• Journal of Periodontal and Implant Science
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• v.32 no.4
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• pp.827-836
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• 2002

The present study was performed to evaluate how cellular and humoral immune responses were perturbed by immunization of mixed periodontal bacterial biofilms. Each group of mice was immunizared with 1) Poqhyromonas gingivalis (P. gingivaliis) grown as a planktonic culture, 2) Fusobacterium nucleatum (F. nucleatum), 3) P. gingivalis grown as a biofilm, or 4) mixed P. gingivalis plus F. nucleatum grown as a biofilm culture, respectively. Immune mouse sera were collected from each mouse. Spleens were harvested to isolate T cells and consequently stimulated with antigen presenting cells and P. gingivalis whole cell antigen to establish P. gingivalis-specific T cell lines. There were no significant differences in the mean anti- gingivalis IgG antibody titers among mouse groups. Immunization of mice with pure P. gingivalis biofilm or mixed P gingivalis plus F. nucleatum biofilm resulted in significant reduction o f antibody avidity and opsonophagocytois function. INF-$\gamma$production by P. gingivalis-specific T cell lines was also substantially recluced in mouse groups immunized with the biofilm. It was concluded that P. gingivalis biofilm perturbs the cellular and humoral immune responses in periodontal disease.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.110-115
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• 2004

This study was undertaken to develop PCR primers for the simultaneous detection of Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans, using two species-specific reverse primers in combination with a single conserved forward primer. These primers target the variable and conserved regions of the 16S rDNA. The primer specificity was tested against (i) four F. nucleatum and three A. actinomycetemcomitans strains and (ii) seven representatives of the different species of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of F. nucleatum and A. actinomycetemcomitans. The data indicate that species-specific amplicons could be obtained for all the F. nucleatum and A. actinomycetemcomitans strains tested, which were not found in the seven other species. The multiplex PCR could detect as little as 4 fg of chromosomal DNA of F. nucleatum and A. actinomycetemcomitans simultaneously. These findings suggest that these PCR primers are highly sensitive and are suitable for applications in epidemiological studies, diagnosis, and monitoring F. nucleatum and A. actinomycetemcomitans after the treatment of periodontitis.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.401-411
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• 2005

The aim of this study was to determine the minimal inhibitory concentration(MIC) of cefixime, which is a 3rd generation of cefalosporin, against 6 species of putative periodontopathogens; Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Tannerella forsythia and Porphyromonas gingivalis. The efficacy of cefixime was examined by comparing it with that of several antibiotics(amoxicillin, $Augmentin^{(R)}$ ciprofloxacin, metronidazole, and tetracycline), which were used as the control. The MIC was measured using a microdilution method. The MIC of cefixime against the putative periodotopathogens, as a single use regimen, was relatively lower than that of the other antibiotics. The MIC of cefixime/metronidazole against P. intermedia ChDC KB14, P. nigrescens ChDC KB50, F. nucleatum ChDC PV-F37, F. nucleatum ChDC F130, and F. nucleatum ChDC F175, as a simultaneous regimen, was lower than that of the other antibiotics. The concentration of cefixime in the crevicular fluid of volunteers who received 250mg every 12 hours for 3 days was $9{\mu}g/ml$ after 9 hours. In conclusion, cefixime showed good antimicrobial activity in a single treatment or as a combined therapy with amoxicillin, $Augmentin^{(R)}$ or metronidazole against 6 periodontopathogens.

• Journal of Periodontal and Implant Science
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• v.31 no.4
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• pp.661-668
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• 2001

