THE MOLECULAR ANALYSIS OF THE BIOFILM OF PROXIMAL INCIPIENT

CARIES IN YOUNG PERMANENT TEETH

A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By Ross Harris Fishman, DMD

The Ohio State University 2009

Master’s Examination Committee: Approved by Dr. Ann L. Griffen, Advisor Dr. Ashok Kumar ______Dr. Eugene J. Leys Advisor Graduate Program in Dentistry

ABSTRACT

Dental caries is the most common chronic disease of childhood and is the biggest unmet healthcare need among America’s children. To date, effective biological interventions to prevent caries have not been developed. Dental plaque contains several hundred different organisms, many of which are poorly studied. DNA-based methods provide the most comprehensive and precise bacterial identification, and this technology now makes it possible to investigate uncultivated species of oral flora. The purpose of this study is to use molecular methods to identify and differentiate bacterial species present in childhood incipient caries and in health. Fifteen caries-free subjects and 15 subjects with incipient caries (8 to 16 years of age) were selected and matched based on age, sex, and race. For the caries-free subjects, plaque samples were collected from interproximal sites of healthy enamel. For the subjects with caries, plaque samples were collected from interproximal sites of 1) healthy enamel and 2) enamel with incipient

ii caries. DNA was isolated from the samples, and the 16S ribosomal genes were amplified via PCR and cloned. A total of 2,854 clones were identified, and 131 species were detected. Statistical analysis showed no significant differences between the biofilm of healthy teeth and teeth with incipient caries. Although this data confirms the relative stability of the oral microbial ecosystem, it also promotes questions of what drives the caries process from the incipient stage to cavitation. Local tooth factors and saliva may play a more important role than previously thought. Dental caries appears to involve complex bacterial communities, therefore, limiting the scope of future research to S. mutans or Lactobacilli may not be sufficient to gain a complete understanding of the dynamic disease process. As more studies and literature emerge further defining the roles of microorganisms and identifying behavioral risk factors associated with increased caries, information from these studies could affect treatment strategies and provide alternative targets for biological interventions

iii VITA

July 6, 1978 ……………………………………………………Born – Philadelphia, PA.

2000 ………………………………………………...………….B.S. Microbiology University of Florida

2004 …………………………………….……………………...D.M.D. University of Florida

PUBLICATIONS

1. Fishman R, Guelmann M, Bimstein E. Children’s selection of posterior restorative materials. J Clin Pediatr Dent. 2006 Fall;31(1):1-4.

FIELDS OF STUDY

Major Field: Dentistry

iv TABLE OF CONTENTS

Page

Abstract……… …………………………………………………………………………………..ii

Vita ……………………………………………………………………………………………….iv

List of Figures…………………………………………………………………...………………..vi

Introduction……………………………………………………………………………………….1

Materials and Methods……………………………………………………………………………6

Results …………………………..……………………………………………………………….12

Discussion ……………………………………………………………………………………….21

Bibliography …………………………………………………………………………………….30

v LIST OF FIGURES

Figure Page

1. Samples matched based on sex, age, race ………………...…………………………………..10

2. Summary of clinical study groups and sample sizes ………………………………………....11

3. All species identified at all sites combined …………………………………………...... …...14

4. Mean levels of all species in healthy and caries subjects at all sites …………………………17

5. Mean levels of all genera in healthy and caries subjects at all sites ………………………….18

8

Mean levels of D. invisus and S. intermedius constellatus~arginosus in healthy
:JR






caries subjects at all sites………………………………………………………………………19











7. Mean levels of S. sanguinis, and S. mutans in healthy and caries subjects at all sites……...... 20

vi INTRODUCTION

Caries is the most prevalent unmet healthcare need of children and is the most common chronic childhood disease (1, 2). Caries is a multi-factorial disease involving a susceptible host, cariogenic bacteria, and a substrate in the form of a fermentable carbohydrate. However, this seemingly simple triad is not without complexity. The susceptible host factor varies in regard to the quality of enamel, the salivary flow, and the saliva’s pH buffering capacity. The cariogenic bacteria vary in their acid tolerance and their acidogenicity, and the substrate varies based on the diet of the host in terms of frequency and quantity of fermentable carbohydrate. Dental caries is the demineralization of tooth structure by high concentrations of organic acids produced from carbohydrate metabolism by bacteria in plaque (3).

Classic caries literature has focused on Streptococcus mutans (Sm) and

Lactobacillus (Lb) as probable etiologic agents. In the 1920s and 1930s, these two organisms were cultivated from carious lesions and research into the etiology of caries was just beginning to be published. From the 1930s to the 1960s, the dominant theory of the etiology of caries was the nonspecific plaque hypothesis which held that all plaque was equally pathogenic; hence, research did not necessarily hone in on these organisms.

