Quantitative Sequencing and its Application to Studies of the Human Small-Intestine Microbiota

Author: Barlow, Jacob T.

Year: 2022

Degree: Dissertation (Ph.D.)

Advisor: Ismagilov, Rustem F.

Committee Members: Mazmanian, Sarkis K.; Thomson, Matthew; Cai, Long; Ismagilov, Rustem F.

Option: Bioengineering

DOI: 10.7907/ca28-fk21

Abstract

Our understanding of the interplay between microbial species and the hosts they live on and in is continually expanding. New insights have focused not only microorganisms that drive specific disease states but also those that help maintain human health. As research drives towards mechanistic understanding of host-microbe relationships new quantitative tools are needed to help interrogate these complex interactions. Chapter I of this thesis discusses formulation of a method for rapid detection of antibiotic resistance in Neisseria gonorrhoeae. Our approach identified RNA signatures from transcriptional profiling of Neisseria gonorrhoeae after 10-minute antibiotic exposure. Utilization of these RNA markers allowed for rapid identification of antibiotic susceptibility or resistance to the antibiotic ciprofloxacin. Chapter II shifts focus to the development of a quantitative sequencing technique for the measurement of absolute taxon abundances in complex microbial communities. Combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing allowed for simultaneous quantitative profiling of all bacterial taxa in host-associated microbial communities. We extensively characterized our quantitative sequencing methodology in the presence of high host nucleic acid levels and low microbial loads to understand the limits of quantification and detection in complex sample types. Last, Chapter III applies the quantitative sequencing technology from Chapter II to investigate the microbial community of the human small intestine, specifically the duodenum. Data from the duodenum of 250 individuals revealed a wide range of total microbial loads and a distinct subset of microbes, termed disruptor taxa, that were associated with small intestinal bacterial overgrowth (SIBO) and GI symptom severity.

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