Maternal Diet Habits and the Salivary Microbiome of Caries-Free Children THESIS Presented in Partial Fulfillment of the Requirem

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

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

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.

Dedication

This document is dedicated to my husband, Mark and to my Dad, Mom, sister, and

brother who supported me through all the years.

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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.

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

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

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

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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

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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 multispecies 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 to the environment, the oral cavity and rest of the gastrointestinal tract is colonized, usually within 24 hours of birth5, 7. After colonization, the number of oral bacteria increases with exposure to external sources from the environment. However, the rate and extent of colonization of the gastrointestinal tract after birth can be affected by multiple factors such as cesarean delivery, type of feeding delivery, or administration of medicines such as antibiotics 7-8.

Bacteria adhere to mucosal and other surfaces of the gastrointestinal tract so they are not sloughed away by mechanical forces or the movement of saliva. Bacteria adhere to surfaces via adhesins and receptors. Adhesins are adhesive structures on the surfaces of microorganisms formed of adhesive appendages called fimbriae9-10. Receptors are complementary adhesive structures on the surfaces of host cells that adhesins of bacteria

2 use to attach to9-10. Certain bacteria will only attach to certain types of surfaces. As predentate infants, only soft tissue surfaces are available for bacteria to attach, unless natal or neonatal teeth are present. The first and most abundant species that colonize the soft tissue in the oral cavity of newborns have been found to be Streptococcus salivarius,

Streptococcus mitis, and Streptococcus oralis11. However, as a child ages and teeth erupt, different habitats are present for a more diverse group of bacteria to colonize and therefore a shift in the presence of bacterial species occurs.

The presence of teeth increases the diversity of the possible host sites and thus increases the complexity of bacterial composition in the oral cavity in many ways. There is a significant change in bacterial presence from a predentate state where there is a sloughing soft tissue surface to a non-shedding hard tissue surface once teeth erupt.

When a tooth erupts, the embryologic cystic covering is lost and it is replaced by a bacteria-free, protein based acellular membrane called the enamel pellicle 12-13. This membrane can be disrupted or removed by mechanical cleansing but has been shown to start reforming on the surface of teeth within minutes 12, 14. This enamel pellicle has several functions, with the most notable of the two being the chemical and mechanical protection of tooth enamel as well as the determination of the initial attachment of bacteria to the tooth surface12. Once bacteria attach to the pellicle, a complex aggregate of microorganisms adhere to each other on the surface of the tooth15. The intricate biofilm that develops express behavior very differently from free-floating bacteria and can vary dramatically based on the location and maturity of the biofilm3, 15. Studies show the composition of bacterial communities is determined primarily on the body location or

“habitat” and that although the bacteria in these different locations may serve the same purpose they can vary from person to person3. As the biofilm on a tooth matures and what is known as “plaque” begins to accumulate, it has been found that there is a shift from gram-positive and streptococcus-rich bacteria to gram-negative anaerobic organisms3. It is the bacteria in these biofilms that are the main microbiological agents of tooth decay. The number, type, and interplay between these bacteria determine their virulence and ability to cause dental disease.

While the composition of oral bacteria is multifactorial and can be affected by these changing attachment sites, oral bacteria is transmissible and which bacteria colonize the oral cavity has a lot to do with the number and frequency of inoculations that occur.

Certain bacteria, such as mutans streptococci have been found to be transmitted via vertical transmission from mother to child16-19. In seventeen reports, vertical transmission of Streptococcus mutans from mother to their infants has been documented with identical genotypes of bacteria noted in 24-100% of the pairs20. This is an important factor to consider when assessing caries susceptibility if highly virulent bacteria linked to dental disease are transmitted. The number and type of bacteria in a mother’s mouth can directly affect the composition of a child’s oral flora and thus affect their susceptibility to dental decay. Horizontal transmission between siblings or children in a daycare center have also been shown as a source of transmission of oral bacteria21-22. A longitudinal study by Stahringer et al of bacterial communities in saliva of twins from early adolescence to early adulthood found that although the bacterial composition of twins saliva resemble each other more closely than the whole population at any time point, they

4 become less similar to each other with time and when no longer cohabitating16. This highlights the fact that the composition of one’s oral bacteria is highly influenced on environmental factors and exposure to different sources of bacterial origin. This is an important factor to take into account when assessing caries risk factors.

