Selection for AMR in complex microbial communities at sub-therapeutic antibiotic concentrations

抗菌素耐药性（AMR）是人类和动物病原体中日益严重的问题，并已被首席医疗官强调为对公共卫生的严重威胁。越来越多的证据表明，农业中的抗生素使用可能导致临床出现AMR，政府的5年AMR战略强调提高对AMR的认识和理解是一个关键的优先事项。本项目将利用实验室进化实验研究AMR在复杂微生物群落中的进化。首次在临床上重要的条件致病菌E.大肠杆菌，将在存在和不存在复杂的细菌群落的情况下研究。该菌群将来自猪的粪便，并将在厌氧发酵罐中孵育，这些发酵罐以前曾被用作简单的肠道模型。传统的研究细菌AMR的方法通常只考虑一个孤立的物种。这种方法可以用于研究基于突变的抗生素适应;然而，它没有考虑水平基因转移的影响，其中抗性基因是从微生物群落中获得的。单一物种实验也没有考虑对抗生素具有不同内在抗性的不同细菌物种之间的竞争。在存在和不存在复杂微生物群落的情况下进行的竞争实验将有助于我们了解AMR的进化是如何发生的。我们将实验微生物群落暴露于不同浓度的抗生素，这些抗生素代表了口服抗生素治疗期间在动物和人类肠道中发现的亚治疗浓度。这将使人们深入了解微生物种群内发生的复杂相互作用如何影响AMR的进化，相对于单一物种实验中AMR的选择。传统上认为暴露于低浓度抗生素对AMR的选择并不重要。然而，最近的数据表明，选择发生在比以前认为的低得多的浓度下，Gaze实验室的初步数据表明，在较低的亚治疗浓度下，对AMR革兰氏阴性机会性病原体的选择可能比在更接近用于治疗感染的浓度下更大。我们还将研究单一抗生素选择对所有已知抗性基因的相对丰度和多样性的影响。这将提供由于复杂群落中细菌基因组中的遗传联系而对AMR基因进行间接共选择的数据，以及能够在细菌之间转移多个基因的移动的遗传元件的数据。细胞分选和下一代测序技术将识别所有已知的转移到遗传标记的E。大肠杆菌进行抗生素筛选。相反，我们也将研究AMR基因从标记的E.大肠杆菌与复杂群落中的所有其他细菌。

Antimicrobial resistance (AMR) is an increasing problem in human and animal pathogens, and has been highlighted as a serious threat to public health by the Chief Medical Officer. There is increasing evidence that antibiotic usage in agriculture may be contributing to the emergence of AMR in the clinic and the government's 5 year AMR strategy highlights improved knowledge and understanding of AMR as a key priority. This project will investigate the evolution of AMR in complex microbial communities using laboratory evolution experiments. For the first time, selection for AMR in a clinically important opportunistic pathogen, E. coli, will be studied in the presence and absence of a complex community of bacteria. The community will be from pig faeces, and will be incubated in anaerobic fermenters that have previously been used as simple gut models.Traditional approaches to studying AMR in bacteria often consider only one species in isolation. This approach can be useful in studying mutation based adaptation to antibiotics; however it does not consider the impact of horizontal gene transfer where resistance genes are acquired from the microbial community. Single species experiments also fail to consider competition between different species of bacteria that have differing intrinsic resistance to antibiotics. Competition experiments in the presence and absence of a complex microbial community will help us understand how evolution of AMR occurs. We will expose experimental microbial communities to different concentrations of antibiotic representing the sub-therapeutic concentrations found in the animal and human gut during oral antibiotic therapy. This will give insights into the way complex interactions that occur within microbial populations affect evolution of AMR, relative to selection for AMR in single species experiments. Exposure to low concentrations of antibiotics was traditionally thought to be unimportant in selection for AMR. However recent data suggests that selection occurs at much lower concentrations than previously thought, and preliminary data in Gaze's lab suggests that selection for AMR gram-negative opportunistic pathogens can be greater at lower sub-therapeutic concentrations than at higher concentrations closer to those used to treat infections. We will also investigate the effects of selection by a single antibiotic on relative abundance and diversity of all known resistance genes. This will provide data on indirect co-selection for AMR genes due to genetic linkages in the genomes of bacteria in the complex community and on mobile genetic elements capable of transferring multiple genes between bacteria. Cell sorting and next generation sequencing techniques will identify all known AMR genes transferred to a genetically tagged E. coli under antibiotic selection. Conversely, we will also investigate AMR gene transfer from a tagged E. coli to all other bacteria within the complex community.