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1.  Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis 
Journal of Clinical Microbiology  1999;37(11):3504-3508.
Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.
PMCID: PMC85679  PMID: 10523542
2.  Are Putative Periodontal Pathogens Reliable Diagnostic Markers?▿  
Journal of Clinical Microbiology  2009;47(6):1705-1711.
Periodontitis is one of the most common chronic inflammatory diseases. A number of putative bacterial pathogens have been associated with the disease and are used as diagnostic markers. In the present study, we compared the prevalence of oral bacterial species in the subgingival biofilm of generalized aggressive periodontitis (GAP) (n = 44) and chronic periodontitis (CP) (n = 46) patients with that of a periodontitis-resistant control group (PR) (n = 21). The control group consisted of subjects at least 65 years of age with only minimal or no periodontitis and no history of periodontal treatment. A total of 555 samples from 111 subjects were included in this study. The samples were analyzed by PCR of 16S rRNA gene fragments and subsequent dot blot hybridization using oligonucleotide probes specific for Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, a Treponema denticola-like phylogroup (Treponema phylogroup II), Treponema lecithinolyticum, Campylobacter rectus, Fusobacterium spp., and Fusobacterium nucleatum, as well as Capnocytophaga ochracea. Our data confirm a high prevalence of the putative periodontal pathogens P. gingivalis, P. intermedia, and T. forsythia in the periodontitis groups. However, these species were also frequently detected in the PR group. For most of the species tested, the prevalence was more associated with increased probing depth than with the subject group. T. lecithinolyticum was the only periodontopathogenic species showing significant differences both between GAP and CP patients and between GAP patients and PR subjects. C. ochracea was associated with the PR subjects, regardless of the probing depth. These results indicate that T. lecithinolyticum may be a diagnostic marker for GAP and C. ochracea for periodontal health. They also suggest that current presumptions of the association of specific bacteria with periodontal health and disease require further evaluation.
PMCID: PMC2691128  PMID: 19386852
3.  Clinical evaluation of salivary periodontal pathogen levels by real-time polymerase chain reaction in patients before dental implant treatment 
Clinical Oral Implants Research  2014;25(1):977-982.
Periodontal pathogens in dental plaque are the main causative agents of periodontitis and peri-implantitis. Detection of the presence of such periodontal pathogens early would serve as a useful tool in the diagnosis and treatment of this disease. Therefore, the purpose of this study was to investigate whether the periodontal pathogen levels in saliva were correlated with the periodontal status of patients receiving implant treatment.
Materials and Methods
A total of 291 patients visiting Tokyo Dental College Chiba Hospital were divided into four groups: a no-periodontitis (np) group, a mild-periodontitis (mip) group, a moderate-periodontitis (mop) group, and a severe-periodontitis (sp) group. The levels of the following five periodontal pathogens in saliva were evaluated using real-time polymerase chain reaction: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Prevotella intermedia.
The levels of P. gingivalis and T. forsythia were significantly higher in mop group than in np group (P <  0.05). The levels of all periodontal pathogens tested except A. actinomycetemcomitans were significantly higher in sp group than in np group (P <  0.05).
The detection levels of the periodontal pathogens targeted in saliva samples were correlated with the periodontal status. This suggests that using saliva to screen for periodontopathic bacteria offers an easier-to-use clinical tool than the paper point method in the diagnosis and treatment of periodontitis and peri-implantitis.
PMCID: PMC4232322  PMID: 23745964
clinical research; diagnosis; microbiology; periodontology
4.  Effect of smoking on subgingival microflora of patients with periodontitis in Japan 
BMC Oral Health  2011;11:1.
Smoking is a risk factor for periodontitis. To clarify the contribution of smoking to periodontitis, it is essential to assess the relationship between smoking and the subgingival microflora. The aim of this study was to gain an insight into the influence of smoking on the microflora of Japanese patients with periodontitis.
Sixty-seven Japanese patients with chronic periodontitis (19 to 83 years old, 23 women and 44 men) were enrolled in the present study. They consisted of 30 smokers and 37 non-smokers. Periodontal parameters including probing pocket depth (PPD) and bleeding on probing (BOP) and oral hygiene status were recorded. Detection of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum/periodonticum, Treponema denticola and Campylobacter rectus in subgingival plaque samples was performed by polymerase chain reaction. Association between the detection of periodontopathic bacteria and smoking status was analyzed by multiple logistic regression analysis and chi-square test.