Dental caries is one of the most prevalent preventable diseases in children ages 5-

17 years old according to a report by the United States Surgeon General in 200023. An

NHANES review found that more than 80 percent of the pediatric population is affected by dental caries by age 1724-25. Dental decay in children less than five years old is defined as early childhood caries (ECC) 26. As noted by Savage et al (2004) almost 19% of children 24 to 40 months of age have ECC and the prevalence of ECC has even been reported to be as high as 90% in children from low socioeconomic backgrounds27.

Studies have highlighted the consequences of dental caries reach far beyond decayed teeth, noting that children that experience decay have been shown to miss more school and have decreased body weight27. In addition, the economic burden of dental caries is substantial. The Centers for Medicare and Medicaid Services have estimated the child dental services expenditures in the US is expected to reach almost 49.5 billion dollars by

2017.

The multi-species bacterial community of the oral cavity is dynamic and complex and many of the bacteria are not culturable which can limit identification and analysis.

About 280 bacterial species from the oral cavity have been identified through culture although more sophisticated genetic studies such as amplified 16S rRNA analysis have identified over 500 different taxa1, 28. It is estimated only about half of the bacterial

5 species in the oral cavity can be cultivated, whereas newer cultivation-independent methods such as 16s rRNA gene-based cloning studies have shown that there are up to

600 or 700 species1, 29. New molecular techniques such as pyrosequencing can allow the microbial makeup of oral flora to be better identified30. Pyrosequencing relies on detection of pyrophosphate release with the reaction taking place with a single strand of

DNA. It can be used to refine the knowledge of the makeup of the oral cavity by providing a large number of sequence reads in a single run resulting in a large sampling depth and allowing detection of both dominant and rare bacteria31. Pyrosequencing will be used in this study to analyze saliva samples from both children and their mothers.

The makeup of oral microflora can be influenced by both bacterial factors such as transmission frequency or bacterial virulence and host factors such as age, diet, and oral hygiene32. Caries is a complex and multifactorial disease as illustrated by the conceptual model designed by Fisher-Owens et al (2007)32. One of the three core oral health factors in Fisher’s conceptual model is diet32. Infants and young children do not have the ability to make dietary decisions and therefore must eat and drink what their parents provide for them. Thus, these dietary choices can have a direct impact on multiple health factors, including dental disease, in their children. High-risk dietary practices appear to be established early, by around 12 months old, and are maintained throughout early childhood33-34. Obtaining a perspective on how a mother’s diet affects oral flora and the development of dental disease has implications for more accurate caries risk analysis and more effective preventive and therapeutic measures for dental disease. The objective of

6 this research study is to examine maternal diet habits and child feeding practice in its relation to the salivary microbiome of mother-child dyads in different dentition states.

Materials and Methods

This cross-sectional clinical study involved obtaining samples as well as surveying study participants. Subjects for the study were recruited from the dental clinic at Nationwide Children’s Hospital as well as from one Close to Home primary care site affiliated with Nationwide Children’s Hospital. The patients were identified by the study investigator when they presented to either of the two clinics for a routine check-up. The goal was to identify, sample, and survey 60 pairs of children and their biological mothers.

Inclusion criteria for study participants included English-speaking parents that had children with no chronic medical conditions. The study included children ages 0 to

18 years old that were predentate or had primary, mixed, or permanent dentition with no history of decay and were currently caries-free. Exclusion criteria included non-English speaking parents or patients to facilitate answering questions necessary to complete the survey to maintain consistency in the case of the absence of an interpreter. Children with significant chronic medical conditions and those requiring regular medications for chronic conditions were also excluded due to the possible impact on bacterial composition obtained in samples. Children with a history of decay or current active decay were excluded because the presence of different bacteria can occur based on the status of the enamel of the teeth sampled, depending on the extent of decay for example in areas of demineralization versus dentinal decay. We did not exclude a mother-child

8 pair if the mother had a history of caries or currently had dental decay. Caries status of the child was determined by the standardized study investigator through a clinical and if applicable radiographic exam during a routine check-up appointment. A summary of inclusion and exclusion criteria are summarized in Table 1.

Inclusion Criteria Exclusion Criteria Biological mother-child pair Non-English speaking Child with no Children with significant chronic medical conditions chronic medical conditions Child with no history/current decay Child with history/active decay Children 0-18yo Children >18yo Table 1: Study Inclusion and Exclusion Criteria

Once a mother-child pair that fit the inclusion criteria was identified, consent and if applicable assent was obtained for participation in this non-incentive clinical study.

