Salivary detection of periodontopathic bacteria in periodontally healthy children

BACKGROUND: Salivary occurrence of periodontopathic bacteria is of interest especially in children as a risk indicator for the transmission, development and control of periodontal disease. We assessed the prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola as microbial complexes in the saliva of children with mixed dentition and healthy gingiva. MATERIALS AND METHODS: Paraffin-stimulated saliva samples were collected from 41 children (22 boys and 19 girls), aged 6-13 years old. Gingival health was determined during the initial screening exam. The test bacteria were identified using a 16S rRNA-based PCR analysis. RESULTS: P. nigrescens was the most frequent species (80%), followed by T. denticola (32%), A. actinomycetemcomitans (24%) and P. gingivalis (12%). P. intermedia and T. forsythia were not detected. P. nigrescens was also common species in combinations. Paired and triple bacterial combinations were found in 24% and 20% of all children, respectively. There was no positive association between bacterial combinations in colonization and subject's gender (P>0.05, Fisher exact test). CONCLUSION: The salivary presence of P. nigrescens, T. denticola, A. actinomycetemcomitans and P. gingivalis but not P. intermedia and T. forsythia can occur in childhood without clinical signs of gingival disease. Thus, the possible risk of bacterial transmissions through saliva and, the need to screen for periodontal pathogens should be considered before mixed dentition.     

Detection of DNA from periodontal pathogenic bacteria in biofilm obtained from waterlines in dental units

Direct person-to-person transmission of periodontal bacteria through saliva has recently been widely reported and dental units have been demonstrated to retract saliva from patients under treatment and to release it into the mouths of subjects undergoing the next operation. In this study the presence of a group of periodontal pathogenic bacteria inside waterlines in dental units was investigated using polymerase chain reaction (PCR) based methods. Briefly, 18 dental units of three different manufacturers were studied. Dental units were divided into two groups according to their prevalent use in routine practice. The first group consisted of nine dental units characterized by the frequent use of high-speed dental hand-pieces directly inside the mouth and in contact with patients' saliva. The second group, as a control, consisted of nine dental units where high-speed dental hand-pieces were not in use. A one cm section of the waterline tubing connected to the high-speed hand-piece was removed from each dental unit to evaluate the presence of DNA of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Treponema denticola. Two specimens were positive for Prevotella intermedia DNA. All the positive results were from samples obtained from dental units categorised in the first group. These findings clearly suggest that dental units have the potential to transmit periodontal pathogens. Manufacturers should be invited to design dental units that incorporate automated devices to disinfect DUWLs between patients with minimal effort by dental staff.     

甘草提取物对四种牙周常见致病菌的抑菌作用

目的体外评价甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌的抑制效果。方法以牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌作为供试菌,采用液体稀释法,考察甘草提取物对这四种细菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);并采用不同浓度的甘草提取物溶液,绘制甘草提取物对四种牙周致病菌的时间-杀菌曲线。结果甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌的MIC值分别为1.50、1.50、0.75和1.50mg/mL,MBC值分别为6、3、3和3mg/mL。当甘草提取物达到对四种细菌的MBC值时,对于牙龈卟啉单胞菌、中间普氏菌、伴放线放线杆菌可在2h后可达到杀菌效果,对于具核梭杆菌可在4h后达到杀菌效果。结论甘草提取物对以上四种牙周常见致病菌具有良好的抑菌及杀菌作用。     

Hemagglutinin/Adhesin domains of Porphyromonas gingivalis play key roles in coaggregation with Treponema denticola

Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.     

Rapid and simple detection of eight major periodontal pathogens by the loop-mediated isothermal amplification method

Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64 degrees C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.     

