Identification of periodontopathic bacteria in gingival tissue of Japanese periodontitis patients

INTRODUCTION: The identification of invading periodontopathic bacteria in tissues is important to determine their role in the pathogenesis of periodontal disease. The objective of this study was to identify periodontopathic bacteria in diseased gingival tissue of periodontitis patients. METHODS: Subgingival plaque and gingival tissue were collected from 32 generalized chronic periodontitis (CP), 16 generalized aggressive periodontitis (GAgP) and eight localized aggressive periodontitis (LAgP) patients. Detection frequencies and quantities of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Tannerella forsythensis were investigated by polymerase chain reaction. The prevalences of Streptococcus oralis and Streptococcus sobrinus were also examined and the distribution of A. actinomycetemcomitans serotypes was observed. RESULTS: P. gingivalis and T. forsythensis were detected in approximately 70% of tissue samples and 50% of plaque samples in the three periodontitis groups. Prevalence of A. actinomycetemcomitans in tissue samples was higher in the LAgP (63%) group than in either the CP (16%) or the GAgP (38%) group. A. actinomycetemcomitans serotype c was detected in 50% of LAgP patients. Detection frequencies of S. oralis and S. sobrinus were markedly low in both plaque and tissue samples from all three periodontitis groups. Amounts of P. gingivalis, A. actinomycetemcomitans and T. forsythensis in the tissue samples were not different among the three periodontitis groups. CONCLUSION: P. gingivalis, A. actinomycetemcomitans and T. forsythensis can localize in diseased gingival tissue and may be involved in periodontal tissue destruction. Serotype c is the predominant serotype of A. actinomycetemcomitans in Japanese LAgP patients.     

Effects of endotoxins from periodontopathic bacteria on the collagen metabolism of cultured normal human gingival fibroblasts

The present study was performed to investigate the effects of endotoxins from periodontopathic bacteria on collagen metabolism. Endotoxins were extracted from Bacteroides gingivalis 381 and Bacteroides intermedius ATCC 25611 using the hot-phenol method. A commercially available endotoxin from Escherichia coli 0111: B4 was also used as a control. Human gingival fibroblasts (Gin-1, ATCC CRL 1292) were maintained with DMEM containing 10% FBS. When the fibroblasts became confluent they were exposed to each of the endotoxins in various concentrations (0, 5, 10, 15, 20 micrograms/ml). The effects of these endotoxins on the collagen metabolism of the fibroblasts were assessed on the basis of collagen synthesis and collagenase activity. The former was assessed by 3H-proline incorporation and bacterial collagenase digestable protein. The latter was assessed by fibril assay. The results were as follows: There was no change in glucose consumption, cell viability or morphology under the light microscope when the concentration of endotoxin was 20 micrograms/ml or less. 3H-proline incorporation into protein and collagen synthesis were inclined to decrease. Endotoxin from B. gingivalis resulted in an acceleration of collagenase activity. There findings indicate that the endotoxins from these bacteria might affect collagen metabolism early in gingivitis in vivo.     

Identification of bacteria using DNA probes

Since the microbial specificity of periodontal diseases is well established, having a valid microbial diagnostic test is essential for a correct and a coherent treatment planning. DNA probe will be used to identify and quantify the oral pathogens, most commonly associated with periodontitis. This test utilizes innovative DNA probe technology to identify unique segments of DNA in each of the following bacteria: Bacteroides gingivalis, Bacteroides intermedius, Actinobacillus actinomycetemcomitans, Wollinella recta, Fusobacterium nucleatum and the spirochete, Treponema denticola.     

The relationship between physiologic halitosis and periodontopathic bacteria of the tongue and gingival sulcus

To determine the influence of oral status on halitosis, the relationship between halitosis and periodontopathic bacteria present in plaque on the tongue and the subgingival sulcus was examined in 62 periodontally healthy adults. Halitosis indicators used were the organoleptic score; gas chromatography results [total volatile sulfur compounds (VSCs) = H₂S + CH₃SH + (CH₃)₂S]; Halimeter values; and the results of three clinical tests, plaque control record (PlCR), plaque index (PlI), and tongue coat status. Significant correlations with organoleptic scores was observed for PlCR, PlI, tongue coat status, VSC amounts, and Halimeter values, indicating that halitosis in periodontally healthy subjects tended to originate from tongue plaque deposits. Polymerase chain reaction analysis was used to detect six periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Treponema denticola) from the tongue and subgingival plaque. Significant effects on the organoleptic scores, tongue coat status, total VSC, H₂S and CH₃SH amounts, and Halimeter values were observed only for T. denticola and F. nucleatum and only in the tongue plaque, not in the subgingival plaque. Thus, therapies developed to inhibit the growth of these bacteria may lead to future treatments of halitosis.     

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.     

Multi-center clinical evaluation of a chairside method for detecting certain periodontopathic bacteria in periodontal disease

The association of bacteroides gingivalis, Bacteroides forsythus, Treponema denticola, and Actinobacillus actinomycetemcomitans among others with periodontal disease offers the opportunity for the development of diagnostic tests that are based upon the detection and/or quantification of one or more of these organisms or their by-products in the plaque. Three of the putative periodontal pathogens namely, T. denticola, B. gingivalis, and B. forsythus, can hydrolyze the synthetic trypsin substrate, N-benzoyl-DL-arginine-2-naphthylamide (BANA) forming a color reaction. The present investigation evaluated a commercially developed solid state assay for BANA hydrolysis that can be read after 15 minutes incubation at chairside. A total of 702 subgingival plaque samples were collected from 117 patients seen at four university dental clinics and placed on reagent cards. The color development on the cards was compared to the presence of T. denticola and B. gingivalis in the plaque, and with the clinical appearance of the sampled sites. This multi-center study demonstrated that antibodies to B. gingivalis and T. denticola could detect these organisms by an ELISA in the majority of the subgingival plaque samples. Comparable information could be obtained when the same plaques were evaluated by the reagent card format for BANA hydrolysis. The ELISA and reagent card were comparable in their ability to distinguish between clinically healthy and diseased sites. Both diagnostic procedures detected the periodontopathogens in plaques from sites that were judged clinically healthy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hen egg-white lysozyme inhibits biological activities of lipopolysaccharides from periodontopathic bacteria