Anti-P. gingivalis immune sera were obtained from mice immunized with either P. gingivalis alone, or F. nucleaturm followed by P. gingivalis. Two groups of immune sera were examined for binding capacity to P. gingivalis biofilm by confocal laser scanning microscope, Antibody avidity index was also determined for each immune sera. The results indicated that prior immunization of mice with F. nucleaturm impaired P. gingivalis-specific immune sera in binding capacity to biofilm and antibody avidity to P. gingivalis. Elevated antibody responses in patients with destructive periodontal disease has often been related to suboptimal level of protective antibody $(opsonophagocytosis)^{1-3)}$ while post-immune sera obtained with experimental animals using a single periodontal pathogen demonstrated satisfactory levels of protective function against the homologous bacterial $challenge^{4,5)}$.The reason is unclear why elevated IgG responses in periodontal patients to periodontal pathogens do not necessarily reflect their protective function. Such an immune deviation might be derived from the fact that destructive periodontal disease is cumulative result of immunopathologic processes responding to an array of different colonizing microorganisms sequentially infecting in the subgingival environmental niche. Fusobacterium nucleaturm is one of the key pathogens in gingivitis, in the transitional phase of conversion of gingivitis into destructive periodontitk, and in adult $periodontitis^{6-8)}$. It also plays a central role in coaggregation with other important microbial species in subgingival $area^{6,9,10)}$ as well as in $biofilm^{11)}$, especially with Porphyromonas gingjvalis in synergism of virulence in human periodontal disease or in animal $models^{12-14)}$. This organism has also been reported to have immune modulating activity for secondary immune response to Actinobacillus $actinomycetemcomitans^{15)}$. It is presumed that sequential colonization and intermicrobial coaggregation between intermediate and late colonizers could potentially modulate the immune responses and development of specific T cell phenotypes in periodontal lesions. We have recently demonstrated the skewed polarization of P. gingivalis-specific helper T cell clones in mice immunized with F. nucleaturm followed by P. $gingivalis.^{16)}$. Consequently F. nucleaturm may initially prime the immune cells and modify their responses to the successive organism, P. gingivalis. This could explain why one frequently observes non-protective serum antibodies to P. gingivalis in periodontal patients in contrast with those obtained from animals that were immunized with $P.gingivalis\;alone^{17)}$. The present study was performed to investigate the immune modulating effect of F. nucleatum on serum binding to experimental biofilms and the avidity of anti-P. gingivalis antibody.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.197-202
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• 2010

Most oral microorganisms exist as biofilms which initiate formation via the attachment of an early colonizer to host proteins on the tooth surface. Fusobacterium nucleatum act as a bridge between early and late colonizers. Dental biofilms eventually comprise dental pathogens such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. To evaluate the effects of mutual interactions between oral bacteria on the growth of biofilms, periodontopathogens were co-cultured with a $0.4\;{\mu}m$ barrier. Streptococcus gordonii inhibited the growth of F. nucleatum and periodontopathogens. However, F. nucleatum, P. gingivalis and T. denticola activated the growth of other bacteria. A co-culture system of early and late colonizers could be a useful tool to further understand bacterial interactions during the development of dental biofilm.

• International Journal of Oral Biology
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• v.39 no.2
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• pp.115-120
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• 2014

Minimal inhibitory concentration (MIC) is the lowest concentration of antibiotics that inhibits the visible growth of a microorganism. It has been reported that sub-MIC of antibiotics may result in morphological alterations along with biochemical and physiological changes in bacteria. The purpose of this study was to examine morphological changes of periodontal pathogens after treatment with sub-MIC antibiotics. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis were used in this study. The MIC for amoxicillin, doxycycline, metronidazole, penicillin, and tetracycline were determined by broth dilution method. The bacterial morphology was observed with bright field microscope after incubating with sub-MIC antibiotics. The length of A. actinomycetemcomitans and F. nucleatum were increased after incubation with metronidazole; penicillin and amoxicillin. P. gingivalis were increased after incubating with metronidazole and penicillin. However, F. nucleatum showed decreased length after incubation with doxycycline and tetracycline. In this study, we observed that sub-MIC antibiotics can affect the morphology of periodontal pathogens.

• International Journal of Oral Biology
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• v.43 no.4
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• pp.193-200
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• 2018

The purpose of this study is to determine if natural extracts could be used as an additive in oral health food made with Weissella cibaria CMU (oraCMU). Natural extracts of green tea, mulberry leaf, licorice, and propolis, which are reported to have antimicrobial activities, were selected and used in this study. The minimum inhibitory concentrations (MIC) of extracts on periodontal pathogens such as Fusobacterium nucleatum and Porphyromonas gingivalis and their synergy effects with oraCMU by the fractional inhibitory concentrations methods were measured. From the results obtained, all the extracts showed no effect on the growth of oraCMU. Green tea extract showed the best antibacterial activity with MIC of 1.8 mg/ml against both F. nucleatum and P. gingivalis. In addition, green tea extract had a synergistic effect with oraCMU against F. nucleatum. Therefore, these results suggested that green tea extract is available as an additive in oral health food made with oraCMU.

