DNA probe detection of Eikenella corrodens, Wolinella recta and Fusobacterium nucleatum in subgingival plaque.

This cross-sectional study used species-specific DNA probes to examine subgingival plaque specimens for the presence of Eikenella corrodens, Wolinella recta, and Fusobacterium nucleatum in adults with untreated periodontitis or gingivitis and in healthy controls. W. recta and F. nucleatum were more prevalent in diseased sites from the periodontitis group when compared with the controls (81% vs 22% and 83% vs 20% respectively). E. corrodens was detected in 62% of the control sites and 81% of the periodontitis sites. Because the control sites commonly contained this organism, E. corrodens may not be useful in differentiating between health and disease. In addition, the relationship between the prevalence of W. recta and F. nucleatum and the prevalence of the established periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides intermedius and Bacteroides gingivalis, was examined. Positive detection of W. recta and F. nucleatum correlated closely with the presence of A. actinomycetemcomitans, B. intermedius and B. gingivalis. Therefore, W. recta and F. nucleatum do not appear to be unique indicators of periodontal disease.

DNA probe detection of periodontal pathogens in HIV-associated periodontal lesions.

Recent studies have shown that an atypical gingivitis and a rapidly progressive periodontal disease may be early-occurring opportunistic infections associated with human immunodeficiency virus (HIV) infection. This study examined the prevalence of selected periodontal pathogens associated with these HIV-related periodontal lesions. Subgingival plaque samples were obtained from both HIV-seronegative and HIV-seropositive homosexual men and from presumably uninfected heterosexual men. DNA probes were used to detect Actinobacillus actinomycetemcomitans, Bacteroides intermedius, Bacteroides gingivalis, Eikenella corrodens and Wolinella recta in the plaque. The healthy sites in both the seronegative and seropositive homosexual groups showed a greater prevalence of all test bacteria, except for E. corrodens, than did the heterosexual group. HIV-associated periodontitis sites showed a microbial profile qualitatively similar to that of conventional periodontitis, except that B. gingivalis was more prevalent in conventional periodontitis. In contrast, HIV-associated gingivitis sites exhibited a greater prevalence of all bacteria tested than conventional gingivitis sites. In fact, HIV gingivitis generally showed a bacterial profile similar to that of the HIV periodontitis lesions, except that W. recta was significantly more prevalent in HIV periodontitis. These data suggest that the HIV gingivitis lesion is a precursor to HIV periodontitis. Thus, early identification and prophylactic treatment of high-risk individuals may prevent the destruction of periodontal tissues.

Localization of interleukin-1 beta in human periodontal tissue.

Interleukin-1 beta (IL-1 beta) is the predominant form of IL-1 produced by macrophages. IL-1 beta possesses numerous and diverse biological activities. Several of these activities, including fibroblast proliferation, potentiation of the immune response, and stimulation of bone resorption may be of relevance to the pathogenesis of periodontal disease. This study was designed to examine the presence of IL-1 beta in human periodontal tissue. An antiserum directed against the N-terminal segment (117-131) of human IL-1 beta was used to detect IL-1 beta using immunofluorescent staining techniques. IL-1 beta positive staining cells were observed in both normal and diseased tissue and were limited to the lamina propria. Brightly staining cells were increased by almost 3-fold in periodontally diseased tissue when compared to normal tissue. Low intensity staining cells were equally distributed in the normal and diseased specimens. We propose that IL-1 beta and IL-1 beta produced by cells in periodontal tissues may be related to the pathological processes associated with periodontal disease.

DNA probes in the diagnosis of periodontal microorganisms.

The need for a rapid and sensitive microbiological assay has become necessary for both research and clinical diagnostic purposes. This need has become clear as a result of extensive documentation linking specific bacterial species and periodontal destruction. DNA probe technology provides both a sensitive and specific assay and alleviates the concern for transport of fastidious microorganisms. The DNA probe procedure includes (1) disruption of bacterial cells with denaturation of DNA, (2) immobilization of DNA onto a nitrocellulose filter, (3) blocking unbound nitrocellulose with non-specific DNA, (4) hybridization of the filter with 32P-labelled probe, (5) washing and detection of bound probe. At our laboratory, microbiological analysis with whole-genomic and cloned DNA probes has been used on thousands of plaque specimens in several large-scale research projects. In one study, levels (greater than or equal to 10(5)) of Actinobacillus actinomycetemcomitans were significantly higher in subjects under 21 yr than in subjects with adult periodontitis (over 21 yr old). In another study, the distribution of periodontal pathogens throughout the mouth was examined. High levels were selectively found at sites with probing depths of 5 mm or more and bleeding on probing, directing specimen collection to these sites. In contrast, random selection of sites for sampling was found to be a poor method for detecting high levels of pathogens. These data suggest appropriate sites for specimen collection for further research and diagnostic purposes.

