Access Type

Open Access Embargo

Date of Award

January 2015

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Robert A. Akins

Abstract

A thesis presented on the characterization of oral microbiota in xerostomic versus non-xerostomic volunteers and in daily samples following standard oral hygiene practices. Xerostomia is a difficult and burdensome disease that can be very difficult to diagnose. Understanding the oral microbiota between these diseased and healthy (non-xerostomic) can give us great insight on new treatments and/or prevention. Goals of the study included determining whether there substantial differences in oral microbial populations between the two groups, and whether varying nightly oral hygiene practices had an impact on next-morning oral microbiota titers or composition. Microbial loads were determined by qPCR using broad-spectrum primers. Microbial compositions were estimated based on melt curve analysis of amplicons that spanned the internal transcribed spacer between small and large ribosomal RNA genes, and by qPCR using phylogenetic branch-specific primers. The project succeeded in developing and optimizing a storage media that allowed 30 day room temperature storage, and an DNA extraction method that outperformed commercial kits.

The xerostomia versus control study used three sequential daily saliva samples, collected from 18 xerostomia and from 18 healthy, control volunteers. Fungal populations and several potentially novel species were found to be more significantly prevalent in xerostomia patients as compared to healthy (P = 0.001). Surprisingly, total bacterial titers and overall compositions at the branch levels were not dramatically different in control versus xerostomia groups. 6. However, several subgroups of bacteria (Lachnospiraceae and Bacteroidaceae) were reduced by 5 to 20 fold on average, and specific species were less prevalent; among xerostomic patients, and none were elevated. These studies suggest fungal species may play a role in the poorer oral hygiene of xerostomic patients and that more detailed analysis using next generation sequencing is warranted.

Mouthwashes and toothpastes are composed of several different ingredients, many of which purported to have anti-caries or anti-gingivitis activities. However, the quantitative impact of these is not well studied. The objective to this part of the thesis was to examine the shift in populations after a specific oral hygiene practice repeated over 5 nights and assayed from saliva the next mornings. This essentially uses the mouth as in incubator for microbial regrowth. A total of 30 saliva samples were collected from 17 individuals the morning after a given nightly oral hygiene practice, including no routine, Listerine mouthwash only, Crest mouthwash only, Crest toothpaste only, and Colgate toothpaste only. These samples were analyzed using qPCR and sequencing. Total bacterial loads returned to approximately the same levels after the 4 routines compared to no routine. Overall, Crest toothpaste and mouthwash routines reduced titers more in more individuals. However, individual routines did have an impact by reducing next-morning saliva bacterial compositions and titers of specific groups, but these reductions were highly specific to the individual and the routine. This suggests that we have highly individualized responses to common oral hygiene products, and that tailoring our choice of these to optimize specific bacterial group reductions could improve oral health.

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