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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.  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
4.  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
5.  Etiological Analysis of Initial Colonization of Periodontal Pathogens in Oral Cavity▿  
Journal of Clinical Microbiology  2008;46(4):1322-1329.
It is unclear when the initial colonization by periodontal pathogens occurs in the oral cavity. Therefore, we report here the association between specific age groups and the time when the initial colonization by periodontal pathogens occurs in the oral cavity in such groups. Findings are based on an epidemiological analysis of the prevalence of five periodontal pathogens in the oral cavities of a wide range of age populations, from newborn to elderly, who were randomly selected in a geographic region of Brazil. These periodontal pathogens include Campylobacter rectus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Tannerella forsythia and were analyzed in the bacterial samples isolated from gingival sulcus, the dorsum of the tongue, and cheek mucosa of diverse age groups, using a bacterial DNA-specific PCR method. Results indicated that there are distinct age-related groups where initial colonization by the five periodontal pathogens examined in this study can be detected and that the presence of teeth is a permissive factor for colonization by P. gingivalis, P. intermedia, and T. forsythia. Although it remains unclear exactly how or when target pathogens colonize healthy subjects, an understanding of age-related groups does provide a potentially useful tool in the early detection and prevention of periodontitis in healthy individuals.
PMCID: PMC2292974  PMID: 18287326
6.  Traditional Medicinal Plant Extracts and Natural Products with Activity against Oral Bacteria: Potential Application in the Prevention and Treatment of Oral Diseases 
Oral diseases are major health problems with dental caries and periodontal diseases among the most important preventable global infectious diseases. Oral health influences the general quality of life and poor oral health is linked to chronic conditions and systemic diseases. The association between oral diseases and the oral microbiota is well established. Of the more than 750 species of bacteria that inhabit the oral cavity, a number are implicated in oral diseases. The development of dental caries involves acidogenic and aciduric Gram-positive bacteria (mutans streptococci, lactobacilli and actinomycetes). Periodontal diseases have been linked to anaerobic Gram-negative bacteria (Porphyromonas gingivalis, Actinobacillus, Prevotella and Fusobacterium). Given the incidence of oral disease, increased resistance by bacteria to antibiotics, adverse affects of some antibacterial agents currently used in dentistry and financial considerations in developing countries, there is a need for alternative prevention and treatment options that are safe, effective and economical. While several agents are commercially available, these chemicals can alter oral microbiota and have undesirable side-effects such as vomiting, diarrhea and tooth staining. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicines are considered as good alternatives. In this review, plant extracts or phytochemicals that inhibit the growth of oral pathogens, reduce the development of biofilms and dental plaque, influence the adhesion of bacteria to surfaces and reduce the symptoms of oral diseases will be discussed further. Clinical studies that have investigated the safety and efficacy of such plant-derived medicines will also be described.
PMCID: PMC3145422  PMID: 19596745
7.  Quantitative analysis of periodontal pathogens present in the saliva of geriatric subjects 
At present, information regarding periodontal disease in geriatric patients is scarce. The purpose of this study was to quantify the periodontal pathogens present in the saliva of Korean geriatric patients and assess the relationship between the bacterial levels and the periodontal condition.
Six putative periodontal pathogens were quantified by using a real-time polymerase chain reaction assay in geriatric patient groups (>60 years) with mild chronic periodontitis (MCP), moderate chronic periodontitis (MoCP), and severe chronic periodontitis (SCP). The copy numbers of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Prevotella intermedia were measured.
It was found that the bacterial copy numbers increased as the severity of the disease increased from MCP to SCP, except for P. intermedia. For P. intermedia, it was found that samples in the MCP group yielded the largest amount. It was also found that the quantities of P. gingivalis, T. forsythia, and T. denticola, the so-called "red complex" bacteria, were lower than those of F. nucleatum, A. actinomycetemcomitans, and P. intermedia in all of the samples.
Collectively, the results of this study suggest that the levels of P. gingivalis, T. forsythia, F. nucleatum, and T. denticola present in saliva are associated with the severity of periodontal disease in geriatric patients.
PMCID: PMC3769597  PMID: 24040571
Chronic periodontitis; Disease progression; Geriatrics; Microbiology; Real-time polymerase chain reaction; Saliva
8.  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
9.  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
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.  Characterization of inhibitory effects of suspected periodontopathogens on osteogenesis in vitro. 
