Takuichi Sato

Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real-time PCR

BACKGROUND AND OBJECTIVEnQualitative and quantitative changes of the subgingival plaque biofilm microflora in periodontal pockets are thought to be associated with the development and progression of periodontitis. The aims of the present study were to quantify the proportions of nine periodontitis-associated bacterial species and four Streptococcus species in subgingival plaque, and to evaluate their relationship with periodontitis quantitatively.nnnMATERIAL AND METHODSnSubgingival plaque samples were obtained from 12 periodontally healthy subjects and from 28 patients with periodontitis. The amounts of total and target bacteria were measured by quantitative real-time PCR using universal and species-specific primers, respectively.nnnRESULTSnThe proportion of total obligate anaerobes was found to be higher in subjects with periodontitis than in periodontally healthy subjects (p < 0.05). Among obligate anaerobes, Tannerella forsythia (2.04 +/- 5.27%, p < 0.05), Porphyromonas gingivalis (0.54 +/- 1.41%) and Eubacterium saphenum (0.30 +/- 0.96%) were detected at high proportions in subjects with periodontitis, but not in periodontally healthy subjects. By contrast, the proportion of total streptococci was lower in subjects with periodontitis (p < 0.05). Specifically, the proportion of T. forsythia, P. gingivalis or E. saphenum increased (>or= 2.78%) and the proportion of Streptococcus species decreased to virtually undetectable levels, in subjects with periodontitis.nnnCONCLUSIONnObligate anaerobes, including T. forthysia, P. gingivalis and E. saphenum, were identified predominantly in microflora from subjects with periodontitis, whereas Streptococcus species were identified predominantly in microflora from periodontally healthy subjects, suggesting a change in the subgingival environment that resulted in conditions more suitable for the survival of obligate anaerobes. The proportion of these obligate anaerobes in the subgingival plaque of subjects with periodontitis appears to be associated with the status of human periodontitis.

Profiling of dental plaque microflora on root caries lesions and the protein-degrading activity of these bacteria

PURPOSEnTo profile plaque microflora on root-caries lesions, and to examine the protein-denaturing activity as a pilot study.nnnMETHODSnSix subjects with root-caries were investigated. Plaque samples on root caries lesions (R), as well as from healthy supragingival sites (S) and periodontal pockets (> or = 5 mm) (P) were collected and cultured anaerobically on blood agar plates. The isolated bacteria were identified by 16S rRNA sequencing analysis, and examined for the protein-denaturing activity using the skim-milk plates and the SDS-PAGE, and for the acidogenicity using the FAB broth containing 1% glucose.nnnRESULTSnPropionibacterium, Actinomyces, Streptococcus, Lactobacillus and Bifidobacterium were predominant in R, while Actinomyces, Streptococcus, Veillonella and Capnocytophaga in S, and Actinomyces, Prevotella, Actinobaculum, Streptococcus, Olsenella and Eubacterium were predominant in P. Proteolytic bacteria comprised 40%, 26% and 57% of microflora in R, S and P, respectively. The skim-milk plates distinguished between protein-degrading and protein-coagulating bacteria, which comprised 7 and 33%, 0 and 26%, and 17 and 40% of microflora, in R, S and P, respectively. The SDS-PAGE analysis revealed that protein-degrading isolates were capable of degrading collagen molecules. Furthermore, the final culture pHs of protein-degrading and -coagulating bacteria were 5.0-5.4 and 3.8-3.9, respectively. The latter pH was low enough to denature proteins in skim milk. The microbial composition of R was distinct from those of S and P.

Detection and identification of non-Candida albicans species in human oral lichen planus.

Candida species were detected and identified in samples from the buccal mucosa, dorsal surface of the tongue and supragingival plaque of subjects with oral lichen planus (OLP). The Candida in the samples were cultured on selection agars, and identified by sequence analyses of 18S, 5.8S and 25/28S rRNA. The isolation frequency of Candida was higher in subjects with OLP than in those with healthy oral mucosa. Non‐C. albicans were only isolated from people with OLP. These results support the notion that subjects with OLP are more likely to have oral colonization with Candida, and that non‐C. albicans are specifically present in subjects with this condition.

Porphyromonas pogonae sp. nov., an anaerobic but low concentration oxygen adapted coccobacillus isolated from lizards (Pogona vitticeps) or human clinical specimens, and emended description of the genus Porphyromonas Shah and Collins 1988.

