원발성 치근단 치주염을 갖는 감염근관에서 증상유무에 따른 세균분포의 pyrosequencing 분석
Microbial profile of asymptomatic and symptomatic teeth with primary endodontic infections by pyrosequencing
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연구목적: 본 연구는 원발성 치근단 치주염(primary apical periodontitis)을 갖는 치아에서 임상증상 유무에 따른 미생물군집의 차이를 GS FLX Titanium pyrosequencing을 이용하여 species level까지 분석하였다. 연구 재료 및 방법: 원발성 치근단 치주염을 갖는 6개의 표본에서 pysequencing을 시행하였다. 중합효소 연쇄반응(PCR)에의해 얻어진 small-subunit ribosomal RNA의 초가변 영역(hypervariable region)의 amplicon을 이용하여 GS FLX Titanium pyrosequencing 을 시행하였다. 결과: 평균적으로 무증상군 및 증상군에서 각각 10,639 및 45,455개의 16S rRNA sequence을 얻었으며 평균길이는440bases였다. Ribosomal Database Project Classifier을 이용한 분석결과 142종의 genera 및 13종의 phylum 수준에서의 세균종을 검출하였다. 검출된 13개의 phyla 가운데 Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria,Proteobacteria, Spirochetes, and Synergistetes 종이 상대적으로 호발하였으며, genus 수준에서는 Pyramidobacter,Streptococcus, Leptotrichia이 무증상의 근관의 50%를 차지하였으며, Neisseria, Propionibacterium, Tessaracoccus 균종은 증상이 있는 근관의 69%를 차지하였다. Operational taxonomic units (3%)로 나눈 결과 증상이 없는 치아에서450개, 증상이 있는 치아에서 1,997개의 species가 발견되었다. 증상이 있는 치아에서 통계적으로 유의성 있게 많은 수의세균이 검출되었다(p < 0.05). 결론: GS FLX Titanium Pyrosequencing 기법을 통해 원발성 감염근관에서 이전에 검출하지 못했던 다양한 근관내 분포세균을 검출할 수 있었다.
Abstract
Objectives: The purpose of this in vivo study was to investigate the microbial diversity in symptomatic and asymptomatic canals with primary endodontic infections by using GS FLX Titanium pyrosequencing. Materials and Methods: Sequencing was performed on 6 teeth (symptomatic, n = 3; asymptomatic, n = 3)with primary endodontic infections. Amplicons from hypervariable region of the small-subunit ribosomal RNA gene were generated by polymerized chain reaction (PCR), and sequenced by means of the GS FLX Titanium pyrosequencing. Results: On average, 10,639 and 45,455 16S rRNA sequences for asymptomatic and symptomatic teeth were obtained, respectively. Based on Ribosomal Database Project Classifier analysis, pyrosequencing identified the 141 bacterial genera in 13 phyla. The vast majority of sequences belonged to one of the seven phyla: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochetes, and Synergistetes. In genus level, Pyramidobacter, Streptococcus, and Leptotrichia constituted about 50% of microbial profile in asymptomatic teeth, whereas Neisseria, Propionibacterium, and Tessaracoccus were frequently found in symptomatic teeth (69%). Grouping the sequences in operational taxonomic units (3%) yielded 450 and 1,997 species level phylotypes in asymptomatic and symptomatic teeth, respectively. The total bacteria counts were significantly higher in symptomatic teeth than that of asymptomatic teeth (p < 0.05). Conclusions: GS FLX Titanium pyrosequencing could reveal a previously unidentified high bacterial diversity in primary endodontic infections.
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