DNA probe detection of key periodontal pathogens in juveniles.

Recognition of juvenile forms of periodontitis have been shown to be directly linked with specific Gram-negative rods, primarily Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius. However, clinical application of these laboratory findings have generally been restricted to the research environment. The purpose of this study was to explore the relationship of these three pathogenic species in children using the reliable and accurate new technology of DNA probes. Results indicated that the pathogenic oral flora did not differ significantly between subjects with and without gingival inflammation. Therefore, tracking of pathogens using clinical parameters of inflammation are unreliable and accurate monitoring requires microbiological testing in order to properly identify the presence or absence of pathogens.

Comparison of cultural methods and DNA probe analyses for the detection of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in subgingival plaque samples.

The purpose of this study was to compare DNA probe analyses to cultural methods for detecting three periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius, in human subgingival plaque. Subgingival sites from patients diagnosed as either healthy or showing evidence of gingivitis or juvenile or adult periodontitis were sampled using two paper points. The number of these pathogens from one paper point was determined using microbiologic media and speciated by biochemical tests. Results were then compared to bacterial numbers obtained from the other paper point using species-specific DNA probes. In 60 samples from the disease group, DNA probe analysis demonstrated 100% effectiveness in detecting A. actinomycetemcomitans and B. intermedius and 91% effectiveness in detecting B. gingivalis at culture positive levels (greater than or equal to 10(3) cells). In addition, probe assays frequently identified these pathogens in samples that were culture negative. Probe analysis revealed a better correlation between presence of a pathogen and clinical evidence of disease on an individual patient basis. In contrast, most samples taken from sites of healthy individuals showed undetectable levels of all three pathogens as determined by both techniques. These results suggest that DNA probe technology is at least equivalent and often superior to cultural methods for detecting A. actinomycetemcomitans, B. gingivalis, and B. intermedius in human subgingival plaque samples.

Synergism between parathyroid hormone and interleukin 1 in stimulating bone resorption in organ culture.

The interaction of interleukin 1 (IL-1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL-1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL-1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1 alpha and interleukin 1 beta were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL-1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL-1 may be an important mechanism controlling localized bone resorption. A role for IL-1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL-1 can produce resorptive effects by synergistic interaction with PTH.

Synergistic interactions between interleukin 1, tumor necrosis factor, and lymphotoxin in bone resorption.

Cytokines with bone-resorbing activity include IL 1 beta (pI 7), IL 1 alpha (pI 5), tumor necrosis factor (TNF), and lymphotoxin (LT). Possible interaction between IL 1 beta, the major mediator with osteoclast-activating factor (OAF) activity, and other cytokines was studied. By itself, IL 1 beta was 13-fold more potent than IL 1 alpha and 1000-fold more potent than either TNF or LT in stimulating bone resorption. Suboptimal concentrations of IL 1 beta or IL 1 alpha in combination with suboptimal concentrations of TNF or LT resulted in synergistic bone-resorptive responses (1.5 to 10 times the expected responses if their effects were additive). Synergy between either form of IL 1 and TNF or LT resulted in a twofold increase in activity of IL 1, and a 100-fold increase in activity of TNF or LT. However, even with optimal synergy, IL 1 beta remained 20-fold more potent in inducing bone resorption than TNF or LT. Because IL 1 beta is considerably more potent than TNF and LT in stimulating bone resorption either alone or under synergistic conditions, it is unlikely that TNF and LT are responsible for more than a minor proportion of the total bone-resorbing activity formerly referred to as OAF.

Evidence suggesting multiple binding sites in experimental pellicles for Streptococcus mutans JBP.

This study presents evidence suggesting that multiple binding sites exist for S. mutans JBP (serotype c) in experimental salivary pellicles formed on hydroxyapatite surfaces. Adsorption isotherms were performed using S. mutans JBP cells at concentrations ranging from 1-1000 (x 10(7) streptococci per mL to pellicles prepared from whole clarified saliva and from saliva which had been previously absorbed with JBP cells. The isotherms were analyzed using a one- and a two-site model. Adsorption of S. mutans JBP cells to pellicles formed from untreated saliva was statistically significantly better described by the two-site model, and the two classes of binding sites present had widely different affinities. Also, there were approximately one-third fewer high-affinity sites than low-affinity sites. In contrast, adsorption of S. mutans JBP cells to pellicles formed from JBP-absorbed saliva was better described by the one-site model, and the sites present were of low affinity. Thus, the absorption process appeared to remove or alter specific salivary molecules which comprise the high-affinity binding sites for S. mutans JBP cells.

Effect of monoclonal antibodies against lipoteichoic acid from the oral bacterium Streptococcus mutans on its adhesion and plaque-accumulation in vitro.

