Evaluating the Protective Capacity of Secreted Products from Bacteroides ovatus against Clostridioides difficile Infection
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Authors
Greenlaw, Jill
Date
2024-08-01
Type
thesis
Language
eng
Keyword
Clostridioides difficile , Clostridioides difficile infection , Defined microbial community , Gastrointestinal microbiota , Bacterial secreted products
Alternative Title
Abstract
Clostridioides difficile (CD) is the leading cause of healthcare-associated diarrhea and colitis. CD infection (CDI) is mediated by two exotoxins, toxin A (TcdA) and toxin B (TcdB). The pathogenesis of CDI is closely linked to disruption of the gastrointestinal microbiota; most commonly resulting from antibiotic exposure. Although antibiotics cure 70% of CDI, ~30% of patients do not respond to antibiotics and develop CDI recurrence. Recent treatment strategies have focused on repairing the gastrointestinal microbiota to prevent CDI recurrence using laboratory-cultured defined microbial communities. Our lab recently developed a 4-member microbial community (DMC-4) where we observed that soluble factors from DMC-4 protected mice against CDI. Here, we investigate if soluble factors produced by B. ovatus, a member of DMC-4, protect against CD.
Bacterial-free conditioned media (CM) of B. ovatus was created using centrifugation, and filter-sterilization. TcdA/TcdB activity were evaluated using fibroblast cytotoxicity assays and western blots were used to assess the degradation of TcdA and TcdB by CM. CM was tested in vivo using an established CDI mouse model.
B. ovatus CM neutralized TcdA and TcdB activity following incubation with CM compared to toxin controls (p<0.05 for both). Size-based separation of CM into products >10 and <10kDa revealed that both fractions were capable of neutralizing toxin activity on cells (p<0.05 for all). Incubation of CM with TcdA and TcdB led to the disappearance of toxins on western blots, suggesting that a protease may be neutralizing CD toxins. Although CM demonstrated promise in vitro, in a pilot study using a CDI mouse model, oral administration of CM did not prevent death, weight-loss, colonic shortening or colonic damage. Furthermore, additional experiments demonstrated the loss of toxin neutralizing activity by CM following incubation at a pH of 3, suggesting that its lack of efficacy in vivo may be attributable to the inability to withstand the low pH of the murine stomach.
The in vitro results from this study demonstrate the potential of secreted products from B. ovatus as a therapeutic strategy against CDI and provides insight into the mechanism of action by which DMC-4 protects against CDI.
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Attribution-NonCommercial-NoDerivatives 4.0 International
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial-NoDerivatives 4.0 International