Joseph J. Zambon

Cigarette Smoking Increases the Risk for Subgingival Infection With Periodontal Pathogens

Cigarette smoking has been found to increase the risk for periodontitis. The present study examined the association between cigarette smoking and subgingival infection with periodontal pathogens to determine if smokers are more likely to be infected with certain periodontal pathogens than non-smokers. Self-reported data on 1,426 subjects, aged 25 to 74, from the Erie County Study were obtained including data on 798 subjects who were current or former smokers. Mean clinical attachment loss was used to estimate the severity of periodontal destruction. Subgingival infection with target periodontal pathogens was determined by indirect immunofluorescence microscopy. Smokers harbored significantly higher levels and were at significantly greater risk of infection with Bacteroides forsythus than non-smokers. Adjusting for disease severity, the risk of subgingival infection with B. forsythus in current smokers was 2.3 times that of former smokers or non-smokers. The relative risk of B. forsythus infection also increased 1.18 times for every category of smoking as the amount of smoking measured in packyears increased from very light to heavy. Adjusting for disease severity, Porphyromonas gingivalis was also more likely to subgingivally infect smokers than non-smokers; however, there was not a significantly higher relative risk for infection with this bacterium. The data from this study indicate that cigarette smoking increases the likelihood of subgingival infection with certain periodontal pathogens. This may partly explain the increased risk for periodontitis seen in smokers. J Periodontol 1996;67:1050-1054.

Response to Periodontal Therapy in Diabetics and Smokers

Diabetics and smokers are two patient groups at high risk for periodontal disease who also exhibit impaired wound healing and, therefore, constitute two different groups in whom the relationship between host-parasite interaction, outcome of periodontal therapy, and systemic factors is best represented. The results of two independent clinical trials involving treatment of periodontal disease in diabetics and smokers are presented. A new treatment regimen-for the management of periodontal disease associated with diabetes mellitus is proposed. This treatment approach incorporates both antimicrobial agents and pharmacological modulation of the host response. Elimination of periodontal infection and reduction of periodontal inflammation in diabetic patients resulted in a significant short-term reduction in the concentration of glycosylated hemoglobin (HbA1c ). Control of chronic infections and modulation of the host response offer a new therapeutic approach in the management of patients with both diabetes and periodontal disease. The effect of smoking on periodontal healing is also discussed. The clinical and microbiological response of smokers to non-surgical periodontal therapy is compared to non-smokers. In addition, possible mechanisms whereby diabetes mellitus and cigarette smoking increase the severity of periodontal disease are discussed. J Periodontol 1996;67:1094-1102.

The Origin of Periodontal Infections

Periodontal diseases are recognized as bacterial infections, and some forms are associated with specific organisms, such as Actinobacillus actinomycetemcomitans in juvenile periodontitis, and Bacteroides gingivalis and others in adult periodontitis. The source of the periodontal organisms, whether they are part of the indigenous or resident flora and overgrow to become opportunistic oral pathogens, or whether they are exogenous oral pathogens, is important to determine. The chain of periodontal infection, microbial agent(s) and their transmission, and host response are reviewed with respect to the role of A. actinomycetemcomitans in localized juvenile periodontitis and B. gingivalis in adult periodontitis. The present data lead us to hypothesize that some periodontal organisms may be exogenous pathogens. Prevention of periodontal diseases may be influenced by the knowledge of whether various forms are caused by opportunistic organisms or exogenous pathogens. If exogenous pathogens are responsible, prevention can be directed to intercepting transmission, thereby preventing colonization. On the other hand, if the organisms are opportunistic pathogens, prevention might be directed at interfering with initial acquisition of the flora earlier in life, as well as suppressing them to low levels consistent with health. For those exogenous periodontal infections, attempts at eradication and prevention of re-infection are likely to be effective. If the organisms are part of the indigenous flora, there is little hope of complete elimination of the organism. Criteria for distinguishing exogenous periodontal pathogens from opportunistic periodontal pathogens include the prediction that exogenous pathogens would be transient members of the oral flora associated with periodontal disease, likely to be comprised of one or a few clonal types, and intrinsically virulent. In contrast, opportunistic periodontal pathogens would likely be members of the indigenous flora and would overgrow. They would likely be comprised of many clonal types, and have an intrinsically low level of virulence.

Actinobacillus actinomycetemcomitans in the pathogenesis of human periodontal disease.

The present report reviews data implicating Actinobacillus actinomycetemcomitans in the etiology of human periodontal disease. Recent data are also presented relative to: (1) serological studies of this microorganism using monoclonal antibodies and the serodiagnosis of A. actinomycetemcomitans infections; (2) characterization of the serotype antigens; (3) studies of the serotype distribution of A. actinomycetemcomitans in extra-oral infections; and (4) examination of the correlation between A. actinomycetemcomitans colony morphology and fimbriae.