However, in the 1960s, a surge of literature focusing on these bacteria emerged, and then again, in the 1980s, more studies were published implicating these bacteria as the

1 causative agents of caries (4-9). The school of thought began to turn to the specific plaque hypothesis, which held that out of the diverse collection of bacteria in plaque, only a very limited number of species are involved in disease. However, in recent years, scientists have begun to question both of these hypotheses and have suggested that Sm and Lb are associated with caries, but that a cause and effect relationship due to their mere presence is too simplistic to describe the caries process. In the 1990s, Marsh proposed the ecological plaque hypothesis which suggests that plaque is a biofilm, and the oral cavity has many different ecological niches (10). Under this theory, potentially cariogenic bacteria could be present in health, but at levels that are not clinically relevant.

However, stresses to the environment such as repeated conditions of low pH can select for bacteria which are acidophilic and have high cariogenic potential.

Sm and Lb have been the most studied cariogenic organisms because 1) they have been repeatedly cultivated from sites associated with caries, and 2) they have characteristics which lend themselves to the disease process. Streptococcus mutans , when paired with their similar cousin Streptococcus sobrinus form the group Mutans streptococci (MS), in particular have been defined as cariogenic because they are acidogenic and acid tolerant, they are able to adhere to the enamel surface and accumulate, and they can store polysaccharides to use in the absence of fermentable carbohydrate (11). Lb are mucosal colonizers and do not avidly colonize teeth; however, they are highly acidogenic and acid tolerant. They have been frequently isolated from established carious lesions (12, 13). Coaggregation of bacteria has been hypothesized to explain how Lb appears in cultures of advanced lesions, and ecological succession also

2 provides an explanation for the appearance of Lb in later stages of disease (14). The most simplified overview of the ecological succession of bacteria in the oral cavity of an individual with caries can be summarized in four phases. The first phase involves the appearance of S. salvarius prior to tooth-eruption. The second phase is the appearance of

Sm at the introduction of a non-shedding surface such as a tooth or obturator. The appearance of Sm and S. Sobrinus and/or Sm and A. odontolyticus as early carious lesions are detected is the third phase, and final phase involves the presence of Lb as progression of the disease is noted (15). Hence, Sm has been implicated in the initiation of caries, and Lb has been implicated in the progression of caries.

While understanding the roles of Sm and Lb in the caries process is helpful, it does not give us the full picture of the disease process. Caries can occur in the absence of, or in the presence of, a low concentration of Sm (16). Several studies have been published in which subjects with early caries had low levels or no detectable Sm (17, 18).

A 2009 study demonstrated, for example, that the proportion of S. sobrinus to Sm had a higher correlation with children’s caries prevalence, significantly greater than Sm alone

(19). These studies demonstrated that Sm is not the single agent of caries and other bacteria must play a role in the etiology. A group of bacteria other than MS or Lb which are sufficiently acidogenic and acid tolerant to be cariogenic were identified: S. mitis,

S. milleri, S. gordonii, and S. oralis (3). These bacteria have been commonly grouped as the low pH, non-MS group. These organisms have cariogenic properties and are abundant in plaque (20). For these reasons, Kleinberg asserts that MS are not the sole etiology of caries (20). He further proposes that organisms which are non-alkali

3 producers are more cariogenic than organisms capable of raising the pH of plaque by metabolizing urea and/or arginine into ammonia (20). He identifies the following organisms as non-PH raising: most strains of S. mutans, S. mitis, A. viscosus, A. israelii,

L. acidophilus, L. salivarius, L. casei, N. sicca, N. subflava, and B. Catarrhali (20). He affirms that these organisms may play a significant role in maintaining a low plaque pH and in shifting the balance towards demineralization (20).

Until recently, nearly all investigations into the microbial pathogenesis of caries have been conducted by cultivation of bacteria. The major limitation of this method is that roughly 50% of the oral microflora is not cultivable, and many bacteria are difficult to identify and enumerate by cultivation (21). Hence, only about 260 species of oral bacteria have been cultivated while the actual diversity has been estimated at approximately 600 species (22). Oral biofilms are complex structures containing microorganisms with interdependent relationships and interactions that have not been replicated in vitro. In order to obtain a more complete profile, molecular methods for identification and enumeration of oral bacteria have been developed (22, 23). Using the techniques of PCR, cloning, and sequencing of the 16S rRNA genes make it possible to study the microbiota of dental caries more precisely (19, 24). In 2002, Becker et al. were the first to publish the results of utilizing these methods to study the bacteria associated with childhood caries, and they were able to identify 294 phylotypes of which 10 were novel phylotypes (24). The most recent study by Aas et al. found 22 novel phylotypes using molecular methods (18).

4 To date, only a handful of studies have looked at the biofilm of incipient carious lesions and even fewer have utilized molecular methods to do so. Becker’s study found incipient “white-spot” lesions to have elevated Sm, Veillonella , and Actinomyces species levels along with a complex flora, but only two samples were analyzed (24). Incipient caries in the study by Aas et al. were found to have higher isolation frequencies of

Veillonella, Actinomyces , Streptococcus sanguinis , and Fusobacterium (18). This study, however, sampled incipient caries in patients who had concurrent dentinal lesions, some of which were severe.

