Date of Award

Spring 5-2017

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Tinchun Chu, Ph.D

Committee Member

Angela V. Klaus, PH.D

Committee Member

Daniel B. Nichols, Ph.D

Keywords

antibacterial, biofilm, spore, tea polyphenol

Abstract

Tea, one of the most common beverages, originates from the leaves of the Camellia sinensis plant. Two major groups of tea are fermented black tea and unfermented green tea. Theaflavins (TFs) are the major polyphenols present in black tea, while mono-palmitoyl-epigallocatechin-gallate (pEGCG) is a modified green tea polyphenol. In this study, the antibacterial effects of TF and pEGCG were evaluated against six selected bacteria, Staphylococcus epidermidis, Streptococcus mutans, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris, using an antibacterial assay. A viability assay using SYTOX® staining and flow cytometry was also used to determine the effect of these compounds on S. epidermidis and S. mutans. The anti-biofilm effects of the compounds were also investigated using Congo red assay, resazurin assay, polymerase chain reaction and flow cytometry. Finally, TF and pEGCG were also evaluated on the effects of inhibiting sporulation and germination in Bacillus spp. The results indicate that 0.2% TF and 0.2% pEGCG contain strong antibacterial effects against all bacteria tested with an IC50 range of approximately 0.05-0.1%. In addition, the viability assay showed that both compounds effectively inhibit the growth of the bacteria as early as 3 hours and can maintain their effect for 24 hours. Results from anti-biofilm assays showed that TF and pEGCG are also highly effective in inhibiting the formation of biofilm. Qualitatively, the compounds prevented the formation of biofilm, indicated by the absence of black colonies on the Congo red assay. Quantitatively 0.5% and 1% concentrations of both TF and pEGCG inhibited biofilm formation in the four biofilm forming bacteria tested. PCR and gel electrophoresis was also performed to study the effect of TF and pEGCG on biofilm forming genes in S. epidermidis and S. mutans. Results indicated the presence of bands in the control sets, but absent in the treated samples for both sets of genes, aap and brpA. In addition, flow cytometry was used to further understand the effects of these compounds on S. epidermidis and S. mutans. Results showed that 0.5% TF is able to inhibit approximately 86% of biofilm formation in S. epidermidis and 88% in S. mutans, and 1% pEGCG is able to inhibit about 53% in S. epidermidis and 85% in S. mutans. Finally, results from sporulation and germination assays indicate that both compounds are capable of inhibiting these processes in spore-forming bacteria. 1% TF and 1% pEGCG inhibit sporulation as well as the germination process. 1% TF inhibits approximately 77% of germination whereas 1% pEGCG inhibits 99.9% of germination in B. cereus. These results suggest that tea polyphenols are not only viable antibacterial alternatives, but also potentially promising anti-biofilm and anti-spore agents.

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