Citation
Subgingival Microflora in Smokers and Non-Smokers in Response to Initial Therapy

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Title:
Subgingival Microflora in Smokers and Non-Smokers in Response to Initial Therapy
Creator:
Strange, Leah R
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (51 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Dental Sciences
Dentistry
Committee Chair:
NEIVA,RODRIGO
Committee Co-Chair:
YILMAZ,OZLEM
Committee Members:
NEIVA,KATHLEEN
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Actinomyces ( jstor )
Fusobacterium nucleatum ( jstor )
Microorganisms ( jstor )
Periodontal diseases ( jstor )
Periodontitis ( jstor )
Prevotella ( jstor )
Species ( jstor )
Streptococcus ( jstor )
Teeth ( jstor )
Veillonella ( jstor )
Dentistry -- Dissertations, Academic -- UF
microflora
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Dental Sciences thesis, M.S.

Notes

Abstract:
Smoking has been identified as a risk factor for periodontal disease1, and differences in the microflora of smokers as compared to smokers have been recognized2. It was the purpose of this investigation to determine if there was a difference in the microflora of smokers and never-smokers using deep sequencing in response to initial therapy. The subgingival plaque samples of smokers and never-smokers were taken at baseline and at 6 months after scaling and root planing. Clinical parameters were also recorded at these time points. Sites were classified as being healthy or diseased at baseline. Sites were also classified as having had a loss of attachment, a good response to treatment, and a lack of a response to treatment. The samples were analyzed using deep sequencing techniques. Results revealed great similarity between the groups in regards to the clinical parameters. Deep sequencing revealed great differences between the groups in regards to the microbiological profile of the various classified sites. Furthermore, great heterogeneity was seen when the microflora of smokers was compared to smokers and when the microflora of never-smokers was compared to never-smokers. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
Local:
Adviser: NEIVA,RODRIGO.
Local:
Co-adviser: YILMAZ,OZLEM.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31
Statement of Responsibility:
by Leah R Strange.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
5/31/2015
Classification:
LD1780 2014 ( lcc )

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SUBGINGIVAL MICROFLORA IN SMOKERS AND NON SMOKERS IN RESPONSE TO INITIAL THERAPY By LEAH RENEE STRANGE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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2014 Leah Strange

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To my amazing family whose love and support made this possible

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4 ACKNOWLEDGMENTS I want to thank my husband and my son for all of their continuous love, support, and inspiration through this journey I want to thank my parents and my family for their unending encouragement. Also, I must thank my sweet dog who has been by my side for countless hours of studying and writing. Also, I want to thanks Drs. Gary Wang and Clay Walker and their labs for their support and resources. Finally, I thank the faculty members of the University of Florida especially Drs. Shannon Wallet and Rodrigo Neiva, for their tremendous efforts and dedication to my education and our field.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 12 The Healthy Periodontium ................................ ................................ ...................... 12 The Diseased Periodontium ................................ ................................ .................... 13 Risk Factors for Disease ................................ ................................ ......................... 13 The Periodontium of Smokers ................................ ................................ ................ 14 Treatment of the Diseased Periodontium ................................ ................................ 14 Bacteria of Smoking ................................ ................................ ................................ 15 Participant Population ................................ ................................ ............................. 17 Clinical Procedures ................................ ................................ ................................ 18 Sample Storage and Preparation ................................ ................................ ............ 19 Sample Analysis ................................ ................................ ................................ ..... 19 Statistical Analysis ................................ ................................ ................................ .. 20 2 RESULTS ................................ ................................ ................................ ............... 21 Clinical Parameters ................................ ................................ ................................ 21 Microbiological Findings ................................ ................................ .......................... 23 3 DI SCUSSION ................................ ................................ ................................ ......... 42 LIST OF REFERENCES ................................ ................................ ............................... 46 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 51

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6 LIST OF TABLES Table page 4 1 Experimental groups ................................ ................................ ........................... 32 4 2 Clinical parameters of selected sites ................................ ................................ .. 33 4 3 Proportion of organisms in healthy and diseased sites prior to treatment of never smoker and smoker periodontal patients ................................ .................. 34 4 4 Proportion of organisms in sites that displayed periodontal breakdown in never smoker and smoker periodontal patients ................................ .................. 35 4 5 Proportion of organisms in diseased sites that responded well to treatment in the course of treatment in never smoker and smoker periodontal patients ........ 36 4 6 Proportion of organisms in diseased sites with a lack of significant improvement over the course of disease in never smoker and smoker periodontal patie nts ................................ ................................ ............................ 37

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7 LIST OF FIGURES Figure page 4 1 Probing depths for smoker and never smoker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. ................................ .. 38 4 2 Gingival margin for smoker and never smoker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. ................................ .. 39 4 3 Clinical attachm ent loss for smoker and never smoker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. ..................... 40 4 4 Plaque index for smoker and never smoker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. ................................ .. 41

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8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SUBGINGIVAL MICROFLORA IN SMOKERS AND NON SMOKERS IN RESPONSE TO INITIAL THERAPY By Leah Renee Strange May 2014 Chair: Rodrigo Neiva Major: Dental Sciences Smoking has been identified as a risk factor for periodontal disease 1 and d ifferences in the microflora of smokers as compared to sm okers have been recognized 2 It was the purpose of this investigation to determine if there was a difference in the microflora of smokers and never smokers using deep sequencing in response to initial therapy. The subgingival plaque samples of smokers and never smokers were taken at baseline and at 6 months after scaling and root planing. Clinical parameters were also recorded at th e s e time points. Sites were classified as being healthy or diseased at baseline. Sites were also classified as having had a loss of attachment, a good response to treatment, and a lack of a response to treatment. The samples were analyzed using deep sequencing techniques. Results revealed great simi larity between the groups in regards to the clinical parameters. Deep sequencing revealed great differences between the groups in regards to the microbiological profile of the various classified sites. Furthermore, great heterogeneity was seen when the microflora of smokers was compared to smokers and when the microflora of never smokers was compared to never smokers.

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9 CHAPTER 1 INTRODUCTION Chronic periodontitis is inflammation of the gingiva extending into the adjacent attachment apparatus. The disease is characterized by a loss of clinical attachment from destruction of the periodontal ligame nt and loss of supporting bone 3 Periodontal disease is a very common problem. NHANES 2009 2010 data found that over 47% of ad ults had periodontitis 4 There is evidence to link periodontitis to pre term, low birth weight babies 5 cardiovascular disease 6 and diabetes 7 If left untreated, chronic periodontitis can result in tooth loss. Although the dise ase is dependent on a susceptible host, there are known risk factors for periodontal disease. Smoking is one of the most significant risk factors for periodontal disease development and progression 8 9 Smoking has also been associated with poorer outcomes to non surgical therap y 9 13 and tooth loss 14 15 Many microorganisms are involved in periodontitis, and studies have shown that s moking has an influence on the microbiota of the periodontium Immunofluorescence showed smokers were more likely to be positive for Actinobaci llus actinomycetecomitans (now known as Aggregatibacter actinomycetemcomitans) Porphyromonas gingivalis, and Tanerella forsythia 16 Also, smokers have been found to have an increased risk for harboring Treponema denticola in periodontal pockets 17 Another study showed s mokers to have more E. nodatum, F nucleatum ss vincentii, P intermedia, P micros, P nigrescens, B f orsythia, P gingivalis, and T denticola 18 Furthermore, current smokers harbor more periodontal pathogens after non surgical

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10 periodontal therapy 19 21 Other studies have failed to show differences between smokers and non smokers 22 23 Most studies to date have been cross sectional investigations of patients with chronic periodontitis comparing smokers and non smokers; therefore, the microbial profiles of sites at risk for developing disease are not known. Furthermore, although more than 700 oral bacterial species have now been reported by cultivation and culture independent molecular methods, studies on most oral microorganisms have been limited. Most studies hav e relied on conventional microbiologic or immunofluorescent methods or bacterium specific PCR to quantify specific pathogens. Limitations of these techniques have precluded detailed analyses of the entire oral microbial communities and their relationships to disease. More recently, culture independent molecular methods targeting the small subunit ribosomal RNA (rRNA) sequences 24 checkerboard DNA DNA hybridization, and microarray based methods 25 have provided a more complete view of the oral microbial communities. However, even with extensive dideoxy Sanger sequencing of 16S rRNA genes, the immense diversity and abundance of oral microbiota pose a major challenge to study less abundant species, some of which may be important for pathogenesis. Recently, open ended techniques have been used in the study of chronic peri odontitis, giving us a more comprehensive understanding of the microbiota associated with periodontal disease. Numerous additional bacterial species have been associated with periodontitis using 454 pyrosequencing of the 16S rRNA genes 26 Also, smoking has been associated with operational taxonomic units classified to genera Fusobacterium, Prevotella, and Selenomonas in chronic periodontiti s patients using