The study investigator documented the number and type of teeth present and assigned a plaque score to each child (not applicable in predentate infants) based on the zero to three scale developed by Silness and Loe. Then, three different samples were obtained from both the child and their mother. A saliva sample was obtained by using a single toothette brush placed underneath the tongue of the child and mother. After 30 seconds of soaking up saliva the toothette was then placed back in its original packaging or in a test tube for storage. A supragingival plaque sample was then obtained from the facial surface of anterior teeth by swiping a small bond brush along the teeth of the child and mother and then placed in a sealed small test tube. A subgingival plaque sample was also obtained by placing several paper points in the sulcus of anterior teeth of the child and mother and 9 then stored in a small sealed test tube. All three of the child and mother samples were stored in a separate larger sealed bag and then placed in a -200C degree freezer until DNA isolation and pyrosequencing analysis. A single standardized study investigator obtained the samples and stored them to maintain consistency throughout the study. All samples were collected prior to running samples so they could all be run at one time. For the purposes of this study, the saliva samples were the only ones analyzed with reported results while the supragingival and subgingival plaque samples were banked for future analysis.

DNA isolation

DNA was isolated from each sample by placing the paper points in a 1.5mL sterilized collection tube with 200μL of PBS then agitated for 20-30 minutes. The paper points were then removed and placed into a 0.5mL collection tube with a hole punctured in the base and this tube was returned to the 1.5mL tube to be centrifuged for 3 minutes to allow the fluid from the paper points to return to the PBS solution and the paper points were saved in -80°C. 180μL of Buffer ATL and 40μL of Proteinase K were added to the

PBS/Bacteria solution, and agitated for 15 seconds to mix the solutions. The collection tube was then incubated at 56oC in a hot water bath for at least 2 hours. Following incubation, 200μL of Buffer AL was added to the collection tube, agitated for 15 seconds, and incubated at 70oC in a dry bath for 10 minutes.

Once this was completed, 200μL of 100% Ethanol was added to the tube, agitated for 15 seconds, the solution was then transferred to a QIAamp Spin Column nested inside of a 2.0mL collection tube and centrifuged for 1 minute. The filtrate was then discarded

10 and 500μL of Buffer AW1 was placed into the spin column and centrifuged for 1 minute, filtrate was discarded, 500μL of Buffer AW2 was added to the spin column and centrifuged for 3 minutes. Once completed the collection tube was discarded, and the spin column was placed in a new sterile 2.0mL collection tube. 50μL of Buffer EB/AE was added to the column, incubated at room temperature for 5 minutes and centrifuged for 1 minute, the sample was then moved to a sterile 0.2mL collection tube.

Pyrosequencing

Multiplexed bacterial tag-encoded FLX amplicon pyrosequencing was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN, USA) as previously described by Dowd et al. 2008 in a commercial facility (Research and Testing

Laboratories, Lubbock, TX, USA)35. Briefly, a single step PCR with broad-range universal primers and 22 cycles of amplification was used to amplify the 16S rRNA genes as well as to introduce adaptor sequences and sample-specific bar-code oligonucleotide tags into the DNA. Two regions of the 16S rRNA genes were sequenced: V1–V3 and V7–V9. The primers used for sequencing have been previously described by Kumar et al 201136. Adaptor sequences were trimmed from raw data with

98% or more of bases demonstrating a quality control of 30 and sequences binned into individual sample collections based on bar-code sequence tags, which were then trimmed. The resulting files were denoised with Pyronoise and depleted of chimeras using B2C237. Sequences <500 basepairs were discarded and the rest were clustered into species-level operational taxonomic units (s-OTUs) at 97% sequence similarity and assigned a taxonomic identity by alignment to locally hosted version of the Greengenes

11 database using the Blastn algorithm38. Phylogenetic trees were generated and visualized using FastTree39. Unifrac and community diversity metrics were computed as previously described by Lozupone et al. 200740. All analysis were conducted using the QIIME

(quantitative insights into microbial ecology) pipeline41.

Questionnaire

For the questionnaire, a pre-designed survey was used as the basis for the dietary assessment portion of the study. The survey was originally designed for the MOMs

(Making Meals Special) study in the Ohio State University College of Nursing. This survey was used for childhood obesity studies and oral microflora studies. This questionnaire was edited and condensed based on feedback from a pilot study using the questionnaire. The survey consisted of patient identifiers including inclusion and exclusion criteria information. There were six sections to the questionnaire including history and demographics, nutritional intake, eating habits, meal planning, emotions and eating, and child feeding practices.

