Progression of periodontal inflammation in adolescents is associated with increased number of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Fusobacterium nucleatum.

OBJECTIVE: The study aims to evaluate the change of related subgingival periodontopathogens among different stage of gingivitis in adolescent and assess the relationship between periodontopathogens and the progression of periodontal inflammation. METHODS: A total of 77 subgingival plaque samples from 35 adolescent individuals were divided into three groups including gingivitis group (mild, 15 samples; moderate, 16 samples; severe, 15 samples), chronic periodontitis group (15 samples) and healthy group (15 samples). Real-time PCR was used to quantitate Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Fusobacterium nucleatum in subgingival plaque samples. RESULTS: All species, except for F. nucleatum, were detected in samples from gingivitis and periodontitis groups in significantly greater number than in those from healthy group (P < 0.05). In gingivitis groups, the number of P. gingivalis, T. forsythensis, and F. nucleatum in moderate and severe gingivitis groups was significantly higher than in mild gingivitis group (P < 0.05). After merging moderate gingivitis and severe gingivitis groups into moderate-to-severe gingivitis group, the four periodontopathogens were detected in samples from periodontitis group in significantly greater number than in those from moderate-to-severe gingivitis group (P < 0.05). CONCLUSION: The number of P. gingivalis, P. intermedia, T. forsythensis, and F. nucleatum in subgingival plaque increases with progression of periodontal inflammation in adolescents. Examination of periodontopathogens number in adolescents may be of some value for monitoring of periodontal disease development.

Antimicrobial effect of acidified nitrate and nitrite on six common oral pathogens in vitro.

BACKGROUND: Salivary nitrate is positively correlated with plasma nitrate and its level is 9 times the plasma level after nitrate loading. Nitrate in saliva is known to be reduced to nitrite by oral bacteria. Nitrate and nitrite levels in saliva are 3 - 5 times those in serum in physiological conditions respectively in our previous study. The biological functions of high salivary nitrate and nitrite are still not well understood. The aim of this in vitro study was to investigate the antimicrobial effects of nitrate and nitrite on main oral pathogens under acidic conditions. METHODS: Six common oral pathogens including Streptococcus mutans NCTC 10449, Lactobacillus acidophilus ATCC 4646, Porphyromonas gingivalis ATCC 33277, Capnocytophaga gingivalis ATCC 33624, Fusobacterium nucleatum ATCC 10953, and Candida albicans ATCC 10231 were cultured in liquid medium. Sodium nitrate or sodium nitrite was added to the medium to final concentrations of 0, 0.5, 1, 2, and 10 mmol/L. All of the microorganisms were incubated for 24 to 48 hours. The optical densities (OD) of cell suspensions were determined and the cultures were transferred to solid nutrient broth medium to observe the minimum inhibitory concentration and minimum bactericidal/fungicidal concentration for the six tested pathogens. RESULTS: Nitrite at concentrations of 0.5 to 10 mmol/L had an inhibitory effect on all tested organisms at low pH values. The antimicrobial effect of nitrite increased with the acidity of the medium. Streptococcus mutans NCTC 10449 was highly sensitive to nitrite at low pH values. Lactobacillus acidophilus ATCC 4646 and Candida albicans ATCC 10231 were relatively resistant to acidified nitrite. Nitrate at the given concentrations and under acidic conditions had no inhibitory effect on the growth of any of the tested pathogens. CONCLUSION: Nitrite, at a concentration equal to that in human saliva, is both cytocidal and cytostatic to six principal oral pathogens in vitro, whereas nitrate at a similar concentration has no antimicrobial effect on these organisms.

[Construction of the hemagglutinin-2-deficient mutant of Porphyromonas gingivalis].

OBJECTIVE: To construct and identify the Porphyromonas gingivalis(Pg)ATCC33277 hemagglutinin-2(HA-2)-deficient mutant. METHODS: The genomic DNA of Pg was isolated from PgATCC33277. The up/down stream genes of HA-2-HA(u), HA(l) were amplified by PCR, and inserted into pSY118 separately which contains a 2.1 kb antibiotic resistance ermF-ermAM cassette. The resultant recombinant plasmid-pSY118-HA was linearized as the gene targating fragment HA-ermF-ermAM and used in the electroporation of PgATCC33277. The Pg HA-2-deficient mutant was screened by allelic exchange. The test of aggregation of red blood cells was used to investigate the function change between PgHA2-deficient mutant and the wild type of PgATCC33277. RESULTS: The PgHA-2-deficient mutant was identified by PCR. The ability of Pg HA-2-deficient mutant to aggregate red blood cell was significantly decreased compared with the wild type. CONCLUSIONS: HA-2-deficient mutant of Pg ATCC33277 was constructed successfully, which lays a foundation for further study of its biological function.

[Sequence analysis of hemin-binding peptide derived from recombinant hemagglutinin-2 of Porphyromonas gingivalis].

OBJECTIVE: To determine the sequence of the active peptide derived from recombinant hemagglutinin (rHA-2) of Porphyromonas gingivalis (Pg). METHODS: The HA-2 gene from PgATCC33277 was cloned, expressed in Escherichia coli (Ec) BL21 (DE3), and purified. The purified recombinant protein was evaluated for its ability to bind hemin-linked agarose. The active peptide was subjected to endoproteinase-mediated sequence analysis. RESULTS: The protein expressed in Ec BL21 (DE3) was identified as PgHA-2 by plasmid sequence analysis, Western blotting, and mass spectrometry. The recombinant protein was confirmed functional by its ability to bind hemin. The sequence of the active peptide of rHA-2 was determined to be DHYAVMISKTGTNAG. CONCLUSIONS: The availability of sequence of the active peptide of rHA-2 provides a foundation for the development of immunoprophylactic and therapeutic agents against this human pathogen.

