In vivo induction of proinflammatory cytokines in mouse tissue by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans

Periodontitis is a chronic inflammatory disease initiated by a multitude of bacteria. Persistent infection leads to generation of various inflammatory mediators, resulting in tissue destruction and osteoclastic resorption of the alveolar bone. This study describes a novel in vivo murine calvarial model to assess the effects of oral pathogens on the expression of three proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha] which are involved in bone resorption. We chose Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans as prototype oral pathogens. We also tested the effects of Streptococcus gordonii, an oral commensal supragingival microorganism, considered a non-pathogen. Live bacteria were injected into subcutaneous tissue overlying the parietal bone of mice calvaria for 6 days. At the end of the experimental period, tissues overlying the calvaria were removed and analyzed for proinflammatory cytokine expression by Northern blotting. Cytokine mRNA was not detected in the tissue over the calvaria of control animals. In contrast, P. gingivalis and A. actinomycetemcomitans elicited mRNA expression of all three cytokines, TNFalpha being the highest (TNFalpha > > IL-1beta > IL-6). P. gingivalis was more potent than A. actinomycetemcomitans in inducing cytokine expression. In contrast, S. gordonii induced only low levels of mRNA for IL-1beta and TNFalpha but no IL-6 mRNA induction. These results suggest that oral microorganisms with access to host tissues elicit a battery of proinflammatory cytokines. There were clear differences in profiles and, interestingly, a commensal bacterium also stimulated bone resorptive cytokine expression in host tissues.     

Interactions between periodontopathogenic bacteria and cytokines

Cytokines produced in response to plaque bacteria clearly play a key role in the periodontal diseases. However, we know very little about the interactions between cytokines and periodontopathogenic bacteria. The aims of this study were to determine whether the key pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-6 could affect the growth of Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis and to determine whether these organisms could hydrolyse IL-1β, IL-6 or the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Culture medium containing up to 100 ng/ml of IL-1β or IL-6 was inoculated with A. actinomycetemcomitans (serotypes a, b and c) or P. gingivalis and growth was monitored by measuring changes in electrical conductivity every 3 min for up to 48 h. IL-1β, IL-6 or IL-1ra were added to culture supernatants and incubated for up to 24 h. Samples were taken at various times, analysed by SDS-PAGE and the separated proteins transferred by Western blotting to PVDF membranes and probed with anticytokine antibodies. None of the cytokines tested had any effect on the rate of growth or yield of A. actinomycetemcomitans or P. gingivalis . Supernatants from P. gingivalis cultures, but not those from A. actinomycetemcomitans , hydrolysed IL-1β, IL-6 and IL-1ra. The hydrolysate from the P. gingivalis supernatant-treated IL-1β was unable to stimulate the release of IL-6 from human gingival fibroblasts showing that it had lost biological activity. These results suggest that P. gingivalis can perturb the cytokine network, not only by stimulating the release of cytokines from host cells, but also by removing them from its local environment.     

The relationship between gingivitis and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in children

BACKGROUND: Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are closely associated with the onset and severity of adult periodontal disease. However, little is known regarding the colonization by, and host antibody response to, these microorganisms in children. METHODS: Plaque and sera were obtained from 40 healthy children, 2 to 18 years old. Gingival health was assessed by the periodontal disease index (PDI), papillary bleeding score (BS) and the modified total papillary margin attachment index (M-PMA). P. gingivalis and A. actinomycetemcomitans in plaque samples were detected by slot immunoblotting (SIB). Serum antibody levels against these microorganisms were evaluated using ELISA. RESULTS: More than 60% of the children had detectable levels of P. gingivalis in their plaque. Those having detectable levels had more gingival inflammation than those having none; however, these differences were significant only in children over the age of 12 years (PDI, BS). In contrast, while 75% of the children had detectable A. actinomycetemcomitans, there were significant differences in gingival inflammation associated with colonization in children from 3 to 7 years of age (PDI) and over 12 years of age (M-PMA). Serum antibody levels to P. gingivalis were inversely correlated with gingival inflammation in all age groups, while A. actinomycetemcomitans titers were positively correlated with gingival inflammation only in the children over 12 years. No significant relationship between the presence of either A. actinomycetemcomitans or P. gingivalis and antibodies to them was found. CONCLUSIONS: Our findings show that P. gingivalis and A. actinomycetemcomitans are readily detected as early as 3 years of age and that their presence is associated with the onset and severity of gingivitis.     

