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The Reconstruction and Analysis of Oral Microbiome Composition Using Dental Calculus from the Mississippi State Asylum (1855-1935), Jackson, Ms

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The Reconstruction and Analysis of Oral Microbiome Composition Using Dental Calculus from the Mississippi State Asylum (1855-1935), Jackson, Ms Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2016 The Reconstruction and Analysis of Oral Microbiome Composition Using Dental Calculus from the Mississippi State Asylum (1855-1935), Jackson, Ms Jonathan Robert Belanich Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Belanich, Jonathan Robert, "The Reconstruction and Analysis of Oral Microbiome Composition Using Dental Calculus from the Mississippi State Asylum (1855-1935), Jackson, Ms" (2016). Theses and Dissertations. 4599. https://scholarsjunction.msstate.edu/td/4599 This Graduate Thesis - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Template B v3.0 (beta): Created by J. Nail 06/2015 The reconstruction and analysis of oral microbiome composition using dental calculus from the Mississippi State Asylum (1855-1935), Jackson, MS By TITLE PAGE Jonathan Robert Belanich A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Arts in Applied Anthropology in the Department of Anthropology and Middle Eastern Cultures Mississippi State, Mississippi August 2016 Copyright by COPYRIGHT PAGE Jonathan Robert Belanich 2016 The reconstruction and analysis of oral microbiome composition using dental calculus from the Mississippi State Asylum (1855-1935), Jackson, MS By APPROVAL PAGE Jonathan Robert Belanich Approved: ____________________________________ Molly K. Zuckerman (Major Professor) ____________________________________ Heather Jordan (Committee Member) ____________________________________ Darcy Shane Miller (Committee Member) ____________________________________ Nicholas P. Herrmann (Committee Member) ____________________________________ David M. Hoffman (Graduate Coordinator) ____________________________________ Rick Travis Interim Dean College of Arts & Sciences Name: Jonathan Robert Belanich ABSTRACT Date of Degree: August 12, 2016 Institution: Mississippi State University Major Field: Applied Anthropology Major Professor: Dr. Molly K. Zuckerman Title of Study: The reconstruction and analysis of oral microbiome composition using dental calculus from the Mississippi State Asylum (1855-1935), Jackson, MS Pages in Study: 82 Candidate for Degree of Master of Arts The human oral microbiome is the total amount of microbial biodiversity present in the oral cavity and, given its relevance to human health and disease, has recently become a foci for study. By analyzing dental calculus, and sequencing the bacterial DNA, it is possible to reconstruct and examine the oral microbiomes of past individuals. In this study, dental calculus was sampled from (N=4) skeletons recovered from the cemetery of the mid 19th- 20th, century Mississippi State Asylum in Jackson, MS. Bacterial DNA isolation and shotgun sequencing were successful, with 16S analyses yielding an average of 96 identified species. All samples were significantly different from each other at all taxonomic levels (p <0.0001). Targeted examinations for opportunistically pathogenic oral bacteria were performed, but no detectable bacterial DNA was found in the samples. This study is the first to reconstruct the oral microbiomes of a subsample of an historic institutionalized population. DEDICATION This is dedicated to my fountain pens. It is additionally dedicated to my parents, John and Donna Belanich, who were incredibly supportive throughout my undergraduate years, and remain so during my ever-increasing graduate career. ii ACKNOWLEDGEMENTS The author would like to specifically acknowledge Drs. Molly Zuckerman (Department of Anthropology and Middle Eastern Cultures (AMEC)) and Heather Jordan (Department of Biological Sciences) for the edits of previous drafts of the thesis proposal, and thesis, as well as Drs. Nicholas Herrmann (Texas State University) and Shane Miller (AMEC), the additional members of this thesis committee. Thanks are also extended to Dr. Jason Rosch (Infectious Disease Department, St. Jude) for the sequencing of the DNA samples. Additional thanks to Amanda Lawrence (Institute for Imaging & Analytical Technologies) and Dr. Gerald Baker (Department of Biochemistry, Molecular Biology, Entomology, & Plant Pathology) for their assistance with the Scanning Electron Microscopy and imaging, and Dr. Christopher Lynn and the HBERG writing group at The University of Alabama for the comments on previous drafts of this thesis. Additionally, Jesseca A. Paulsen is thanked for her support during the process of writing and revising. iii TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER I. INTRODUCTION ................................................................................................1 Research Questions ...............................................................................................4 Significance of Research .......................................................................................5 II. BACKGROUND INFORMATION .....................................................................7 The Human Microbiome .......................................................................................8 Bacteria and the Oral Microbiome ..................................................................8 Biofilms .........................................................................................................10 Dental Calculus Formation ............................................................................11 Ancient microbial DNA ................................................................................11 History of the MSA .............................................................................................13 Individual Burials ..........................................................................................14 The Human Oral Microbiome Database ..............................................................15 III. MATERIALS AND METHODS ........................................................................16 Hypotheses ..........................................................................................................16 Hypothesis 1: .................................................................................................16 Hypothesis 2: .................................................................................................17 Hypothesis 3: .................................................................................................17 Hypothesis 4: .................................................................................................18 Hypothesis 5: .................................................................................................18 Sampling methods ...............................................................................................19 Scanning Electron Microscopy Sampling ...........................................................20 Microbial DNA Preparation ................................................................................21 DNA Isolation Kits ........................................................................................21 Targeted Polymerase Chain Reaction and gel electrophoresis ......................22 iv Metagenomic Sequencing .............................................................................23 Illumina BaseSpace Analysis ........................................................................23 Scanning Electron Microscopy ............................................................................23 Human Oral Microbiome Database Biodiversity ................................................24 Statistical Analyses ..............................................................................................25 IV. RESULTS ...........................................................................................................26 DNA Isolation Results .........................................................................................26 16S Metagenomic Analysis .................................................................................27 Burials ...........................................................................................................28 Negative Control ...........................................................................................28 Scanning Electron Microscopy ............................................................................29 Chi-Square Test of Independence .......................................................................34 Microbes in Burials .............................................................................................35 Specific Pathogenic Bacteria .........................................................................35 Targeted Polymerase Chain Reaction ...........................................................35
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