The specific aim of this study is to identify and compare the microbial community profile associated with initial proximal caries in the early permanent dentition using an open-ended molecular approach. The precise identification and enumeration of the bacteria in plaque is essential to the further elucidation of the bacterial etiology of caries and to those organisms associated with dental health. As these species are identified, information from these studies could profoundly affect the direction of future work.

Moreover, alternative targets for biological interventions could be identified, and new therapeutic interventions and/or prevention strategies could be developed to help combat and/or prevent caries.

5 MATERIALS AND METHODS

Subject Selection: Fifteen caries subjects and 15 healthy control subjects were recruited from the Nationwide Children’s Hospital Dental Clinic, Columbus, Ohio.

Verbal assent was obtained from all subjects along with parental permission for this IRB approved study. The subjects ranged in age from 8-16 years. The general exclusion criteria included: a history of antibiotic use or a professional cleaning within the previous

30 days, and siblings were excluded from participating in the same study group. The inclusion criterion for the caries subjects were: at least one initial proximal carious lesion confined to enamel on a permanent tooth detected via radiograph taken during the normal course of treatment, and no other current caries extending to or beyond the dentino- enamel junction in the primary or permanent dentition. The inclusion criteria for the healthy subjects were: no history of previous caries and no current caries in the primary or permanent dentition. Hence, subjects with restorations or missing teeth extracted due to caries were excluded. The existence of sealants, however, did not exclude the subject from the healthy group.

Subject Pairing: Caries and caries-free subjects were sampled and matched on the basis of age, race, and sex (Figure 1).

6 Data Collection : Demographic information including age, race, and sex was collected.

Sampling Procedure:

Interproximal Plaque : Plaque was obtained from proximal areas by flossing the proximal surface of the teeth with black floss (POH, Tulsa, OK). The black floss enabled

clear visualization of the plaque sample. The section of floss containing the plaque was cut, and each sample was placed in a sterile 1.5 mL microcentrifuge tube. All samples were transported to the laboratory and frozen until analyzed.

Sampling Sites : The healthy subjects were sampled at a maximum of 5 interproximal sites. The caries subjects were sampled at a maximum of 5 interproximal sites with incipient caries and 5 interproximal caries-free sites. All sampled sites were in proximal contact with another tooth. Only one sample from adjacent sites in proximal contact (ie the mesial of one tooth contacting the distal of another) was taken. Figure 2 summarizes subjects and sampling sites.

Saliva sample: All subjects expectorated 3-5ml of un-stimulated saliva into 25ml tubes. These samples were transported to the laboratory and frozen for future research.

Molecular Analysis

The laboratory portion of this project was carried out at The Ohio State University

College of Dentistry. Bacterial DNA was isolated by soaking the sample in 180 ul of

ATL buffer (Qiagen, Valencia, CA) and 20 ul of Proteinase K (Promega, Madison WI).

The samples were then beaten with 0.25g of 0.1mm glass beads (Biospec Products, Inc.,

Bartlesville, OK) for 60 seconds at 4800 rpm in a bead beater. The liquid was aspirated

7 and transferred to a sterile 1.5 ml tube. The DNA was cleaned using a QiaAmp DNA

Minikit column (Qiagen) following the manufacturer’s instructions. The final step yielded an elution filtrate which contained DNA. The elution was examined using a nanospectrometer which reads the absorbance of DNA at 260 nm to confirm the presence of DNA. PCR amplification was performed using a universal forward primer (5’-GAG

AGT TTG ATY CTG GCT CAG-3’) and a universal reverse primer (5’-GAA GGA GGT

GWT CCA RCC GCA-3’). The amplifications were screened by electrophoresis in a

1% agarose gel, and the DNA was cleaned again with QiaAmp columns following the manufacturer’s instructions.

Cloning of the PCR amplified DNA was performed using a TOPO TA Cloning

Kit (Invitrogen, San Diego, CA) following the manufacturer’s instructions.

Transformation was completed using competent E. coli TOP 10 cells provided by the manufacturer. The transformed cells were plated onto Luria-Bertani agar plates

◦ supplemented with kanamycin and incubated overnight at 37 C. The size of inserts

(approximately 1500 base pairs) was determined by PCR using flanking vector primers followed by gel electrophoresis on 1.5% agarose gel. Prior to sequencing, the PCR amplified 16S rDNA fragments were purified and concentrated using Microcon 100

(Amicon), followed by the QIAquick purification (Qiagen).

A minimum of 50 inserts of the correct size of approximately 1,500 bases were sequenced per subject for a total of 2,854 clones. Sequences were BLASTED against a locally hosted oral microbiome 16S database to determine the identity of the clones.

