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

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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.