The state of small amount bacteria in oral cavity biofilm

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15592204
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Social dentistry
• Research Institution: Hokkaido University
• Principal Investigator: HONGO Hirohisa Hokkaido University, Oral health science, instructor, 大学院・歯学研究科, 助手 (00281816)
• Co-Investigator(Kenkyū-buntansha): MORITA Manabu Hokkaido University, Oral health science, professor, 大学院・歯学研究科, 教授 (40157904) ; HONDA Okahito Hokkaido University, Oral health science, professor, 大学院・歯学研究科, 助教授 (30109475)
• Project Period (FY): 2003 – 2004
• Keywords: Biofilm / Scanning electron microscopy / fimbriae / In situ hybridization / 16SrRNA

Research Abstract

We have received the acceptance of the ethical committee of Hokkaido University Hospital to collect the biofilm from oral cavity of patients in the the Hospital.
We observed the biofilm by scanning electron microscopy and tried to detect small amount of bacteria in the biofilm. We kept a part of biofilm frozen for incubation and extract DNA from another part of biofilm for PCR analysis.
We could observe spirillum, rods, cocci and fungus by scanning electron microscopy(SEM). We could also observe the flmbriae like structure of P.gingivalis by SEM.
The fimbriae have been believed to be related to the binding ability of P.gingivalis, however, they had not been ovserved by SEM. So, we examined by SEM colonies of parent strain of P.gangivalis (ATCC33277) and pgmA-knockout (mutant) strains, and we have succeeded to observe the fimbriae of P.gingivalis clearly and three-dimentionally by SEM. Bodies in the colonies were connected by dense fimbriae in the parent strain.
To compare binding abilities of the two strains, we used hemagglutination titers. The hemagglutination titer of the mutant strain was only 1/10 of that of the parent strain. In the aggregates of erythrocytes and P.gingivalis, the erythrocytes were bridged by long fimbriae extending from the bodies. From these SEM images, we confirmed that the length and number of the fimbriae play important roles in making biofilms or colonization. The SEM images of the colony gave us an imagination about the role of the fimbriae. The space formed by long fimbriae between the bodies would be used as routes for nutrients and endotoxins.
In order to detect the bacteria having strong virulence, we made probes complement to 16SrRNA of Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Acinetobacter, Haemophilus influenzae etc. We could detect Pseudomonas aeruginosa by In situ hybridization, however, we have not detected other bateria, so we have need to make an improvement in detection of bacteria, and continue the study.
We presented papers on the results on our study to 83_<rd> m International Association of Dental Research (Baltimore, USA).