Other than the last section involving child feeding practices, the questions were aimed at obtaining information on the mother’s dietary habits and opinions. For specifics on nutritional intake and eating habits, the number of times a day or times a week were asked to be identified with no limitation on value. For specifics on the mother’s opinions about meal planning and emotions and eating the Likert scale ranging from strongly disagree to strongly agree was utilized. Several yes/no questions or circle an option questions were also used in some of the sections. Mothers completing the survey were

12 able to select or write-in not applicable for certain questions as well. The information from the questionnaires was compiled into a database designed in Excel and analyzed.

Results

A total of sixty mother-child pairs were identified and included in the study. One- hundred percent of the sixty children were caries free with no clinically or radiographic active carious lesions or past history of treatment for carious lesions. The one hundred and twenty subjects were divided into four groups based on the dentition status of the child sampled. These four groups included predentate infants or children with primary, mixed, or permanent teeth. 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. The ages of the child subjects ranged from 6 days to 17 years, 1 month, 16 days (with an average age of 6 years and 8 months). There were a total of 34 males and 26 females sampled with a fairly even distribution of males to females in each group (57% males to

43% females). Refer to Table 2 for demographic data for the child subjects.

Group # Subjects #Male/#Female Age Range Average Age Predentate 11 7/4 6days - 1yr9mo6days 4mo22days 11mo22days - Primary 20 11/9 5yr5mo9days 3yr1mo 5yr6mo26days - Mixed 14 8/6 12yr1mo30days 8yr9mo 11yr0mo12days - Permanent 15 8/7 17yr1mo16days 14yrs 6days - Total 60 34/26 17yr1mo16days 6yr8mo Table 2: Child Demographic Data

Every mother sampled for each of the sixty caries-free children was the biological mother of the child. Over half of the mothers were African American (53%) with

Caucasians (32%) and Hispanics (13%) the next most common. The ethnicities of the children sampled were identical to the mother’s and should be considered the same for demographic purposes. Although the children included in the study were caries-free with no history of caries, the mothers’ caries status was not an inclusion/exclusion criteria.

The current caries status of the mothers was documented as a self-report. Of the sixty mothers, 52% reported having no active caries and 48% stated they had current active caries. Of those 31 mothers that did not have active caries, 98% reported having had a restoration or other treatment for caries in the past. Most of the mothers were non- smokers with 44 out of the 60 (73%) being a current non-smoker. Only 16 of the mothers were current smokers. A total of 26 mothers (43%) had some high school education, received their high school diploma, or obtained their general equivalency diploma.

Almost half of the mothers had some college or their college degree (47%) with four attending graduate school. Most of the mothers were either married living with the child’s father (35%) or single (35%) living alone or co-habitating with their partner

(16%). A large percent of the mothers had other children that co-habitated with the child that was included in the study with almost 68% of the mothers reporting co-habitating children. Many of the mother’s sampled did not report their age, so it is not identified in this study. Refer to Table 3 for demographic data for the mothers included in this study.

Caries Status Education No Caries 31 52% Some College/Degree 28 47% Active Caries 29 48% Highschool/Diploma 26 43% Smoking status Graduate School 4 7% Non-Smokers 44 73% Did not report 2 3% Smokers 16 27% Relationship Status Ethnicity Married 21 35% African American 32 53% Single 21 35% Caucasian 19 32% Live with Partner 10 16% Hispanic 8 13% Divorced 5 8% Asian 1 2% Engaged 1 2% Co-habitating Children 41 68% Widowed 1 2% Co-habitating Grandparents 8 13% Separated 1 2% Table 3: Mother Demographic Data

Brushing status and frequency of each child was reported by their mothers. Only one mother in the predentate groups reported rag wiping after feeding every day while the other ten reported no oral care at this time. In the primary dentition group, a majority of mothers reported both the child and the parent brushing the child’s teeth (70%) every day. This is a contrast to the mixed and permanent dentitions where the mothers reported the child being the sole brusher 64% and 93% respectively. In mixed and permanent dentitions, there was also an increased incidence of mothers reporting the brushing frequency as only a few times a week at 14% and 7% respectively versus the primary dentition where the mothers reported 100% of the time brushing frequency was every day. Plaque scores were documented for each child at the time of sampling for the three dentate groups (predentate infants were excluded since they have no teeth). The plaque score ranged from 0 to 3. In each of the three dentate groups, the majority of children

16 had a plaque score of 1 with 60% in the primary dentition group, 64% in the mixed dentition group, and 53% in the permanent dentition group. A chi-squared analysis of the plaque scores in each dentition group showed no significant difference between the three groups with a p value of 0.464. A summary of the brushing status, frequency, and plaque scores for the four groups are found in Table 4.