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11 pyrosequencing, a difference not detected using culture and qPCR techniques 2 Using deep sequencing technology will show if the microbiological differences persist after non surgical periodontal therapy. To determine how smoking influences the microbiota of periodontal diseas e and the response to per iodontal therapy, smokers and never smokers were recruited to undergo treatment of chronic periodontitis. Subgingival plaque samples were collected at baseline and at 6 months after scaling and root planing. The samples were sequen ced using deep sequencing technology The data indicate that the periodontal micro flora of smokers and never smokers is different. There were minimal similarities when the groups were compared for sites that were healthy at baseline, sites that were dise ased at baseline, sites that broke down over time, sites that responded well to therapy, and sites that did not respond well to therapy. In fact, when comparing sites within the smoker and never smoker groups, there were minimal similarities. This indicate s that the periodontal microflora is variable, Hypothesis: Smoking has an effect on the subgingival microflora of sites that are periodontally healthy, sites that are diseased, sites that respond well to therapy, sites that do not respond well to therapy, and sites that br eak down during the observation time.

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12 CHAPTER 2 BACKGROUND The Healthy Periodontium The periodontium consists of root cementum, periodontal ligament, bone lining the tooth s ocket, and that part of the gingiva facing the tooth. These structures support the teeth and protect the oral cavity from disease processes. The dentogingival junction is comprised epithelial a nd connective tissue components, and it is the interface of the periodontium and microbial insult. The integrity of these tissues is critical for periodontal health as they provide a barrier against pathogens The epithelium is subdivided into gingival, sulcular, and junctional epithelial layers, and the connective ti ssue is divided into superficial and deep compartments 27 The junctional epithelium is found at the base of the gingival sulcus. It has been extensi vely studied and found to be about 0.97 mm long on average 28 It has a layer of cells that face the tooth that actually attach to the tooth, a complex c alled the epithelial attachment. The junctional epithelium has various immune system cells including defensins, IL 8, and IL 27 The connective tissue compartments lie just apical to the junctional epithelium. The average dimension of the connective tissue attachment is 1.07 mm 28 and it is the most consistent dimension in studies 29 It is highly vascular and has an inflammatory cell infiltrate in all circumstance s. Inflammatory cells such as neutrophils and T lymphocyt es continually migrate from the area and cross into the sulcus 27

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13 The Diseased Periodo ntium Gingivitis is inflammation of the periodontal tissues without loss of periodontal attachment. Periodontitis involves loss of periodontal support. Gingivitis and periodontitis are due to the host response to a biofilm of bacteria l plaque and its byproducts. Bacteria may perturb the integrity of the junctional epithelium which is the first step towards disease progression The inflammatory response to this leads to degradation of the underlying connective tissue and junctional epi thelial attachments. The junctional epithelium and connective tissue attachment migrate apically, leading to the formation of a periodontal pocket. Bone resorption may follow in this inflammatory process 27 Numerous bacteria have been implicated in the periodontal disease process. Some of these clusters of bacteria have been known since 1998, and they have received much attention in the literature. DNA DNA hybridization has indicated that a certain set of bacteria consisting of Porphyromonas gingivalis, Treponema denticola, and Tanerella forsythi a are closely associated with pocket depth and bleeding on probing. This group has been termed the red complex 30 and many studies have looked at these bacteria as markers of periodontal disease. Risk Factors for Disease A risk factor is an environmental exposure or inborn characteristic which is known to be associated with disease related conditions. Cigarette smoking is a we ll established risk factor for periodontitis and it is associated with a two to eight fold increased risk for attachment and/or bone loss 31 The relationship between smoking and periodontitis is dose dependent, with heavier smokers having more disea se than light

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14 smokers 32 33 D iabetes mellitus is also a major risk factor for periodontitis 34 Other prominent risk factors include specific bacteria, age, and male gender 35 The Periodontium of Smokers As previously mentioned, the periodontal microflora of smokers has been found to be different from that of non smokers. Smoking has additional effe cts on the periodontium These differences may result in an increased susceptibility to disease. The oxygen saturation of hemoglobin in gingiva has been found to be reduced in smokers, suggesting impaired gingival microcirculation. This could affect the c omposition of the microflora 36 37 Clinically, s mokers exhibit less bleeding upon periodontal probing, suggesting suppressed vascular ity 38 Smoking also affects the immune response resulting in modification of humoral and cellular immune systems 32 39 40 Furthermore, n icotine and its metabolite cotinine have been shown to be toxic to fi broblasts 41 and nicotine exposure causes fibroblasts to produce more collagenase 42 Treatment of the Diseased Periodontium Numerous treatment modalities are used to treat periodontitis. Therapy usually aims to halt the progression of periodontal attachment loss resolve inflammation, and regenerate destroyed tissues whenever possible 35 The removal of local etiologic irritating factors such as plaque, bacteria, and calculus is accomplished by scaling and root planing using hand instruments and ultrasonics. The benefit of scalin g and root planing is from the removal of subgingival plaque and calculus and disruption of the bacterial microflora 43 This along with regular periodontal maintenance has resulted in long term periodontal stability for many patients with chronic periodontitis 44 46

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15 Antibiotics aimed at controlling the pathoge nic organisms have also been used for the treatment of chronic periodontitis. Currently, systemic antibiotics are considered to be unnecessary due to the ability of the disease to be controlled with conventional therapy. Local antibiotics in conjunction wi th biofilm disruption may be used in sites that 35 but their clinical significance is questioned 47 Surgical p eriodontal therapy is also used to treat chronic periodontitis primarily to facilitate mechanical instrumentation of the roots by improved access. Flap reflection has shown to be more likely to render a root surface free of calculus 48 It is also used to reduce periodontal probing depths and regenerate periodontal tissues 35 Surgical treatment inc luding the modified Widman flap and osseous resective surgery has been shown to be effective in the long term treatment of chronic periodon titis 44 45 49 Bacteria of Smoking Most studies have concluded that the periodontal microflora of smokers is different from that of non smokers. Immunofluorescence showed smokers were more likely to be positive for Actinobacillus actinomycetecomitans (now known as Aggregatibacter actinomycetemcomitans) Porphyromonas gingivalis, and Tanerella forsythia 16 Also, smokers have been found to have an increased risk for harboring Treponema denticola in periodontal pockets 17 Another study showed smokers to have more E nodatum, F nucleatum ss vincentii, P intermedia, P micros, P nigrescens, B fo rsythia, P gingivalis, and T denticola 18 Furthermore, current smokers harbor more periodontal pathogens after non surgical periodontal therapy 19 21 Numerous additional bacterial species have been associated with periodontitis using 454 pyrosequencing of the 16S rRNA genes 26 Smoking has be en associated with operational taxonomic units

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16 classified to genera Fusobacterium, Prevotella, and Selenomonas in chronic periodontitis patients using pyrosequencing 2 Other studi es have failed to show differences between smokers and non smokers 22 23

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17 CHAPTER 3 MATERIALS AND METHOD S Participant Population The study was a prospective cohort study conducted at the University of Florida Periodontology Clinic and/or predoctoral DMD clinics in which microbial compositions of subgingival microbiota from current and never smokers were determined over the course of 6 months and correlated with treatment response. 5 smokers and 5 never smokers were recruited to participate in the study. At least 3 samples were taken from each subject at each visit. The aim was to sample and record data from diseased sites in the maxillary molars, maxillary premolars, maxillary anteriors, mandibular molars, mandibular premolars, and mandibular anteriors Pe riodontal examinations and the collection of specimens were performed at the gradu ate periodontal clinic. Subjects (both smokers and n ever smokers) were recruited from the periodontal clinic or the DMD clinics at the University of Florida or the greater Gainesville community. Smokers were defined as individuals who ha d smoked > 10 cigarettes per day for a minimum of 5 years by self report. Patients were included in the study if they met the following criteria: age > 18, a minimum of 20 natural teeth, and willingness to receive periodontal treatment. Exclusion criteria included : diabetes, pregnancy, lactation, recent systemic antibiotic use including subgingival antimicrobials (within 6 months prior to baseline examination), periodontal treatment within the previous 12 months, systemic condition that could affect the progression or treatment of periodontal disease or would require prophylactic antibiotics for dental procedures, known immune deficiency such as HIV, organ transplantation or

