Utilization of Probiotics to Compete with Clostridioides difficile for Nutrient-Niches in a Variety of in vitro Contexts

Authors

April Elizabeth Johnson, University of Nebraska-LincolnFollow

First Advisor

Jennifer Auchtung

Committee Members

Amanda Ramer-Tait, Devin Rose

Date of this Version

7-2024

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Food Science and Technology

Under the supervision of Professor Jennifer Auchtung

Lincoln, Nebraska, July 2024

Comments

Copyright 2024, April Elizabeth Johnson. Used by permission

Abstract

Clostridium difficile is a major threat to public health and primarily affects at risk groups such as elderly, immunocompromised individuals, and individuals on antibiotics. Many decades of research have been conducted to figure out ways to exclude or reduce C. difficile colonization and toxin production. Two major proposed mechanisms for colonization resistance are production of secondary bile salts and competition for nutrients.

In Chapter 2, we found that antibiotic treatment disrupted bile salt metabolism increasing the levels of the primary bile salt cholate and decreasing the levels of deoxycholate, a secondary bile salt. However, bile salts were not required to inhibit C. difficile colonization in vitro. It was also determined that C. difficile mutants unable to metabolize proline through Stickland fermentation due to a disruption of proline reductase ΔprdB colonized complex fecal communities cultured in continuous-flow minibioreactor arrays at significantly lower rates relative to the wild type strain for 4 out of 6 fecal communities tested. This data emphasized the importance of further understanding colonization resistance mechanisms independent of bile salts.

In Chapter 3, I utilized rationally selected probiotic mixtures to test whether mixtures of probiotics alone or in conjunction with prebiotic fibers could reduce C. difficile and its toxin levels in pure culture and in the presence of antibiotic-treated complex fecal communities. I found administration of probiotic mixes Lacto 11 and Lacto 5, along with the prebiotic fiber inulin, lead to the highest levels of C. difficile inhibition in pure culture and also contributed to decreased levels of toxin activity. While low pH is known to inhibit C. difficile growth, there was no correlation found between the pH and C. difficile levels in Lacto 11 communities cultured in pure culture in the presence of inulin, which points to pH-independent mechanisms for inhibition. While I tested whether proline metabolism could contribute to inhibition in pure culture with Lacto 11 and Lacto 5, the results were inconclusive due to lack of proline-dependent metabolism in controls. Finally, I observed that supplementation with the prebiotic fiber inulin reduced susceptibility to C. difficile colonization in antibiotic-treated complex fecal communities. However, this was not enhanced by treatment with probiotic mixes. While there was partial inhibition of C. difficile colonization in one of two complex fecal communities treated with Lacto 11 in the absence of prebiotics, further studies will be needed to understand features of responder and non-responder communities to probiotic treatment. Nevertheless, this work provides further support for understanding nutrient niches colonized by C. difficile and using this information to try to decrease the likelihood of C. difficile colonization for potentially susceptible individuals.

Advisor: Jennifer Auchtung