Classification of black-pigmented anaerobes by pyrolysis mass spectrometry (PMS) and conventional tests (CTs)

Abstract Clinical (66: dental 53; vaginal 4; wound 9) and reference (5^∗) strains of pigmented Gram-negative anaerobic bacilli were examined in pyrolysis mass spectrometry (PMS) and conventional tests (CTs). The strains were identified in CTs as: Prevotella intermedia (48^∗); Pr. melaninogenica (1); Pr. corporis (7); Porphyromonas asaccharolytica (12^∗); P. endodontalis (1^∗) and P. gingivalis (2^∗). Numerical classification based on CTs resolved five clusters comprising strains identified as (I) Pr. corporis , (II) Pr. melaninogenica , (III) Pr. intermedia , (IV) P. gingivalis and (V) P. asaccharolytica and P. endodontalis . Numerical classification based on PMS showed a similar division, with decreasing homogeneity in the order Pr. intermedia, Pr. corporis, P. asaccharolytica , in agreement with the ordering of homogeneity for these species in CTs. PMS clusters corresponding the Porphyromonas spp. were clearly distinct from those of Prevotella spp. PMS and CT classifications disagreed on cluster membership for only six of the strains. PMS identification from blind challenge sets agreed with conventional identification for 64 of 67 strains.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

Black-pigmented Gram-negative anaerobes in periodontitis

Abstract Black-pigmented Gram-negative anaerobes have been associated with periodontal disease and tooth loss since they were first isolated by Burdon in 1928. Porphyromonas gingivalis , which is usually not isolated from children, adolescents or adults with no periodontal breakdown, has been recognized as one of the most important periodontopathogens. Its presence is strongly correlated with deep periodontal pockets, which are assumed to be its main habitat. Correlations have been shown also with attachment loss, clinical inflammation and serum antibody levels, indicating an aetiological role in the periodontal disease. Their pathogenicity in animal models resembling periodontal disease is documented. They are frequently isolated from periodontal abscesses. The relationship between Prevotella intermedia and periodontal disease is not clear. It is frequently isolated from advanced periodontitis, often as the only black-pigmented Gram-negative anaerobic species; however, the prevalence in adults with no periodontal breakdown is high. It is found frequently in periodontal abscesses and in acute necrotizing and ulcerative gingivitis. Serogroup I is found predominantly in deep periodontal pockets, whereas all serogroups (I–III) are found in shallow pockets and gingivitis. No conclusive difference in pathogenicity between serogroups has been found. Pr. melaninogenica, Pr. denticola and Pr. loescheii are frequently found in the gingival crevice in preschool children and other age groups with gingivitis, but are seldom found in deep periodontal pockets.     

Quantitative detection of periodontopathogens by real-time PCR

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.     

Occurrence of plasmids in black-pigmented Gram-negative anaerobes

Abstract Black-pigmented Gram-negative anaerobes are causative agents of pyogenic infections and are closely linked to various forms of periodontal diseases. Whereas many studies have shown a high incidence of plasmids in intestinal Bacteroides spp., there have been only a few reports of plasmid analyses in pigmented Gram-negative anaerobes. According to previous reports and confirmed in this study, plasmids can be present in Porphyromonas asaccharolytica, Prevotella intermedia, Pr. melaninogenica , and B. levii but have not been detected in P. gingivalis or other black-pigmented species. There were no correlations between plasmids and phenotypes such as resistance to antibiotics or bacteriocinogenicity. The highest carriage rate was found in isolates from cases of chronic otitis media, but the relationship between this site of infection and a high incidence of plasmids could be incidental. The size of plasmids ranged from 1.5 to 29 MDa. Plasmids with molecular weight > 10 MDa were described for the first time in these organisms. Repeated plasmid analyses showed that the plasmid patterns were generally stable.     

Antimicrobial activities of black-pigmented Gram-negative anaerobes

Abstract The antimicrobial activities of Prevotella intermedia and Porphyromonas gingivalis isolates were tested against other species of Gram-positive and Gram-negative anaerobes as well as against each other. Generally, Pr. intermedia possessed significantly higher antimicrobial activity than P. gingivalis . The strongest activity of P. gingivalis towards Gram-negative anaerobes was directed against Pr. intermedia . Cross-sensitivity between both species was observed with strains from different lesions. Antimicrobial activity towards strains of the same species was detected only with Pr. intermedia . No correlations were found between plasmid content and antimicrobial activity. It was concluded that the inhibitory potency of Pr. intermedia could be one reason for the high proportion of black-pigmented Gram-negative anaerobes in the subgingival flora of periodontitis lesions.     

Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries

The oral bacterial flora in the saliva from two patients with periodontitis and from a periodontally healthy subject were compared using a sequence analysis of 16S rDNA libraries without cultivation. 16S rDNAs were amplified from salivary DNA by PCR and cloned. Randomly selected clones were partially sequenced. On the basis of sequence similarities, the clones were classified into several clusters corresponding to the major phylum of the domain Bacteria. The major phylum in the libraries was the low G+C Gram-positive bacteria. There was no clonal sequence affiliated with periodontopathic bacteria in the salivary sample from the healthy subject, while a number of periodontal pathogens such as Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis and Treponema socranskii were detected in the salivary samples from the patients with periodontitis. In addition, a number of previously uncharacterized and uncultured microorganisms were recognized. These organisms may have some role in periodontal disease. This study reveals some potential for a molecular-biological technique to analyze the oral microflora associated with periodontal disease, including previously uncharacterized and uncultured microorganisms, without cultivation.     

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.     

Black-pigmented Gram-negative anaerobes in endodontic infections

Abstract Necrotic dental root canal infections are polymicrobial infections dominated by anaerobic bacteria. The number of different species in one canal is usually low, approx. 4–7 species. The species isolated most frequently belong to the genera Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Eubacterium and Streptococcus . The frequency of isolation of black-pigmented Gram-negative anaerobes in endodontic infections varies from 25% to >50%. Pr. intermedia is the most commonly found pigmented species, followed by Pr. denticola and two Porphyromonas species, P. gingivalis and P. endodontalis . Several studies have shown that P. gingivalis and P. endodontalis are closely related to the presence of acute symptoms in endodontic infections, whereas other black-pigmented Gram-negative anaerobes are not. However, several other species may also be involved in acute infections. Moreover, Porphyromonas species have occasionally been isolated from cases with no symptoms. Although Porphyromonas spp. are clearly related to symptoms at the beginning of therapy, they are not important for the prognosis of the treatment.     

Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species

Abstract Dioxigenin-labelled synthetic DNA probes directed against the 16S rRNA were used for the direct detection of the periodontopathogenic bacteria Prevotella intermedia and Porphyromonas gingivalis in subgingival plaque by applying a DNA-RNA dot-blot hybridization procedure. The test was evaluated with 134 plaque samples from 26 patients with adult periodontitis or rapidly progressive periodontitis. The lower limit of detection was 10⁴–10⁵ bacteria/specimen. A semiquantitative assessment of the two species in each sample and in the corresponding periodontal site was achieved by this technique. It is possible to examine 80–90 samples within two days with low material costs.     

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。     

Characterization of a bacteriocin produced by Prevotella nigrescens ATCC 25261

AIMS: To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts. METHODS AND RESULTS: An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6.5 and 9.5, at 100 degrees C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts. CONCLUSION: Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry.     

Emended descriptions of Prevotella denticola, Prevotella loescheii, Prevotella veroralis, and Prevotella melaninogenica.

During studies of human periodontal disease, a number of bacterial strains were encountered that, on the basis of results of standard biochemical tests, appeared to be Prevotella buccalis, Prevotella denticola, Prevotella melaninogenica, or Prevotella loescheii. However, use of the standard biochemical tests, cellular fatty acid analyses, and the polyacrylamide gel electrophoresis patterns of soluble proteins resulted in conflicting identifications of these strains. The results of tests for cellobiose fermentation, inulin fermentation, and pigment production were responsible for most of the discordant results. Cellular fatty acid analyses in which the Microbial Identification System was used did not differentiate these strains from validly described species, even though separate library entries were created for them. DNA reassociation determinations in which the S1 nuclease procedure was used showed that cellobiose fermentation and pigment production are variable among strains of P. melaninogenica and P. denticola and that fermentation of xylan is not a reliable characteristic for differentiating P. buccalis from Prevotella veroralis. In contrast to previous indications, most strains of P. veroralis do not ferment xylan. These species can be differentiated by DNA-DNA reassociation and by cellular fatty acid analysis, using the Microbial Identification System, but differentiation by currently described phenotypic characteristics is not reliable. Similarly, P. loescheii and the genetically distinct (but closely related) D1C-20 group cannot be distinguished reliably from each other or from P. veroralis, P. denticola, and P. melaninogenica on the basis of currently described phenotypic tests other than cellular fatty acid composition or, for some species, electrophoretic patterns of soluble whole-cell proteins.     

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.     