During the process of identifying a Gram-negative coccobacillus isolated from a human clinical specimen, we found that the isolates 16S rRNA gene had very close sequence identity with that of a variant Porphyromonas isolated from polymicrobial infections in the central bearded dragon, a species of lizard [2]. The 16S rRNA gene sequences of the human isolate and of six isolates from lizards were nearly identical (99.9-100%). Phylogenetic analysis placed all of these isolates in a single phylogenetic cluster well separated from other species in the genus Porphyromonas. The closest species was Porphyromonas catoniae with 90.7-90.9% sequence identity, although there was less than 6% DNA similarity between the P. catoniae type strain and our representative isolates from lizards (PAGU 1787(T)) and human (PAGU 1776). These isolates could grow under anaerobic or microaerobic conditions (6% O2 atmosphere). The isolates were positive for catalase and very strong β-hemolytic activity, but did not show black or brown pigmentation. Biochemically, the isolates could be differentiated from closely related species by pyroglutamic acid arylamidase and glycine arylamidase activity, and some others. The fermentation products mainly included succinic acid and propionic acid. The major fatty acids detected in cells of the isolates were iso-C15:0, anteiso-C15:0, and 3OH-iso-C17:0. The G+C content was 43.0 ± 0.62 mol%. The species name Porphyromonas pogonae sp. nov. is proposed for these isolates with the type strain of PAGU 1787(T) (=MI 10-1288(T)=JCM 19732(T)=ATCC BAA-2643(T)).

Cultivable Anaerobic Microbiota of Infected Root Canals

Objective. Periapical periodontitis is an infectious and inflammatory disease of the periapical tissues caused by oral bacteria invading the root canal. In the present study, profiling of the microbiota in infected root canals was performed using anaerobic culture and molecular biological techniques for bacterial identification. Methods. Informed consent was obtained from all subjects (age ranges, 34–71 years). Nine infected root canals with periapical lesions from 7 subjects were included. Samples from infected root canals were collected, followed by anaerobic culture on CDC blood agar plates. After 7 days, colony forming units (CFU) were counted and isolated bacteria were identified by 16S rRNA gene sequencing. Results. The mean bacterial count (CFU) in root canals was (0.5 ± 1.1) × 106 (range 8.0 × 101–3.1 × 106), and anaerobic bacteria were predominant (89.8%). The predominant isolates were Olsenella (25.4%), Mogibacterium (17.7%), Pseudoramibacter (17.7%), Propionibacterium (11.9%) and Parvimonas (5.9%). Conclusion. The combination of anaerobic culture and molecular biological techniques makes it possible to analyze rapidly the microbiota in infected root canals. The overwhelming majority of the isolates from infected root canals were found to be anaerobic bacteria, suggesting that the environment in root canals is anaerobic and therefore support the growth of anaerobes.

Microflora profiling of infected root canal before and after treatment using culture-independent methods

This study aimed to profile the microflora in infected root canals before and after root canal treatment using culture-independent methods. Six infected root canals in single-rooted teeth with periapical lesions from five subjects were included. Quantification of total bacteria was performed by real-time PCR with primers targeting 16S rRNA genes. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species level was performed by comparative analysis with the GenBank database. The concentration of extracted DNA before treatment was higher than that after root canal treatment, although the difference was not statistically significant. Sequence analysis revealed that oral bacteria such as Fusobacterium, Streptococcus, Olsenella, and Pseudoramibacter detected in cases before root canal treatment disappeared after treatment. These results suggest that the root canal microflora are distinct before and after root canal treatment, and that treatment changes the microflora in both quantity and quality.