Brushing Status Brushing Frequency Plaque Score Predentate Predentate Predentate N/A N/A Rag wiping 1 9% Every day 1 9% Primary Primary Primary PI = 0 2 10% Both 14 70% Every day 20 100% PI = 1 12 60% Adult only 4 20% Few times a week 0 0% PI = 2 3 15% Child only 2 10% Mixed PI = 3 3 15% Mixed Every day 12 86% Mixed Both 5 36% Few times a week 2 14% PI = 0 0 0% Adult only 0 0% Permanent PI = 1 9 64% Child only 9 64% Every day 14 93% PI = 2 3 21% Permanent Few times a week 1 7% PI = 3 2 14% Both 1 7% Permanent Adult only 0 0% PI = 0 1 7% Child only 14 93% PI = 1 8 53% PI = 2 6 40% PI = 3 0 0% Table 4: Child Brushing Status/Frequency and Plaque Score (p=0.464)

Significant dietary variables identified in the survey were examined using a chi- squared analysis to determine significance between the mothers in each dentition group.

For example, 97% of mothers drank water more than four times a week and only 45% drank soda. The majority of mothers also ate fruit and vegetables (62% and 80% respectively) more than four times a week instead of carbohydrate based foods such as potatoes, pasta or rice (17% and 32% respectively). All but two variables showed no

17 statistical difference between the mothers in each group at a level of significance of p<0.05. See Table 5 for specific maternal diet analysis, correlated p-values, and the percentage distribution for mother’s responses. Specifically, predentate children’s mothers reported a statistically significantly higher response to skipping meals than the other groups (see Table 6, p=0.05). In addition, predentate mothers reported a statistically significantly higher response to snacking instead of eating a meal (see Table

7, p=0.05).

Maternal Diet Analysis Survey Question p Value ≥4x/wk ≤3x/wk How often do you drink fruit juices 0.476 48% 52% Not counting juice, how often do you eat fruit 0.278 62% 38% How often do you eat green salad 0.990 42% 58% How often do you eat potatoes 0.540 17% 83% How often do you eat vegetables 0.875 80% 20% How often do you eat dairy products 0.245 83% 17% How often do you eat pasta or rice 0.828 32% 68% How often do you eat protein or meat 0.957 83% 17% How often do you drink soda ? 45% 55% How often do you drink milk ? 70% 30% How often do you drink water ? 97% 3% How often do you eat breakfast 0.261 68% 32% How often do you eat food while driving 0.584 8% 92% How often do eat food together with your family 0.457 82% 18% *How often do you skip meals 0.050 27% 73% *How often do you snack instead of eating a meal 0.050 32% 68% How often do you eat snack while watching television 0.080 43% 57% Agree Disagree Neutral My emotions affect what and how much I eat 0.332 35% 45% 20% When I am upset, I eat for comfort 0.140 13% 65% 22% When I am in a bad mood, I eat more and what I want 0.571 15% 72% 13% When I am upset or stressed, I stop or cannot eat 0.281 30% 50% 20% When I am bored, I snack more 0.858 33% 42% 25% Table 5: Maternal Diet Analysis Data and Correlated p-Values

How often do you skip meals Group >4x's/wk <3x's/wk Predentate 6 5 Primary 4 16 Mixed 0 14 Permanent 6 9 Table 6: Statistically Significant Maternal Diet Variable 1 (p=0.050)

How often do you snack instead of eating a meal Group >4x's/wk <3x's/wk Predentate 8 3 Primary 5 15 Mixed 3 11 Permanent 3 12 Table 7: Statistically Significant Maternal Diet Variable 2 (p=0.050)

Biological samples were obtained from all one hundred and twenty subjects. For predentate infants, only a saliva sample was obtained. However, for all the mothers as well as all of the other primary, mixed, and permanent dentition groups a saliva, a supragingival, and a subgingival plaque sample was obtained. For this study and analysis, only the saliva sample was analyzed and the supragingival and subgingival plaque samples were banked for future analysis. DNA was isolated from each of the 120 saliva samples and through pyrosequencing over 1.3 million sequences were determined after filtering for sequencing quality. The pyrosequencing technique provided species level identification based on 500 base-pairs in two identifiable variable regions of the 16S ribosome (V1-V3 and V7-V9) with a similarity of greater than or equal to 97% required to identify the species. In total, the sequences represent 9 phyla, 14 classes, 29 orders, 52 families, 103 genera, and 332 species-level operational taxonomic units (s-OTU’s) (see

Figure 1).