8 Statistical Analysis

The clones were enumerated by species or genera, and percentage of total microbial community was calculated. An independent, two sample t-test was used to compare levels of each species in proximal healthy sites in caries-free subjects (PH-CF) to proximal healthy sites in subjects with proximal incipient caries (PH-IC). A paired t- test was used to compare levels of each species in PH-IC sites to proximal incipient caries (PC-IC) sites in the same subject. The Bonferroni correction was applied to adjust the alpha level for both t-tests since multiple comparisons were made (with tests run for

50 species, the alpha level was lowered to 0.001). All statistics were performed using

JMP, version 7 (SAS Corporation, Cary, NC).

9 Sample Age Pairs Sex Race Type (years) Caries -Free Child Female 10.92 Caucasian Match 1 Child with Caries Female 10.56 Caucasian Caries-Free Child Male 11.96 Caucasian Match 2 Child with Caries Male 11.34 Caucasian Caries-Free Child Male 10.33 African-American Match 3 Child with Caries Male 10.86 African-American Caries-Free Child Female 8.50 More than one Match 4 Child with Caries Female 8.93 More than one Caries-Free Child Female 9.04 Caucasian Match 5 Child with Caries Female 9.49 Caucasian Caries-Free Child Male 9.10 African-American Match 6 Child with Caries Male 10.73 African-American Caries-Free Child Female 12.62 African-American Match 7 Child with Caries Female 11.76 African-American Caries-Free Child Male 16.36 African-American Match 8 Child with Caries Male 15.60 African-American Caries-Free Child Female 11.06 Caucasian Match 9 Child with Caries Female 13.54 Caucasian Caries-Free Child Female 10.86 African-American Match 10 Child with Caries Female 9.89 African-American Caries-Free Child Female 13.00 African-American Match 11 Child with Caries Female 12.06 African-American Caries -Free Child Male 13.41 Caucasian Match 12 Child with Caries Male 12.34 Caucasian Caries-Free Child Female 16.00 African-American Match 13 Child with Caries Female 15.54 African-American Caries-Free Child Male 13.59 African-American Match 14 Child with Caries Male 15.29 African-American Caries-Free Child Female 14.00 African-American Match 15 Child with Caries Female 13.52 African-American

Mean Age Child with Caries 12.10 (SD 2.2) years

Mean Age Caries-Free Child 12.05 (SD 2.4) years

Average Characteristics of Sample 60% Female % Sex 40% Male 60% African-American % Race 33% Caucasian 7% More Than One

Figure 1. Samples Paired Based on Sex, Age, Race

10

Total Type of Samples Number Group (for each sample, up to 5 proximal sites will be of collected) Subjects 1. Healthy proximal sites

2. Proximal sites with incipient lesions Incipient Caries 15

3. Saliva sample (only one sample)

1. Healthy proximal sites (one site from each quadrant) Caries-Free 15

2. Saliva sample (only one sample)

Figure 2 . Summary of Clinical Study Groups and Sample Sizes

11 RESULTS

All subjects with incipient caries were matched to a healthy subject on the basis of age, race, and sex (Figure 1). The mean difference in ages between the children with incipient caries and the caries-free was 0.04 (SD 2.2) years. The difference in age was not significant, as determined by a t-test. The mean age of all the subjects was 12.08 (SD 2.3) years. The subject matches were comprised of African-American children (60%),

Caucasian children (33%) and one pair of biracial children (7%), who each had one

Caucasian and one African-American parent (Figure 1). Sixty percent of the samples were from females and 40% from males. One plaque sample from each of 15 caries-free control subjects and 2 plaque samples (one from a healthy site and one from an early carious site) from each of 15 subjects with only incipient caries were selected for cloning and sequencing of 16S ribosomal DNA.

A minimum of 50 clones were sequenced from each sample yielding a total of

2,854 clones. One hundred and thirty-one species, representing 52 genera were identified

(Figure 3). Twenty-one species represented 78% of the total population (Figure 3).

Figure 4 lists the predominant species in order of decreasing frequency for each sample type, and Figure 5 lists the predominant genera in order of decreasing frequency for each sample type.

12 Statistical analysis of the bacterial species associated with sites of PH-CF children and PH-IC children revealed no significant differences. Statistical analysis of the bacterial species associated with PH-IC sites and PC-IC sites in the same child revealed no significant differences. By this logic, there were no statistical differences between the sites of PH-CF children and PC-IC children.

The most prevalent species found in all three sites were Streptococcus mitis pneumonia infantis~oralis group , followed by the Veionella atypical dispar parvula group, Neisseria meningitidis group, and Dialister invusis (Figure 4). Well studied bacteria, Streptococcus sanguinis, Streptococcus mutans, and Actinomyces viscosus naeslundii , were sixth, seventh, and twelfth most common respectively (Figures 3, 4).

Despite the lack of statistical significance, there were some noteworthy trends and observations. The levels of Dialister invusis increased in PH-IC sites compared with sites in PH-CF individuals, and increased further at PC-IC sites. The opposite trend is seen with the Streptococcus intermedius constellagus~anginosis group where these bacterium tend to decrease from PH-CF sites to PC-IC sites (Figure 6). Streptococcus sanguinis was present as often in incipient sites as in healthy sites while Streptococcus mutans also was almost equally distributed in healthy enamel as in early demineralization (Figure 7).