Rapid Identification of Periodontal Pathogens in Subgingival Dental Plaque: Comparison of Indirect Immunofluorescence Microscopy with Bacterial Culture for Detection of Bacteroides gingivalis*

A large body of research implicates Bacteroides gingivalis in the etiology of adult periodontitis, however, the application of this information to clinical diagnosis and treatment has been hampered by the need for a simple, rapid, and reliable means of detecting this microorganism. In the present study, indirect immunofluorescence microscopy using species specific, polyclonal antisera and a monoclonal antibody was evaluated in the clinical identification and quantitation of B. gingivalis in human subgingival dental plaque. One hundred and twenty subgingival plaque samples were obtained from predetermined sites by means of sterile paper points from 20 human subjects including 10 adult periodontitis patients and 10 periodontally normal subjects. The proportions of cultivable B. gingivalis in each sample were determined following anaerobic culture on nonselective blood agar media and selective media containing kanamycin. These results were then compared to quantitative estimates of B. gingivalis by indirect immunofluorescent microscopic evaluation of heat-fixed plaque smears. Using both immunofluorescence microscopy and bacterial culture, the present study confirms the importance of B. gingivalis in adult periodontitis previously described by culture. The organism was cultivable from 70% of the adult periodontitis patients but not from any of the normal adults. In contrast, indirect immunofluorescence microscopy detected the organism in up to 40% of the subgingival sites in 100% of the adult periodontitis patients as well as four sites in the periodontally normal subjects. The sensitivity of indirect immunofluorescence microscopy compared to culture ranged from 91 to 100% while the specificity varied from 87 to 89%. The numbers of B. gingivalis identified by immunofluorescence microscopy were directly proportional to the severity of periodontal disease as measured by clinical indices. Linear regression analysis of the immunofluorescent data suggests that the probability of a subject having adult periodontitis approaches 100% when B. gingivalis comprises 9% or more of the subgingival microflora. The present study indicates that indirect immunofluorescence microscopy using speciesspecific serodiagnostic reagents is useful in the clinical detection of B. gingivalis in human subgingival dental plaque. The data also suggests that certain cases of adult periodontitis can be diagnosed solely on the basis of this laboratory test.

Rapid Identification of Periodontal Pathogens in Subgingival Plaque: Comparison of Indirect Immunofluorescence Microscopy with Bacterial Culture for Detection of Actinobacillus actinomycetemcomitans

The sensitivity of indirect immunofluorescence microscopy using specific polyclonal or monoclonal serodiagnostic reagents for Actinobacillus actinomycetemcomitans in subgingival dental plaque ranged from 82-100% as compared with culture on selective or non-selective media. This bacterium was found in 100% of the periodontally diseased sites examined in localized juvenile periodontitis patients and was statistically related to clinical indices of periodontal disease including the Gingival Index, Plaque Index, and Pocket Depth. Indirect immunofluorescence microscopy is a useful technique for the rapid and reliable determination of A. actinomycetemcomitans in human subgingival dental plaque which may be applied to the clinical diagnosis, treatment, and monitoring of periodontitis associated with A. actinomycetemcomitans.

The Microbiology of Early-Onset Periodontitis: Association of Highly Toxic Actinobacillus actinomycetemcomitans Strains With Localized Juvenile Periodontitis

Recent studies of the dental plaque bacteria associated with the various forms of early-onset periodontitis confirm the importance of target periodontal pathogens such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia, and Porphyromonas gingivalis in these diseases. A. actinomycetemcomitans strains exhibit a wide range of variability in leukotoxin production. By virtue of a unique promoter for the leukotoxin (ltx) operon, highly leukotoxic A. actinomycetemcomitans strains (e.g., JP2) express 10- to 20-times greater levels of leukotoxin than minimally toxic strains (e.g., 652). In dot blot hybridization and polymerase chain reaction (PCR) assays, the distribution of leukotoxic A. actinomycetemcomitans was examined among 165 fresh isolates and strains from our culture collection obtained from 91 human patients and non-human primates. Highly leukotoxic A. actinomycetemcomitans strains were found in 22% of the subjects and represented 28% of the isolates examined. This is a much higher prevalence than reported in a similar survey of A. actinomycetemcomitans strains from Northern Europe. Patients harboring the highly leukotoxic strains were much younger (mean age 12.7 years) than those harboring minimally toxic A. actinomycetemcomitans (mean age 25.5 years). In addition, patients with localized juvenile periodontitis were shown to have a substantially higher prevalence of highly leukotoxic strains than healthy individuals or those with adult periodontitis. Fifty-seven percent of the localized juvenile periodontitis patients harbored these strains and 64% of the isolates obtained from these patients were highly toxic A. actinomycetemcomitans. No highly toxic strains were identified from healthy individuals or from patients with adult periodontitis. The polymerase chain reaction assay could readily identify and distinguish the ltx promoters from highly toxic and minimally toxic A. actinomycetemcomitans in whole plaque samples. These data point to the importance of specific A. actinomycetemcomitans strains, as characterized by their expression of high levels of leukotoxin, in the pathogenesis of certain types of early-onset periodontitis and, possibly, other forms of rapidly progressing periodontitis. J Periodontol 1996;67:282-290.