Bacterial diversity in the human saliva from different ages

To obtain primary idea on oral bacterium species that are generally present in periodotally healthy Koreans, the oral bacterial flora in the saliva of four periodontally healthy Koreans at different ages (5, 32, 35, 65) was investigated in this study. For this investigation, 16SrRNA gene clone libraries were generated from the saliva of the four healthy Koreans, and 50 clones were randomly selected from each saliva clone library and sequenced. Totally, 37 different kinds of bacterial 16S rRNA gene sequences were identified based on sequence homology search through GenBank database. The 37 kinds of saliva clone sequences were classified to 14 genera and 2 uncultured and 1 unidentified bacteria. Among the 14 identified genera, Streptococcus, Prevotella, and Veillonella were common genera, and Streptococcus was dominant genus that accounted for 7 different species. Among the seven Streptococcus species, S. salivarius appeared as the most common species. More numbers of species belonging to the genera Streptococcus and Prevotella was present in saliva from ages 32 and 35. While saliva from ages 5 and 65 showed more numbers of species belonging to the genera Rothia, including potential pathogenic species. Overall, saliva of a young child and a senior showed higher bacterial diversity than that of young adults.     

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.     

Microbiota in the oral subgingival biofilm is associated with obesity in adolescence

To test the hypothesis whether microbiota in oral biofilm is linked with obesity in adolescents we designed this cross-sectional study. Obese adolescents (n = 29) with a mean age of 14.7 years and normal weight subjects (n = 58) matched by age and gender were examined with respect to visible plaque index (VPI%) and gingival inflammation (bleeding on probing (BOP%)). Stimulated saliva was collected. They answered a questionnaire concerning medical history, medication, oral hygiene habits, smoking habits, and sociodemographic background. Microbiological samples taken from the gingival crevice was analyzed by checkerboard DNA-DNA hybridization technique. The sum of bacterial cells in subgingival biofilm was significantly associated with obesity (P < 0.001). The link between sum of bacterial cells and obesity was not confounded by any of the studied variables (chronic disease, medication, VPI%, BOP%, flow rate of whole saliva, or meal frequency). Totally 23 bacterial species were present in approximately threefold higher amounts, on average, in obese subjects compared with normal weight controls. Of the Proteobacteria phylum, Campylobacter rectus and Neisseria mucosa were present in sixfold higher amounts among obese subjects. The association between obesity and sum of bacterial cells in oral subgingival biofilm indicates a possible link between oral microbiota and obesity in adolescents.     

Properties of hemagglutination by Prevotella melaninogenica

Although Prevotella melaninogenica belongs to the commensal oral microbiota, some strains possess putative virulence factors. For example, we have previously described fimbriated, hemagglutinating strains of P. melaninogenica, isolated from patients with periodontal disease. The aim of this investigation was to compare some chemical and physical properties of hemagglutination (HA) of P. melaninogenica with those of other pigmented gram-negative anaerobes. HA of 13 P. melaninogenica strains proved to be considerably weaker than that of the major periodontal pathogen, Porphyromonas gingivalis. Vigorous shaking reduced HA of shaken cells but the shaken supernatant had the same hemagglutinating activity as non-shaken cells. The hemagglutinating agent on P. melaninogenica seemed to be a protein, which can be separated from the cell and binds to lactose-, galactose-, and raffinose-containing carbohydrates on the erythrocytes. Adherence to epithelial cells did not differ significantly between the hemagglutinating and non-hemagglutinating strains of P. melaninogenica. Although P. melaninogenica is able to agglutinate erythrocytes, this potential virulence factor is of a considerably lower magnitude than that of major periodontal pathogens.     

Periodontal microflora of HIV infected patients with periodontitis

The aim of this study was to determine the microbial profile of periodontal lesions in HIV seropositive patients and to compare it with rapidly progressing periodontal lesions in systemically healthy patients. The subgingival microflora of 20 CDC II, 20 CDC III, 20 CDC IV/V and 20 systemically healthy patients with rapidly progressing periodontitis was examined. Four sites with greatest probing depth in each patient were selected for microbiological sampling. The samples were cultured aerobically and anaerobically for bacterial isolation using selective and non-selective media. Isolates were characterized to species level by conventional biochemical tests and various identification kits. The microflora of periodontitis lesions within the three stages of the HIV infection was similar to that of progressing periodontitis in systemically healthy adults including Campylobacter rectus, Capnocytophaga spp., Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Selenomonas spp. and Peptostreptococcus micros. However, HIV seropositive periodontitis lesions harboured a range of exogenous pathogens rarely associated with common types of periodontitis including Staphylococcus aureus, Enterobacter cloaca, Pseudomonas aeruginosa, Candida albicans, Enterococcus faecalis, Enterococcus avium, Clostridium difficile, Aspergillus fumigatus, Klebsiella pneumoniae and Mycoplasma incognitum. The lack of immune effector and regulatory cells in HIV infected patients could in fact explain the increase of some opportunistic pathogens and the characteristic and rapidly progressing nature of the periodontal disease in these patients.     

Association between Polycystic Ovary Syndrome,Oral Microbiota and Systemic Antibody Responses

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.