Maternal Diet Habits and the Salivary Microbiome of Caries-Free Children THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Dr. Stephanie Chambers Furlong Graduate Program in Dentistry The Ohio State University 2013 Master's Examination Committee: Dr. Sarat Thikkurissy “Advisor” Dr. Purnima Kumar Dr. Homa Amini Copyright by Dr. Stephanie Chambers Furlong 2013 Abstract This cross-sectional clinical study examines maternal diet habits and child feeding practices in relation to the mother-child bacterial makeup. Mother-child dyads of caries- free children in four age cohorts between 0-18 years were included in this study. Mothers answered a 65-question survey on their own eating habits as well as child feeding and oral hygiene practices. Children and mothers also provided a saliva and plaque sample for analysis of microbial colonies. A total of sixty mother-child pairs were identified and included in the study. Of the 60 pairs, 11 were predentate infants, 20 had only primary teeth, 14 were in the mixed dentition state, and 15 had all permanent teeth. All but two diet variables showed no statistical difference between the mothers in each group at a level of significance of p<0.05. ANOVA analysis of the average s-OTU count showed the predentate group had a significantly lower bacterial diversity than the other groups (p<0.05). ANOVA analysis of the Bray-Curtis Similarity Index of the mother/child dyads showed no statistically significant difference between the groups (p<0.05). On average, this similarity index showed that each child shared on average about 50% of their salivary microbial profile with their mother. These differences are attributed to the different stages of dentition development as well as the impact of both vertical and horizontal transmission. ii Dedication This document is dedicated to my husband, Mark and to my Dad, Mom, sister, and brother who supported me through all the years. iii Acknowledgments I would like to thank all those who have helped me in the past two years with this thesis project. It was a long and arduous process but I could not have gotten through it without their support. First, I would like to express my gratitude to Dr. Sarat Thikkurissy for being my thesis advisor. He not only assisted in the development and completion of this project, but helped remind me to maintain a balance between my academic and personal responsibilities. In addition, I would like to thank Dr. Purnima Kumar for her constant positivity and encouragement along the way. She was a great colleague and I am thankful for the relationship we developed throughout this process. Matthew Mason was also an integral part of this project. He performed most of the bacterial analysis and took the time out of his extremely busy schedule to be sure I understood everything. I am grateful for the understanding of Dr. Homa Amini who helped coordinate my schedule to ensure I had the time and opportunity to work on this project and also for her input and support along the way. I would also like to extend a special thanks to the hygienists at Nationwide Children’s Hospital who took the time to help identify potential study subjects and ensure I obtained all the information I needed. “You can do anything, but you can’t do everything” and therefore I would like to thank everyone for their collaboration to complete this project. iv Vita 2002................................................................TC Roberson High School 2006................................................................B.S. Biology, University of North Carolina 2011................................................................D.D.S., University of North Carolina 2013................................................................Pediatric Dentistry, The Ohio State University Fields of Study Major Field: Dentistry v Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v Table of Contents ............................................................................................................... vi List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Introduction ......................................................................................................................... 1 Materials and Methods ........................................................................................................ 8 Results ............................................................................................................................... 14 Discussion ......................................................................................................................... 28 Summary and Conclusions ............................................................................................... 36 References ......................................................................................................................... 37 vi List of Tables Table 1: Study Inclusion and Exclusion Criteria ................................................................ 9 Table 2: Child Demographic Data .................................................................................... 14 Table 3: Mother Demographic Data ................................................................................. 16 Table 4: Child Brushing Status/Frequency and Plaque Score (p=0.464) ......................... 17 Table 5: Maternal Diet Analysis Data and Correlated p-Values ...................................... 19 Table 6: Statistically Significant Maternal Diet Variable 1 (p=0.050) ............................. 19 Table 7: Statistically Significant Maternal Diet Variable 2 (p=0.050) ............................. 20 Table 8: Total s-OTU’s by Group ..................................................................................... 22 vii List of Figures Figure 1: Bacterial Taxa................................................................................................... 21 Figure 2: Average Number of s-OTU’s by Group (p<0.0001) ........................................ 24 Figure 3: Shannon Diversity Index (p<0.001) ................................................................. 25 Figure 4: Bray-Curtis Similarity Index ............................................................................ 26 Figure 5: Phylogenetic Tree ............................................................................................. 27 viii Introduction Bacteria are essential to human life. They are vital to many basic human functions such as preventing disease, assisting in nutrition acquisition, training our immune systems, and many others. Thousands of bacteria inhabit the human body. These bacteria exist in both a pathologic as well as symbiotic function. The network of bacteria is often specific to the anatomic location. In the oral cavity, this collection of bacteria have been termed the oral microbiome 1. The investigators for the human microbiome project have adopted the term microbiome that was coined by Joshua Lederberg in 2001 1-2. Lederberg defined microbiome as “the ecological community of commensal, symbiotic, and pathogenic microorganisms that share our body space.” 1-2 The oral microbiome has also been referred to as the oral microflora or oral microbiota. However, in this study, we will refer to the collective bacterial community in the oral cavity as the oral microbiome. Bacteria in the oral cavity form an intricate symbiosis which means both the bacteria and the host mutually benefit by interacting and living together. The multi- species bacterial community that makes up the oral microbiome is dynamic and complex. In order to appreciate the symbiotic relationship of the human oral microbiome, it is necessary to understand how it develops, how innate and external factors affect it, and how it may change over time and location3. In addition, it is essential to define and 1 identify the healthy state of the oral microbiome in order to determine its role in the predisposition or prevention against disease. The development of the oral microbiome is a complex and dynamic multifactorial process. To date, there are more than five hundred different bacterial species that are known to reside in the oral cavity4. Estimates suggest this represents only about half of the actual number of bacteria present. However, in many cases humans are not born with these bacteria inherently residing within our oral cavity. Most infants, except those with chorioamnionitis - a condition where the fetal membranes become inflamed due to bacterial infiltration during a prolonged labor, are born with a sterile gastrointestinal tract5-6. It is not until birth that bacterial colonization in humans occurs. As an infant emerges through the birth canal or immediately after delivery when exposed