Investigating the use of bacteria and their products for the treatment of Clostridioides difficile infection

Abstract

Clostridioides difficile (CD) infection (CDI) is the leading cause of antibiotic-associated infectious diarrhea. Antibiotic-mediated disruption of the gastrointestinal microbiota (GIM) is the single-greatest risk factor for the development of CDI. Despite the role of antibiotics in the pathogenesis of CDI, antibiotic therapy is still the current standard of care for treating CDI. However, antibiotic therapy is associated with up to 25% of patients failing treatment and developing recurrent CDI. Novel, safe and effective treatments have recently emerged that focus on correcting the perturbations in the GIM by using bacteria to repopulate the gut. However, mechanisms associated with bacterial repopulation of the gastrointestinal tract remain unclear. My thesis aims to investigate a potential mechanism underlying the efficacy of bacterial communities in the treatment of CDI. Using in vitro models of CDI, I first investigated the efficacy of individual bacterial strains within an established defined microbial community (DMC). I found that one strain provided significant protection against CDI by producing a soluble factor able to degrade CD toxins. These findings prompted further investigation into secreted products, particularly proteases. By using size-exclusion chromatography and bacterial genomics, we were able to identify, clone, express and characterize proteases of interest. A purified serine protease high-temperature requirement – A (HtrA) neutralized CD toxins in vitro. Next, I investigated the efficacy of HtrA in vivo using a mouse model of CDI. I found that prophylactic oral treatment with HtrA protected mice from developing CD disease and led to increased survival rates compared to mice without treatment. Next generation sequencing (NGS) revealed that HtrA does not alter the GIM population of mice. The work conducted in this thesis provides a foundation for exploring HtrA as an effective, safe and novel orally delivered non-antibiotic therapeutic against CDI.