A statistically significant association was found between having a PPD ≥ 4 mm and detection of T. denticola, P. intermedia, T. forsythia, or C. rectus, with odds ratios ranging from 2.17 to 3.54. A significant association was noted between BOP and the detection of C. rectus or P. intermedia, and smoking, with odds ratios ranging from 1.99 to 5.62. Prevalence of C. rectus was higher in smokers than non-smokers, whereas that of A. actinomycetemcomitans was lower in smokers.
Within limits, the analysis of the subgingival microbial flora in smokers and non-smokers with chronic periodontitis suggests a relevant association between smoking and colonization by the specific periodontal pathogens including C. rectus.
PMCID: PMC3020163  PMID: 21208407
5.  The presence of cariogenic and periodontal pathogens in the oral cavity of one-year-old infants delivered pre-term with very low birthweights: a case control study 
BMC Oral Health  2014;14:109.
Recently, the dental literature has focused mainly on the microbial colonization of healthy full-term infants and their mothers or caretakers. However, oral microbial acquisition by premature infants has not been adequately investigated, and the correlation between pre-term birth and the presence of cariogenic and periodontal pathogens has not been determined. The aim of this study was to identify the presence and quantities of representative cariogenic and periodontal pathogens in the oral cavities of 12-month-old infants and compare the occurrence of these microbes between a cohort of pre-term infants with very low birthweights and a control cohort comprising full-term infants.
The research cohort was composed of 69 one-year-old infants, of whom 24 were born prematurely with very low birthweights and 45 of whom were carried to full term. Information regarding the infants’ gestational age, mode of delivery, general health status, birthweight and antibiotic use were obtained from hospital records and through oral interviews. At 12 months of age, both groups of infants were examined, and unstimulated saliva samples from the dorsum of the tongue and dental plaque samples were collected. The microorganisms (Streptococcus mutans, Lactobacillus spp., Actinomyces spp., Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Peptostreptococcus micros, Prevotella intermedia, Fusobacterium nucleatum) were identified and their quantities were evaluated using a PCR-based method. The chi-squared and Fisher’s factorial tests were used for the statistical evaluations.
The infants had a high prevalence of cariogenic microbes and of Fusosbacterium nucleatum and Aggregatibacter actinomycetemcomitans. Cariogenic microbes were detected in 91.7% of the very low birthweight infants and in all full-term infants. Periodontal pathogens were present in 83% of the pre-term infants and in 96% of the full-term infants. A significant difference was found between the cohorts in terms of the presence of S. mutans. Most of the very low birthweight infants had negative values of this microbe, while the full-term infants had positive values.
This study confirms the early transmission of representative cariogenic and periodontal pathogens to the oral cavity of one-year-old infants and a higher prevalence of S. mutans in full-term infants than in premature infants.
PMCID: PMC4236672  PMID: 25178742
Cariogenic microbes; Dental caries; Periodontal pathogens; Pre-term infants; S. mutans
6.  Evaluation of efficacy of tetracycline fibers in conjunction with scaling and root planing in patients with chronic periodontitis 
The objective of this study was to compare the efficacy of scaling and root planing (SRP) alone versus tetracycline fiber therapy used adjunctively with SRP in the treatment of chronic periodontitis sites in maintenance patients.
Materials and Methods:
A total of 30 patients with a diagnosis of chronic periodontitis (60 localized chronic periodontitis sites) in the age group of 35 to 55 were selected. None of these patients had received any surgical or non-surgical periodontal therapy and had sites of periodontal pockets measuring 4—7 mm clinically and demonstrated radiographic evidence of moderate bone loss. Two non-adjacent sites in separate quadrants were selected in each patient for monitoring based on criteria that the sites had localized chronic periodontitis. Plaque index (PI) (sillness and loe) and Gingival-bleeding index (GI) (loe and sillness) were measured at baseline and 15th, 30th, 60th, and 90thday. Clinical pocket depth (PD) and microbial analysis (MA) were analyzed at baseline and 90th day.
At 0 and 3 months adjunctive tetracycline fiber therapy was significantly better in reducing PI, GBI, (P<0.001) than S and RP alone. In comparison, the reduction in the PD was non-significant at 0 and 3 months (P<0.001). The microbial analysis showed significant reduction in Porphyromonas gingivalis and Prevotella intermedia though there was no significant reduction in the Actinobacillus actinomycetemcomitans.