Differential expression of interleukin-8 and intercellular adhesion molecule-1 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis infection

Little is known regarding the molecules expressed by gingival epithelial cells that are involved in initiating and maintaining inflammation following the interaction with periodontal pathogens. Thus, we investigated the effect of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis infection on the expression of neutrophil chemoattractant interleukin 8 (IL-8) and the adhesion molecule intercellular adhesion molecule-1 by gingival epithelial cells. The data revealed that both IL-8 and intercellular adhesion molecule-1 expression increased after infection with A. actinomycetemcomitans (IL-8: 2- to 7-fold; intercellular adhesior molecule-1: 2.5- to 3.7-fold). IL-8 secretion reached a maximal level 6 h after the infection and the expression subsequently decreased to basal level. The increased cell surface intercellular adhesion molecule-1 expression started at 4 h after infection and reached a maximal level 14 h after the infection. In contrast, the expression of both molecules rapidly decreased 2 h after challenge with P. gingivalis. This opposite influence of A. actinomycetemcomitans and P. gingivalis infection on the expression of IL-8 and intercellular adhesion molecule-1 by gingival epithelial cells suggests that A. actinomycetemcomitans infection may initiate the recruitment of neutrophils, whereas the P. gingivalis infection may retard this process and therefore demonstrate a distinct perspective of virulence.     

Epithelial cell pro-inflammatory cytokine response differs across dental plaque bacterial species

Aim: The dental plaque is comprised of numerous bacterial species, which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines, which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of interleukin (IL)-1 β , IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential.
Materials and Methods: HGECs were challenged with the four bacterial species, live or heat killed, at various multiplicity of infections and the elicited IL-1 β , IL-6, IL-8 and IL-10 responses were assayed by enzyme-linked immunosorbent assay.
Results: Primary HGECs challenged with live P. gingivalis produced high levels of IL-1 β , while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory.
Conclusion: We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary HGECs that may reflect their individual virulence or commensal status.     

The host cytokine response to Porphyromonas gingivalis is modified by gingipains

Background/aims:  Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1β (IL-1β) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis , a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses.
Methods:  HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1β, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot.
Results:  We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1β but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective.
Conclusion:  We conclude that P. gingivalis , through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities.     

Intragingival occurrence of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis in active destructive periodontal lesions

A total of six active and six nonactive sites from six untreated periodontitis patients were examined for intragingival presence of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis. The active destructive periodontal disease was determined by the "tolerance method." The method of immunoperoxidase was used in the identification of intragingival microorganisms in active and nonactive periodontal sites. Light microscopic sections of gingival tissues consecutive to those with gram stain, revealing presence of bacteria (substantiated by electron microscopy), were stained with peroxidase-labeled antibodies against A. actinomycetemcomitans and B. gingivalis. B. gingivalis was found to be significantly elevated in the connective tissue of active sites when compared to nonactive sites. A statistically significant border-line difference was found between active and nonactive sites in the connective tissue invaded by A. actinomycetemcomitans. Our findings plus the well established periodontopathic potential of A. actinomycetemcomitans and B. gingivalis support the concept that these bacteria are important invasive pathogenic agents in periodontitis.     

Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates macrophages to produce interleukin-1 and tumor necrosis factor mRNA and protein