Infection and Immunity  1995;63(9):3287-3296.
By using an in vitro bone-forming culture system, the chick periosteal osteogenesis (CPO) model, the direct effects on osteogenesis of sonicated extracts derived from oral bacteria were examined. Both extracts from bacterial species having strong associations with periodontal diseases (Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Prevotella intermedia, hereinafter referred to as suspected periodontopathogens) and extracts from species not correlated with periodontal disease (Streptococcus sanguis, Veillonella atypica, and Prevotella denticola, hereinafter referred to as nonpathogenic bacteria) were tested. All bacterial cultures were grown under standard anaerobic culture conditions. Sonicated bacterial extracts were prepared from the bacterial pellet. These were added in various proportions to the CPO cultures. Parameters of osteogenesis, including alkaline phosphatase activity, calcium and P(i) accumulation, and collagen synthesis, were measured in 6-day-old cultures. Compared with controls grown in the absence of bacterial products, osteogenesis was inhibited significantly in cultures treated with extracts derived from the suspected periodontopathogens. No osteogenic inhibition was observed in cultures treated with extracts from the nonpathogenic bacteria. These results suggest that the ability to inhibit osteogenesis in vitro may be a pathogenic property shared by a limited group of species. Further characterization of the P. gingivalis extracts revealed that both proteinaceous and nonproteinaceous products, including lipopolysaccharide, were able to inhibit osteogenesis. P. gingivalis extract-mediated inhibition of osteogenesis in CPO cultures was blocked by indomethacin, implicating prostaglandins in the regulation of the bacterial effects. The bacterial extracts had either reversible or irreversible inhibitory effects on osteogenesis when added after differentiation or before/during differentiation of bone cells, respectively.
PMCID: PMC173453  PMID: 7642257
12.  Association of Bacteroides forsythus and a Novel Bacteroides Phylotype with Periodontitis 
Journal of Clinical Microbiology  2002;40(3):821-825.
Chronic periodontitis is a common infectious disease in the adult population. The etiology is clearly bacterial, and a small number of bacterial species have been consistently associated with periodontitis, including Bacteroides forsythus and Porphyromonas gingivalis. Comparatively little attention has been paid to the identification of health-associated and potentially beneficial bacterial species that may reside in the gingival sulcus. The purpose of the present study was to examine the relationship of the presence of B. forsythus and a newly identified Bacteroides phylotype, oral clone BU063, to periodontal health status. The study was accomplished with a set of samples that were collected from subjects with periodontitis and healthy controls. These samples had previously been analyzed for the presence of P. gingivalis. An oral sampling strategy that included every tooth and a PCR-based detection method were used to maximize detection sensitivity. The presence of B. forsythus in the oral cavity was strongly associated with periodontitis, and its nearest genetic neighbor, oral clone BU063, was associated with oral health (P < 0.0001 for both). Colonization with P. gingivalis was independent of the presence of either Bacteroides species, but the two Bacteroides species were found together less often than would be expected by chance (P < 0.0001). This suggests the presence of a specific exclusionary mechanism between the two Bacteroides species. Comparisons between these two organisms may prove useful for studies that determine how B. forsythus functions in the disease process. In addition, oral clone BU063 deserves further study as a possible preventive or therapeutic intervention for periodontitis.
PMCID: PMC120258  PMID: 11880400
13.  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
14.  Photoactive Porphyrin Derivative with Broad-Spectrum Activity against Oral Pathogens In Vitro 
Antimicrobial Agents and Chemotherapy  2000;44(12):3364-3367.
Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (≥5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6–pentalysine conjugate to more effectively reduce the pathogenic bacteria in the periodontal pocket may be a significant tool for the treatment of periodontal disease.
PMCID: PMC90206  PMID: 11083641
15.  Cloning, characterization, and antigen specificity of T-lymphocyte subsets extracted from gingival tissue of chronic adult periodontitis patients. 
Infection and Immunity  1995;63(6):2147-2153.
