Clostridioides difficile Colonization Is Not Mediated by Bile Salts and Utilizes Stickland Fermentation of Proline in an in vitro Model

Authors

• Xiaoyun Huang, University of Nebraska-Lincoln
• April E. Johnson, University of Nebraska-Lincoln
• Joshua N. Brehm, Texas A&M University-College Station.
• Thi Van Thanh Do, University of Nebraska-Lincoln
• Thomas A. Auchtung, University of Nebraska-Lincoln
• Hugh C. McCullough, University of Nebraska-Lincoln
• Armando I. Lerma, University of Nebraska-Lincoln
• Sigmund J. Haidacher, Baylor College of Medicine and Texas Children’s Hospital
• Kathleen M. Hoch, Baylor College of Medicine and Texas Children’s Hospital
• Thomas D. Horvath, Baylor College of Medicine and Texas Children’s Hospital and University of Houston
• Joseph A. Sorg, Texas A&M University-College Station
• Anthony M. Haag, Baylor College of Medicine and Texas Children’s Hospital
• Jennifer M. Auchtung, University of Nebraska-LincolnFollow

ORCID IDs

Sorg https://orcid.org/0000-0001-7822-2656

Auchtung https://orcid.org/0000-0003-3038-583X

Document Type

Article

Date of this Version

2025

Citation

mSphere (2025) 10: e01049-24

doi: 10.1128/msphere.01049-24

Comments

Open access

License: CC BY 4.0

Abstract

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include the conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model that simulates the nutrient conditions of the distal colon, we investigated how treatment with 6 clinically used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate. However, changes in bile salt did not correlate with increased C. difficile susceptibility. Furthermore, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to the persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (prdB−) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. The inability to ferment glycine or leucine as electron acceptors, however, was not sufficient to limit colonization in any communities. The data provide further support for the importance of bile salt-independent mechanisms in regulating the colonization of C. difficile.