Overall, these results indicate that fiber therapy significantly enhanced the effectiveness of SRP in the management of chronic periodontitis.
PMCID: PMC3498710  PMID: 23162335
Periodontitis; scaling and root planing; tetracycline fiber
7.  Association between Selected Oral Pathogens and Gastric Precancerous Lesions 
PLoS ONE  2013;8(1):e51604.
We examined whether colonization of selected oral pathogens is associated with gastric precancerous lesions in a cross-sectional study. A total of 119 participants were included, of which 37 were cases of chronic atrophic gastritis, intestinal metaplasia, or dysplasia. An oral examination was performed to measure periodontal indices. Plaque and saliva samples were tested with real-time quantitative PCR for DNA levels of pathogens related to periodontal disease (Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Actinobacillus actinomycetemcomitans) and dental caries (Streptococcus mutans and S. sobrinus). There were no consistent associations between DNA levels of selected bacterial species and gastric precancerous lesions, although an elevated but non-significant odds ratio (OR) for gastric precancerous lesions was observed in relation to increasing colonization of A. actinomycetemcomitans (OR = 1.36 for one standard deviation increase, 95% Confidence Interval = 0.87–2.12), P. gingivalis (OR = 1.12, 0.67–1.88) and T. denticola (OR = 1.34, 0.83–2.12) measured in plaque. To assess the influence of specific long-term infection, stratified analyses by levels of periodontal indices were conducted. A. actinomycetemcomitans was significantly associated with gastric precancerous lesions (OR = 2.51, 1.13–5.56) among those with ≥ median of percent tooth sites with PD≥3 mm, compared with no association among those below the median (OR = 0.86, 0.43–1.72). A significantly stronger relationship was observed between the cumulative bacterial burden score of periodontal disease-related pathogens and gastric precancerous lesions among those with higher versus lower levels of periodontal disease indices (p-values for interactions: 0.03–0.06). Among individuals with periodontal disease, high levels of colonization of periodontal pathogens are associated with an increased risk of gastric precancerous lesions.
PMCID: PMC3538744  PMID: 23308100
8.  Actinobacillus actinomycetemcomitans in Human Periodontal Disease: a Cross-Sectional Microbiological Investigation 
Infection and Immunity  1980;29(3):1013-1020.
Actinobacillus actinomycetemcomitans is a facultative gram-negative bacterium which has been associated with severe oral and nonoral infections. This study examined its occurrence in the oral cavities of 10 normal juveniles, 11 normal adults, 10 juvenile periodontitis patients, and 12 adult periodontitis patients. Four deep periodontal pockets and two normal periodontal sites were sampled in the diseased patients, and six normal periodontal sites were sampled in the healthy individuals. In all subjects samples were obtained from the cheek, tongue, and saliva. Samples from a total of 172 normal periodontal sites, 83 deep periodontal pockets, 42 cheek mucosae, 42 tongue dorsa, and 42 salivas were examined. Isolation was performed by using a medium for selective isolation of A. actinomycetemcomitans (Trypticase soy agar [BBL Microbiology Systems] supplemented with 10% serum and 75 μg of bacitracin per ml). The carrier rates were 20% for normal juveniles, 36% for normal adults, 50% for adult periodontitis patients, and 90% for juvenile periodontitis patients. A. actinomycetemcomitans was on average recovered in about fivefold-higher numbers from infected deep periodontal pockets than from infected normal subgingival areas. Samples of periodontal pockets generally contained 100-fold-more cells of A. actinomycetemcomitans than did samples of the cheek, tongue, and saliva. A. actinomycetemcomitans is commonly isolated from patients with juvenile periodontitis, often isolated from patients with adult periodontitis, and occasionally isolated from normal juveniles and adults. Its primary oral ecological niche appears to be dental plaque and periodontal pockets.
PMCID: PMC551232  PMID: 6968718
9.  Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus. 
Journal of Clinical Microbiology  1992;30(2):427-433.
Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed.
PMCID: PMC265072  PMID: 1311335
10.  Proficiencies of Three Anaerobic Culture Systems for Recovering Periodontal Pathogenic Bacteria 
Journal of Clinical Microbiology  1999;37(1):171-174.