• Journal of Oral Medicine and Pain
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• v.30 no.1
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• pp.1-14
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• 2005

The maintenance of good oral health in adults is often hindered by oral malodor and periodontal diseases which are known to be commonly caused by some species of Gram-negative anaerobic bacteria, with low sensitivity to common synthetic antibiotics or antibacterial chemical agents. Therefore the development of a nonharmful natural antibacterial oral rinsing remedy against the causative bacteria is thought to be very important. The purpose of this study is to obtain the basic data for development of a nonharmful natural antibacterial oral rinsing remedy using colloidal silver. The author applied colloidal silver solution with concentration of 10, 30, 50, 80 ppm to some strains in species of Prevotella intermedia, Porphyromonas gingivalis, Fusobaterium nucleatum, and evaluated the effects of colloidal silver on the growth of experimental bacterial strains in aspects of minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and growth pattern after incubation for 24, 48, 72 hours. The obtained results were as follows: MIC of colloidal silver solution against experimental strains was 30 ppm in P. intermedia, 10 or 30 ppm in P. gingivalis, and 30, 50, or 80 ppm in F. nucleatum. And MBC of colloidal silver solution against experimental strains was 30 ppm in P. intermedia, 30 or 50 ppm in P. gingivalis, 30 or 80 ppm in F. nucleatum. Therefore it was concluded that colloidal silver exhibited bacteriostatic or/and bacteriocidal effects against some experimental strain. And the inhibition of growth of experimental strains were markedly or considerably exhibited under 30 ppm$\sim$50 ppm of colloidal silver solution for 48 hours$\sim$72 hours in P. intermedia, 10 ppm$\sim$30 ppm for 24 hours$\sim$48 hours in P. gingivalis, 30 ppm for 24 hours in F. nucleatum. These results indicate that the colloidal silver inhibited effectively the growth of some species of Gram-negative anaerobic bacteria by exhibition of bacteriostatic or/and bacteriocidal effects, and can be used as a possible major ingredient of the nonharmful natural antibacterial oral rinsing remedy to oral malodor and periodontal diseases.

• Journal of dental hygiene science
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• v.15 no.2
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• pp.209-219
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• 2015

In order to explore an effect of interaction of Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis that are bacteria relevant to periodontal disease on its growth, the bacteria were incubated in trypticase soy hemin menadione broth at $37^{\circ}C$ $CO_2$ incubator for 7 days through anaerobic jar by single and co-culture with heat treated dead bacteria under anaerobic gas pack. In order to confirm growth level, absorbance was measured and for confirming colony structure and form, it was observed with scanning electron microscope. In order to confirm an effect on pathogenicity of P. gingivalis, real time reverse transcriptase polymerase chain reaction was implemented for expression analysis for rgpA gene that produces HRgpA which is gingipain. As a result, the following conclusion was obtained. Colony formation of S. gordonii and P. gingivalis was increased by other dead bacteria and in case of F. nucleatum, its colony formation was showed an aspect of being increased by dead bacterium of P. gingivalis but decreased by dead bacterium of S. gordonii. Therefore, it is considered that the strains being used for this study would affect interactively through bacterial cell itself as well as their interaction factor at the time of colony formation.

• International Journal of Oral Biology
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• v.41 no.4
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• pp.237-242
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• 2016

Interleukin-1b ($IL-1{\beta}$), a proinflammatory cytokine, regulates the innate immune responses against bacterial infection. Mature $IL-1{\beta}$ is produced from $pro-IL-1{\beta}$ by activated caspase-1, which in turn is activated by the inflammasome complex formation. In this study, we compared the inflammasome mRNA expression induced by S. sanguinis, S. oralis, F. nucleatum and P. intermedia. Among the tested bacteria, S. sanguinis induced the highest $IL-1{\beta}$ secretion. S. oralis, F. nucleatum and P. intermedia induced very weak $IL-1{\beta}$ secretion. S. sanguinis mostly induced the NLRP3 mRNA expressions. Although F. nucleatum did not induce high $IL-1{\beta}$ secretion, it induced high expression levels of AIM2, NLRP2, and NLRP3. No specific inflammasomes were induced by S. oralis and P.intermedia. Studying the inflammasome complex activation induced by oral bacteria may thus enhance our understanding of the pathogenesis of oral diseases.