Actinobacillus actinomycetemcomitans is associated with periodontal disease in children and adults. We report that low concentrations of lipopolysaccharide (LPS) from A. actinomycetemcomitans stimulated human macrophages to increase dramatically their accumulation of mRNA coding for interleukin-1 alpha (IL-1 alpha), IL-1 beta as well as tumor necrosis factor (TNF). Protein levels of IL-1 and TNF alpha also increased. Levels of these mRNAs increased by 4-5 fold as compared with unstimulated macrophages when these cells were cultured with as little as 2 ng/ml LPS from A. actinomycetemcomitans. Polymyxin binds and blocks the action of LPS; polymyxin inhibited the ability of LPS from A. actinomycetemcomitans to increase levels of IL-1 beta mRNA. The LPS of A. actinomycetemcomitans stimulated increased levels of IL-1 beta mRNA in the presence of cycloheximide, showing that stimulation by this LPS did not require new synthesis of protein. Furthermore, dexamethasone inhibited the ability of LPS from A. actinomycetemcomitans to stimulate the accumulation of mRNA coding for IL-1 beta. A. actinomycetemcomitans is an invasive microorganism of the gingiva; high intragingival numbers correlate with sites undergoing local destruction of the periodontium. IL-1 alpha, IL-1 beta, and TNF are potent monokines that mediate inflammation and resorption of bone. Out studies suggest that macrophages migrating to these gingival sites of A. actinomycetemcomitans infection will be stimulated by LPS of A. actinomycetemcomitans to produce IL-1 alpha, IL-1 beta and TNF. These cytokines will mediate gingival inflammation and stimulate resorption of alveolar bone.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Gingival fluid IL-1 and IL-6 levels in refractory periodontitis

Abstract Selected gingival bacteria and cytokine profiles associated with patients who did not respond to conventional periodontal therapy (refractory) were evaluated. 10 subjects with a high incidence of post-active treatment clinical attachment loss (>2% sites/year lost ≥ 3 mm) were compared to 10 age-, race-, and supragingival plaque-matched patients with low post-treatment clinical attachment loss (<0.5% sites/year) relative to the following parameters at 2 sites/patient with the deepest probing depths: (1) presence of 3 selected periodontal pathogens (Actinobacillus antinomycetemcomitans, Porphyromonas gingivalis. Eikenella corrodens) in subgingival plaque as determined by selective culturing, and (2) gingival crevicular fluid (GCF) levels of 3 cytokines associated with bone resorption (IL-1 alpha, IL-1 beta, IL-6) as determined by two-site ELISA. Results indicated no significant differences in any clinical measurement (except incidence of clinical attachment loss), in the presence of any bacterial pathogen, or in GCF cytokine levels between refractory subject sites versus stable subject sites. However, when sites producing the greatest total GCF cytokine/patient were compared, sites from refractory patients produced significantly more IL-6 (30.1 ± 4.0 versus 15.4 ± 2.8 nM, p<0.01). The subgingival presence of each of the 3 bacterial pathogens was associated with elevated GCF IL-1 concentrations. These data suggest that gingival IL-1 and IL-6 production is different in response to local and systemic factors associated with periodontitis, and that IL-6 may play a role in the identification and mechanisms of refractory periodontitis.     

Differential regulation of innate immune response genes in gingival epithelial cells stimulated with Aggregatibacter actinomycetemcomitans

Background and Objective:  The gingival epithelium provides the first line of defense against colonization by periodontal pathogens, both as a physical barrier and by the production of inducible innate immune mediators such as β-defensins and pro-inflammatory cytokines. The gram-negative bacterium Aggregatibacter actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, although the bacterium is found widely in the healthy population. We hypothesized that gingival epithelial cell-derived innate immune mediators triggered in response to A. actinomycetemcomitans infection may play an important role in increased susceptibility to infection.
Material and Methods:  Primary cultures of human gingival epithelial cells were cultured in the presence of A. actinomycetemcomitans . Total mRNA was examined for the presence of innate immune markers using RT-PCR.
Results:  We show here that the mRNA levels of human β-defensin 2 and interleukin-8 are elevated by live cultures of a clinical isolate of A. actinomycetemcomitans in cultured gingival epithelial cells from healthy individuals, but not by A. actinomycetemcomitans lipopolysaccharide. Cells from a patient with localized aggressive periodontitis, however, did not respond to this bacterial stimulation. In contrast, the pro-inflammatory cytokine interleukin-19 was induced in cells from both localized aggressive periodontitis and healthy subjects. Examination of Toll-like receptors and associated adapter molecules indicated lower levels of Toll-like receptor 2 mRNA in the localized aggressive periodontitis patient-derived cells compared with cells from healthy subjects.
Conclusion:  These results suggest that a differential expression of innate immune response genes to A. actinomycetemcomitans in the gingival epithelium could be an underlying factor of susceptibility to localized aggressive periodontitis.     

