Author ORCID Identifier

https://orcid.org/0000-0001-7152-8564

Date of Award

Spring 4-3-2025

Document Type

Thesis (Ph.D.)

Department or Program

Microbiology and Immunology

First Advisor

George O'Toole

Abstract

The microbiome of the cystic fibrosis (CF) intestine is altered significantly in structure and function beginning in early life. Key taxa associated with health, digestion and immune-training are reduced in abundance, including but not limited to Bacteroides, Faecalibacterium, Akkermansia, Ruminococcus and other short-chain fatty acid producers. Conversely, there is an expansion of microbes with pathogenic potential, such as Escherichia, Veillonella, Pseudomonas and Staphylococcus. The shifts in microbiota composition direct community-wide changes with differences in beta diversity by genotype, whereby children with CF (cwCF) exhibit significantly different gut microbial compositions compared to nonCF children. Additionally, shifts in microbiota structure often translate to shifts in microbiota function, with microbial metabolism as a large focus in most studies. Taken together, an imbalance in the intestinal microbiome as observed early in life in cwCF contributes to an observed delay in maturation of their intestinal microbiome compared to nonCF cohorts, underscoring the need to identify key drivers of microbial imbalances which may or may not overlap with other intestinal diseases (i.e., inflammatory bowel disease).

In this thesis, I introduce a novel growth medium to represent the nutritional environment of the CF colon. Through the implementation of this medium in growth assays, I show that physiological features relevant to the CF intestine (i.e., excess mucin, fats, bile, altered pH, alternative electron acceptors, inflammatory by-products, antibiotic perturbation) are sufficient in directing community-wide changes in microbiome structure that mimic CF intestinal microbiota signatures. Furthermore, these taxonomical shifts translate to altered microbial metabolism, with short chain fatty acid abundance as proxy.

I apply this medium to study the mechanism of altered microbial interactions, with a focus on Escherichia coli and Bacteroides vulgatus. In vitro growth assays analyzed via linear regression identify significant drivers of dysbiosis in our system: excess bile and excess fat (in the form of glycerol). Genetic mutants of E. coli involved in microbial antagonism of B. vulgatus in the context of glycerol reveal a significant role for colibactin biosynthesis, linking a genotoxin associated with colorectal cancer to signatures of gut microbial dysbiosis in children with CF.

The findings presented in this thesis begin to elucidate host physiological features and microbial genetic factors involved in modulating markers of dysbiosis across CF cohorts. While many altered physiologies and treatments inherent to the CF genotype have been correlated with microbial imbalances, there are limited mechanistic studies in the field. Development and implementation of the novel in vitro medium introduced in this thesis sets the stage for future in vitro studies to further our understanding of CF gut biology.

Original Citation

Barrack, K. E., Hampton, T. H., Valls, R. A., Surve, S. V., Gardner, T. B., Sanville, J. L., Madan, J. C., O’Toole, G. A. (2024). An in vitro medium for modeling gut dysbiosis associated with cystic fibrosis. J Bacteriol. 2024 Jan 3:e0028623. doi: 10.1128/jb.00286-23. Epub ahead of print. PMID: 38169295.

Price, C. E.*, Valls, R. A.*, Ramsey, A. R., Loeven, N. A., Jones, J., Barrack, K. E., Schwartzman, J., Royce, D., Cramer, R. A., Madan, J., Ross, B., Bliska, J. B., O’Toole, G. A. (2024). Intestinal Bacteroides modulates inflammation, systemic cytokines and the microbial ecology via propionate in a mouse model of cystic fibrosis. mBio. 2024 Jan 5:e0314423. doi: 10.1128/mbio.03144-23. Epub ahead of print. PMID: 38179971.

McManus, T. G., Fort, M. W., Ray, G. S., Barrack, K. E., Sparks, M. B., McGuire, K. J., O’Toole, G. A. (2023). Study on agitation for biofilm eradication and reduction (SABER): Evaluation of mechanical brushing with sonication and radiofrequency. In press (Acta Orthopaedica)

Price, C. E., Hampton, T. H., Valls, R. A., Barrack, K. E., O'Toole, G. A., Madan, J. C., Coker, M. O. (2023). Development of the intestinal microbiome in cystic fibrosis in early life. mSphere. 2023 Aug 24;8(4):e0004623. doi: 10.1128/msphere.00046-23. Epub 2023 Jul 5. PMID: 37404016; PMCID: PMC10449510.

Valls, R. A., Hampton, T. H., Price, C. E., Barrack, K. E., O’Toole, G. A., Coker, M. O., & Madan, J. C. (2022). Predicting clinical outcomes in infants with cystic fibrosis from stool microbiota using random forest algorithms. BioRxiv, 2022.08.06.503028. https://doi.org/10.1101/2022.08.06.503028

Katharios-Lanwermeyer, S., Koval, S. A., Barrack, K. E., & O’Toole, G. A. (2022). The diguanylate cyclase YfiN of Pseudomonas aeruginosa regulates biofilm maintenance in response to peroxide. J Bacteriol. 2022 Jan 18;204(1):e0039621. doi: 10.1128/JB.00396-21. Epub 2021 Oct 25. PMID: 34694901; PMCID: PMC8765426.

Verster A.J, Salerno P.E., Valls R.A., Barrack K.E., Price C.E., McClure E.A., Madan J.C., O’Toole G.A., Sanville J.L., Ross B.D. (2025). Persistent delay in maturation of the developing gut microbiota in infants with cystic fibrosis. mBio 2025 Feb 13:e03420-24. https://doi.org/10.1128/mbio.03420-24.

Sudo, K., Delmas-Eliason, A., Soucy, S., Barrack, K. E., Liu, J., Balasubramanian, A., Shu, C. J., James, M. J., Hegner, C., Dionne, H. D., Rodriguez-Palacios, A., Krause, H. M., O’Toole, G. A., Karpen, S. J., Dawson, P., Schultz, D., Sundrud, M. S. (2024). Quantifying forms and functions of intestinal bile acid pools in mice. Cell Mol Gastroenterol Hepatol. 2024 Aug 21:101392. doi: 10.1016/j.jcmgh.2024.101392. Epub ahead of print. PMID: 39179177.

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