Anaerobic culture is employed routinely in the primary isolation of periodontal pathogenic bacteria. However, little or no data exist on the relative abilities of the Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.), the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and the AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) systems to grow important periodontal species, including Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Bacteroides forsythus, Eubacterium species, Campylobacter species, Fusobacterium species, and Peptostreptococcus micros. A total of 78 specimens from advanced periodontitis lesions were collected anaerobically, plated on enriched blood agar medium, and incubated at 35°C for 5 to 7 days in each anaerobic culture system. The three culture systems were equally efficient in isolating Porphyromonas gingivalis and Prevotella intermedia/nigrescens. The Coy anaerobic chamber yielded the highest proportional recoveries of Campylobacter (P = 0.0001; nonparametric analysis of variance) and Eubacterium (P = 0.009). The Coy anaerobic chamber and the GasPak system demonstrated higher proportional recoveries of Bacteroides forsythus (P = 0.0006) and Peptostreptococcus micros (P = 0.0001) than the AnaeroPack system. The AnaeroPack system was most efficient in growing Fusobacterium species (P = 0.0001). Overall, the Coy anaerobic chamber and the GasPak system showed the highest proportional recoveries of putative periodontal pathogens, but the recoveries by the various anaerobic test systems varied considerably from sample to sample.
PMCID: PMC84198  PMID: 9854085
11.  Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells 
Infection and Immunity  2000;68(6):3140-3146.
Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.
PMCID: PMC97547  PMID: 10816455
12.  High incidence of Aggregatibacter actinomycetemcomitans infection in patients with cerebral infarction and diabetic renal failure: a cross-sectional study 
BMC Infectious Diseases  2013;13:557.
Recent epidemiological studies suggest that periodontitis is a major risk factor for renal failure and cerebral infarction. The aim of this study was to evaluate the association among periodontitis, renal failure, and cerebral infarction, focusing on microbiological and immunological features.
Twenty-one patients treated with hemodialysis (HD) were enrolled in this study. They were 8 with diabetic nephropathy and 13 with non-diabetic nephropathy. Blood examination, periodontal examination, brain magnetic resonance image (MRI), and dental radiography were performed on all patients. Subgingival plaque, saliva, and blood samples were analyzed for the periodontal pathogens, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), and Prevotella intermedia (P. intermedia) using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA).
We found that the patients with diabetic nephropathy had more A. actinomycetemcomitans compared with non-diabetic nephropathy (P = 0.038) in dental plaque. Furthermore, the patients with diabetic nephropathy showed a significantly higher incidence of cerebral infarction compared with those with non-diabetic nephropathy (P = 0.029). Clinical oral and radiographic scores tended to be higher among patients in the diabetic nephropathy group than in the non-diabetic nephropathy group.
Periodontal pathogens, particularly A. actinomycetemcomitans, may play a role, at least a part, in the development of cerebral infarction in Japanese HD patients with diabetic nephropathy.
PMCID: PMC4222637  PMID: 24267704
Periodontitis; Aggregatibacter actinomycetemcomitans; Diabetic nephropathy; Cerebral infarction
13.  A microbiological study of Papillon-Lefévre syndrome in two patients 
Journal of Clinical Pathology  2001;54(5):371-376.
Aim—To analyse the microflora of subgingival plaque from patients with Papillon-Lefévre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction.
Methods—Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies.
Results—The culture results showed that most isolates were capnophilic and facultatively anaerobic species—mainly Capnocytophaga spp and Streptococcus spp. The latter included S constellatus, S oralis, and S sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P melaninogenica, and P nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers.
Conclusions—Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.
Key Words: Papillon-Lefévre syndrome • periodontopathogens
PMCID: PMC1731428  PMID: 11328836
14.  Relationship between the Pathogenic Representatives of Periodontal Pockets Microbiocenosis in Patients with Periodontitis with Varying Degrees of Severity  
Acta Naturae  2011;3(2):99-102.
Periodontitis is a common disease that is considered to be a manifestation of the distortion of the ratio between the normal and conditionally pathogenic microflora of periodontal pockets. In this study, the ratio between the six most important periodontal pathogens and the total microflora of the periodontal pocket in healthy individuals and patients with varying severity of periodontitis was ascertained by quantitative real-time PCR. It was ascertained that the relative content ofPorphyromonas gingivalis,Prevotella intermedia, andTannerella forsythensis(Bacteroides forsythus) persistently develops in the total microflora of the periodontal pocket upon progressing periodontitis; this value is higher than that in the control group by more than two orders of magnitude upon a severe degree of chronic generalized periodontitis.