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18 cancer, or being on any immunosuppressive medications (e.g. prednisone), since these factors have been implicated in altering oral microbiota. Sites were considered to be healthy at baseline if they had a probing depth of 3 mm or less and no attachment loss. Sites were considered to be diseased at baseline if they had a 5 6 mm probing depth and at least 2 mm of clinical attachment loss. Sites were considered to have had breakdown if they lost at least 2 mm of clinical attachment between the baseline visit and the 6 month visit Sites were considered to have responded well to therapy if they gaine d at least 2 mm in clinical attachment from the baseline visit to the 6 month visit Sites were considered to have not responded well to therapy if the probing depth was initially at least 5 mm with at least 2 mm of CAL and had no change in probing depth o r attachment level from baseline to 6 months Clinical Procedures The medical and dental histories of the potential subjects were evaluated and a periodontal evaluation was completed. All subjects unde rwent an informed consent process before being enrolle d in the study. Eligible subjects had clinical parameters evaluated including probing depth, plaque index 50 and gingival margin position and plaque samples taken at baseline. Diseased sites rec eive d scaling and root planing treatment. Subjects were re examined at 3 months and 6 months, and the selected sites were re sampled 6 months post scaling and root planing. Subgingival biofilms were sampled using sterile endodontic points. Each sample was transferred to a sterile tube containing sample buffer (MO BIO, Carlsbad, CA), placed immediately on ice, and stored at 80C until processing for DNA extraction and microbiome analysis.

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19 Sample Storage and Preparation Subgingival specimens were collected f rom assigned pockets longitudinally according to the clinical sampling protocols and standards in the NIH Roadmap Human Microbiome Project 51 Plaques and b iofilm on supragingival surfaces were removed with sterile gauze prior to the collection of subgingival specimens. Subgingival biofilm was sampled using sterile endodontic points. Each sample was transferred to a sterile tube containing sample buffer (MO B IO, Carlsbad, CA), placed immediately on ice, and stored at 80C until processing. All specimens were coded. Sample Analysis DNA was extracted from subgingival biofilm using a bead beating, solvent extraction method (UltraClean microbial DNA isolation kit, MO BIO, Carlsbad, CA). For all extractions, a saline only negative control was performed in parallel, as well as a spiked mixing control to exclude the presence of PCR inhibitors. The V1 V3 region of bacterial 16S rRNA gene se gments from extracted DNA using broad range rRNA PCR primers 27F and 534R was amplified. The reverse primer (534R) include d a barcode sequence unique to each specimen, thereby allowing PCR amplicons to be multiplexed and sequenced simultaneously. The ampli cons were then gel purified, pooled, and subjected to pyrosequencing on the Roche/454 GS FLX platfor m 52 using the new Titanium chemistry T he number of sequence reads was about 1,000,000 reads per plate with read length of ~500 nt. With DNA bar coding, about 500 specimens on a single plate were r ead, yield ing ~2,000 reads per sample, an ample amount for bacterial community analysi s 53 The 5 most prevalent species at baseline and 6 months were reported for each site.

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20 Statistical Analysis Clinical data were expressed as means and standard deviations and tested for significant differences between smokers and non smokers using Prism software. The unpaired t test was used to determine differences between the groups for the clinical parameters at the various time points. The 2 way ANOVA test was used to determine if the clinical parameters changed over the course of time. P values were accepted as

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21 CHAPTER 4 RESULTS A total of 10 subjects 5 smokers and 5 never smokers, participated in this study. Patients were either periodontally healthy or had a diagnosis of chronic periodontitis 54 All of them had plaque samples that were analyzed using deep sequencing. The demographics of the participant population are summarized in Table 4 1. Briefly, among the smokers, there were 2 males and 3 females while the never smokers were comprised of 3 males and 2 females. The average age of smokers was 51.8 4.6 years and the average age of never smokers was 42.6 19.1 years. The ages were not statistically different between the groups ( P >0.05). The smoker group was comprised of 1 African American and 4 Caucasians. The never smoker group was comprised of 1 African American, 2 Caucasians, and 2 Asians. The clinical information for the sites included in this study are found in Table 4 2. Clinical Parameters The average probing depths are summarized in Figure 4 1. Analysis of the average probing depth at baseline for smokers and never smokers was 4.559 0.475 mm and 3.5008 0.556 mm, respectively. There was no statistical difference between the groups (u n p probing depth for smokers and never smokers was 4.0122 0.602 mm and 3.1508 0.437 mm, respectively. Here there was a statistically significant difference (2 way ANOVA). Similarly, at s ix months post treatment, the average probing depth for smokers and never smokers was 4.019 0.661 mm and 3.113 0.386 mm, respectively, whereby a statistically significant difference was also observed (2 way ANOVA). Together these data suggest that the never smoker group saw a greater

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22 improvement in PD reduction compared to smokers in response to conventional treatment The average gingival margin values are summarized in Figure 4 2. The average gingival margin at baseline for smokers and never smokers prior to periodontal treatment was 1.4336 1.074 mm and 1.839 1.505 mm, respectively. Three months following therapy, the average gingival margin for smokers and never smokers was 0.8672 1.117 mm and 1.7674 1.352 mm, respectively. Six months fo llowing therapy, the average gingival margin for smokers and never smokers was 1.2364 1.224 mm and 1.7942 1.082 mm, respectively. There was no statistical difference from the baseline values (2 way ANOVA) or between the groups t est) at any time point in regards to the gingival margin values The average clinical attachment loss values are summarized in Figure 4 3. Prior to treatment, the average clinical attachment loss for smokers and never smokers was 3.1256 1.391 mm and 1.63 86 1.956 mm, respectively. At three months post treatment, the average clinical attachment loss for smokers and never smokers was 3.1446 1.556 mm and 1.3834 1.663 mm, respectively, while six months following treatment, the average clinical attachment loss for smokers and never smokers was 2.7826 1.739 mm and 1.319 1.314 mm, respectively. Again, t here was no statistical difference from the baseline values (2 way ANOVA) or between the groups (unpaired test) at any time point in regards t o the clinical attachment loss values The average plaque index values are summarized in Figure 4 4. The average plaque index prior to treatment for smokers and never smokers was 1.0444 0.366 and 0.5358 0.350, respectively. At three months post treatme nt, the average plaque index

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23 for smokers and never smokers was 0.7602 0.319 and 0.63 0.332, respectively. At six months post treatment, the average plaque index for smokers and never smokers was 0.767 0.312 and 0.6212 0.363, respectively. There was no statistical difference from the baseline values (2 way ANOVA) or between the groups test) at any time point in regards to the plaque index at any time point. Microbiological Findings In order to determine if there were differences in the microflora between smokers and never smokers under the condition of periodontal health, 2 healthy sites in smokers and 2 healthy sites in never smokers were paired to compare the microflora using deep sequencing (Table 4 3 ) Interestingly, not only were the 5 most prevalent species different between smokers and never smokers, but the 5 most prevalent species were different between the two never smokers and two smokers evaluated For instance, the health y site from o ne n ever smoker (AD site 22DF ) had a particularly high proportion of Rothia dentocariosa (0.559), with lower p r oportions of Fusobacterium nucleatum subsp. Vincentii (0. 044 ), Porphyromonas endodontalis (0. 036 ), Streptococcus mitis (0.0 3 6), and Actinomyces sp. (0.0 27) The site from the other never smoker (AH site 13MB ) had a high proportion of Prevotella nigrescens (0.177) with lower proportions of Campylobacte r gracilis (0.108) Corynebacterium matruchotii (0.05 6 ) Prevotella oris (0.055), and other Prevotella sp (0.05 2 ). While o ne of the smokers ( AA site 18DF ) also had a high proportion of Rothia dentocariosa (0.161) in the healthy site, it was not as high as the never smoker ( AD ) The other most prevalent species at this site (AA site 18DF ) were Veillonella parvula (0.187 ) Streptococcus gordonii (0. 069 ), Streptococcus intermedius (0.061 ) and

