抗生素耐药性对人类健康构成了严重且仍在增长的威胁。对于大多数抗生素来说，选择抗药性所需的环境暴露水平是未知的。在这里，我们评估了不同的实验方法和方法来解释效应措施，以便确定在水生环境中可能选择抗药性的甲氧苄啶浓度。当在复杂的生物膜中生长时，对抗性大肠杆菌的选择以100 µg / L的速度增加，而在测试范围内（0–100 µg / L）的生物分类组成变化只有很小的趋势。浮游生物共培养149种不同的大肠杆菌从污水中分离出的菌株再次确认选择浓度为100 µg / L。最后，成对竞争实验用改造的进行大肠杆菌携带不同的甲氧苄啶抗性基因的菌株（DFR）及其敏感的对应对象。虽然引入抗性基因的菌株在0和10 µg / L时生长速度比敏感菌株慢，但在10 µg / L时已经发现成本显着降低。通过在同一时间点比较敏感菌株与敏感菌株的比例而不是初始比例来确定最低效应浓度，将反映出抗性因子可以带来的优势，同时忽略了与暴露无关的适应性成本。由于成本可能高度依赖于特定的环境和遗传环境，因此前一种方法可能更适合作为定义暴露极限的基础，以防止选择抗药性。根据目前和其他研究，我们建议1 µg / L是甲氧苄啶在水生环境中的合理保护性暴露极限。