PMCID: PMC3347576  PMID: 22649688
ecosystem; habitat; periodontitis; periodontopathogenic microflora of periodontal pockets; quantitative polymerase chain reaction
15.  Higher Prevalence of Epstein–Barr Virus DNA in Deeper Periodontal Pockets of Chronic Periodontitis in Japanese Patients 
PLoS ONE  2013;8(8):e71990.
Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein–Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤3 mm (shallow) or ≥5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients.
PMCID: PMC3753341  PMID: 23991022
16.  Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis. 
Molecular Pathology  1997;50(6):291-297.
AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in periodontitis, owing to the release of proteinases from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence.
PMCID: PMC379662  PMID: 9536278
17.  Comparisons of Subgingival Microbial Profiles of Refractory Periodontitis, Severe Periodontitis and Periodontal Health using the Human Oral Microbe Identification Microarray (HOMIM) 
Journal of periodontology  2009;80(9):1421-1432.
This study compared the subgingival microbiota of subjects with refractory periodontitis (RP) to those in subjects with treatable periodontitis (GR) or periodontal health (PH) using the Human Oral Microbe Identification Microarray (HOMIM).
At baseline, subgingival plaque samples were taken from 47 periodontitis and 20 PH individuals, and analyzed for the presence of 300 species by HOMIM. The periodontitis subjects were classified as RP (n=17) based on mean attachment loss (AL) and/or >3 sites with AL ≥2.5 mm after SRP, surgery and systemically administered amoxicillin and metronidazole or as GR (n=30) based on mean attachment gain and no sites with AL ≥2.5 mm after treatment. Significant differences in taxa among groups were sought using the Kruskal Wallis and Chi-square tests.
More species were detected in diseased patients (GR or RP) than those without disease (PH). RP subjects were distinguished from GR and PH by a significantly high frequency of putative periodontal pathogens such as, Parvimonas micra, Campylobacter gracilis, Eubacterium nodatum, Selenomonas noxia, Tannerella forsythia, Porphyromonas gingivalis, Prevotella spp., Treponema spp., Eikenella corrodens, as well as “unusual” species (Pseudoramibacter alactolyticus, TM7 spp. oral taxon (OT) 346/356, Bacteroidetes spp. OT 272/274, Solobacterium moorei, Desulfobulbus sp. OT 041, Brevundimonas diminuta, Sphaerocytophaga sp. OT 337, Shuttleworthia satelles, Filifactor alocis, Dialister invisus/pneumosintes, Granulicatella adiacens, Mogibacterium tidmidum, Veillonella atypica, Mycoplasma salivarium, Synergistes sp. cluster II, Acidaminococcaceae [G-1] sp. OT 132/150/155/148/135) [p<0.05]. Species that were more prevalent in PH than in periodontitis patients included Actinomyces sp. OT 170, Actinomyces spp. cluster I, Capnocytophaga sputigena, Cardiobacterium hominis, Haemophilus parainfluenzae, Lautropia mirabilis, Propionibacterium propionicum, Rothia dentocariosa/mucilagenosa, Streptococcus sanguinis (p<0.05).
RP patients present a distinct microbial profile compared to patients in the GR and PH groups as determined by HOMIM.
PMCID: PMC3627366  PMID: 19722792
Refractory periodontitis; subgingival microbiota; periodontal pathogen; HOMIM; periodontal therapy
18.  Association of Three Bacterial Species and Periodontal Status in Chinese Adults: an Epidemiological Approach ▿  
Journal of Clinical Microbiology  2010;49(1):184-188.
Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia are oral pathogens associated with periodontitis. The association between these three bacteria and periodontal disease has been reported in populations of many countries. However, corresponding data in Chinese populations are still lacking. The aim of this study was to detect these pathogens in subgingival plaque collected from 468 subjects with chronic periodontitis in a group of Chinese adults by using a PCR method and to determine the degree of association between the target bacteria and periodontal status based on logistic regression analysis. A. actinomycetemcomitans, P. gingivalis, and T. forsythia were found in 20.5%, 70.7%, and 77.1% of the subjects, respectively. About one-third (36.1%) of subjects had chronic periodontitis. Upon univariate analysis, age, male gender, current smoking status, diabetes, and the presence of A. actinomycetemcomitans or P. gingivalis were positively associated with chronic periodontitis, whereas education and income exhibited inverse associations with chronic periodontitis. Upon multivariate analysis, education, current smoking status, diabetes, and the presence of A. actinomycetemcomitans and P. gingivalis remained significant. The adjusted odds ratios for having chronic periodontitis were 2.5 and 3.4 in subjects positive for A. actinomycetemcomitans and P. gingivalis, respectively. However, no significant association was observed between the presence of T. forsythia and periodontal status. This study assesses the prevalence of periodontal pathogens and chronic periodontitis and the associations with sociodemographic characteristics among this group of Chinese adults. These findings also suggest that PCR should be considered for field oral epidemiologic studies and may be necessary in investigations presenting major logistic challenges.
PMCID: PMC3020465  PMID: 21106792
19.  Assessment of serum antibody patterns and analysis of subgingival microflora of members of a family with a high prevalence of early-onset periodontitis. 
Infection and Immunity  1985;49(3):742-750.
In a study of members of a large family with a high prevalence of early-onset periodontitis, we sampled the subgingival microflora and characterized 40 isolates from each sample. We surveyed serum samples by enzyme-linked immunosorbent assay for antibodies reacting with any of a panel of 21 periodontal bacteria. The mother and 7 of her 13 children had early-onset periodontitis. Bacteroides gingivalis was not detected in the subgingival flora of any affected or unaffected family member, and Actinobacillus actinomycetemcomitans was isolated from only one affected child. Capnocytophaga ochracea was isolated from five of seven affected children and from none of their normal siblings. We found no significant differences among the floras from family members who had rapidly progressive, juvenile, and prepubertal forms of periodontitis. Elevated levels of serum antibody reacting with one or more of the bacteria tested were found in all family members with disease, but in only one periodontally normal family member. Both children with prepubertal periodontitis had antibodies reacting with C. sputigena, a species not found in their subgingival floras, but with none of the other bacteria tested. All remaining affected family members had antibodies to one or more serotypes of A. actinomycetemcomitans, and four had antibodies reacting with additional bacteria, including C. sputigena, Eikenella corrodens, Fusobacterium nucleatum, and Haemophilus aphrophilus. Sera from patients contained antibodies specific for putative periodontal pathogens not found in their pocket flora, and conversely, putative periodontal pathogens for which no serum antibodies were found frequently comprised a large proportion (10% or more) of the pocket flora. In no case were both the bacterium and its antibody found. These observations are suggestive of sequential infection in the early-onset forms of periodontitis and of induction of protective immunity against reinfection by the same microorganism.
PMCID: PMC261261  PMID: 4030102
20.  Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD) 
PLoS Pathogens  2013;9(9):e1003627.
Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.
Author Summary
Clinical and epidemiological data indicates that chronic periodontal disease (PD), one of the most prevalent infectious inflammatory disease of mankind, is linked to systemic inflammatory diseases such as cardiovascular diseases (CVD), rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD). Nevertheless, the causative mechanisms of association between PD and chronic inflammatory diseases are very poorly understood. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic response to citrullinated epitopes. In present study we show that infection with viable periodontal pathogen Porphyromonas gingivalis but not another oral bacterium (Prevotella intermedia), exacerbated CIA, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique enzyme peptidylarginine deiminase, which converts arginine residues in proteins to citrulline. This knowledge may create new perspectives in the treatment and prevention of RA in susceptible individuals.
PMCID: PMC3771902  PMID: 24068934
21.  Distribution of periodontopathic bacterial species in Japanese children with developmental disabilities 
BMC Oral Health  2009;9:24.
Recent developments in molecular biological techniques have enabled rapid detection of periodontopathic bacterial species in clinical specimens. Accumulated evidence suggests that detection of specific bacterial species enables identification of subjects at high risk for the onset of periodontitis. We investigated the distribution of 10 selected periodontopathic bacterial species in dental plaque specimens obtained from children with disabilities who were attending daycare centers.
A total of 187 children (136 boys, 51 girls) aged 1-6 years old and diagnosed with such disabilities as mental retardation, cerebral palsy, and autism, participated in the study. Subgingival dental plaque specimens were collected from the buccal side of the maxillary left second primary molar after a clinical examination. Bacterial DNA was extracted from the specimens and PCR analyses were carried out to detect 10 selected periodontopathic species using specific primers for each. In addition, statistical analyses were performed to analyze the correlations among clinical parameters and the detected species.