Transmission of periodontal disease-associated bacteria from teeth to osseointegrated implant regions

PURPOSE: The presence of periodontopathic bacteria is a risk factor for peri-implantitis. The present study examined colonization by periodontopathic bacteria and their transmission from periodontal pockets to osseointegrated implant sulcus. MATERIALS AND METHODS: Plaque samples were collected from 105 sites in the 15 patients who participated in the study. Colonization by these bacteria was examined by polymerase chain reaction (PCR) and culture. The transmission of periodontopathic bacteria from periodontal sites of natural teeth to the implant sulcus was analyzed by pulsed field gel electrophoresis (PFGE). RESULTS: The PCR detection rates of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Treponema denticola were 80.0%, 53.3%, 46.7%, 60.0% and 40.0%, respectively. Colonizations by P gingivalis and A actinomycetemcomitans were statistically correlated with periodontal pockets and implant sulcus regions (P < .01). The PFGE patterns of the P gingivalis strains isolated from each patient were identical, but differed from those from other patients. The PFGE patterns of P intermedia strains were identical in 2 out of 3 patients. DISCUSSION: These analyses indicated that there appeared to be transmission of P gingivalis and P intermedia from the periodontal pocket to the peri-implant region. CONCLUSION: Elimination of these periodontal pathogens from the patient's oral cavity before administering dental implant treatment may inhibit colonization by these pathogens and reduce the risk of peri-implantitis.     

Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases

Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans seem to be major pathogens in advancing periodontitis in man. First, these organisms are recovered in higher prevalence and proportions from progressive periodontitis lesions than from quiescent periodontal sites. Second, antibody levels against B. gingivalis and A. actinomycetemcomitans are markedly elevated in serum and gingival crevice fluid of periodontitis patients compared to normal controls. Third, B. gingivalis and B. intermedius elaborate potent proteases and A. actinomycetemcomitans various noxious substances which have the potential to perturb important host defenses and to disintegrate key constituents of the periodontal tissues. Monitoring these bacteria in advanced periodontal lesions may greatly assist the assessment of treatment efficacy and risk of further periodontal breakdown.     

Treponema medium glycoconjugate inhibits activation of human gingival fibroblasts stimulated with phenol-water extracts of periodontopathic bacteria

Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.     

Cyclooxygenase-2 is upregulated in inflamed gingival tissues

BACKGROUND: Increased release of prostaglandins (PG) within periodontal tissues is considered to play a pathogenetic role during periodontal disease progression. The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX). Currently there are 2 known isoforms of the enzyme. COX-1 is constitutively expressed in various tissues whereas COX-2 is an inducible enzyme believed to be responsible for PG synthesis at sites of inflammation. The purpose of this study was to compare COX-2 expression in inflamed and healthy human gingiva and further explore some of the pathogenetic mechanisms which may lead to elevated COX-2 expression in vivo. METHODS: Thirty-two gingival biopsies were obtained during routine oral surgical procedures and were processed histologically using hematoxylin and eosin to determine the degree of inflammation. Of these biopsies, 7 with low and 7 with high histological levels of inflammation were further processed immunohistochemically in order to assess the levels of COX-2 expression in situ. To explore some potential mechanisms of COX-2 upregulation, gingival connective tissue primary cell cultures were established and challenged with periodontal bacteria or proinflammatory cytokines in vitro. The levels of COX-2 expression were analyzed by Western blot of cell lysates. COX-2 activity was assessed by quantifying prostaglandin E2 (PGE2) levels in culture supernatants by competitive EIA. RESULTS: We have shown by immunohistochemistry that COX-2 expression was significantly higher (P < 0.01) in tissues with higher levels of inflammatory infiltrates. Expression of COX-2 was detected in gingival epithelium, endothelial cells as well as cells with fibroblast morphology. In vitro studies indicated that gingival fibroblasts (GF) did not express COX-2 constitutively. However, when these cells were challenged with interleukin (IL)-1 beta or bacterial cells (A. actinomycetemcomitans JP2 or B. forsythus ATCC 43037), COX-2 expression as well as COX-2 activity were upregulated. COX-2 expression was upregulated as early as 2 hours post IL-1 beta challenge and was accompanied by a sustained PGE2 release in the culture supernatants. Cyclosporin A (CsA) did not inhibit COX-2 expression induced by bacterial challenge. In contrast, NS-398, a selective inhibitor of COX-2 activity, almost completely abolished PGE2 synthesis by these cells in response to bacterial or cytokine challenge. CONCLUSIONS: We conclude that COX-2 expression is significantly upregulated in inflamed periodontal tissues. Both inflammatory cytokines such as IL-1 beta and bacterial constituents may be responsible for the enhanced COX-2 expression and PGE2 synthesis in vivo.     