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24 Campylobacter gracilis (0.054) Again the proportion of microbes within the healthy site of the second smoking participant (AU site 27DF ) differed fro m the first (AA site 18DF ). Specifically, Streptococcus parasanguinis (0.187) was the most prevalent followed by Eikenella corrodens (0.137), Atopobium sp (0.109) Solobacteriummoorei (0.047) and Peptostreptococcus anaerobius (0.047). Tog e ther these da ta suggest that it is unclear if there is a stable subgingival biome under the conditions of health regardless of smoking status. In order to determine if there were differences in the microflora between smokers and never smokers under the condition of pe riodont itis 2 diseased sites in smokers and 2 sites in never s mokers were paired to compare the microflora using deep sequencing (Table 4 3 ) Both sites of never smokers had Veillonella parvula in the top 5 most prevalent species ( AL site 6MF was 0.057 and AL site 18DF was 0.298). The other most prevalent species of one of the never smoker diseased sites (AL site 6MF) were Streptococcus sp (0.313), Streptococcus mitis (0.238), Streptococcus oralis (0.168), and Actinomyces sp. (0.026). The other site (AL site 18DF) was comprised of Campylobacter gracilis (0.116), Prevotella veroralis (0.056), and Fusobacterium nucleatum subsp. Animalis (0.053), and Prevotella sp. (0.050). Both diseased at baseline sites of smokers had Streptococcus sp. (0.264 in AA site 19MF and 0.123 in AA site 30MF ) and Streptococcus mitis (0.143 in AA 19 site MF and 0.131 in AA site 30MF ). Other prevalent species of one of the smoker diseased sites (AA site 19MF) were Rothia mucilaginosa (0.074 ), Veillonella parvula (0.068) and Eikenella corrodens (0.038) The other diseased site from the smoker group (AA site

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25 30MF) had species Actinomyces sp (0.223), Corynebacterium matruchotii (0.085), and Streptococcus gordonii (0.077). Together, these microbiological findings of the di seased sites at baseline suggest that there is little consistency in the microbiome of diseased sites, regardless of smoking status. The only species that was fairly common in the diseased sites of both groups was Streptococcus sp. In order to determine i f there were differences in the microflora between smokers and never smokers in sites that broke down from baseline to 6 months after treatment, 2 sites in smokers and 2 sites in never s mokers that had periodontal breakdown were paired to compare the micro flora using deep sequencing (Table 4 4 ). For one of the never smoker sites (AL site 9DF), the microflora was predominated by Neisseria sp at baseline (0.369 ) and at 6 months post initial treatment ( 0.387 ) The other species of this site (AL site 9DF) had no similarities. At baseline, the other species included Streptococcus sanguis (0.130), Streptococcus mitis (0.084), Bergevella sp. (0.048), and Fusobacterium nucleatum subsp. Polymorphum (0.048). At 6 months after initial treatment, the other species included Actinomyces naeslundii (0.053), Veillonella parvula (0.035), Campylobacter gracilis (0.030), and Actinomyces sp. (0.029). The other n ever smoker site that had breakdown (AL site 25MF) had Cory nebacterium matruchotii as the most prevalent species at baseline (0.208) and at 6 months after initial treatment (0.194). For this site (AL site 25MF), Leptotrichia sp. was also seen in the top 5 most prevalent at baseline (0.108) and at 6 months after tr eatment (0.080). Furthermore, P orphyromonas catoniae was seen in the top 5 most prevalent species at baseline (0.071) and at 6 months after initial treatment (0.061). The other most prevalent species

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26 at baseline for this site (AL site 25MF) were Streptococ cus mitis (0.147) and Captnocytophaga sp. (0.069). The other most prevalent species for this site ( AL site 25MF) at 6 months after initial treatment were Actinoomyces sp. (0.075) and Fusobacterium nucleatum subsp. Polymorphum (0.063). For one of the smok er sites with periodontal breakdown (AA site 28MF) Prevotella intermedia was among the top 5 most prevalent species at baseline (0.131) and at 6 months after initial treatment (0.159). The other most common species at baseline for this site (AA site 28MF) were Streptococcus sp. (0.152), Bacteroidetes[G 2] sp. (0.101), Fusobacterium nucleatum subsp. Vincentii (0.081), and Peptostreptococcaceae[13][G 1] sp (0.081). At 6 months after initial treatment, the most common species for the site (AA site 28MF) were Synergistetes[G 3] sp. (0.089), Tannerella forsythia (0.085), Synergistetes[G 3] sp. (0.053), and Treponema lecithinolyticum (0.049). The other site in the smoker group that broke down (AR site 3F) had Aggregatibacter segnis as the most prevalent spec ies at baseline (0.096) and at 6 months after initial treatment (0.081). Also, Prevotella buccae was among the top 5 most prevalent species at baseline (0.078) and at 6 months after initial treatment (0.078). The other most prevalent species for this site (AR site 3F) at baseline were Clostridiales[F 2][G 1] sp. (0.096), Streptococcus sanguis (0.039), and Veillonella parvula (0.039). The other most prevalent species for this site (AR site 3F) at 6 months after initial treatment were Peptoniphilus lacrimalis (0.076), Peptostreptococcus anaerobius (0.074), and Parvimonas sp. (0.051). Interestingly these microbiological findings of the sites that exhibited breakdown had little consistency in the microbiome, regardless of smoking status. Sites in both

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27 group s that had breakdown did have species that persisted, but these were differen t within the smoker and never smoker groups and differe nt between the smoker and never smoker groups. All sites that exhibited breakdown for the smoker and never smoker groups had Streptococcus sp. at baseline. Actinomyces sp. was among the 5 most prevalent species at 6 months after initial treatment in both of the never smoker sites that broke down. No other single species was among the top 5 most prevalent species 6 months after periodontal treatment in any of these sites that had breakdown In order to determine if there were differences in the microflora between smokers and never smokers in sites that responded well to initial periodontal treatment, 2 sites in smokers and 2 si tes in never s mokers that responded well to periodontal treatment were paired to compare the microflora at baseline and at 6 months after initial therapy using deep sequencing (Table 4 5 ). For the never smoker group, one site (AL site 6MF) had Veillonella parvula among the top 5 most prevalent species at baseline (0.057) and at 6 months (0.091). The other most prevalent species at baseline for the site (AL site 6MF) were Streptococcus sp. (0.313), Streptococcus mitis (0.238), Streptococcus oralis (0.168), and Actinomyces sp. (0.026). The other most prevalent species at 6 months for the site (AL site 6MF) were Fusobacterium sp. (0.146), Captnocytophaga sp. (0.097), Fusobacterium nucleatum subsp. Vincentii (0.058), and Kingella oralis (0.050). The other site of the never smoker that exhibited a good response to treatment (AL site 22DL) group had Corynebacterium matruchotii among the most prevalent at baseline (0.204) and at 6 months (0.119). Also, that same site (AL site 22DL) had Capnocytophaga sp. among the most prevalent at baseline (0.192) and 6 months (0.060). The other most prevalent species at baseline were Porphyromonas catoniae

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28 (0.092), Fusobacterium nucleatum subsp. Polymorphum (0.065), and Capnocytophaga leadbetteri (0.047). At 6 months after periodo ntal therapy the other most common species were Lautropia mirabilis (0.308), Neisseria bacilliformis (0.103), and Fusobacterium sp. (0.036). Both sites had Fusobacterium sp. among the most prevalent at 6 months. One of the sites (AR site 10MF) that respon ded well to therapy in the smoker group had totally different bacteria comprising the most prevalent species at baseline and at 6 months after treatment The most prevalent species for that site (AR site 10MF) at baseline were Fusobacterium sp (0.197), Ba cteroidetes[G 2] sp. (0.131), Synergistetes[G 3] sp. (0.091), Prevotella pleuritidis [NV] (0.084), and Porphyromonas sp. (0.083). The most prevalent species at 6 months after periodontal treatment for that site (AR site 10MF) were Peptostreptococcus anaerobius (0.148), Eikenella corrodens (0.137), Aggregatibacter segnis (0.099), Parvimonas sp. (0.096), and Solobacterium moorei (0.090). The other site (AT site 28ML) that responded well to therapy had 4 repeating species in the top 5 most prevalent. Eik enella corrodens was the most prevalent at baseline (0.198) and at 6 months after treatment (0.243). Peptostreptococcus anaerobius was the second most prevalent at baseline (0.149) and at 6 months after initial treatment (0.186). Streptococcus sanguis was the third most prevalent at baseline (0.076) and the fourth most prevalent at 6 months after treatment (0.096). Solobacterium moorei was the fourth most prevalent at baseline (0.074) and fifth most prevalent at 6 months after periodontal treatment (0.050). Synergistetes[G 3] sp was also among the top 5 most prevalent at baseline (0.061). Parvimonas sp. was