[Bacteriological analysis of subgingival plaque in adolescents].

OBJECTIVE: To investigate the changes in characteristics of periodontal pathogens in subgingival plaque of patients with puberty gingivitis and its relevance with clinical symptoms. METHODS: A total of 108 subgingival plaque samples were collected from 30 patients with puberty gingivitis (Group G), 9 cases of chronic periodontitis (Group P) and 15 healthy controls (Group H). The age of the 54 participants was from 11 to 17. The black-pigmented bacteria (BPB), Fusobacterium nucleatum (Fn), Actinobacillus actinomycetemcomitans (Aa), Actinomyces were detected using bacterial culture. The probing depth (PD), gingival index (GI), bleeding index (BI) and attachment loss (AL) were also recorded. RESULTS: In all these three groups, the detection rates of black-pigmented bacteria were: 3%, 30% and 100%; Fn were: 30%, 68% and 94%, statistically significantly different (P < 0.01). The lgCFU/ml of black-pigmented bacteria and Actinomyces was higher in mild-moderate group [(3.8 +/- 0.7) and (5.3 +/- 0.9)] than in Group H (P < 0.001). The lgCFU/ml of black-pigmented bacteria and Fn significantly was higher in severe inflammation group [(4.7 +/- 1.2) and (4.4 +/- 0.8)] than in the mild-moderate group (P < 0.01). The lgCFU/ml of black-pigmented bacteria, Fn and Aa was higher in severe gingivitis group [(6.6 +/- 1.0), (5.5 +/- 1.0) and (4.2 +/- 1.7)] than in mild gingivitis group (P < 0.01). The detection rate and lgCFU/ml of black-pigmented bacteria, Fn and Aa were both positively correlated with BI, PD and AL. CONCLUSIONS: In the stage of severe gingivitis, the periodontal pathogens increased markedly, suggesting that risk of further destruction of periodontal tissue may exist.

[Alterations of salivary flow rate and oral main pathogens in miniature pig with bilateral parotid gland atrophy].

OBJECTIVE: To observe the effect of bilateral parotid gland atrophy on the whole saliva flow rate and the growth of main oral pathogens in different sites of oral cavity. METHODS: Ten healthy miniature pigs were divided into two groups. The parotid glands of test group (n = 5) were bilaterally ablated by methyl violet. Another healthy five miniature pigs served as the control group. Whole saliva was collected and the whole saliva flow rate detected in both groups at 12 and 24 months respectively after parotid atrophy. The total numbers of oral main pathogens in the first molar, cuspid sub-gingival bacteria plaque and whole saliva were also detected. RESULTS: The whole saliva flow rate was significantly decreased at both 12 and 24 months respectively after atrophy of bilateral parotid gland in miniature pig. Pathogens including Streptococcus mutans, Porphyromonas gingivalis and Fusobacterium nucleatum in different sites oral cavity were increased after bilateral parotid gland atrophy. CONCLUSIONS: Bilateral ablation of the parotid glands led to a significant decrease of whole saliva flow rate. The total numbers of main oral pathogens were increased in different sites of oral cavity.

[Cloning and polymorphism analysis of prtH gene from Porphyromonas gingivalis].

OBJECTIVE: To clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis. METHODS: Using PCR, the prtH was amplified and cloned into pGEM-T vector. To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe. RESULTS: Recombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay. Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns. Strain 47A-1 seemed even not to contain prtH gene. CONCLUSIONS: Different prtH gene sequences exist in different P.g strains. This polymorphism may indicate various potential virulent effects during the infection and pathogenesis. Established PCR protocol is sensitive for identification of prtH gene.

[Phenotypic variation in Actinobacillus actinomycetemcomitans].

OBJECTIVE: To investigate the colony variation in Actinobacillus actinomycetemcomitans (Aa) from rough to smooth and recognize its different morphology during laboratory translations. METHODS: Primary strains isolated from subgingival plaque of two juvenile periodontitis patients were repeatedly subcultured on agar plates and broth; for broth culture, every generation was translated in broth and on solid medium separately to observe the corresponding morphologies of Aa grow in broth. RESULTS: Three smooth strains of Aa from the broth culture were obtained. The process was about 7-8 generations: colonies changed from a small and adherence phenotype to a bigger and sediment ones and finally the culture supernatant became turbid; the corresponding morphologies grow on agar exhibiting an adherent, small rough colony phenotype which had a star-shaped internal structure converted gradually to a kind of bigger, opaque, nonadherent, smooth phenotype, then the colony extended out from the margin of the colony and finally converted to a flat, almost parent morphology and the same time the star-like inner structure converted to a simpler and smaller type and finally disappeared. We could not get completely smooth variants of Aa from agar. CONCLUSIONS: The variation in colony morphology of Aa from rough to smooth is a process, in which the colony was gradually wetter and bigger and at the same time gradually lost the inner structure. During this process three colony morphologies at least can be seen, including rough, opaque smooth and almost translucent smooth.