Engineering a synthetic gut model to explore microbial infections

Our intestines are home to over one thousand bacterial species, often referred to as the gut microbiota. This microbiota is profoundly beneficial to our bodies, with disturbances to this community associated with pathogenic infection and diseases such as diabetes, obesity and inflammatory bowel disease. In this project we aimed to engineer a representative microbiota, with an emphasis on tracking individual species. Utilising propidium monoazide (PMA)-qPCR, we were able to track the nine individual species of our representative gut microbiota within a mixed biofilm setup for 72 hours. Clostridioides difficile is an opportunistic pathogen, which targets the gut during times of microbiota dysbiosis. We have used our tracking technology to see how a representative microbiota reacts to a C. difficile invasion. We found that the representative gut microbiota could inhibit C. difficile colonisation, with a pre-established microbiota biofilm creating the most significant effect. Given the strong links between antibiotic-associated microbiota disturbance and the increased risk of C. difficile infection, we tested the impact of vancomycin treatment on the nine species mix. Surprisingly, we found that the microbiota in biofilm conditions could withstand high doses of vancomycin (20 μg/ml). We developed a gut epithelial model, the E-VDC, that supports the growth of strictly anaerobic bacteria. We used this model to investigate C. difficile infections in the presence of the gut commensal, Bacteroides dorei. In dual biofilm conditions, we found B. dorei to have an inhibitory effect on C. difficile. This effect carries over into the adherent community within our gut model, however we found the inhibition was less pronounced. To investigate how B. dorei was inhibiting C. difficile we performed mass-spectrometry based proteomics. A total of 39 proteins were found to be differentially expressed between single and cocultures of C. difficile and B. dorei. For C. difficile we found that the majority of the proteins affected by the presence of B. dorei were stress related. Interestingly, when with B. dorei, C. difficile was producing more of the protein HpdB, an enzyme needed for the production of p-cresol a highly bacteriostatic compound thought to influence the gut microbiota