Host β‐globin gene fragments in crevicular fluid as a biomarker in periodontal health and disease

BACKGROUND AND OBJECTIVEnLeukocytes and epithelium are the first line of defense in preventing bacterial invasion into periodontium. Some of these cells die in gingival crevicular fluid, whereupon their DNA is spilled out. The present study was designed to investigate the profile of host beta-globin gene fragments in the gingival crevicular fluid of various periodontal conditions.nnnMATERIAL AND METHODSnGingival crevicular fluid from 40 teeth with chronic periodontitis, 30 with gingivitis and 22 that were clinically healthy were centrifuged (3,000 g, 10 min). The supernatant (cell-free gingival crevicular fluid) was centrifuged again (13,000 g, 10 min), resulting in the pellet and the supernatant as debris and debris-free fractions, respectively. Specific primers for amplifying 110 bp, 536 bp and 2 kb amplicons of human beta-globin gene were used to investigate host DNA by quantitative and qualitative polymerase chain reaction.nnnRESULTSnThe periodontitis group showed the largest amount of host beta-globin gene fragments, while the healthy group had the lowest. In the debris and debris-free fractions, the 536 bp and 2 kb amplicons were more often detected in the periodontitis group than in the other groups. Interestingly, the presence of 2 kb amplicon in the debris fraction could be used to discriminate periodontitis from gingivitis and healthy groups because we found it in 85% of periodontitis samples but only in 13% of gingivitis samples, and it was absent in the healthy group.nnnCONCLUSIONnThis study shows the different DNA profiles of cell-free gingival crevicular fluid in periodontal health and disease. It suggests that the quantity and quality of host DNA are dependent on the disease conditions. Therefore, the beta-globin gene fragments in cell-free gingival crevicular fluid may be a potential biomarker of periodontal disease progression.

Rapid Quantification of Bacteria in Infected Root Canals Using Fluorescence Reagents and a Membrane Filter: A Pilot Study on Its Clinical Application to the Evaluation of the Outcomes of Endodontic Treatment

Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23–79 years). Samples from infected root canals were collected at the beginning of the treatment (termed #25 First), the end of the first day of treatment (termed #55 First), and the next appointment day (termed #55 Second). Then, the bacterial count (CFU) was measured using fluorescence reagents (4′,6′-diamidino-2-phenylindole and propidium iodide) and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of “#25 First” was (1.0 ± 1.4) × 105. As the root canal treatment progressed, the CFU decreased as 7.9 × 103 (#55 First) and 4.3 × 102 (#55 Second). Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis).

Porphyromonas bronchialis sp. nov. Isolated from Intraoperative Bronchial Fluids of a Patient with Non-Small Cell Lung Cancer

Porphyromonas strains, including Porphyromonas-like strains, have been isolated from oral and various other systemic infections. The characterization of such strains is a crucial issue, because such information contributes to both the taxonomy of anaerobic bacteria and the clinical aspects of infectious diseases. We previously isolated four Porphyromonas-like strains from intraoperative bronchial fluids of a patient with non-small cell lung cancer. This study aimed to characterize the genetic, biochemical and chemotaxonomic aspects of these isolates. Each strain only grew under anaerobic conditions and their colony morphology was convex, 0.1-1.0 mm in diameter, light gray, and slightly glistening colony, with no black or brown pigmentation on blood agar plates after five-day incubation. The pigmentation was helpful to differentiate the isolates from other Porphyromonas, as most of Porphyromonas species show the pigmentation. In the 16S rRNA gene phylogenetic analysis (98% sequence identity of isolates indicates the same species), the four isolates were closely related to one another (99.7-100.0%), but not related to Porphyromonas (P.) catoniae, the closest species (96.9%). In addition, the DNA-DNA hybridization data revealed less than 16% similarity values between a representative isolate and the P. catoniae, indicating that the strains were genetically independent. Biochemically, the isolates could be differentiated from closely related species, i.e., P. catoniae, P. gingivalis, P. gulae, and P. pogonae, with trypsin activity (negative only in the isolates) and leucine arylamidase activity (positive only in the isolates). We therefore propose a new species to include these isolates: Porphyromonas bronchialis sp. nov.

Subgingival Plaque Biofilm Microflora of Elderly Subjects

This study aimed to quantify Porphyromonas gingivalis (Pg) in subgingival plaque of periodontitis (P) and periodontally healthy (H) subjects in the elderly. Subsequently, fimA genotypes were determined, and compared between P and H sites in the same subjects. The proportion of Pg was higher in P than in H. The fimA genotypes of P were identified mainly as II and Ib, while those of H were as I–IV. In four out of five subjects in whom Pg was detected in H sites, the fimA genotype was identical between P and H sites, but the proportion of Pg was significantly higher in P sites than in H sites (P < 0.05). This study suggests that the increase of Pg and the specific fimA genotypes are associated with P in the elderly, and that Pg possibly transmits within the oral cavity.