Finally, the Lactobacillus genus was the 26 th most prevalent genera with Lactobacillus gasseri the 53 rd most common species (Figures 5, 3).

13 Species % of all Rank clones Abiotrophia defective 1.383 18 Achromobacter xylosoxidans 0.036 97 Acidaminococcaceae G-1 sp oral taxon 132 0.036 98 Acidaminococcaceae G-1 sp oral taxon 135 0.036 99 Acidaminococcaceae G-1 sp oral taxon 148 0.073 81 Acidaminococcaceae G-1 sp oral taxon 150 0.291 49 Acidaminococcaceae G -1 sp oral taxon 155 0.182 58 Actinobaculum sp oral clone EL030 0.073 82 Actinomyces gerencseriae 0.146 63 Actinomyces massiliensis 0.036 100 Actinomyces odontolyticus lingnae meyeri 0.218 53 Actinomyces orihominis dentalis 0.073 83 Actinomyces sp oral clone IP073 0.509 33 Actinomyces viscosus group A 0.036 101 Actinomyces viscosus naeslundii 2.402 12 Aggregatibacter segnis 0.109 67 Atopobium parvulum 1.419 17 Atopobium rimae 1.055 20 Bacillus silvestris 0.073 84 Bifidobacterium scardovii dentium 0.255 51 Campylobacter concisus 0.109 68 Campylobacter gracilis 3.603 5 Campylobacter showae rectus 0.509 34 Capnocytophaga gingivalis 0.328 47 Capnocytophaga granulose 0.364 44 Capnocytophaga leadbetteri 0.036 102 Capnocytophaga ochracea 0.109 69 Capnocytophaga sp 0.182 59 Capnocytophaga sputigena 0.073 85 Cardiobacterium hominis 0.182 60 Catonella morbid 0.073 86 Catonella sp oral clone BR063 0.036 103 Centipeda periodontii 0.146 64 Corynebacterium durum 0.255 52 Corynebacterium matruchotii 1.128 19 Cryptobacterium curtum 0.473 37 Dialister invisus 4.803 4 Dialister pneumosintes 0.073 87 Eikenella corrodens 0.546 29 Eubacterium brachy 0.036 104 Eubacterium sp group C 1.82 14 Eubacterium sp oral clone CK047 0.036 105

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Eubacterium sp oral clone DO008 0.109 70 Eubacterium sp oral clone EI074 0.291 50 Eubacterium sulci infirmum 0.073 88 Eubacterium yurii subsp margaretiae 0.036 106 Firmicutes oral clone F058 0.146 65 Fusobacterium nucleatum 0.109 71 Fusobacterium periodonticum 0.036 107 Gemella haemolysans 2.802 8 Gemella morbillorum 0.437 40 Gemella sanguinis 0.036 108 Granulicatella adiacens 1.456 16 Granulicatella elegans 0.582 26 Haemophilus genomosp P3 oral clone MB3_C38 0.036 109 Haemophilus parainfluenzae group A 0.509 35 Haemophilus parainfluenzae group B 0.036 110 Haemophilus pittmaniae 0.036 111 Johnsonella ignava 0.036 112 Kingella denitrificans 0.473 38 Kingella oralis 0.691 25 Lachnospiraceae sp oral clone C1_DO016 0.546 30 Lactobacillus gasseri 0.218 54 Lactobacillus salivarius 0.182 61 Lactobacillus vaginalis 0.036 113 Lautropia FX006 0.036 114 Lautropia mirabilis 2.802 9 Leptotrichia hofstadii 0.036 115 Leptotrichia sp oral clone BU064 0.036 116 Megasphaera micronuciformis 0.546 31 Megasphaera sp group B 0.109 72 Micrococcus luteus 0.036 117 Mitsuokella sp oral taxon 131 0.036 118 Mitsuokella sp oral taxon 521 0.036 119 Mogibacterium timidum pumilum neglectum diversum 0.073 89 vescu Moraxellaceae sp 0.728 24 Neisseria bacilliformis 0.073 90 Neisseria meningitidis group 9.061 3 NOVEL Lachnospiraceae RF01 0.073 91 Olsenella uli 0.109 73 Oribacterium sinus 0.036 120 Oribacterium sp group B 1.674 15 Parvimonas micra 0.364 45 Peptoniphilus lacrimalis 0.036 121 Peptostreptococcus stomatis 0.073 92