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29 also among the top 5 most prevalent at 6 months after initial periodontal treatment (0.110). Interestingly these microbiological findings of the sites that exhibited a good response to periodontal therapy had minimal similarities in the microbiome at baseline and at 6 months after treatment when comparing smokers and never smokers Specifically, only Fusobacterium sp. was found in both never smokers and smokers, but this was seen at 6 months in the never smokers and at baseline in the smoker group. On the other hand, within the smoker and never smoker groups, there was some consistency. Both sites (AL site 6MF and AL site 22DL) of the never smokers that responded well to treatment had Fusobacterium sp. and Capnocytophaga sp at 6 months after periodontal treatment. Both sites of the smokers that responded well to treatment (AR site 10MF and AT site 22DL) had Peptostreptococcus anaerobius Eikenella corrode ns Parvimonas sp and Solobacterium moorei among the most prevalent species at 6 months after periodontal treatment. In order to determine if there were differences in the microflora between smokers and never smokers in sites that did not respond well to initial periodontal treatment, 2 sites in smokers and 2 sites in never s mokers treatment were paired to compare the microflora at baseline and at 6 months after initial therapy using deep sequencing (Table 4 6 ). For the never smokers, one site (AD site 13DF) had Fusobacterium nucleatum subsp. Vincentii as the most prevalent species at baseline (0.184) and at 6 months (0.278). Pophyromonas endodontalis was also seen in the top 5 most prevalent species at baseline (0.0 76) and 6 months after periodontal therapy (0.116) of that site (AD site 13DF) Prevotella tannerae was also seen among

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30 the top 5 most prevalent species at baseline (0.049) and at 6 months after periodontal treatment (0.109) of that site (AD site 13DF) Pr evotella intermedia also was seen in the top 5 most prevalent species at baseline (0.157) and at 6 months after periodontal treatment (0.046) for that site (AD site 13DF) Prevotella sp (0.046) was also seen at baseline for the site (AD site 13DF) and Ca mpylobacter gracilis (0.053) was also seen at 6 months for that site (AD site 13DF) The other site that did not respond well to therapy in the never smoker group (AL site 18DF) had Veillonella parvula as the most prevalent species at baseline (0.298) and 6 months post initial treatment (0.129). Campylobacter gracilis was the second most prevalent species at baseline (0.116) and at 6 months after periodontal treatment (0.111) in that site (AL site 18DF) Prevotella sp. was also prevalent at baseline (0.050) and at 6 months after treatment (0.064). Prevotella veroralis and Fusobacterium nucleatum subsp. Animalis were also prevalent at baseline [(0.056) and (0.053), respectively] for the site (AL site 18DF) Capnocytophag a sp. was also prevalent at 6 months after initial treatment (0.070) as was Streptococcus mutans (0.059) for that site (AL site 18DF) For the in the smoker group one site (AA site 19MF) had Streptococcus mitis among the top 5 most prevalent at baseline (0.143) and at 6 months after initial periodontal treatment (0.048). The site (AA site 19MF) also had Veillonella parvula among the top 5 most prevalent at baseline (0.068) and 6 months after treatment (0.117). Al so prevalent at baseline in the site (AA site 19MF) were Streptococcus sp. (0.264), Rothia mucilaginosa (0.074 ), and Eikenella corrodens (0.038). At 6 months after treatment the other most prevalent species were Actinomyces sp. (0.14 7), Veillonella atypica (0.132), and Prevotella oris

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31 (0.072). The other site (AT site 6MF) had the top 4 most prevalent species to be the same. Peptostreptococcus anaerobius was the most prevalent species at baseline (0.286) and 6 months after treatment (0 .293) for the site (AT site 6MF) Streptococcus sanguis was the second most prevalent species at baseline (0.229) and 6 months post initial treatment (0.227) for that site (AT site 6MF) Parvimonas sp. was the third most prevalent species at baseline (0.19 6) and 6 months after initial treatment (0.096) for the site (AT site 6MF) The site (AT site 6MF) had Peptoniphilus lacrimalis as the fourth most prevalent species a t baseline (0.078) and 6 months post treatment (0.086). Eikenella corrodens was also preva lent at baseline (0.060), and Haemophilus influenza was also prevalent at 6 months post initial treatment (0.079) for that site (AT site 6MF) Interestingly these microbiological findings of the sites that exhibited a good response to periodontal therapy had minimal similarities in the microbiome at baseline and at 6 months after treatment when comparing smokers and n ever smokers. All of the sites had bacteria that were seen both at baseline and at 6 months after treatment, but for the most part, these wer e different among th e sites. One site (AL site 18DF) o f the never smokers and one site (AA site 19MF) of the smokers had Veillonella parvula at respond well to treatment in th e never smokers had Prevotella sp. at baseline. This species was not observed in the smoker group. Also, both sites (AA site 19MF and AT site 6MF) of the smokers that did not respond well to treatment had Eikenella corrodens at baseline whic h was not obser ved in the never smoker group.

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32 Table 4 1. Exper imental g roups Smokers (5) Never Smokers (5) Males/Females 2/3 3/2 Mean Age SD 51.8 4.6 42.6 19.1 Ethnicity 4 Caucasians 1 African American 2 Caucasians 2 Asians 1 African American

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33 Table 4 2. Clinical parameters of selected s ites Subject Visit # Tooth Position PD CAL PI AD 1 22DF 3 0 0 AH 1 13MB 3 0 0 AA 1 18DF 3 0 1 AU 1 27DF 3 0 0 AL 1 6MF 5 5 0 AL 1 18DF 5 2 2 AA 1 19MF 5 2 0 AA 1 30MF 5 4 1 AL 1 9DF 2 3 0 AL 3 9DF 3 5 0 AL 1 25MF 2 2 2 AL 3 25MF 3 5 1 AA 1 28MF 4 4 1 AA 3 28MF 4 7 0 AR 1 3F 2 0 0 AR 3 3F 3 2 0 AL 1 6MF 5 5 0 AL 3 6MF 3 1 1 AL 1 22DL 5 4 1 AL 3 22DL 3 1 2 AR 1 10MF 5 4 1 AR 3 10MF 3 2 0 AT 1 28ML 6 4 1 AT 3 28ML 3 1 0 AD 1 13DF 6 10 1 AD 3 13DF 5 10 0 AL 1 18DF 5 2 2 AL 3 18DF 4 1 2 AA 1 19MF 5 2 0 AA 3 19MF 4 2 1 AT 1 6MF 7 9 2 AT 3 6MF 6 8 1

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34 Table 4 3 Proportion of organisms in healthy and diseased sites prior to treatment of never s moker and smoker periodontal patients Baseline Healthy Sites Never smokers Smokers Pt AD AH Pt AA AU Site 22DF 13MB Site 18DF 27DF Rothia dentocariosa 0.559 Prevotella nigrescens 0.177 Veillonella parvula 0.187 Streptococcusparas anguinis 0.187 Fusobacterium nucleatum subsp. Vincentii 0.044 Campylobacter gracilis 0.108 Rothia dentocariosa 0.161 Eikenella corrodens 0.137 Porphyromonas endodontalis 0.036 Corynebacterium matruchotii 0.056 Streptococcus gordonii 0.069 Atopobium sp. 0.109 Streptococcus mitis 0.036 Prevotell a oris 0.055 Streptococcus intermedius 0.061 Peptostreptococcusa naerobius 0.047 Actinomyces sp. 0.027 Prevotella sp. 0.052 Campylobacter gracilis 0.054 Solobacteriummoore i 0.047 Diseased Sites Never smokers Smokers Pt AL AL Pt AA AA Site 6MF 18DF Site 19MF 30MF Streptococcus sp. 0.313 Veillonella parvula 0.298 Streptococcus sp. 0.264 Actinomyces sp. 0.223 Streptococcus mitis 0.238 Campylobacter gracilis 0.116 Streptococcus mitis 0.143 Streptococcus mitis 0.131 Streptococcus oralis 0.168 Prevotella veroralis 0.056 Rothia mucilaginosa 0.074 Streptococcus sp. 0.123 Veillonella parvula 0.057 Fusobacterium nucleatum subsp. Animalis 0.053 Veillonella parvula 0.068 Corynebacterium matruchotii 0.085 Actinomyces sp. 0.026 Prevotella sp. 0.050 Eikenella corrodens 0.038 Streptococcus gordonii 0.077

