Investigating the microbial “dark matter” of the human oral microbiome using cultivation-dependent and –independent approaches
Date of Award
Doctor of Philosophy
Heidi Goodrich-Blair, Erik Zinser, Frank Loeffler, Stephen Kania
The advancement of sequencing technology has provided a renewed appreciation for the diversity of life on earth with most environments being dominated by yet-uncultured microorganisms. Analysis of 16S rRNA gene sequencing libraries of the original Human Microbiome Project dataset revealed that approximately 70% of sequences belonged to bacteria that were uncultured in the laboratory and current metagenomic analysis of the human microbiome has proposed 77% of taxa lacked sequenced genomes. As the human microbiome is important for healthy host physiology, these findings emphasize just how little we currently know about the microorganisms that reside in the human body. Understanding the microorganisms that make up the normal human microbiome is the first logical step in determining, during dysbiosis and disease, how the community changes as a whole so as to permit investigation into preventative and/or treatment measures during disease. For example, many disease-associated lineages of bacteria in periodontal disease remain uncultured in the laboratory and therefore limited insight is available as to whether or not these organisms play a role in disease etiology or their presence is a secondary effect of the disease state. Therefore, cultivating more microorganisms from the human microbiome is critical as it enables hypothesis-driven experimentation into the roles of those organisms in human health and disease. The work presented here represents a two-fold approach for cultivation of bacteria from the human oral microbiome by both classical means and by the development of a novel, “reverse-genomics” approach. First, I identified a specific interaction between Desulfobulbus oralis and Fusobacterium nucleatum that resulted in the isolation of the first Desulfobulbus genus member from the human oral cavity and I characterized this bacterium both physiologically and genomically. Next, I investigated what causes the recalcitrance of some oral bacteria to cultivation and what contributes to their dependence on other bacteria, such as F. nucleatum. Lastly, we leveraged existing public sequence data to target specific TM7 bacteria from within the microbial community for both single-cell genomic characterization and cultivation. Collectively this work demonstrates how cultivation-dependent and cultivation-independent methodology can be combined to investigate the “dark microbial matter” in the human oral cavity.
Cross, Karissa, "Investigating the microbial “dark matter” of the human oral microbiome using cultivation-dependent and –independent approaches. " PhD diss., University of Tennessee, 2019.