Chronic periodontitis is characterized by dense infiltrations of B and T lymphocytes within the gingival connective tissue. Distinct anaerobic gram-negative bacteria as well as autoimmunity to collagen have been reported to play a role in the etiology and the pathogenesis of this disease. Here we describe the cloning and characterization of CD4+ and CD8+ T lymphocytes isolated from inflamed gingival tissue obtained from four patients with chronic periodontitis. Clones were raised with phytohemagglutinin and interleukin-2 and tested for proliferation in response to whole-cell antigens of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, human collagen type I, and two bacterial heat shock proteins. CD4+ T-cell clones reactive with collagen type I were obtained from all four patients. Eighty percent of these clones had phenotypes resembling the mouse type 2 T helper (Th) phenotype, i.e., they produced high levels of interleukin-4 and low levels of gamma interferon. No collagen-type-I-reactive CD8+ clones were obtained. Bacterial-antigen-reactive CD4+ and/or CD8+ T-cell clones were also obtained from each patient, and the majority of the clones showed a Th0-like cytokine pattern and produced equal amounts of interleukin-4 and gamma interferon. Although most clones were reactive with P. intermedia, it seems that the immune response is not strictly directed against this particular microorganism, as clones reactive with one of the other bacteria were also obtained from two patients. We propose that collagen-specific CD4+ Th2-like T cells contribute to the chronicity of periodontitis but that their modes of activation might be controlled by Th0-like T cells specific for periodontitis-associated bacteria.
PMCID: PMC173279  PMID: 7539406
16.  Effect of controlled-release Periochip™ on clinical and microbiological parameters in patients of chronic periodontitis 
The aim of the present study was to evaluate and compare the clinical and microbiological effectiveness of Periochip™ as an adjunct to scaling and root planing (SRP) with SRP alone in patients with chronic periodontitis.
Materials and Methods:
This randomized, split mouth, 3-month clinical and microbiological trial included 30 sites in 15 patients aged 30-50 years diagnosed with chronic periodontitis. In each patient, two bilateral pockets probing 5-7 mm were randomly assigned to test and control groups. The test group received SRP plus Periochip™, whereas the control group received SRP alone. Clinical indices and anaerobic culture analysis was done at baseline, 1 month, and 3 months interval. Total bacterial count and analysis of four major periodontopathogenic bacteria Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) was done.
Significant improvement was obtained in all clinical variables in the test group as compared to the control group over the study period. Total colony counts were significantly reduced in the test group as compared to control over the period of time. At baseline Aa was recovered from 4 test group sites and 5 control group sites, Pg from 15 test group and 14 control group sites, Pi from 5 test group and 2 control group sites, Fn from 7 test and 7 control group sites. At 3 months, Aa was recovered from 1 test group and 4 control group sites, Pg from 4 test group and 8 control group sites, Pi from 1 test group and 1 control group site, Fn from 3 test and 4 control group sites.
Periochip™ placement as an adjunct to SRP, showed promising results, when compared to SRP alone. Healthy microflora can be maintained for a longer period of time and delay in the repopulation by periodontopathic microorganisms was observed.
PMCID: PMC3808015  PMID: 24174754
Chlorhexidine/therapeutic use; controlled clinical trial; subgingival periodontopathic anaerobes
17.  Determination of periodontopathogens in patients with Cri du chat syndrome 
Objectives: Cri du chat syndrome is a genetic alteration associated with some oral pathologies. However, it has not been described previously any clinical relationship between the periodontal disease and the syndrome. The purpose of this comparative study was to compare periodontopathogenic flora in a group with Cri du chat syndrome and another without the síndrome, to assess a potential microbiological predisposition to suffer a periodontitis. Study Design: The study compared nineteen subjects with Cri du chat Syndrome with a control group of nineteen patients without it. All patients were clinically evaluated by periodontal probing, valuing the pocket depth, the clinical attachmente level and bleeding on probing. There were no significant differences between both groups. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola were detected by multiplex-PCR using 16S rDNA (microIDENT). Results: When A. actinomycetemcomitans, P. gingivalis, P. intermedia and T. denticola were compared, no statistically significant differences were found between the two groups (p>0.05). The value of T. forsythia was significantly higher for Cri du chat syndrome (31.6%) than for the control group (5.3%). The odds ratio for T. forsythia was 8.3. Conclusions: In the present study T. forsythia is associated with Cri du chat syndrome subjects and not with healthy subjects.