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35 Table 4 4. Proportion of organisms in sites that displayed periodontal breakdown in never smoker and smoker periodontal patients Never smokers Pt AL AL Pt AL AL Site 9DF 9DF Site 25MF 25MF Visit 1 3 Visit 1 3 Neisseria sp. 0.3 69 Neisseria sp. 0.387 Corynebacterium matruchotii 0.208 Corynebacterium matruchotii 0.194 Streptococcus sanguis 0. 130 Actinomyces naeslundii 0.053 Streptococcus mitis 0.147 Leptotrichia sp. 0.080 Streptococcus mitis 0.0 84 Veillonella parvula 0.035 Leptotrichia sp. 0.108 Actinomyces sp. 0.075 Bergeyella sp. 0.0 48 Campylobacter gracilis 0.030 Porphyromonas catoniae 0.071 Fusobacterium nucleatum subsp. Polymorphum 0.063 Fusobacterium nucleatum subsp. Polymorphum 0.0 48 Actinomyces sp. 0.029 Capnocytophaga sp. 0.069 Porphyromonas catoniae 0.061 Smokers Pt AA AA Pt AR AR Site 28MF 28MF Site 3F 3F Visit 1 3 Visit 1 3 Streptococcus sp. 0.152 Prevotella intermedia 0.159 Aggregatibacter segnis 0.096 Aggregatibacter segnis 0.081 Prevotella intermedia 0.131 Synergistetes[G 3] sp. 0.089 Clostridiales[F 2][G 1] sp. 0.096 Prevotella buccae 0.078 Bacteroidetes[G 2] sp. 0.101 Tannerella forsythia 0.085 Prevotella buccae 0.078 Peptoniphilus lacrimalis 0.076 Fusobacterium nucleatum subsp. Vincentii 0.081 Synergistetes[G 3] sp. 0.053 Streptococcus sanguis 0.039 Peptostreptococcus anaerobius 0.074 Peptostreptococc aceae[13][G 1] sp. 0.081 Treponema lecithinolyticum 0.049 Veillonella parvula 0.039 Parvimonas sp. 0.051

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36 Table 4 5. Proportion of organisms in diseased sites that responded well to treatment in the course of treatment in never smoker and smoker periodontal patients Never smokers Pt AL AL Pt AL AL Site 6MF 6MF Site 22DL 22DL Visit 1 3 Visit 1 3 Streptococcus sp. 0.313 Fusobacterium sp. 0.146 Corynebacterium matruchotii 0.204 Lautropia mirabilis 0.308 Streptococcus mitis 0.238 Capnocytophaga sp. 0.097 Capnocytophaga sp. 0.192 Corynebacterium matruchotii 0.119 Streptococcus oralis 0.168 Veillonella parvula 0.091 Porphyromonas catoniae 0.092 Neisseria bacilliformis 0.103 Veillonella parvula 0.057 Fusobacterium nucleatum subsp. Vincentii 0.058 Fusobacterium nucleatum subsp. Polymorphum 0.065 Capnocytophaga sp. 0.060 Actinomyces sp. 0.026 Kingella oralis 0.050 Capnocytophaga leadbetteri 0.047 Fusobacterium sp. 0.036 Smokers Pt AR AR Pt AT AT Site 10MF 10MF Site 28ML 28ML Visit 1 3 Visit 1 3 Fusobacterium sp. 0.19 7 Peptostreptococc us anaerobius 0.148 Eikenella corrodens 0.198 Eikenella corrodens 0.243 Bacteroidetes[G 2] sp. 0.13 1 Eikenella corrodens 0.137 Peptostreptococcus anaerobius 0.149 Peptostreptococcu s anaerobius 0.186 Synergistetes[G 3] sp. 0.09 1 Aggregatibacter segnis 0.099 Streptococcus sanguis 0.076 Parvimonas sp. 0.110 Prevotella pleuritidis [NV] 0.08 4 Parvimonas sp. 0.096 Solobacterium moorei 0.074 Streptococcus sanguis 0.096 Porphyromonas sp. 0.08 3 Solobacterium moorei 0.090 Synergistetes[G 3] sp. 0.061 Solobacterium moorei 0.050

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37 Table 4 6 Proportion of organisms in diseased sites with a lack of significant improvement over the course of disease in never smoker and smoker periodontal patients Never smokers Pt AD AD Pt AL AL Site 13DF 13DF Site 18DF 18DF Visit 1 3 Visit 1 3 Fusobacterium nucleatum subsp. Vincentii 0.184 Fusobacterium nucleatum subsp. Vincentii 0.278 Veillonella parvula 0.298 Veillonella parvula 0.129 Prevotella intermedia 0.157 Porphyromonas endodontalis 0.116 Campylobacter gracilis 0.116 Campylobacter gracilis 0.111 Porphyromonas endodontalis 0.076 Prevotella tannerae 0.109 Prevotella veroralis 0.056 Capnocytophaga sp. 0.070 Prevotella tannerae 0.049 Campylobacter gracilis 0.053 Fusobacterium nucleatum subsp. Animalis 0.053 Prevotella sp. 0.064 Prevotella sp. 0.046 Prevotella intermedia 0.046 Prevotella sp. 0.050 Streptococcus mutans 0.059 Smokers Pt AA AA Pt AT AT Site 19MF 19MF Site 6MF 6MF Visit 1 3 Visit 1 3 Streptococcus sp. 0.264 Actinomyces sp. 0.147 Peptostreptococcus anaerobius 0.286 Peptostreptococcus anaerobius 0.293 Streptococcus mitis 0.143 Veillonella atypica 0.132 Streptococcus sanguis 0.229 Streptococcus sanguis 0.227 Rothia mucilaginosa 0.074 Veillonella parvula 0.117 Parvimonas sp. 0.196 Parvimonas sp. 0.096 Veillonella parvula 0.068 Prevotella oris 0.072 Peptoniphilus lacrimalis 0.078 Peptoniphilus lacrimalis 0.086 Eikenella corrodens 0.038 Streptococcus mitis 0.048 Eikenella corrodens 0.060 Haemophilus influenzae 0.079

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38 Figure 4 1 P robing d epths for s moker and n ever s moker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. All selected sites in smokers (red) and never smokers (blue) were probed at the time points indicated. Note statistical difference between the groups at 3 and 6 months post initial t reatm ent. *p value > 0.05.

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39 Figure 4 2 Gingival margin for smoker and never s moker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. All selected sites in smokers (red) and never smokers (bl ue) were evaluated for the gingival margin position at the time points indicated. Note no statistical difference between the groups at any time point.

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40 Figure 4 3 Clinical attachment l oss for s moker and never s moker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. All selected sites in smokers (red) and never smokers (blue) were evaluated for the clinical attachment loss at the time points indicated. Note no statistical difference be tween the groups at any time point.

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41 Figure 4 4 Plaque i ndex for s moker and never s moker groups at baseline, 3 months post initial treatment, and 6 months post initial treatment. All selected sites in smokers (red) and never smokers (blue) were evaluated for the plaque index at the time points indicated. Note no statistical difference between the groups at any time point.