Figure 1: Bacterial Taxa

Total species-level operational taxonomic units (s-OTUs) for each of the four child groups and the mother group was determined (see Table 8). For each sample in each group, the average s-OTU was determined in order to obtain a distribution of the statistical analysis for significance between the groups (see Figure 2). An ANOVA test of the mean and distribution was used to determine there was statistical significance between the groups with a p-value <0.0001. The Tukey Test was then used to show the predentate group had a statistically significantly less s-OTU count compared to the other four groups (p<0.05).

Total s-OTUs by Group Dentition State s-OTU Count

Predentate 159

Primary 235

Mixed 231

Permanent 232

Mothers 310 Table 8: Total s-OTU’s by Group

The presence and abundance of the species in each group was also analyzed using the Shannon-Diversity Index (see Figure 3). This index takes into account both the type and number of species present (ie. 1 species present 100 times or 100 species present only

1 time) in order to obtain a relative view of the bacterial species in each group. An

ANOVA analysis showed a statistical significance between the groups with a p- value<0.001. The Tukey Test was then used to show the predentate group had a significantly lower bacterial diversity than the other groups (p<0.05).

In order to compare the mother/child dyads as a pair, a Bray-Curtis Similarity

Index was used (see Figure 4). On this chart, zero would represent no similarity in the microbial profile between a mother and child, while a 1 would indicate the microbial profiles are identical. ANOVA analysis of each dentition group pairs showed no statistically significant difference between the groups. However, the predentate group exhibited more similar bacterial species with their mothers than the other groups but not to a level of statistical significance. On average, this similarity index showed that each child shared on average about 50% of their salivary microbial profile with their mother. 22

The 16s rDNA of the detected s-OTU’s in the dataset were used to generate a phylogenetic tree (see Figure 5). The phylogenetic tree diagrams the relationship between each bacterial species identified based on the genetic similarities between the species. The tree is organized based on occurrence (not abundance) and color coded by group (inside to outside: teal represents predentate, green represents primary, dark blue represents mixed, light blue represents permanent, and navy represents mothers). For the predentate group, every species except for one (Acinetobacter Baumanni) that was found in predentates was also found in the maternal group. In the other dentition groups, there was an increased occurrence of bacterial species such as, corynebacterium diphtheria and haemophilus simiae, which were found in the primary, mixed, or permanent dentition groups but not in any of the mothers. It is evident however, with progression from each stage (predentate to primary to mixed to permanent to mothers) the diversity of the bacterial community increases and the number of different types of bacteria present grow.

Noteworthy, are the presence of several lactobacillus species (a cariogenic bacteria noted in more advanced carious lesions) that are only found in mothers and none of the child dentition groups. There are several bacteria included that were isolated but are unclassified due to the fact that sequence quality could not get to the species level of specificity or these bacterium have not yet been named.

Figure 2: Average Number of s-OTU’s by Group (p<0.0001)

Figure 3: Shannon Diversity Index (p<0.001)

Figure 4: Bray-Curtis Similarity Index

Figure 5: Phylogenetic Tree

Discussion

The purpose of this study was to examine the salivary microbiome and its relationship to maternal diet in specific mother-child pairs based on different stages of dentition development. Child subjects were categorized based on dentition state rather than chronological age in order to obtain a more specific view on how one’s oral flora changes in relation to the biological habitat development (ie transition from predentate to primary teeth to a mixed dentition state to young permanent teeth). In normal healthy children, different stages of dental development progress successively as a child ages.

However, the range of normal eruption and/or exfoliation of teeth can vary greatly, thus each child was categorized based on clinical presentation of each dentition state rather than age. Each child subject included in the study also had to be caries-free in order to minimize the confounding factors of possibly obtaining different bacteria that can occur in different disease states. A view of a normal, disease-free oral flora in different specific dentition states was desired; therefore, children with active caries or a history of caries was an exclusion factor in this study. In addition, each child subject could not have a significant medical issue or take medications for a chronic disease in order to eliminate the possibility of these factors altering the child’s normal flora.

The mothers used in the study had to be the biological mother for each child subject. By using a biological pair, we assume continuous interaction of the mother-child

28 pair throughout childhood and are able to longitudinally assess the changes in the oral microbiome of children over time and dentition state in relation to their mothers more accurately. In addition, documentation of co-habitation of siblings or extended family was also obtained. We can then make inferences on vertical and horizontal transmission of bacteria and the impact each type of transmission has upon the development of a child’s own unique oral microbiome. There were no inclusion/exclusion requirements for the mothers in terms of caries status or health status in this study. Therefore, there is a possibility of confounding local and host factors that can vary between the individuals in the mothers group.