Effectiveness of ultrasonic instruments in the therapy of severe periodontitis: a comparative clinical-microbiological assessment with curettes

Patients with deep periodontal pockets were treated with either Vector System (TG) or manual instruments (CG). Clinical assessments by supragingival plaque (PL+), gingival index (GI), bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL) and subgingival plaque collection for microbiological analysis were made prior to and after treatment. Multiplex Polymerase Chain Reaction was used to determine the presence of Actinobacillus actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola. GI, PD, CAL and the number of BOP+ sites underwent a significant reduction over time in both groups. When compared to baseline, the pair-wise analyses showed significantly lower PD and CAL at 6 months in the CG and significant reductions in the GI, PD, CAL and a number of BOP+ sites at 3 and 6 months in the TG. For microbiological results, significant reductions were seen for C. rectus and R. gingivalis in the CG and for T. forsythensis, E. corrodens and T. denticola in the TG. The total bacterial count underwent a reduction in both groups. Both ultrasonic and manual debridement are equally effective in non-surgical periodontal therapy of severe periodontitis in terms of clinical and microbiological effects.     

Detection of Tannerella forsythia and/or Prevotella intermedia might be useful for microbial predictive markers for the outcome of initial periodontal treatment in Koreans

A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans.     

Quantification of Subgingival Bacterial Pathogens at Different Stages of Periodontal Diseases

Anaerobic gram-negative oral bacteria such as Treponema denticola, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Campylobacter rectus, and Fusobacterium nucleatum are closely associated with periodontal diseases. We measured the relative population (bacterial levels) of these oral pathogens in subgingival tissues of patients at different stages of Korean chronic periodontal diseases. We divided the individuals into those with chronic gingivitis (G), moderate periodontitis (P1), severe periodontitis (P2), and normal individuals (N) (n?=?20 for each group) and subgingival tissue samples were collected. We used real-time PCR with TaqMan probes to evaluate the change of periodontal pathogens among different stages of periodontitis. Bacterial levels of A. actinomycetemcomitans and C. rectus are significantly increased in individuals with chronic gingivitis and moderate periodontitis, but unchanged in severe periodontitis patients. These results suggest that analyzing certain bacterial levels among total oral pathogens may facilitate understanding of the role of periodontal bacteria in the early stages of periodontitis.     

PCR直接检测龈下菌斑主要可疑牙周致病菌

目的：应用PCR方法直接检测龈下菌斑主要可疑牙周致病菌与牙周病活动部位的关系,探讨其方法的可行性并探讨其主要可疑牙周致病菌的分布规律。方法：应用聚合酶链反应(polymerase chain reaction,PCR)直接检测龈下菌斑主要可疑致病菌16s RNA保守区域片段。40名受试者包括牙周病患者20人,每人同口取一个牙周病活动部位,一个相对健康或牙周病静止对照部位;成人健康者20人,每人各取一个标本。结果：龈下菌斑5种可疑牙周致病菌在牙周病活动部位的检出率牙龈卟啉菌为86％,福赛类杆菌为95％,螺旋体为86％,中间普氏菌和黑色普氏菌分别为95％和33％,均显著高于同口部位对照组和健康对照组。结论：PCR直接检测菌斑牙龈卟啉单胞菌、中间普氏菌、福赛类杆菌、齿密螺旋体及黑色普氏菌匀与牙周炎活动部位相关。     

Prevalence of putative periodontopathogens in subgingival dental plaques from gingivitis lesions in Korean orthodontic patients

The objective of this study was to detect and compare the presence of periodontopathogens in the subgingival plaques of gingivitis lesions in adults who wore fixed orthodontic appliances, as opposed to adults who did not wear any orthodontic appliances. Thirty-six individuals participated in this study. Nineteen of these subjects did not wear any orthodontic appliances, and these subjects comprised the control group. The other 17 individuals had been wearing fixed orthodontic appliances for at least 3 months each. After a periodontal examination, we collected subgingival plaque samples from the gingivitis lesions of each patient. Using PCR based on 16S rDNA, we detected the presence of 6 putative periodontopathogenic species, Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia (formerly Bacteroides forsythus), Prevotella nigrescens, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. With regard to the presence of individual periodontopathogens, we found that T. forsythia, T. denticola, and P. nigrescens were significantly more common in the samples obtained from the orthodontic patients than in the samples obtained from the non-orthodontic patient controls. Our results indicate that the local changes associated with the wearing of fixed orthodontic appliances may affect the prevalence of periodontopathogens in subgingival dental plaques.     