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Porphyromonas catoniae 0.437 41 Prevotella denticola 0.073 93 Prevotella genomosp C1 0.109 74 Prevotella nigrescens 0.036 122 Prevotella oris 0.182 62 Prevotella oulorum 0.036 123 Prevotella sp oral clone BR014 0.036 124 Prevotella veroralis melaninogenica 0.073 94 Propionibacterium BN085 0.073 95 Propionibacterium propionicum 0.109 75 Rothia aeria 0.509 36 Rothia dentocariosa 0.437 42 Rothia mucilaginosa 0.109 76 Scardovia inopinata 0.036 125 Scardovia sp C1 2.001 13 Selenomonas dianae infelix 0.473 39 Selenomonas flueggei 0.546 32 Selenomonas IK004 0.109 77 Selenomonas infelix 0.109 78 Selenomonas noxia 0.764 23 Selenomonas sp group C 0.036 126 Selenomonas sp group D 0.218 55 Selenomonas sp group E 0.218 56 Selenomonas sputigena 0.582 27 Shuttleworthia satelles 0.364 46 Staphylococcus haemolyticus hominis warneri aureus 0.328 48 Streptococcus agalactiae 0.036 127 Streptococcus anginosus A 0.036 128 Streptococcus anginosus B 0.4 43 Streptococcus australis~sanguinis 0.109 79 Streptococcus cristatus 0.91 22 Streptococcus gordonii 2.766 10 Streptococcus intermedius constellatus~anginosus 2.666 11 Streptococcus mitis pneumoniae infantis~oralis 15.393 1 Streptococcus mutans 2.95 7 Streptococcus oligofermentans sinensis 0.582 28 Streptococcus parasanguinis~oralis 0.109 80 Streptococcus peroris 0.036 129 Streptococcus sanguinis 3.13 6 Streptococcus sobrinus 0.036 130 Streptococcus vestibularis salivarius 1.055 21 Tannerella oral clone BU063 0.073 96 Treponema socranskii subsp buccale paredis socranskii 0.218 57 Veillonella atypica dispar parvula 13.064 2 Veillonella sp oral clone HB016 0.146 66 Vogesella indigofera 0.036 131

16 Figure 4. Mean levels of all species in healthy and incipient caries subjects at all sites.

17 Figure 5. Mean levels of all genera in healthy and incipient caries subjects at all sites.

18 8

V:C .71 V 8 V:C .7]: 1VJ V:C .71 V :`1V]: 1VJ 8 :`1V1 V :`1V ]: 1VJ 8

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8

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19 8 V:C .71 V V:C .7]: 1VJ 8 V:C .71 V :`1V]: 1VJ 8 :`1V1 V :`1V ]: 1VJ 8

8

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Figure 7. Mean levels of S. sanguinis, and S. mutans in healthy and caries subjects at all sites.

20 DISCUSSION

The results of this study support the findings of other studies which indicate that the plaque biofilm is made up of a diverse array of species (18, 21, 24). In this particular study, 131 individual species and 52 genera were identified. In the future, the sample size will be increased to gain more power.

The result of no significant differences in the bacterial population between PH-

CF, PH-IC, and PC-IC sites was surprising. All previous research has shown during caries initiation and progression there are differences in bacterial species than in health

(4, 5, 8-30). Our finding suggests that the degree, stage, and rate of progression of caries may be closely related to the microbial community composition. A relatively undisturbed ecosystem is easily corrected, and this is consistent with clinical impressions that an incipient caries pattern can be arrested (31).

Oral biofilms are fairly stable, even in the presence of minor environmental stress

(32). Studies have shown that the benign oral flora can decrease the damage caused by strong acid production of their pathogenic cohorts. This is done by converting these acids into weaker ones, or neutralizing the acid entirely via ammonia production (20, 33). Oral bacteria can also maintain vitality for a substantial period of time without a normal nutrient source by either utilizing previously stored polysaccharides or switching metabolic pathways (11, 16, 34). Anaerobic organisms can cope with the toxic effects of oxygen by interacting with oxygen-consuming species (35). These examples of stability,

21 referred to by Alexander as microbial homeostasis, result not from any indifference but rather a dynamic balance of numerous inter-microbial and host-microbial interactions

(36, 32).

Our study lends credence to Marsh’s ecological plaque hypothesis given that cariogenic bacteria were identified but their influence may be overwhelmed by a myriad of other benign oral flora (10). Since the microbial compositions of incipient caries were relatively similar to that of health, one must ask the question, “What then drives the caries process?” Although numerous variables play a role, the answer may involve the complex interactions between the normally stable bacterial community and two other important host issues, local tooth factors and saliva.

Local tooth factors such as enamel morphology and gingival crevicular fluid all may affect the biofilm and explain why some incipient caries progress. No one can advertise that their teeth have a perfect enamel surface composed of crystals and prisms that is “so round, so firm, and so fully packed” (37). As early as 1929, Mellanby emphasized that more often than not enamel presents irregularities caused by barely visible surface defects (38). With the use of scanning electron microscopy, modern technology has shown that significantly more cavitations exist in proximal surface incipient lesions than previously reported (39). Kielbassa et al found that even 10% of radiographically sound surfaces on extracted teeth had visual breakdowns (39). Thus, the formation of a surface defect is an important moment clinically because now the biofilm is protected within a microcavity. Unless the patient is able to clean this area, the incipient caries process will continue (40). This protected environment tends to shift the

22 flora towards anaerobic and acid-producing species. Theories on how these defects arise include developmental/mineralization abnormalities, mechanical microtraumas during mastication and interdental wear, or even careless, heavy probing by dentists (40).

