Date of Award

Summer 8-14-2017

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Timothy J. Bowden

Second Committee Member

Melody Neely

Third Committee Member

Ian Bricknell

Additional Committee Members

John Singer

Abstract

Streptococcus spp. are a major concern to healthcare, agriculture, and aquaculture industries worldwide. Of notable concern are S. iniae and S. agalactiae, which contribute to significant loss in the aquaculture field, particularly to tilapia grown in Southeast Asia where low water quality and high stocking density allows these pathogens to thrive. The initial identification of S. iniae came from dolphins located in the Amazon river, and S. agalactiae was first documented in humans and bovine species before being observed in fish. Two strains of S. iniae, one strain of S. agalactiae serotype 1a, and two strains of S. agalactiae serotype 1b were used to the impact of specific carbohydrates on chain formation and capsule production when they are the only carbohydrate source available. Chain formation and the capsule layer play important roles in pathogenesis of streptococci. Most research on capsule production and chain formation focuses on the use of mutant strains with gene deletions. While this shows how genes impact the physiology of the pathogen, it does not show how a wild type would respond to its environment. It is the theme of this thesis to change the physiology of S. iniae and S. agalactiae by manipulating the nutritional environment it is cultured in. Chaining was analyzed both in standard broth media, and broth media containing a subinhibitory concentration of lysozyme, which hydrolyses the cell wall peptidoglycan. Capsule was analyzed qualitatively using a continuous Percoll gradient to measure the density of the cells. Furthermore, a quantification protocol was employed to study the amount of capsule produced by cultured streptococci. It appeared that during in vitro subculture, S. agalactiae serotype 1b had become less virulent. This decrease in virulence appeared to have decreased the production of capsule, which may be an indication of attenuation due to in vitro culture methods. The bacterium appears to have reverted to a more virulent form upon the addition of lysozyme to the broth media. The result was similar for S. agalactiae serotype 1b, but to a much lesser degree due to the naturally increased expression of capsule regulatory genes. Furthermore, S. iniae was presumed to act much like pneumococcal cells, which start with long chains to increase its invasive ability, then decreases its chain length to avoid opsonophagocytosis. This change in physiology was thought to be due to the addition of lysozyme, which is a constituent of host phagocytic cells. This study provides insight into the impact of carbohydrates on the physiology of S. iniae and S. agalactiae, and opens the door for more research opportunities.

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