Lysozyme has a bactericidal activity for some strains of Gram-positive bacteria, by enzymatic cleavage of peptidoglycans that constitute the cell wall. Hen egg-white lysozyme (HEL) was tested in vitro for effects on biological activities of lipopolysaccharides from periodontopathic Gram-negative bacteria, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromonas gingivalis . HEL inhibited a wide range of activities of these lipopolysaccharides, including activation of Limulus amoebocyte lysate, stimulation of human leukocytes to secrete tumour necrosis factor-alpha, polyclonal activation of mouse B cells, and promotion of osteoclastic differentiation in mouse bone marrow cultures. The anti-endotoxic activity of HEL may be worthy of being intended for periodontal therapy.     

The prevalence of BANA-hydrolyzing periodontopathic bacteria in smokers

Smoking has been identified as a risk factor for development of periodontal disease and a strong indicator for treatment failure in periodontal patients. This study examined 172 patients categorized as current smokers (n=55), previous smokers (n=38) or individuals that had never smoked (n=79). A total of 670 interproximal plaques collected with a wooden toothpick were analyzed for hydrolysis of the synthetic trypsin substrate benzoyl-DL-arginine naphthylamide (BANA). About 95% of the BANA hydrolysis by plaque is due to the presence of one or more of the periodontopathogens, P. gingivalis, T. denticola or B. forsythus. Gingival health was measured using the papillary bleeding score (PBS). Current smokers had less gingival bleeding than previous smokers or those who had never smoked (20% versus 41% and 25%, respectively). Plaque removed from non-bleeding sites in current smokers were 11x more likely to have a positive BANA reaction when compared to plaque removed from non-bleeding sites in individuals who never smoked. A significant positive relationship exists between smoking and colonization by the BANA periodontopathogens. Smoking may select for these periodontopathic species in the plaque and may be one reason why smoking is a risk factor in periodontal disease development.     

Local Shwartzman activity of lipopolysaccharides from several selected strains of suspected periodontopathic bacteria

Lipopolysaccharides (LPSs) were isolated from Fusobacterium species, Bacteroides species, Actinobacillus actinomycetemcomitans and Escherichia coli by the phenol water procedure, and their capacity to induce the local Shwartzman reaction was determined. Preparatory and provoking injections of homologous LPS preparations caused LPSs from E. coli and F. nucleatum to induce a hemorrhagic necrosis, indicating a typical Shwartzman reaction. On the other hand, LPSs from A. actinomycetemcomitans and B. gingivalis induced a weak inflammatory lesion, but no necrotic lesion. The provoking activities of LPSs from F. nucleatum and A. actinomycetemcomitans in inducing an inflammatory lesion were comparable to that of LPS from E. coli , but the activity of LPS from B. gingivalis was low. The preparatory activities of LPSs from B. gingivalis and B. oralis in inducing an inflammatory lesion were comparable to those of LPSs from F. nucleatum, F. necrophorum and A. actinomycetemcomitans .     

Prevalence of periodontopathic bacteria in aggressive periodontitis patients in a Chilean population

BACKGROUND: Actinobacillus actinomycetemcomitans is considered a major etiologic agent of aggressive periodontitis (AgP). Other periodontopathic bacteria such as Porphyromonas gingivalis are also suspected of participating in aggressive periodontitis although the evidence to support this is controversial. The aim of the present study was to determine the prevalence of eight periodontopathic bacteria in Chilean patients with AgP. METHODS: Subgingival plaque samples were collected from 36 aggressive, 30 localized, and six generalized periodontitis patients. Samples from 17 advanced chronic periodontitis (CP) patients were taken as controls. Samples collected from the four deepest periodontal pockets in each patient were pooled in prereduced transport fluid (RTF) and cultured. Periodontal bacteria were primarily identified by colony morphology under stereoscopic microscope and rapid biochemical tests. The identity of some bacterial isolates was confirmed by colony polymerase chain reaction (PCR). RESULTS: AgP showed a significatively higher prevalence of C. rectus than CP (P = 0.036). The only statistical difference found was for C. rectus. Patients with AgP showed a higher, but not statistically significant, prevalence of P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. A similar prevalence in both groups of patients was observed for F. nucleatum and P. intermedia/nigrescens, and A. actinomycetemcomitans was less prevalent in AgP than CP patients. In localized AgP, P. intermedia/nigrescens, E. corrodens, F. nucleatum, and P. micros were the more prevalent pathogens in contrast to generalized AgP patients who harbored A. actinomycetemcomitans, P. gingivalis, and Capnocytophaga sp. as the most prevalent bacteria. CONCLUSIONS: C. rectus, P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. were the most predominant periodontopathic bacteria of AgP in this Chilean population, but the only statistical difference found here between AgP and CP was for C. rectus, suggesting that the differences in clinical appearance may be caused by factors other than the microbiological composition of the subgingival plaque of these patients. In this study, the prevalence of A. actinomycetemcomitans was much lower than that of P. gingivalis.