Five monoclonal antibodies directed against Streptococcus mutans strain JBP lipoteichoic acid (LTA) were characterized. They were all similarly reactive with the immunizing LTA-containing extract, with intact Strep. mutans JBP cells and with LTA purified from Lactobacillus casei. Immobilized anti-LTA antibodies removes LTA from LTA-containing extracts. The binding of antibodies to LTA was inhibited by the aqueous extract but not by the organic extract of de-acylated LTA, indicating reactivity with the polyglycerol-phosphate portion of the molecule. Antibodies were reactive with all serotypes of Strep. mutans, as well as with strains of Streptococcus salivarius, Streptococcus sanguis and L. casei, but not with LTA-negative species Streptococcus mitis or Actinomyces viscosus. Anti-LTA antibodies at doses of 0.3 or 3.0 micrograms/ml, had no effect on the adherence of Strep. mutans JBP to experimental salivary pellicles formed on hydroxyapatite, but enhanced adherence 150-300 per cent at 30 micrograms/ml. There was no effect of anti-LTA antibodies in a chemostat model which measured sucrose-dependent plaque accumulation by Strep. mutans. The results argue against a major role for LTA in Strep. mutans adherence or plaque accumulation in vitro.

Influence of sublethal antibiotic concentrations on bacterial adherence to saliva-treated hydroxyapatite.

The influence of growth in the presence of sublethal concentrations of nine antibiotics on the ability of certain potentially odontopathic bacteria to attach to saliva-treated hydroxyapatite surfaces which mimic teeth was studied. Cells of Actinomyces viscosus LY7 and S2, Bacteroides gingivalis 381, Capnocytophaga ochraceus 6, and Actinobacillus actinomycetemcomitans N27 attached in lower numbers to saliva-treated hydroxyapatite when grown in the presence of 50% of the minimum inhibitory concentration of tetracycline. Electron microscopic observations of negatively stained preparations indicated that tetracycline-grown A. viscosus LY7 cells had fewer fimbriae than did untreated cells, which may account for the impaired ability of the treated cells to attach. However, cells of Actinomyces naeslundii L13 and S4 attached in higher numbers when grown in the presence of tetracycline, clindamycin, erythromycin, chloramphenicol, or neomycin. Streptococcus mutans strains H12 and JBP also exhibited increased adherence to saliva-treated hydroxyapatite when grown in the presence of 50 or 25% of the minimum inhibitory concentration of penicillin. Thus, growth in the presence of sublethal antibiotic concentrations could increase as well as decrease the adherence of bacteria to saliva-treated hydroxyapatite. Antibiotic-grown cells of the Actinomyces strains showed enhanced hemagglutination activity, but this did not correlate with their ability to attach to saliva-treated hydroxyapatite. Sublethal concentrations of antibiotics in the growth media also affected the coaggregation reactions of several organisms; the effects were specific for one member of the coaggregation pair.

Influence of growth medium on adsorption of Streptococcus mutans, Actinomyces viscosus, and Actinomyces naeslundii to saliva-treated hydroxyapatite surfaces.

The influence of the growth medium on the ability of strains of Streptococcus mutans, Actinomyces viscosus and A. naeslundii to attach to saliva-treated hydroxyapatite (S-HA) surfaces was studied. Preliminary experiments indicated that cells of each species harvested in lag, log, and early stationary phases of growth adsorbed comparably to S-HA; thus, early stationary phase cells were used in all subsequent assays. Strains were grown in chemically defined medium (CDM), in CDM supplemented with gastric mucin or with filter-sterilized or (60)Co-irradiated saliva from human donors of blood types A, B, or O, and in Trypticase soy broth (BBL Microbiology Systems) and Todd-Hewitt broth. Adherence of S. mutans H12 to S-HA tended to vary when the streptococci were grown in saliva-supplemented CDM, but the number of cells which attached was generally within twofold of that of CDM-grown cells. Attachment of A. viscosus S2 and LY7 and of A. naeslundii S4 and L13 was generally similar when grown in CDM or in CDM supplemented with saliva, but it tended to increase for organisms grown in CDM supplemented with gastric mucin. None of the strains studied appeared to destroy the blood group reactivity of the added salivary components, and they attached equally well to HA treated with homologous or heterogous saliva from that present in the medium in which they were grown. The A. viscosus strains adsorbed in 25 to 40% higher numbers to HA treated with blood type B saliva than with type A saliva, irrespective of the medium used for growth. S. mutans H12 cells displayed alpha- and beta-glucosidase and alpha-galactosidase activity; the Actinomyces strains exhibited these activities plus beta-galactosidase when grown in all media. However, the levels of these glycoside hydrolases did not correlate with cell adsorption to S-HA. The apparent weak influence of the growth medium on attachment of S. mutans was studied further. Strains of S. mutans isolated from the saliva of five human donors were made resistant to streptomycin, grown in CDM, and then added to new saliva samples from the respective donors from which they were obtained. The in vitro-grown cells were found to attach to S-HA comparably to S. mutans cells present naturally in the saliva.