Although a saliva, supragingival and subgingival plaque sample was obtained from each mother-child pair, only the saliva sample was analyzed in this study. Saliva sampling is a non-invasive and well tolerated means of bacterial sampling that provides an overall perspective of the oral microbiome. It provides a representation of multiple ecological oral niches of both hard and soft tissue. By using a saliva sample, we are able to assess the predentate group of children that do not yet have hard surfaces for bacterial attachment. However, saliva is transient and free-flowing and not a site-specific representation of oral bacteria43. The supragingival and subgingival plaque samples that were obtained for future analysis may reveal different bacterial species with different relationships.

There are many different ways to identify bacteria. Traditional bacterial identification methods, such as gram staining and culture, rely on phenotypic characteristics. However, these methods are limited to organisms that can be cultured in

29 vitro and some strains of bacteria are unique and do not fit patterns used to characterize a species. It is estimated only about half of the bacterial species in the oral cavity can be cultivated1, 29. Newer molecular techniques, such as pyrosequencing, overcome these limitations by using a 16s rRNA gene-based cloning system. Therefore, pyrosequencing was used in this study to allow the microbial makeup of the oral microbiome to be better identified30. With this method we are able to identify more bacterial organisms, assess a wider range of bacterial species, and better examine the bacterial community as a whole.

A total of 120 subjects (60 mother-child pairs) were included in this study. The large number and fairly even distribution of each dentition group gives power to the results obtained from the diet and bacterial analysis. In addition, the homogeneity of the individuals used in this study is beneficial to reduce confounding factors. Specifically, individual variables such as medical history, caries status, and brushing frequency were accounted for with no difference between the child subjects. The subjects were identified from only two Nationwide Children’s Hospital clinic sites minimizing geographic variability and the subjects used have a similar socioeconomic status and educational backgrounds. In addition, intra-rater reliability of subject survey and sampling is high because only a single study investigator obtained data and samples for every subject.

These study design aspects lessen the influence of confounding factors in order to reduce error and give power to the study findings.

Diet is a key oral health factor32. Children’s food knowledge, preference, and consumption have been shown to be related to their parents’ preferences, beliefs, and attitudes toward food44. For example, parents’ modeling of healthful dietary behaviors

30 was associated with low-fat eating patterns and lower dietary fat intake45-46. Thus, maternal dietary choices can have a direct correlation with their children’s diet.

Previously studies have shown, high-risk dietary practices appear to be established as early as 12 months old and maintained throughout early childhood33-34. Therefore, poor diet choices can have a significant negative impact on multiple health factors, including dental disease. For the majority of the maternal diet variables analyzed in this study, there was no statistical significance between the dentition groups. This is an important factor to account for when assessing caries-free children. Lack of statistical significance between dietary variables in mothers associated with children in each dentition group adds to the homogeny of the groups. It highlights the similarities of diet choices between the mothers which can be associated with caries-free children. For example, 97% of mothers drank water more than four times a week and only 45% drank soda. The majority of mothers also ate fruit and vegetables (62% and 80% respectively) more than four times a week instead of carbohydrate based foods such as potatoes, pasta or rice

(17% and 32% respectively). Obtaining a perspective on a mother’s dietary practices and its effect on their child’s oral flora has clinical implications for diet analysis and recommendations clinicians can make during anticipatory guidance.

The only two maternal dietary factors that were found to be statistically significantly different involved mothers of predentate infants. Mothers of predentate infants were found to skip more meals and snack instead of eating meals more frequently than mothers of the dentate groups. As new mothers to predentate infants, caloric and energy demands are increased and it makes sense these mothers are having to snack more

31 frequently. In addition, meal planning may be more difficult during this time for this group of new mothers and the convenience of snacking instead of eating a full meal may be attributed to these findings. These results highlight the importance of discussing diet and nutrition especially with new mothers. As diet habits of children have been shown to be directly related to their mothers and are established early in life, it is important to encourage healthy eating habits in mothers that could not only contribute positively to their own oral health but also to the diet and oral health of their children33-34, 44-46.

Periodic review of diet habits and nutritional counseling should be an integral part of anticipatory guidance for mothers and their children.

