Date of Award

8-2018

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Lee H. Lee

Committee Member

Sandra Adams

Committee Member

Julian Keenan

Subject(s)

Green tea--Therapeutic use, Polyphenols, Streptococcus mutans, Pathogenic microorganisms, Epigallocatechin gallate

Abstract

Dental caries, or cavities, is a highly prevalent disease affecting societies of different economic and geographic status across the globe. This disease is caused by dental decay by bacteria fermenting the carbohydrates in food and producing acid end-products that dissolve the enamel. Though there are a few different bacteria that can cause cavities, Streptococcus mutans is the main etiological cause of dental caries. The earliest stages of cavity formation are typically marked with biofilm development, in which the thick slime layer confers bacteria greater adherence to the enamel surface, the ability to adapt to more adverse conditions, and quorum sensing for communicative defense and regulation. S. mutans utilizes sugary foods, such as those containing sucrose, and releases acid end-products that overwhelm the buffering capacity of saliva and drop the pH of the oral cavity to 5.0 or lower. At this point, the enamel begins wearing away and cavities form.

The ESKAPE pathogens represent another healthcare issue, as they are a group of bacteria that are heavily implicated in nosocomial infections and resistance to common antibiotics. The group is made up of the bacteria Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter bacteria species. Due to inappropriate and excessive use of antibiotics over the years, this group is primarily responsible for hospital-acquired infections and treatments have been more challenging to come by due to their resistance mechanisms. These mechanisms include drug inactivation or alteration, modification of drug binding sites, reduced intracellular drug accumulation, and biofilm formation.

Green tea, produced from the plant Camellia sinensis, is a popular beverage throughout the world, and the polyphenolic compounds of the plant have been reported to have many health benefits, including antimicrobial and anti-cariogenic properties. The green tea polyphenols are comprised of several catechins, but Epigallocatechin Gallate (EGCG) is the most abundant. Due to the instability of EGCG in aqueous solution and its tendency to readily oxidize, more stable derivatives must be studied instead. Epigallocatechin Gallate-Sterate (EGCG-S) and Palmitoyl- Epigallocatechin Gallate (P-EGCG) are esterified, stable derivatives. P-EGCG is of particular interest due to its clearing by the FDA in China as a safe food preservative, and thus it could potentially be an oral treatment in the case of S. mutans.

In this study, the inhibitory effects of EGCG-S and P-EGCG at concentrations of 250 μg/mL and 500 μg/mL were studied against the cariogenic S. mutans bacterium. These effects were studied through colony forming unit assays, time course studies, disk diffusion assays, and post-application treatments of the polyphenolic compounds. The time course study was performed in artificial saliva with different concentrations of sucrose (0%, 0.1%, 0.5%, and 1%) in order to simulate the effects of the treatment in the oral cavity filled with sugar. Four different mouthwash products were used in the disk diffusion assay and post-application treatment in order to compare the effects of the mouthwashes and P-EGCG/EGCG-S. The next objective of the study was to investigate the effects of the green tea polyphenol treatments on the growth of S. mutans. To do so, Congo Red assays, Resazurin assays, and Crystal Violet assays were conducted. A separate study was conducted to investigate the effects of EGCG-S against the ESKAPE pathogens, and disk diffusion assays and colony forming unit assays were performed. The disk diffusion assays were conducted synergistically with the tea polyphenolic compounds and several antibiotics in order to study if the effects of the antibiotics could be made more potent by adding EGCG-S.

The results of the study of S. mutans growth suggests that EGCG-S and P-EGCG do inhibit the growth, but with mixed effectiveness. This treatment is comparable to the mouthwashes, although Chlorohexidine appears to be the most effective. S. mutans biofilm formation was inhibited by P-EGCG in the Congo Red assay, but the results of the Resazurin and Crystal Violet assays were inconsistent and inconclusive due to apparent cloudiness and precipitation. EGCG-S appeared to inhibit the growth of the ESKAPE pathogens as evidenced in the colony forming unit assay, although the degree of inhibition was mixed among organisms and concentrations of EGCG-S. The disk diffusion assay demonstrated that EGCG-S made the ESKAPE pathogens more sensitive to several of the antibiotics. Overall, the study suggests that P-EGCG and EGCG-S are effective in inhibiting the growth of S. mutans and the ESKAPE pathogens, but future studies must be conducted in order to ascertain the degree of inhibition and its effectiveness in synergistic treatment with mouthwashes/antibiotics.

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Biology Commons

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