Discipline: Biological Sciences
Denerick Nyquan Simpson - Savannah State University
Co-Author(s): Mitulkumar Patel, Amanda Finger Stadle, Pachiappan Arjunan, Christopher Cutler, and Roger Arce, Augusta University, Augusta, GA
It is well established in the literature that Porphyromonas gingivalis (Pg) is the keystone pathogen in periodontal disease in humans. This comprehensive study was a secondary analysis of a major project aiming to evaluate the effects of Pg grown in a multi species biofilm on alveolar bone resorption in mice. The objective of the present study was to develop a protocol for isolation of bacterial DNA from the oral cavity of the mice, and to measure and identify some commensal bacteria microorganisms in the mice mouth, comparing with the changes in the bacterial profile after antibiotics treatment (sulfamethoxazole and trimethoprim), and after infection with Pg which is found in the human oral cavity and is highly implicated in periodontitis. The study involved the process of isolating DNA from the mouth of mice in an animal model of oral infection mimicking periodontal disease in humans, by using the DNA investigator forensic kit (Qiagen). Periodontitis is a severe health problem which is why animal research is done. Animal models of periodontitis have several advantages with one being the fact that an individual can mimic the human condition (periodontitis) to better understand the disease process better. Four different apparatus for swabbing the gingiva of the mice were tested: cotton swab, sponge swab, paper strips (Periopaper) and paper points. The project was evaluated in three increments. Before antibiotic treatment with periodontal pathogens, one expected to identify regular commensal bacteria and what would be a normal concentration (i.e. “load”) of bacterial DNA. However, after antibiotics, one expected for the amount of bacterial DNA concentration to drop because of the antibiotic activity and after the infection period one expected to identify Pg and/or higher concentrations of commensal bacteria because of the biofilm dynamics after oral infection. Therefore, the aim of the project was to isolate DNA in a 15-20ng/ul range to be able to answer the hypothesis by quantifying total DNA and identifying bacterial genotypes. The concentration of DNA was measured using Nanodrop. After selection of the apparatus that provided the highest concentration of isolated DNA, baseline bacterial DNA was identified using real-time PCR. Cotton swab provided the highest concentration of DNA and it was selected as a standard mode to collect samples. A total of 4 mice were used for sampling. Concentration range of isolated DNA before antibiotics treatment, after antibiotics treatment and after infections was, respectively, 18.2ng/ul – 22.2ng/ul; 10ng/ul – 19.1ng/ul, and 9.2ng/ul–18.9ng/ul. Baseline samples were then investigated for 4 different groups of bacterial DNA amplified by PCR, and it observed the presence of Firmicutes, g-Proteobacteria and Bacillus, besides the Universal Bacteria counts. In conclusion, the preliminary results suggested that cotton swab was the best apparatus for sampling, and it was possible to identify commensal bacteria using this technique. Analyses of samples after antibiotics treatment and after Pg infection are necessary to confirm the effectiveness of the infection protocol.
Funder Acknowledgement(s): Student Training and Research Program, Augusta University; Peach State Louis Stokes Alliance for Minority Participation (PSLSAMP), Savannah State University
Faculty Advisor: Roger Arce, RARCEMUNOZ@augusta.edu
Role: Adequately manipulated and handled mice for experimentation. Successfully isolated bacterial DNA from the mouth of orally-infected mice. Followed through with the procedures and protocol of the Qiagen DNA investigator forensic kit after swabbing mice for total DNA. Identified bacterial genotypes by means of real time PCR reactions.