The key variable assessed through analysis of the salivary microbiome is the four different states of dentition. Many of the past oral microbiological studies have examined specific oral bacteria or assessed the oral microbiome in relation to age48. This is one of the first studies that assesses the development of the oral microbiome from the predentate stage through the three stages of dentition in the in multiple. When assessing the results of the salivary microbiology of each group, there are two main factors to evaluate: the difference in bacterial diversity of each group and the difference in mother-child pairs in each group. First, analysis of the average s-OTU count showed a statistically significant difference between the predentate group and the dentate groups. Specifically, there were significantly less individual bacterial species found in the predentate group. In several microbiological studies, the Shannon-Diversity Index has been used to show differences in both the type and number of bacterial species present47. Using the Shannon-Diversity

Index in this study, a statistically significant difference was found with the predentate

32 group having a lower bacterial diversity than the other dentate groups. Conversely, the presence of varying types of bacteria and the abundance of the species present was significantly higher in the dentate groups. The lower “richness” (type/amount) of bacterial species in predentate children supports previous findings such as those by

Crielaard et al that have shown the salivary microbiome matures with age47. It is evident with the emergence of teeth, there is an increase in the number of attachment sites and potential niches for bacteria to reside. Therefore, with progression from each stage

(predentate to primary to mixed to permanent to mothers) the diversity of the bacterial community increases and the types/amount of bacteria grow48.

The microbiome of each child was evaluated relative to their respective mother’s oral microbiological makeup. Then, the similarities and differences between each pair was compared amongst the four different dentition groups. The Bray-Curtis Similarity

Index showed no statistically significant difference between the pairs in each group.

However, the predentate group shared more than 50% of the same bacterial species with their mothers while the dentate groups shared on average slightly less than 50% of their salivary microbial profile with their mothers. When assessing the results from the phylogenetic tree, every species (except one) that was found in the predentate group was also found in the maternal group. The one species found in the predentate group that was not in any of the mothers was Acinetobacter baumanni, a hospital acquired bacteria that has been found in other oral microbiological studies of young children47. The presence of this particular bacteria is most likely due to the fact that these children were recently exposed to other sources of bacteria while in a hospital setting shortly after birth. With

33 more than 50% similarity of mother-child bacterial profiles and the predentates only having one species present not found in mothers, vertical transmission of bacterial species that has been found in other studies is supported by our findings16-20.

In the dentate groups, there is an increased occurrence of bacterial species that are not found in any of the mothers, such as Corynebacterium diphtheria and Haemophilus simiae. The presence of bacterial species unrelated to their mothers as well as a less than

50% similar bacterial profile supports horizontal transmission of oral bacteria found in other studies21-22. For example, Stahringer et al found that although the bacterial composition of twins saliva resemble each other more closely than the whole population at any time point, they become less similar to each other with time and when no longer cohabitating16. Our findings are similar to these in that both vertical and horizontal transmission impacts the development of the oral microbiome of children at different stages of dentition development. Additionally, our findings show that the microbiome of children is continually maturing. Grielaard et al showed children ages 3-18 years old have a complex oral flora but this community is still not as complex as adults47. In our study, this was also found to be true. The mother’s microbial profile was more diverse with an increased number and type of bacterial species, some of which (ie. lactobacillus) are linked to dental disease. Therefore, although a child’s microbial profile has some relation to their mothers through vertical transmission especially early in life, a child’s oral microbial profile is constantly developing and may evolve individually and independently from their mothers via horizontal transmission over time.

There are several study limitations. First, the survey portion of the study was a self-report by the mothers which can lead to recall bias and social-desirability bias.

Second, as a one-time cross-sectional survey analysis there is no longitudinal diet data.

Third, as a one-time saliva sampling, we are only able to obtain a generalized snap-shot of an individual’s ever-changing microflora. In future studies, a longitudinal analysis of not only how oral microflora changes with dentition state between individuals but also within an individual would be beneficial. A long-term diet diary and oral hygiene analysis of both mothers and children could also provide more information on the relationship between these variables and the developing oral microbiome. Future analysis of the supragingival and subgingival plaque samples for the subjects included in this study are planned and will provide more detailed information about established bacterial communities found in the plaque biofilm.

Summary and Conclusions

In summary, for the majority of the diet variables analyzed, there was no difference between the diet choices of mothers of children in different dentition states.

Since diet is a key to oral health, the similarities of these mother’s diet choices may have associations to their children’s diet and caries-free status. In addition, significant differences were found between the microbial profiles of predentate and dentate children.

These differences are attributed to the different stages of dentition development as well as the impact of both vertical and horizontal transmission. Future studies should focus on longitudinally assessing these variables within an individual over time.

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