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SPME-GC/MS Analysis of Volatile Organic Compounds Produced by Oral Cavity Streptococci

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SPME-GC/MS Analysis of Volatile Organic Compounds Produced by Oral Cavity Streptococci SPME-GC/MS Analysis of Volatile Organic Compounds Produced by Oral Cavity Streptococci by Amanda Dell’Olio B.S. in Chemistry, Northeastern University A thesis submitted to The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Master of Science December 5, 2017 Thesis directed by Dr. Adam B. Hall Director, Core Mass Spectrometry Facility Barnett Institute of Chemical and Biological Analysis 1 Acknowledgements I would like to thank Dr. Adam Hall, my principal investigator, for his insight, expertise, and assistance with the project. I’d particularly like to thank him for assistance with and troubleshooting of the GC/MS and for his reviews of my thesis. I’d also like to thank Violetta Medik and her advisor, Dr. Slava Epstein, for partnering with me on this project and allowing me to undertake the analytical chemistry side of the project while they handled the microbiology side of it. I am grateful to Violetta for teaching me the SPME-GC/MS method, for providing me with bacterial samples to test, and for providing me with some figures to use in my thesis. Thank you to Dr. Adam Hall, Dr. Slava Epstein, and Dr. Paul Vouros for serving on my thesis committee and taking the time to review my thesis. Thank you as well to my other colleagues in the Northeastern University community for giving me the tools and education to complete this thesis. 2 Abstract of Thesis According to the Human Oral Microbiome Project, dental plaque consists of about 700 different species of bacteria.1 Dental plaque is the reason that the average American adult has between ten and seventeen decayed, missing, or filled permanent teeth and most of the population suffers from at least a minor case of gingivitis.1 Metagenomics research has provided useful information about dental plaque composition in various stages of health and disease, but it is unclear what ecological forces cause the establishment and composition of the microbiome. It is known that dental plaque formation is affected by interactions and competitive or mutualistic relationships between different strains of bacteria. This interaction can be driven by the production of volatile organic compounds (VOCs). The ecological roles of these compounds in the headspace was previously overlooked, but it is known that interkingdom interactions and bacterial behavior are affected by bacterial VOCs.2 SPME-GC/MS was used in a series of experiments to determine the VOC profiles of several strains of oral Streptococci to gain insight about what role this may play in the establishment of healthy oral flora. Every strain of bacteria appeared to have a different VOC profile associated with it, but analysis with PCA plots did not reveal any distinct differences that clearly separated strains. It was observed that culturing on agar versus in broth affected the VOC profiles although there were other factors that differed in these experiments such as temperature, so it cannot be confirmed that culture medium alone changes the VOC profile. Co-cultured samples were compared to individual strain samples, and although unique compounds were rarely produced in co-culture compared to the individual strains, some compounds, such as 1-butanol, were often overexpressed in the co-cultures suggesting they may play a role in competitive or synergistic relationships. Time course experiments revealed that the VOC profiles change over time, even just over the course of a day, 3 but there isn’t always a trend in the abundances of antibacterial compounds that could affect bacterial growth. For instance, no trend was observed when looking at acetic acid levels whereas 1-butanol levels seemed to generally increase over time. Still, it is difficult to confirm VOC profiles and abundance levels of all compounds. Hundreds of compounds were identified using the NIST library associated with the ChemStation software, but these compounds often appear to be misidentified. In the VOC profiles of supposedly identical samples analyzed on different days and of triplicate samples analyzed concurrently, differences are seen in the ChemStation NIST library identification of a given peak at the same retention time in the replicate samples. The data looks slightly more similar when viewed using PCA and cluster plots to visualize the results, but there are still some inconsistencies. Using a Kruskal-Wallis test to statistically analyze chromatograms of replicate samples gives a P value that indicates the results are not statistically different, providing validity to the method. Still, to confirm any VOCs of interest as being major contributors to VOC profiles or to the development of healthy oral flora, standards must be analyzed using the same SPME-GC/MS method and the resulting mass spectra must be compared to known mass spectra. No VOCs have been confirmed yet as being key antibacterial compounds, but acetic acid, 1-butanol, and dimethylamine all have antibacterial properties and show varying levels of abundance in the oral Streptococci samples that were analyzed. The following results, while comprehensive, represent a preliminary approach for which further studies should clarify and expand upon these findings. 4 Table of Contents Acknowledgements ......................................................................................................................... 2 Abstract of Thesis ........................................................................................................................... 3 Table of Contents ............................................................................................................................ 5 List of Figures ................................................................................................................................. 7 List of Tables .................................................................................................................................. 9 List of Symbols & Abbreviations ................................................................................................. 10 1. Introduction ........................................................................................................................... 11 2. Background ............................................................................................................................ 12 2.1. Dental Plaque Formation ................................................................................................... 12 2.2. Streptococci........................................................................................................................ 13 2.3. Bacterial VOCs .................................................................................................................. 14 2.4. Solid-Phase Microextraction (SPME)................................................................................ 15 2.5. Gas Chromatography (GC) ................................................................................................ 17 2.6. Mass Spectrometry (MS) ................................................................................................... 18 3. Materials and Methods .......................................................................................................... 19 3.1. Bacterial Samples............................................................................................................... 19 3.2. SPME Procedure ................................................................................................................ 19 3.3. GC/MS Analysis ................................................................................................................ 19 4. Results and Discussion .......................................................................................................... 20 5 4.1. VOC Identification and VOCs of Interest ......................................................................... 20 4.2. Broth versus Agar .............................................................................................................. 26 4.3. Co-culturing ....................................................................................................................... 34 4.4. Time Course Experiments.................................................................................................. 39 4.4.1. 10 Day Time Course of Single Strains and Co-cultures ................................................ 39 4.4.2. 27 Hour Time Course of SA .......................................................................................... 44 4.5. Assessment of Reproducibility .......................................................................................... 47 4.5.1. Day to Day Reproducibility ........................................................................................... 49 4.5.2. 3 Day Time Course Analysis of SR4 and CM7 in Triplicate......................................... 52 4.6. Chemometrics .................................................................................................................... 58 5. Conclusions ........................................................................................................................... 62 6. Future Directions ................................................................................................................... 64 References ..................................................................................................................................... 66 Appendices
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