Keratinocyte growth factor (KGF)-1 and -2 protein and gene expression in human gingival fibroblasts

The onset and progression of periodontal disease is associated with significant changes in the epithelial component of the attachment complex. From the early to the advanced stages of periodontal disease increased epithelial cell proliferation, migration and invasion into the surrounding connective tissue takes place. Concomitantly there is a significant increase in proinflammatory cytokine expression in periodontal tissue and quantitative and qualitative changes in the subgingival microflora, including an increase in gram-negative microorganisms. One of the most significant virulence factors of these bacteria is lipopolysaccharide (LPS) connected to the outer membrane. Two important growth factors controlling epithelial behavior are Keratinocyte Growth Factor-1 (KGF-1) and -2 (KGF-2). Connective tissue cells express these growth factors, but only epithelial cells respond to them. We studied the effect of proinflammatory cytokines and LPS on gingival fibroblast expression of KGF-1 and KGF-2 in vitro. Gingival fibroblasts were found to express KGF-1 and -2 in culture but only KGF-1 protein and gene expression was stimulated by serum, in a concentration-dependent manner by proinflammatory cytokines IL-1alpha, IL-1beta, TNF-alpha and IL-6 and LPS isolated from Porphyromonas gingivalis and Escherichia coli. The local increase in proinflammatory cytokine expression and the accumulation of LPS in disease sites may therefore stimulate gingival fibroblast expression of KGF-1. We hypothesize that this local increase in KGF-1 expression may, via a paracrine mechanism, stimulate local epithelial cell proliferation, migration and invasion during the onset and progression of periodontitis.     

Effects of smoking and treatment status on periodontal bacteria: evidence that smoking influences control of periodontal bacteria at the mucosal surface of the gingival crevice

BACKGROUND: We examined whether smoking status could influence growth of potentially pathogenic bacteria in the periodontal environment of treated and untreated periodontal patients. METHODS: We have previously reported effects of treatment status on marker bacteria in our patients. We established a history of any smoking during 6 months prior to microbiological sampling (F-ME, 16 smokers out of 64; MHM, 70 smokers out of 185). We used a commercial immunoassay to quantitate Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in paper point samples from periodontal sites. RESULTS: Logistic regression showed that in smokers, neither P. gingivalis nor A. actinomycetemcomitans was quantitatively increased, while P intermedia was somewhat increased. Multiple regression demonstrated that smoking disrupts the positive relationship between increasing probing depth and increasing bacterial growth that is found in non-smokers. In smokers, growth of marker bacteria at shallow sites (< or =5 mm) was significantly increased to the levels found at deeper sites (>5 mm) in both smokers and non-smokers. Supragingival plaque biofilm was identified as a reservoir for marker bacteria; smokers and nonsmokers had equal ranges of oral cleanliness. CONCLUSIONS: Smoking-associated periodontitis is not simply a reflection of oral cleanliness. Smoking extends a favorable habitat for bacteria such as P. gingivalis, P. intermedia, and A. actinomycetemcomitans to shallow sites (< or =5 mm). Molecular byproducts of smoking interfere with mechanisms that normally contain growth of damaging bacteria at the surface of the oral mucosa in gingival crevices. In this way, smoking can promote early development of periodontal lesions.