The ability of serum antibodies from gingival crevicular fluid (GCF) to protect tooth surfaces has been debated. Russell et al found certain IgG antibodies interfere with the glucosyltransferase enzymes in S. mutans , thereby reducing their cariogenicity (41).

Crameling et al, however, found no correlation between IgG levels and the amount of S. mutans on the buccal surfaces of molar (42). In addition, the actual flow rate of GCF may alter the nutrient content available to the biofilm, resulting in the selection of cariogenic species (32, 43).

The role of saliva as a host anti-caries factor has long been established (44).

Salivary components (e.g. lysozyme, lactoferrin, sialoperoxidase, antimicrobial peptides), its flow, viscosity, buffering capacity, etc. play a major role in the prevention, initiation, and progression of the disease (10, 45). Since all subjects provided saliva samples, logically, future research should determine whether the saliva of the caries-free children and children with incipient carries differ.

Even though caries progression appears to involve complex interactions between a stable biofilm, the host, and the environment, the specific bacterial findings in this study warrant further explanation. In this study, the levels of S. mutans in all subjects were low and did not appear to rise at early carious sites compared to healthy sites.

While MS has been associated with early caries, there may be more to the initiation process than the presence of this species. Several studies have found carious lesions with

23 either low or no detectable levels of MS (16, 18). The present study confirms the work of van Ruyven in which conspicuously low levels of MS were found in plaque from incipient lesions, even in subjects with high levels of MS in frank carious lesions (16). In other studies, the presence of MS plus S. sobrinus or A. odontolyticus had been associated with the progression of incipient caries (9, 14-16). In the present study, however, A. odontolyticus was the 52 nd most prevalent species, compromising only 0.22% of the total clones, and S. sobrinus was 131 st .

The role of S. mitis in caries has not yet been determined though it has been labeled a low pH, non-MS species. Several studies group some of the low pH, non-MS species together for analysis (24, 25). While these species have been defined on the basis of phenotype, their genetic relationships, at least by 16S, do not parallel the species groupings. These species seem to be genetically indistinct (18), and for this reason, we have grouped the following species together and labeled this grouping the S. mitis pneumonia infantis~oralis group: S. mitis, S. mitis bv2, S. oralis, and S. pneumonia. In this study, when the S. mitis species were grouped, this cluster comprises 15.39% of the clones making this group the most prevalent species (Figure 4). The present study found no difference in the prevalence of the S. mitis pneumonia infantis~oralis group at PH-CF,

PH-IC, and PC-IC sites. This differs from Corby et al. who found an inverse relationship between the levels of S. mitis and caries, while Becker et al. reports that S. mitis levels were significantly higher in subjects with caries (25, 24). The S. mitis group is sufficiently acidogenic to be cariogenic as this group is capable of exhibiting a very high pH drop rate and maintaining a low pH level in the absence of MS or Lb (3). Previously,

24 this group has been implicated in the initiation of caries as several studies have found the levels of these low pH bacteria to increase as individuals transition from a low-caries to a higher-caries status (3, 18, 20, 24). Van Ruyven found this transition to be accompanied by an increase of the total low pH bacteria from about 20 to 60% (16). Moreover,

Kleinberg reports that pH levels below 3.5 have been achieved by combining the S. mitis group with other species such as Neisseria or Veillonella and high levels of glucose (20).

In the present study, the most prevalent four species are the S. mitis group, the Neisseria group, the Veillonella group , and Dialister invisus . Perhaps bacterial mixtures which do not contain MS or Lb may be one of the many factors involved in caries initiation.

The second most prevalent species identified was Veillonella atypical dispar parvula group. The Veillonella genus was isolated in the late 1960s, and its role in plaque and caries has been explored since the 1980s. Veillonella is found in both caries- free individuals and in individuals with active lesions. The species is unable to ferment carbohydrates itself, but it metabolizes the lactic acid produced by other bacteria into weaker organic acids (propionic and acetic). Veillonella coaggregate with many other bacteria because they have a limited ability to adhere to tooth structure (46). Many studies have found Veillonella associated with all types of tooth structure, from intact enamel to deep dentinal lesions (18, 21, 26, 27). The most recent studies have associated one species of Veillonella with dental caries ( V. denticarosi ) and one with health ( V. rogosae ) and have stated that the caries-free sites contained more diversity in the number of Veillonella species than those from carious lesions (27, 28). However, in this study we

25 have grouped all of the Veillonella species together because they are difficult to distinguish as individual species by genetic or other methods.