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42 CHAPTER 5 DISCUSSION Chronic periodontitis is characterized by a loss of periodontal support. It i s due to response leads to the degradation of connective tissue and junctional epithelial attachments. A periodontal pocket forms and bone resorption may follow 27 Numerous bacteria have been implicated in the periodontal disease process, and smoking is a well established risk factor for periodontal disease. It is associated with a two to eight fold increased risk for periodontal attachment and/or bone loss 31 Periodontal therapy aims to reduce the inflammatory response and improve or at least maintain clinical attachment levels. It was the intention of this investigation to compare the subgingival microflora in smokers and never smokers over the course of time in response to initial therapy of scaling and root planing Specifically, samples of the subgingival microflora of healthy sites, diseased sites, sites that responded well to treatment, sites that did not respond well to treatment, and sites that broke down over time were taken and analyzed using deep sequencing techniques. The five most prevalent species were chosen for analysis. Clinical paramete rs of smokers and never smokers were evaluated. Specifically, periodontal probing depths, gingival margin position, plaque index 50 and clinical attachment levels were analyzed. There was no dif ference between the groups in regards to the clinical parameters evaluated with one exception. A significant probing depth reduction was seen in never smokers but not in smokers. This is in accordance with previous studies 20 55 57

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43 Sites that were considered to be periodontally healthy ( probing depth of 3 mm or less and no attachment loss ) at baseline were compared between smokers (2 sites) and never smokers (2 sites). There was significant heterogeneity between the samples when smokers and never smokers were compared. Even when comparing the hea lthy sites of smokers, the 5 most prevalent species of the subgingival microflora was completely different from the other smoker. The same was true for the never smokers. The only species that was common to both groups was Rothia dentocariosa ; however, th is was only seen in one smoker and one never smoker. Other studies corroborate the findings of differences between smokers and never smokers in sites with probing depths of 3mm or less 18 58 Sites that were considered to be periodontally diseased (p robing depth of 5 6 mm and at least 2 mm of clinical attachment loss ) were also compared between smoker s (2 sites) and never smokers (2 sites). Again significant heterogeneity was observed. Three of the four sites had significant numbers of Streptococcus species; however, no single microbe was observed in all 4 periodontally diseased sites. Differences in the microflora of diseased sites in smokers and never smokers have been found in other studies a s well 59 60 An analysis of the sites of smokers and never smokers that had periodontal breakdown ( lo ss of at least 2 mm of clinical attachment ) between the baseline visit and the 6 mont h visit time frame was also done Deep sequencing revealed that a ll of these sites had bacterial species that were present at baseline and 6 months after periodontal treatment, but there were minimal similarities between the groups. The only species that was common to all four sites at baseline was Streptococcus sp. an d none were

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44 common to all four at 6 months after periodontal treatment. The microbial response to scaling and root planing has been proven in other studies to be different between smokers and never smokers 19 21 Sites that were considered to have responded well to therapy ( gain of at least 2 mm in clinical attachment from the baseline visit to the 6 month visit ) were also analyzed using deep sequencing. The sites from 2 smokers and 2 never smokers were used for analysis. At baseline, the 5 most prevalent species of never smokers and smokers were completely different with the exception of one bacterium. Interestingly, 2 of t he most prevalent species were common to both sites in never smokers 6 months after periodontal treatment. Also 4 of the 5 most prevalent species of smokers 6 months after periodontal treatment were common to both sites These findings indicate that the m icroflora of sites that have responded well to treatment is similar within the smoker and never smoker groups, but the microflora is different when smokers and never smokers are compared. Finally, the microbiota of 2 sites of smokers and 2 sites of never smokers that did not respond well to therapy (initial probing depth of at least 5 mm with at least 2 mm of CAL and no change in probing depth or attachment level from baseline to 6 months post treatment) were analyzed using deep sequencing and compared between smokers and never smokers. All sites had at least 3 of their 5 most prevalent species at baseline also present 6 months after initial therapy. Only one bacterium was observed in both the smoker and never smoker group. Veillonella parvula w as present at baseline and 6 months after treatment in one of the smoker sites and one of the never smoker sites. There was significant heterogeneity when smokers and never smokers were compared.

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45 The subgingival microbiota of smokers and never smokers has been compared and contradictory results have been found. Some studies show no difference 22 23 61 Other studies report a higher prevalence of traditional periodontal pathogens 18 59 62 Recently an article was published using pyrosequencing to compare the subgingival microflora in smokers and non smokers. These findings were compared to targeted techniques, specifically culture and qPCR. Culture and qPCR showed smokers and non smokers to have no microbial differences; however, pyrosequencing revealed numerous difference between the groups. Operational taxonomic units classified to genera Fusobacterium, Prevotella, and Selenomonas were more abundant in smo kers. Operational taxonomic units classified to genera Peptococcus and Capnocytophaga were more abundant in non smokers. They also concluded that within periodontal patients, there is a large variation in the composition of the subgingival microbiome 2 This suggests that newer, open ended techniques may be able to better describe the complexity of the periodontal microflora and detect differences that have previously not been disc overed. Numerous microbiological differences for various classifications of sites were detected between smokers and never smokers in this study. The main limitation of this study is the small sample size, so f uture studies on a larger s cale are needed to validate the findings of this investigation This will allow us to better understand the complex microbiology of the periodontal disease process. A better understanding of the etiology will allow us to render more effective treatment.

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46 LIST OF REFERENCES 1. Bergstr m J. Tobacco smoking and chronic destructive periodontal disease. Odontology 2004;92(1):1 8. 2. Bizzarro S, Loos BG, Laine ML, Crielaard W, Zaura E. Subgingival microbiome in smokers and non smokers in periodontitis: an exploratory study using traditional targeted techniques and a next generation sequencing. Journal of Clinical Periodontology 2013;40(5):483 92. 3. Parameter on chronic periodontitis with slight to moderate loss of periodontal support. Am erican Academy of Periodontology. J Periodontol 2000;71(5 Suppl):853 5. 4. Eke PI, Dye BA, Wei L, Thornton Evans GO, Genco RJ. Prevalence of periodontitis in adults in the United States: 2009 and 2010. J Dent Res 2012;91(10):914 20. 5. Sanz M, Kornman K. Periodontitis and adverse pregnancy outcomes: consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic Diseases. J Periodontol 2013;84(4 Suppl):S164 9. 6. Beck JD, Offenbacher S, Williams R, Gibbs P, Garcia R. Periodontitis: a risk fa ctor for coronary heart disease? Ann Periodontol 1998;3(1):127 41. 7. Parameter on periodontitis associated with systemic conditions. American Academy of Periodontology. J Periodontol 2000;71(5 Suppl):876 9. 8. Position paper: tobacco use and the periodo ntal patient. Research, Science and Therapy Committee of the American Academy of Periodontology. J Periodontol 1999;70(11):1419 27. 9. Bergstr m J, Preber H. Tobacco use as a risk factor. J Periodontol 1994;65(5 Suppl):545 50. 10. Preber H, Bergstr m J. The effect of non surgical treatment on periodontal pockets in smokers and non smokers. J Clin Periodontol 1986;13(4):319 23. 11. Renvert S, Dahlen G, Wikstrom M. The clinical and microbiological effects of non surgical periodontal therapy in smokers and non smokers. J Clin Periodontol 1998;25(2):153 7. 12. Kaldahl WB, Johnson GK, Patil KD, Kalkwarf KL. Levels of cigarette consumption and response to periodontal therapy. J Periodontol 1996;67(7):675 81.