Use of molecular genetic systems for diagnostics of inflammatory diseases of oral mucosa and periodontium

Structure of bacterial microflora of periodontal pocket using classical cultural and molecular genetic methods (PCR and RT-PCR assays) was studied in patients with chronic generalized severe or intermediate periodontitis in acute phase and in healthy subjects. Structure of viral and fungal microflora using PCR or ELISA assays was studied also. Markers of Gram-negative bacteria (B. forsithus, T. denticola, A. actinomicetemcommitans, P. intermedia and P. gingivalis) were detected with different rates. Rate of detection of pigment-forming P. intermedia and P. gingivalis was significantly higher when PCR assay was used compared with traditional bacteriologic method. Detection rate of Candida albicans DNA in tooth-gingival fluid was virtually not different from one when cultural method was used. PCR assay is recommended as laboratory method for detection of DNA of microorganisms associated with inflammatory diseases of oral mucosa and periodontium.     

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.     

不同浓度臭氧水对牙周致病菌的体外抑菌作用

摘要：目的 探讨不同浓度臭氧水对4种牙周致病菌的体外抑菌效果。方法 采用定量悬液法,分别用1.02 ppm、2.03 ppm和3.88 ppm三种浓度臭氧水,0 ppm无臭氧水溶液（阴性对照组）及3%双氧水（阳性对照组）,对常见的4种牙周致病菌牙龈卟啉单胞菌（Porphyromonas gingivalis）、具核梭杆菌（Fusobacterium nucleatum）、中间普氏菌（Prevotella intermedia）和黏性放线菌（Actinomyces viscosus）分别作用30 s、60 s后,观察不同作用时间下3种不同浓度臭氧水的抑菌效果。结果 对P. gingivalis、F. nucleatum、P. intermedia和A. viscosus作用30 s和60 s,与阴性对照组比较,1.02 ppm、2.03 ppm和3.88 ppm三种浓度臭氧水均具有明显的抑菌效果,3.88 ppm臭氧水与3%双氧水对4种细菌作用60 s的抑菌效果相同。 结论 实验所用的3种不同浓度臭氧水对牙周致病菌P. gingivalis、F. nucleatum、P. intermedia和A. viscosus均有抑菌作用,浓度越高,抑菌效果越好,臭氧水应用于牙龈牙周疾病临床防治有一定的价值和可行性。     

Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor

Oral malodor is considered to originate primarily from tongue microbiota populations. However, the relationship between oral malodor and tongue microbiota remains unclear. In this study, tongue periodontal pathogens were analyzed via real-time PCR, and the association between oral malodor and tongue periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola, was examined. The subject population consisted of 29 individuals with and 10 healthy persons without oral malodor. Oral malodor was assessed by organoleptic test and volatile sulfur compound (VSC) levels as measured by gas chromatography. Real-time PCR was conducted for anaerobes in tongue biofilm samples employing a LightCycler system; furthermore, bacterial proportion served as a quantitative parameter. Among the five anaerobes, only T. forsythia displayed higher proportions in malodor subjects than corresponding values in healthy controls. Proportions of P. intermedia and P. nigrescens correlated strongly with hydrogen sulfide concentration. Proportions of P. gingivalis and P. nigrescens also exhibited strong correlation with methyl mercaptan concentration. The correlation coefficient between the proportion of the total of the five anaerobes and total VSC level (r = 0.88) was greater than that between bacterial proportion and organoleptic score (r = 0.29). When a linear regression analysis was performed utilizing the proportion of each of the five periodontal pathogens as an independent variable, the explanatory power of these independent variables revealed 81% for total VSC level and 16% for organoleptic score. These results suggest that these five periodontal pathogens on tongue dorsa may contribute greatly to VSC production.     

Porphyromonas gingivalis prevalence related to other micro-organisms in adult refractory periodontitis.

Forty-six adult periodontal patients, selected on the basis of clinical examination, and 46 adult healthy subjects were examined. The subgingival plaque samples from one inflammatory and one non-inflammatory site of each periodontal patient were studied to determine Porphyromonas gingivalis prevalence related to other periodontal micro-organisms and to periodontal tissue destruction. The results showed Porphyromonas gingivalis as the main pathogenic micro-organism isolated in the inflammatory sites together with Bacteroides forsythus. Peptostreptococcus sp., Actinomyces sp. and Prevotella sp. were found as a normal oral flora in the healthy subjects. Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus and Eikenella corrodens were detected both in inflammatory and in non-inflammatory sites of periodontal patients as well as in the healthy subjects.     