The most frequently detected species was Capnocytophaga sputigena (28.3%), followed by Aggregatibacter actinomycetemcomitans (20.9%) and Campylobacter rectus (18.2%). Eikenella corrodens, Capnocytophaga ochracea, and Prevotella nigrescence were detected in approximately 10% of the specimens, whereas Treponema denticola, Tannerella forsythia, and Prevotella intermedia were rarely found, and Porphyromonas gingivalis was not detected in any of the subjects. The total numbers of detected species were positively correlated with the age of the subjects. There were 10 subjects with positive reactions for T. denticola and/or T. forsythia, in whom the total number of bacterial species was significantly higher as compared to the other subjects. Furthermore, subjects possessing C. rectus showed significantly greater values for periodontal pocket depth, gingival index, and total number of species.
We found that approximately one-fourth of the present subjects with disabilities who possessed at least one of T. denticola, T. forsythia, and C. rectus were at possible risk for periodontitis. Follow-up examinations as well as preventive approaches should be utilized for such individuals.
PMCID: PMC2758840  PMID: 19772671
22.  Serum antibodies to periodontal pathogens are a risk factor for Alzheimer’s disease 
Chronic inflammation in periodontal disease has been suggested as a potential risk factor in Alzheimer’s disease. The purpose of this study was to examine serum antibody levels to bacteria of periodontal disease in participants who eventually converted to Alzheimer’s disease (AD) compared to the antibody levels in control subjects.
Serum from 158 participants in the BRAINS (Biologically Resilient Adults in Neurological Studies) research program at the University of Kentucky were analyzed for IgG antibody levels to 7 oral bacteria associated with periodontitis including: Aggregati-bacter actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, Tre-ponema denticola, Fusobacterium nucleatum, Tannerella forsythia, and Prevotella intermedia. All 158 participants were cognitively intact at baseline venous blood draw. Eighty one of the participants developed either mild-cognitive impairment (MCI) or Alz-heimer’s disease (AD) or both, and 77 controls remained cognitively intact in the years of follow up. Antibody levels were compared between controls and AD subjects at baseline draw and after conversion and controls and MCI subjects at baseline draw and after conversion using the Wilcoxon rank-sum test. AD and MCI participants were not directly compared. Linear regression models were used to adjust for potential confounding.
Antibody levels to F. nucleatum and P. intermedia, were significantly increased (α = 0.05) at baseline serum draw in the AD patients compared to controls. These results remained significant when controlling for baseline age, Mini-Mental State Exam (MMSE) score and apolipoprotein epsilon 4 (APOE ε4) status.
This study provides initial data that demonstrate elevated antibodies to periodontal disease bacteria in subjects years prior cognitive impairment and suggests that periodontal disease could potentially contribute to the risk of AD onset/progression. Additional cohort studies profiling oral clinical presentation with systemic response and AD and prospective studies to evaluate any cause-and-effect association are warranted.
PMCID: PMC3712346  PMID: 22546352
Alzheimer’s disease; periodontitis; antibody; periodontal bacteria; periodon-tal disease; mild cognitive impairment
23.  Subgingival bacteria in Ghanaian adolescents with or without progression of attachment loss 
Journal of Oral Microbiology  2014;6:10.3402/jom.v6.23977.
This study describes subgingival bacterial profiles associated with clinical periodontal status in Ghanaian adolescents with or without progression of attachment loss.
Materials and methods
Among 500 adolescents included in a cohort study, 397 returned 2 years later for a periodontal re-examination, including full-mouth CAL measurements. At follow-up, a subgroup of 98 adolescents was also subjected to bacterial sampling with paper points at four periodontal sites (mesial aspect of 11, 26, 31, and 46) and analyzed with the checkerboard DNA–DNA hybridization technique against DNA-probes from nine periodontitis-associated bacterial species.
