Date of Award

Summer 8-4-2024

Embargo Period

8-4-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Melinda "Mindy" Engevik

Second Advisor

Chenthamarakshan Vasu

Third Advisor

Alexander Alekseyenko

Fourth Advisor

Lisa Steed

Fifth Advisor

Caroline Westwater

Abstract

The gut microbiota plays a major role in human diseases. The impact of these microbes on human health comes not only from individual microbes but also from their interactions with each other. A prime example of this is Clostridioides difficile, an intestinal pathobiont that causes severe gastrointestinal illness. C. difficile works cooperatively with other gut bacteria, including Klebsiella pneumoniae. We hypothesized that K. pneumoniae would work synergistically with C. difficile and promote its pathogenesis. We screened over 40 K. pneumoniae strains to assess their interaction with the intestinal mucus layer, a key colonization site for C. difficile. In our screening, we observed that 10% of our clinical isolates exhibited distinct virulence phenotypes when grown on antibiotic selective agar, showing divergent antibiotic responses, biofilm production and motility. We found that all our K. pneumoniae strains had robust adhesion to mucus and flagellated strains of K. pneumoniae chemotaxed towards mucus and individual sugars found in the mucin glycans. We performed computational analysis to examine the mucin glycan degrading capacity of K. pneumoniae and found that it harbored galactosidases and N-acetyl-hexosaminidases. We also found that K. pneumoniae consumed free galactose and N-acetyl-hexosamines and had low level growth with intact mucus. In a larger analysis of gut pathobionts and pathogens, we found that the majority of these microbes did not possess extensive mucin-glycan associated glycosyl hydrolases; suggesting that mucus degradation by most pathobionts is limited. When we grew K. pneumoniae and C. difficile together, we observed both bacteria in co-cultures. We also observed that the presence of K. pneumoniae or its metabolites enhanced the intrinsic green autofluorescence of C. difficile; allowing us to separate the bacteria by flow cytometry. We also found that K. pneumoniae and C. difficile could grow together aerobically, as K. pneumoniae quenched oxygen and supported C. difficile growth in aerobic culture. We observed a similar phenomenon when the bacteria were grown together in bioreactors. Mice that were co-inoculated with C. difficile and K. pneumoniae showed increased gut inflammation and damage. These findings highlight the complexity of microbial interactions in the gut and how they affect human health.

Rights

Copyright is held by the author. All rights reserved.

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