Key words:Cri du Chat syndrome, periodontal health, microbiology, special care dentistry.
PMCID: PMC3854081  PMID: 24121919
18.  Periodontopathogen profile of healthy and oral lichen planus patients with gingivitis or periodontitis 
Oral lichen planus (OLP) is a chronic inflammatory disease that is frequently detected in oral tissues. The aim of our study was to identify the prevalence of the detection of periodontopathogenic microorganisms (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola in OLP patients and to compare with this prevalence of periodontopathogenic microorganisms in healthy non-OLP patients. Our study included 27 (18 chronic periodontitis (OLPP) and 9 gingivitis (OLPG)) patients diagnosed with OLP along with 26 (13 chronic periodontitis (HP) and 13 gingivitis (HG)) healthy non-OLP patients. The multiplex polymerase chain reaction (PCR) with subsequent reverse hybridization method (micro-IDent) was used for identifying periodontopathogenic microorganisms present in subgingival plaque samples. The percentages of detection for A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola in subgingival plaque samples taken from OLP patients (OLPG and OLPP) were 18.5%, 85.1%, 81.4%, 88.8% and 74%, respectively. Meanwhile, in the non-OLP patients (HG and HP), these values were 7.6%, 50%, 46.1%, 73% and 57.7%, respectively. Thus, comparing the non-OLP groups with the OLP groups, the periodontopathogens' percentages of detection in the OLP groups were higher than those in the non-OLP groups. According to our study results, OLP patients have higher levels of infection with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola than non-OLP patients. We argue that the high percentages in patients with OLP may help identify the importance of periodontopathogenic microorganisms in the progress of periodontal diseases of OLP.
PMCID: PMC3707073  PMID: 23743616
Aggregatibacter actinomycetemcomitans; oral lichen planus; Porphyromonas gingivalis; Prevotella intermedia; Tannerella forsythia; Treponema denticola
19.  Rheumatoid arthritis is an autoimmune disease caused by periodontal pathogens 
A statistically significant association between periodontal disease (PD) and systemic diseases has been identified. Rheumatoid arthritis (RA), which is a chronic inflammatory joint disease, exhibits similar characteristics and pathogenesis to PD. The association between RA and PD has been investigated, and numerous publications on this subject exist. Approximately 20 bacterial species have been identified as periodontal pathogens, and these organisms are linked to various types of PD. The most analyzed species of periodontopathic bacteria are Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans. Antibodies and DNA from these oral pathogens have been isolated from the sera and synovial fluids of RA patients. This rapid communication describes the role of periodontal pathogens in the etiopathogenesis of RA.
PMCID: PMC3668087  PMID: 23737674
etiopathogenesis; chronic arthritis; periodontitis; Porphyromonas gingivalis; systemic disease; animal models; antibiotics
20.  Periodontal Disease as a Specific, albeit Chronic, Infection: Diagnosis and Treatment 
Clinical Microbiology Reviews  2001;14(4):727-752.
Periodontal disease is perhaps the most common chronic infection in adults. Evidence has been accumulating for the past 30 years which indicates that almost all forms of periodontal disease are chronic but specific bacterial infections due to the overgrowth in the dental plaque of a finite number of mostly anaerobic species such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola. The success of traditional debridement procedures and/or antimicrobial agents in improving periodontal health can be associated with the reduction in levels of these anaerobes in the dental plaque. These findings suggest that patients and clinicians have a choice in the treatment of this overgrowth, either a debridement and surgery approach or a debridement and antimicrobial treatment approach. However, the antimicrobial approach, while supported by a wealth of scientific evidence, goes contrary to centuries of dental teaching that states that periodontal disease results from a “dirty mouth.” If periodontal disease is demonstrated to be a risk factor for cardiovascular disease and stroke, it will be a modifiable risk factor since periodontal disease can be prevented and treated. Since the antimicrobial approach may be as effective as a surgical approach in the restoration and maintenance of a periodontally healthy dentition, this would give a cardiac or stroke patient and his or her physician a choice in the implementation of treatment seeking to improve the patient's periodontal condition so as to reduce and/or delay future cardiovascular events.
PMCID: PMC89001  PMID: 11585783
21.  Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical, microbiological and serological study 
Arthritis Research & Therapy  2012;14(5):R222.
The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.