The 98 Ghanaian adolescents examined in the present study were similar to the entire group examined at the 2-year follow-up with respect to age, gender, and CAL ≥3 mm. A high detection frequency of Fusobacterium nucleatum and Prevotella intermedia (>99%) using checkerboard analysis was found, while for Aggregatibacter actinomycetemcomitans the detection frequency was <50%. A strong correlation was found at the individual level between the presence of P. intermedia and the total CAL change, and P. intermedia and Porphyromonas gingivalis were strongly correlated with a change in CAL and probing pocket depth (PPD) at the sampled sites. In a linear regression model, a significant discriminating factor for the total CAL change in the dentition during the 2-year follow-up period was obtained for P. intermedia and public school.
This study indicates that subgingival bacterial species other than A. actinomycetemcomitans, for example, P. intermedia, have a significant association with periodontal breakdown (change in CAL) in Ghanaian adolescents with progression of periodontal attachment loss.
PMCID: PMC4013489  PMID: 24834145
subgingival bacteria; periodontitis; attachment loss; progression; adolescent; Ghana
24.  Relationship between gingival crevicular fluid and serum antibody titers in young adults with generalized and localized periodontitis. 
Infection and Immunity  1985;49(3):487-493.
The objective of the present study was to determine the relationship between concentrations of antibodies in serum and those in gingival crevicular fluid (GCF) of patients with juvenile periodontitis and severe periodontitis. Most antigens used to quantitate antibodies were obtained from a panel of bacteria associated with juvenile periodontitis or severe periodontitis. We further investigated variation in antibody titer among different periodontal sites and the extent to which antibody in GCF is locally derived. Titers of antibody, total immunoglobulin G (IgG), and human serum albumin were determined with sensitive radioimmunoassays. The relationship between serum and GCF antibody was complex. Both person-to-person variability and marked variability within the same subject were found among different sites of similar clinical status. The site-to-site variability was found not only for antibody reactive with periodontal organisms, but also for antitetanus toxoid, total IgG, and even human serum albumin. Generally the variability was in the degree of depression of the level in GCF relative to that in serum. However, anti-Bacteroides gingivalis and anti-Actinobacillus actinomycetemcomitans in GCF often exceeded the level in serum. When antibody titers in serum and GCF were calculated per milligram of human serum albumin, most of the apparent depressions of antibody in GCF disappeared. The ratio of antibody in serum to that in GCF approached unity for all organisms except B. gingivalis and A. actinomycetemcomitans Y4, which were markedly elevated. Furthermore, the level of IgG per milligram of human serum albumin in GCF was about twice the level in serum. We believe that human serum albumin reflects serum contribution to the GCF, and we therefore attribute the increased level of IgG per milligram of albumin in GCF to local synthesis. It appears that anti-B. gingivalis and anti-A. actinomycetemcomitans represent an important portion of this local antibody synthesis, since most seropositive patients with severe or juvenile periodontitis had at least one site elevated, and the magnitudes of the elevations were large in many sites. Those sites yielding elevated antibody exhibited no obvious differences in clinical parameters of probeable depth or attachment level as compared with sites in which antibody levels in GCF were similar to serum levels. Elevated antibody in GCF may relate to changes in disease activity that are not detectable by usual clinical measures.
PMCID: PMC261187  PMID: 4030088
25.  Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard? 
Journal of Clinical Microbiology  1992;30(2):418-426.
The development of diagnostic tests for a periodontal infection raises the issue as to what the appropriate reference standard, or "gold standard," should be for the evaluation of a new test. The present research was initiated to compare the ability of several detection methods, i.e., a serial dilution anaerobic culture and/or microscopic procedure, a DNA probe procedure, and immunological reagents using both an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay to detect Treponema denticola, Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans in subgingival plaque samples taken from 204 periodontally diseased tooth sites. The prevalence of the four monitored species varied as a function of both the species and the detection method. Spirochetes were present in 99% of the plaques, whereas A. actinomycetemcomitans was detected at the lowest frequency. The culture method yielded the lowest prevalence values for the three cultivable species. This raised the question as to which results, those obtained by culture or those obtained by the DNA probes and the immunological reagents, were the most reliable. This issue was addressed by looking at the prevalence profile of the monitored organisms, as determined by all the detection methods. If the species was detected by three or four of the detection methods, then it was considered present, whereas if it was absent by three or four of the detection methods, then it was considered absent. This approach showed the DNA probes and immunological reagents to be significantly superior (P less than 0.05) to the culture approach for the detection of P. gingivalis, A. actinomycetemcomitans, and B. forsythus and to be comparable to the microscopic approach in the detection of T. denticola.
PMCID: PMC265071  PMID: 1537912

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