Perturbation and exploitation of host cell cytoskeleton by periodontal pathogens

Some periodontal pathogens disrupt epithelial barriers and cellular adhesion to the extracellular matrix, which affects the cytoskeleton. Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans exploit the cytoskeleton during their uptake by epithelial cells. Treponema denticola perturbs actin and actin-regulating pathways in host cells. Cytoskeletal dysfunction due to pathogenic bacteria may impair physiologic remodeling and wound repair in the periodontium.     

Comparison of culture methods and multiplex PCR for the detection of periodontopathogenic bacteria in biofilm associated with severe forms of periodontitis

Conventional culture methods and Multiplex PCR, both of which we have been used for a long time in our clinical microbiology laboratory, were compared for their ability to detect a selected panel of periodontopathic bacteria: Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. Tests were performed in a single subgingival sample taken from a periodontal diseased site with a probing depth equal to or greater than 6mm. The results were compared site-by-site, taking into account the quality and the presence or absence of pathogens. 529 samples of subgingival plaque were analysed and the prevalence of the six species monitored varied in relation to the species itself and the method of detection. The most represented species is F. nucleatum, with a percentage of positive variability between 44.9% PCR and 46.5% culture test. Generally, the lowest prevalence was determined by culture test, with the exception of E. corrodens and F. nucleatum, which, unlike other bacteria, have been seen in higher percentages in culture with respect to PCR. For both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans, C. rectus, E. corrodens, and P. gingivalis. It becomes weak for F. nucleatum and P. intermedia. Both culture and PCR techniques introduced many methodological problems when applied in oral microbiology, but the ideal technique for accurate detection of pathogens in subgingival plaque samples has yet to be developed.     

Aspartame as a source of essential phenylalanine for the growth of oral anaerobes

Abstract Phenylalanine and aspartic acid requirements were determined for 13 species of oral bacteria using the chemically defined medium OMIZ-W1. None of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Eikenella corrodens, Selenomonas sputigene, Treponema pectinovorum, T. socranskii , or Wolinella recta required either of these amino acid constituents of aspartame ( l -aspartyl- l -phenylalanine methylester). Phenylalanine was essential for the growth of Capnocytophaga gingivalis, Eubacterium timidum, Fusobacterium nucleatum, Porphyromonas gingivalis, T. denticola , and T. vincentii , while aspartic acid was not required. With the exception of E. timidum , all phenylalanine-dependent strains could grow when the free amino acid was replaced by aspartame at concentrations at least 10-fold lower than those used for aspartame as an artificial sweetener.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Periodontal status in an Italian young adult population. Prevalence and relationship with periodontopathic bacteria

To determine the prevalence of periodontitis in an Italian young adult population and the relationship with Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque. A full-mouth periodontal and oral examination was performed in 70 subjects. Dental and behaviour habits were assessed with a standardised questionnaire. Subgingival plaque samples were collected from the deepest pocket of the first molars in each quadrant with a sterile curette. A. actinomycetemcomitans, P. gingivalis and P. intermedia were detected using a multiplex polymerase chain reaction. At subject level, the prevalence of bleeding on probing, calculus, normal pocket depth (PD), PD > 5mm and bacterial positivity were 44.8%, 43.3%, 22.9%, 11.4% and 95.7%, respectively. At quadrant level bacterial prevalence was 79.4%; P. intermedia was the most common bacteria (79.0%); A. actinomycetemcomitans had a prevalence of 40.8%. A significant linear trend across categories of gingival conditions (healthy, bleeding on probing, calculus presence) was detected for P. intermedia (p = 0.0038) and A. actinomycetemcomitans (p = 0.00005) proportions. No significant association was observed between pathogenic bacteria and PD, nor with behavioural attitudes. Gingival conditions are found to be a good predictors (VPP = 85%) for periodontopathic bacteria. For the Italian population, as no data are present, prospective longitudinal studies are needed to examine the relationship between PD and bacteria presence with periodontal disease onset and/or progression.