In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.
A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.
Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.
PMCID: PMC3580533  PMID: 23075462
22.  Recovery of putative pathogens from paper point sampling at different depths of periodontal lesions 
The aim of this study was to compare the recovery of three putative periodontal pathogens from periodontal lesions in samples using paper points inserted to different depths of the lesions.
Twenty 6–8 mm deep periodontal lesions with bleeding on probing were studied. Microbial samples were obtained using paper points inserted to three different depths of the lesions: orifice of lesion; 2 mm into the lesion; and to the base of lesion. Culturing was used for recovery and identification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.
The recovery of each of the three putative periodontal pathogens was similar following sampling at the various depths of the lesions.
The findings may be explained by the fact that the paper points become saturated as they pass through the orifice of the lesion. Absorption of microorganisms will therefore primarily occur at the orifice. It is also conceivable that the pathogens may be present in similar proportions throughout the various depths of the periodontal lesions.
PMCID: PMC3652342  PMID: 23674898
paper point sampling; P. gingivalis; P. intermedia; A. actinomycetemcomitans
23.  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
24.  Occurrence of periodontal pathogens among patients with chronic periodontitis 
Brazilian Journal of Microbiology  2012;43(3):909-916.
The aim of the present study was to evaluate the presence of the periodontal pathogens that form the red complex (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) and Aggregatibacter actinomycetemcomitans in patients with chronic periodontitis. The sample consisted of 29 patients with a clinical and radiographic diagnosis of chronic periodontitis based on the criteria of the American Academy of Periodontology (3). Samples for microbiological analysis were collected from the four sites of greatest probing depth in each patient, totaling 116 samples. These samples were processed using conventional polymerase chain reaction, which achieved the following positive results: 46.6% for P. gingivalis, 41.4% for T. forsythia, 33.6% for T. denticola and 27.6% for A. actinomycetemcomitans. P. gingivalis and T. forsythia were more prevalent (p < 0.05) in periodontal pockets ≥ 8 mm. The combinations T. forsythia + P. gingivalis (23.2%) and T. forsythia + P. gingivalis + T. denticola (20.0%) were more frequent in sites with a probing depth ≥ 8 mm. Associations with the simultaneous presence of A. actinomycetemcomitans + P. gingivalis, A. actinomycetemcomitans + T. forsythia, P. gingivalis + T. forsythia and T. forsythia + T. denticola were statistically significant (p < 0.05). It was concluded that the red complex pathogens are related to chronic periodontitis, presenting a higher occurrence in deep periodontal pockets. Moreover, the simultaneous presence of these bacteria in deep sites suggests a symbiotic relationship between these virulent species, favoring, in this way, a further progression of periodontal disease.
PMCID: PMC3768898  PMID: 24031906
periodontitis; pathogens; Tannerella forsythia; Porphyromonas gingivalis; Treponema denticola; Aggregatibacter actinomycetemcomitans
25.  BANA-Positive Plaque Samples Are Associated with Oral Hygiene Practices and Not CD4+ T Cell Counts in HIV-Positive Patients 
Background. The “red complex” microorganisms, namely, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are considered as potential pathogens causing HIV-associated periodontal diseases. Moreover, it has been recognized that an association exists between CD4+ T cell counts and periodontal disease progression. Objective. To establish whether CD4+ T cell counts or oral hygiene plays a greater role in producing BANA-positive results in HIV-associated periodontal disease. Materials and Methods. One hundred and twenty HIV-positive patients participated in the study, and their CD4+ T cell counts were obtained from their medical records. The six Ramfjord teeth were used for evaluating periodontal clinical indices and subgingival plaque sampling. BANA test was used for the detection and prevalence of the “red complex” bacteria in plaque samples. Results. A majority of 69.17% HIV-positive patients were BANA-positive. No significant associations were found between BANA and CD4+ T cell counts. A highly significant association was found between BANA with probing depth and clinical attachment level (P ≤ 0.0001) and between BANA and the use of interdental aids (P = 0.0168). Conclusion. HIV-associated periodontal diseases are strongly related to oral hygiene practices rather than the effect of CD4+ T cell counts, and the use of interdental aids was marked as a significant predictor of BANA-negative plaque samples.
PMCID: PMC3509373  PMID: 23258979

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