Oral manifestations of HIV infection in homosexual men and intravenous drug users: Study design and relationship of epidemiologic clinical, and immunologic parameters to oral lesions☆

This article describes the baseline findings from a study designed to compare the oral manifestations of HIV infection in homosexual men and intravenous drug users. Both seropositive and seronegative persons were studied. A standard examination instrument was developed to record indexes of oral disease as well as to record the presence of oral lesions. The two groups differed in terms of education, race, socioeconomic status, employment status, housing, and smoking experience. The prevalence and type of oral lesions differed in the two seropositive groups. In seropositive homosexual men, white lesions on the tongue (28.4%) predominated; whereas for the seropositive intravenous drug users, oral candidiasis (43.0%) and gingival marginal erythema (33.3%) were most often detected. We also observed that seronegative intravenous drug users displayed a greater number of oral lesions than seronegative homosexual men. For seropositive homosexual men, lesion presence was significantly associated with decreased levels of CD4; positive associations were seen with current smoking, antiviral drug use, and antibiotic use, and a negative association was observed with current employment. In contrast, only exposure to antiviral drugs was significantly correlated with lesion presence for seropositive intravenous drug users. This baseline analysis from our longitudinal study suggests clear differences in oral manifestations of HIV infection between seropositive homosexual men and intravenous drug users and between seronegative homosexual men and intravenous drug users. Among other parameters, it is apparent that lifestyle, access to health care, and the condition of the oral cavity before infection influence the development of oral lesions in persons with HIV infection.

Effects of Ascorbic Acid Depletion and Supplementation on Periodontal Health and Subgingival Microflora in Humans

This study describes the relationship between varying ascorbate intake, periodontal status, and subgingival microflora as part of a multidisciplinary investigation of ascorbic acid (AA) metabolism in young men housed for 13 weeks in a nutrition suite that provided controlled periods of ascorbic acid depletion and repletion. Twelve medically healthy non-smoking men, aged 25 to 43 years, ate a rotating four-day diet adequate in all nutrients except ascorbic acid. Following an initial baseline period during which the subjects received 250 mgAA/day, the subjects received 5 mgAA/day for a 32-day depletion period. Eight of the 12 subjects participated in a subsequent 56-day repletion period designed to replace the reduced body AA pool slowly. Plasma and leukocyte ascorbate levels, Plaque Index, Gingival Index, probing depths, and attachment level were monitored at the beginning and end of the depletion and repletion periods. Subgingival plaque samples were obtained and examined for selected organisms by indirect immunofluorescence microscopy. A uniform oral hygiene program was reinforced after each examination. Ascorbate concentrations in plasma and leukocytes responded rapidly to changes in vitamin C intake. There were no significant changes in plaque accumulation, probing pocket depth, or attachment level during the study. In contrast, gingival bleeding increased significantly after the period of AA depletion and returned to baseline values after the period of AA repletion. However, no relationship could be demonstrated between either the presence or proportion of target periodontal micro-organisms and measures of gingival bleeding or ascorbate levels.

Arylaminopeptidase Activities of Oral Bacteria

Protease and peptidase enzymes are thought to play a role in the virulence of many oral organisms, especially those associated with periodontal diseases. In order to evaluate the peptidases of periodontopathogens, we compared the arylaminopeptidase activities of Bacteroides gingivalis with those of other oral and non-oral bacteria. Sixty-three bacterial strains representing the prominent cultivable organisms in human periodontal pockets were tested, including representatives of the black-pigmented Bacteroides, Actinobacillus, Actinomyces, Campylobacter, Capnocytophaga, Eikenella, Fusobacterium, Haemophilus, Lactobacillus, Streptococcus, and Veillonella species. Each micro-organism was examined for its ability to hydrolyze 18 synthetic substrates of β-naphthylamide derivatives of amino acids, dipeptides, and tripeptides. Quantitation of the enzyme activity was accomplished by colorimetric measurement of the amounts of released β-naphthylamines. N-CBz-glycyl-glycyl-L-arginine-β-naphthylamide was readily cleaved by B. gingivalis, but slightly or not at all by the other oral strains tested. L-arginine-β-naphthylamide was cleaved by B. gingivalis, Capnocytophaga species, and Streptococcus species, but not readily by the other Bacteroides strains. Some dipeptide substrates tested, such as glycyl-L-arginine- and glycyl-L-proline-β-naphthylamide, were strongly cleaved by B. gingivalis and weakly cleaved by other Bacteroides strains. Since high levels of N-CBz-glycyl-glycyl-L-arginyl-aminopeptidase activity are characteristic of B. gingivalis, its measurement may be valuable in the identification of this organism in clinical samples as an aid in diagnosis and monitoring of periodontal infections. Furthermore, this and other aminopeptidases produced by B. gingivalis and other oral organisms may play a role in the tissue destruction seen in periodontal disease.