Document Type

Dissertation - Open Access

Award Date

2023

Degree Name

Doctor of Philosophy (PhD)

Department / School

Veterinary and Biomedical Sciences

First Advisor

Joy Scaria

Keywords

Bioreactor, Caenorhabditis elegans, Clostridioides diffcile, Colonization resistance, Core human gut bacteria, Gut Microbiome

Abstract

Although fecal transplantation has shown to be effective against Clostridioides difficile infection (CDI), key species responsible for colonization resistance still need to be better identified. There needs to be more understanding of the rules governing the development of a healthy microbiome upon transplantation. The bottom-up approach of assembling simple to complex communities in model systems such as bioreactors is one of the ways to approach this problem. Studying synthetic communities with less complexity can yield a system-level understanding of microbial interactions. In the first study, we assembled a synthetic community of 14 bacterial species belonging to the core human microbiota in a continuous flow mini bioreactor model and determined the succession, stability, and resilience of the community. The resilience of the community was defined using antibiotic perturbation, and colonization resistance capacity was determined by inoculating C.difficile into the bioreactor. Our results show that the community attained stability on day eight, and antibiotic perturbation significantly increased the abundance of B.caccae in the system. However, after the antibiotic withdrawal, the system did not revert to the pre-perturbation state. Furthermore, this non-reverted community could completely exclude C.difficile, indicating the high colonization resistance capacity of the system. To determine whether this increasing microbial complexity with the other microbiome members can increase colonization resistance in-vivo, strains could be poly-associated in the host and challenged again with the pathogen. However, logistical and cost limitations make such approaches difficult to perform in hosts such as germ-free mice. To address this problem, we used C. elegans as a model to determine the colonization resistance capacity of a human gut commensal. C. elegans has been used as a high-throughput model for aerobic bacteria but has yet to be used for strict anaerobes because of a lack of a standardized anaerobic colonization method. In the second study, we aimed to develop C. elegans as a model to study gut microbiota-mediated colonization resistance by establishing an anaerobic bacterial colonization method. For the first time, we can colonize strict human anaerobic gut isolate (B.longum) and a pathogen (C.difficile) in C. elegans. Our C.difficile challenge results in the C .elegans model recapitulating the gene expression changes observed during human CDI. Our method paves the way for using C. elegans as a high-throughput model to study colonization resistance mechanisms of human gut bacterial species against C .difficile. Another essential aspect of gut microbiome research is understanding the role of different mucin in community ecology and related physiology. The role of these integral glycoproteins can be efficiently deciphered using colonization experiments in different knockout models. Our genomebased investigation in C. elegans revealed 6 genes having human orthologs. Expression data and colonization trials in knockouts revealed the role of gly-8 in performing the antimicrobial function. Altogether our studies explored mini-bioreactor array and C. elegans as a model to understand colonization resistance and associated systems interaction better.

Library of Congress Subject Headings

Clostridium difficile.
Clostridium diseases.
Gastrointestinal system -- Microbiology.
Caenorhabditis elegans.
Bioreactors.

Publisher

South Dakota State University

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Available for download on Wednesday, May 15, 2024

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Rights Statement

In Copyright