In this study, the Neisseria meningitidis group is the third most common species at all three sites. No other studies have found a significant association between Neisseria and caries. Neisseria is an aerobic, gram negative proteobacteria and is capable of fermenting sugars. However, Stephan showed that Neisseria do not produce a rapid drop in pH in the presence of glucose, and McKee et al. found that Neisseria could not be maintained under glucose-excess conditions (29, 47). While Neisseria is not very acidogenic, the bacteria are also non-alkalizing bacteria because they do not produce ammonium carbonate from urea which can counteract acidity (29). Thus, Neisseria might be a contributor to a caries-promoting microbial community if combined in plaque with other non-pH raisers such as the S. mitis group (20). The prevalence of Neisseria found in this study is not replicated in any other study which utilizes either culture or molecular methods Moreover, this study was not powered to allow us to obtain similar results for the levels of Neisseria found in other studies.

One of the few bacterial species to even show a trend towards higher quantities at demineralization sites was Dialister invisus , the fourth most common species. Renamed by Downes in 2003, Dialister invisus is an obligately anerobic, gram negative cocci (48).

Dialister species do not ferment carbohydrates and produce very trace amounts of the weak acids acetate and propionate (48). The role of Dialister species in caries is somewhat unknown. Up until now, only one study has noted Dialister species present in tooth surface plaque, and this minimal quantity was found in advanced dentinal lesions

26 (49). They are often, however, isolated from sites involved in endodontic lesions and periodontitis (50, 51).

Our finding of high levels of Dialister in proximal plaque of healthy and incipient caries sites leads us to inquire more about the relationship between supragingival and subgingival bacterial communities. It is well documented that poor plaque control can lead to periodontal disease (52) and previous studies have shown how supragingival plaque can modify subgingival bacteria (53, 54). There is, however, little research that has looked at the dynamic equilibrium between the two populations and/or the inverse relationship of subgingival bacterial effects on supragingival plaque. Recently, a 2009 study by Papaioannu found supragingival and subgingival plaque actually had similar flora (55). This begs the question “what if the proximal tooth surface below the contact and above the gingiva area is more of a transitional environment influenced by both supragingival and subgingival factors?” Further research is needed to learn the answer to this question

The very low prevalence of Lactobaccilli in our study confirms the findings of other studies which associate Lactobacilli only with advanced lesions (17, 18, 24).

Lactobacilli do not appear to play a role in the initiation of caries or in the progression of early carious lesions.

In this study, the mean levels of S. sanguinis were almost equal in healthy individuals and individuals with caries at both healthy and incipient sites. This observation contradicts most other studies which associate higher S. sanguinis levels with health (18, 24, 25, 30). The levels of other species which have been associated with

27 health such as S. intermedius (Figure 6) and Gemella haemolysans , did decrease in caries sites, though the decrease was not statistically significant (18, 25). S. sanguinis, S. intermedius , and G. haemolysans are all able metabolize arginine or urea into ammonia which can help alkalize plaque and keep the pH above the critical demineralization level.

Although not shown by the results in this study, S. sanguinis might be a contributor to health and combat progression of early lesions by its basic pH by-products (11).

There are a few other limitations of this study in addition to a small sample size.

Perhaps the most significant limitation is that the samples are only a snapshot in time.

Studying the biofilm associated with incipient caries presents the challenge of correctly identifying active, incipient carious lesions versus those lesions which have arrested or are beginning the remineralization process. Since all the caries data is pooled together, we currently cannot differentiate which bacterial populations are based on active incipient caries or arrested lesion. A future longitudinal study that re-samples the subjects with incipient caries is crucial to see which lesions are progressing and what their bacterial constituents are. Another limititation is that initial disruptions of microbial homeostatsis may be very subtle. Although our laboratory method is open-ended and specific, it is not very sensitive (56). Studies using next generation sequencing approaches might be better for finding these small shifts as bacterial plaque begins early selection of cariogenic species.

This study is further limited by each subject’s level of plaque maturation. Since the patient pool had a wide range of floss usage and various time intervals since their last prophylaxis, we cannot tell how long the plaque has been present. The early biofilm

28 changes quickly. Plaque that is only two hours old has a different bacterial community profile than the same plaque at 6 hours, let alone the possibility of days or weeks (57).

A final drawback is the difficulty in removing plaque from proximal contacts without inadvertently harnessing bacterial species from the gingival margin. Although great care was taken not to place the black floss in the gingiva, accidental contact was surely possible.

The question still remains: “Why do some children experience devastating dental disease and others do not?” The details regarding the role of each individual species of bacteria in the caries process remain unclear. However, molecular methods are allowing research to delve deeper into the caries-associated plaque ecosystem as well as identify organisms associated with health. Continued work with these methods may lead to more novel species and a better understanding of the ecological succession and intricate relationships of the bacteria associated with caries. In addition, as more factors associated with caries are substantiated, practitioners can educate parents and patients on how to help lower their risk for dental disease. Limiting the scope of future research to quantifying S. mutans , Lactobacilli, and other species may not be sufficient or effective in gaining a complete understanding of the dynamic disease process of caries. As more work emerges about the oral microbial communities, information from these studies could profoundly affect the prevention and treatment strategies we utilize to combat caries.

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