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47 13. Ah MK, Johnson GK, Kaldahl WB, Patil KD, Kalkwa rf KL. The effect of smoking on the response to periodontal therapy. J Clin Periodontol 1994;21(2):91 7. 14. Ravald N, Johansson CS. Tooth loss in periodontally treated patients: a long term study of periodontal disease and root caries. J Clin Periodontol 2012;39(1):73 9. 15. Chambrone LA, Chambrone L. Tooth loss in well maintained patients with chronic periodontitis during long term supportive therapy in Brazil. J Clin Periodontol 2006;33(10):759 64. 16. Zambon JJ, Grossi SG, Machtei EE, Ho AW, Dunford R, Genco RJ. Cigarette smoking increases the risk for subgingival infection with periodontal pathogens. J Periodontol 1996;67(10 Suppl):1050 4. 17. Umeda M, Chen C, Bakker I, Contreras A, Morrison JL, Slots J. Risk indicators for harboring periodontal pat hogens. J Periodontol 1998;69(10):1111 8. 18. Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. J Clin Periodontol 2001;28(5):377 88. 19. Darby IB, Hodge PJ, Riggio MP, Kinane DF. Clinical and microbiological eff ect of scaling and root planing in smoker and non smoker chronic and aggressive periodontitis patients. J Clin Periodontol 2005;32(2):200 6. 20. Grossi SG, Zambon J, Machtei EE, Schifferle R, Andreana S, Genco RJ, et al. Effects of smoking and smoking ces sation on healing after mechanical periodontal therapy. J Am Dent Assoc 1997;128(5):599 607. 21. Haffajee AD, Cugini MA, Dibart S, Smith C, Kent RL, Jr., Socransky SS. The effect of SRP on the clinical and microbiological parameters of periodontal disease s. J Clin Periodontol 1997;24(5):324 34. 22. Darby IB, Hodge PJ, Riggio MP, Kinane DF. Microbial comparison of smoker and non smoker adult and early onset periodontitis patients by polymerase chain reaction. J Clin Periodontol 2000;27(6):417 24. 23. Bost r m L, Bergstr m J, Dahlen G, Linder LE. Smoking and subgingival microflora in periodontal disease. J Clin Periodontol 2001;28(3):212 9. 24. Pace NR, Olsen GJ, Woese CR. Ribosomal RNA phylogeny and the primary lines of evolutionary descent. Cell 1986;45(3 ):325 6. 25. Palmer C, Bik EM, Eisen MB, Eckburg PB, Sana TR, Wolber PK, et al. Rapid quantitative profiling of complex microbial populations. Nucleic Acids Res 2006;34(1):e5.

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48 26. Griffen AL, Beall CJ, Campbell JH, Firestone ND, Kumar PS, Yang ZK, et al. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing. ISME J 2012;6(6):1176 85. 27. Nanci A, Bosshardt DD. Structure of periodontal tissues in health and disease. Periodontol 2000 2006;40:11 28. 28. Gar giulo AW, Wentz FM, Orban B. Dimensions and relations of the dntogingival junction in humans. Journal of Periodontology 1961;32:261 67. 29. Vacek JS, Gher ME, Assad DA, Richardson AC, Giambarresi LI. The dimensions of the human dentogingival junction. Int J Periodontics Restorative Dent 1994;14(2):154 65. 30. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL, Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25(2):134 44. 31. Johnson GK, Guthmiller JM. The impact of cigarette smoking on periodontal disease and treatment. Periodontol 2000 2007;44:178 94. 32. Albandar JM. Global risk factors and risk indicators for periodontal diseases. Periodontol 2000 2002;29:177 206. 33. Martinez Canut P, Lorca A, Magan R. Smoking and period ontal disease severity. J Clin Periodontol 1995;22(10):743 9. 34. Dentino A, Lee S, Mailhot J, Hefti AF. Principles of periodontology. Periodontol 2000 2013;61(1):16 53. 35. Treatment of plaque induced gingivitis, chronic periodontitis, and other clinica l conditions. J Periodontol 2001;72(12):1790 800. 36. Hanioka T, Tanaka M, Ojima M, Takaya K, Matsumori Y, Shizukuishi S. Oxygen sufficiency in the gingiva of smokers and non smokers with periodontal disease. J Periodontol 2000;71(12):1846 51. 37. Haniok a T, Tanaka M, Takaya K, Matsumori Y, Shizukuishi S. Pocket oxygen tension in smokers and non smokers with periodontal disease. J Periodontol 2000;71(4):550 4. 38. Bergstr m J, Bostr m L. Tobacco smoking and periodontal hemorrhagic responsiveness. J Clin Periodontol 2001;28(7):680 5. 39. Bostr m L, Linder LE, Bergstrom J. Clinical expression of TNF alpha in smoking associated periodontal disease. J Clin Periodontol 1998;25(10):767 73.

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49 40. Kinane DF, Chestnutt IG. Smoking and periodontal disease. Crit Rev Oral Biol Med 2000;11(3):356 65. 41. James JA, Sayers NM, Drucker DB, Hull PS. Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts. J Periodontol 1999;70(5):518 25. 42. Tipton DA, Dabbous MK. Effects of nicotine o n proliferation and extracellular matrix production of human gingival fibroblasts in vitro. J Periodontol 1995;66(12):1056 64. 43. Cobb CM. Non surgical pocket therapy: mechanical. Ann Periodontol 1996;1(1):443 90. 44. Ramfjord SP, Caffesse RG, Morrison EC, Hill RW, Kerry GJ, Appleberry EA, et al. 4 modalities of periodontal treatment compared over 5 years. J Clin Periodontol 1987;14(8):445 52. 45. Lindhe J, Westfelt E, Nyman S, Socransky SS, Haffajee AD. Long term effect of surgical/non surgical treatme nt of periodontal disease. J Clin Periodontol 1984;11(7):448 58. 46. Pihlstrom BL, McHugh RB, Oliphant TH, Ortiz Campos C. Comparison of surgical and nonsurgical treatment of periodontal disease. A review of current studies and additional results after 61 /2 years. J Clin Periodontol 1983;10(5):524 41. 47. Bonito AJ, Lux L, Lohr KN. Impact of local adjuncts to scaling and root planing in periodontal disease therapy: a systematic review. J Periodontol 2005;76(8):1227 36. 48. Caffesse RG, Sweeney PL, Smith BA. Scaling and root planing with and without periodontal flap surgery. J Clin Periodontol 1986;13(3):205 10. 49. Kaldahl WB, Kalkwarf KL, Patil KD, Molvar MP, Dyer JK. Long term evaluation of periodontal therapy: II. Incidence of sites breaking down. J P eriodontol 1996;67(2):103 8. 50. Silness J, L e H. Periodontal Disease in Pregnancy. Ii. Correlation between Oral Hygiene and Periodontal Condtion. Acta Odontol Scand 1964;22:121 35. 51. Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Res 2009;19(12):2317 23.

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50 52. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al. Genome sequencing in microfabricated high density picolitre reactors. Nature 2005;437(7057):376 80. 53. Ham ady M, Knight R. Microbial community profiling for human microbiome projects: Tools, techniques, and challenges. Genome Res 2009;19(7):1141 52. 54. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodonto l 1999;4(1):1 6. 55. Mongardini C, van Steenberghe D, Dekeyser C, Quirynen M. One stage full versus partial mouth disinfection in the treatment of chronic adult or generalized early onset periodontitis. I. Long term clinical observations. J Periodontol 1 999;70(6):632 45. 56. Palmer RM, Matthews JP, Wilson RF. Non surgical periodontal treatment with and without adjunctive metronidazole in smokers and non smokers. J Clin Periodontol 1999;26(3):158 63. 57. Preber H, Linder L, Bergstr m J. Periodontal heali ng and periopathogenic microflora in smokers and non smokers. J Clin Periodontol 1995;22(12):946 52. 58. Shiloah J, Patters MR, Waring MB. The prevalence of pathogenic periodontal microflora in healthy young adult smokers. J Periodontol 2000;71(4):562 7. 59. van Winkelhoff AJ, Bosch Tijhof CJ, Winkel EG, van der Reijden WA. Smoking affects the subgingival microflora in periodontitis. J Periodontol 2001;72(5):666 71. 60. Bizzarro S, Loos BG, Laine ML, Crielaard W, Zaura E. Subgingival microbiome in smoker s and non smokers in periodontitis: an exploratory study using traditional targeted techniques and a next generation sequencing. J Clin Periodontol 2013;40(5):483 92. 61. Van der Velden U, Varoufaki A, Hutter JW, Xu L, Timmerman MF, Van Winkelhoff AJ, et al. Effect of smoking and periodontal treatment on the subgingival microflora. J Clin Periodontol 2003;30(7):603 10. 62. Gomes SC, Piccinin FB, Oppermann RV, Susin C, Nonnenmacher CI, Mutters R, et al. Periodontal status in smokers and never smokers: clin ical findings and real time polymerase chain reaction quantification of putative periodontal pathogens. J Periodontol 2006;77(9):1483 90.

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51 BIOGRAPHICAL SKETCH Dr. Leah R. Strange attended Purdue University where she graduated with highest distinction in the fall of 2005 and received a degree in Health Sciences. She attended the University of Florida College of Dentistry where she received her Doctor of Dental Medicine degree in the spring of 2011. Dr. Strange is currently completing her post doctoral residency in Periodontology at the University of Florida. Upon graduation in the summer of 2014, she will work in private practice in Ocala, Florida.


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