Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis

Abstract

Terminal restriction fragment-length polymorphism (T-RFLP) analysis was applied to characterize oral bacterial flora in saliva from 18 healthy subjects and 18 patients with periodontitis. The 16S rRNA genes (rDNAs) of oral bacteria and spirochaetes in saliva were amplified by PCR with a 69carboxy-fluorescein (6-FAM)-labelled universal forward primer (27F) and a universal reverse primer (1492R) or the Spirochaeta-selective reverse primer. The 16S rDNAs were digested with restriction enzymes with 4 bp recognition sites (HhaI or MspI) and analysed by using an automated DNA sequencer. T-RFLP patterns were numerically analysed using a computer program. From analysis of the oral bacterial community, patterns derived from periodontally healthy subjects and patients with periodontitis were grouped into different clusters, though with some uncertainty. Samples from patients with periodontitis tended to cluster into their respective types (aggressive and chronic periodontitis), although this was not very clear. Analysis of spirochaetal community using T-RFLP showed that the patterns derived from patients with periodontitis were grouped more as compared with the analysis of the oral bacterial community. These results suggest that samples from patients with periodontitis contain an unexpected diversity. T-RFLP patterns of 16S rDNAs from saliva samples of two periodontally healthy subjects over a 5-week period showed host-specific relatively stable oral bacterial flora. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of oral bacterial flora and rapid comparison of the community structure between subjects with and without periodontitis. INTRODUCTIONA large number and variety of bacteria exist in the human oral cavity. However, analysis of these bacterial communities has been limited by conventional culture-dependent methods; thus, many oral bacteria remain uncultured and uncharacterized. Consequently, studies of causal microorganisms of oral diseases including periodontal disease are, in general, restricted to cultivable species.Recently, a phylogenetic approach based on 16S rRNA genes (rDNA) has been applied to investigate the diversity of cultivable and non-cultivable species in the human oral cavity without cultivation (Choi et al., 1994(Choi et al., , 1996Dymock et al., 1996;Kroes et al., 1999;Paster et al., 2001;Sakamoto et al., 2000;Spratt et al., 1999). The 16S rDNA clone library method can provide direct sequence information. Paster et al. (2001) demonstrated that the predominant subgingival microbial community consisted of 347 species or phylotypes, based on analysis of 2522 16S rRNA clones. However, analysis of individual 16S rDNA clones is an expensive and extremely inefficient approach for comparison of a multitude of bacterial communities.Terminal restriction fragment-length polymorphism (T-RFLP) is a new molecular approach that allows the assessment of a diversity of complex bacterial communities and rapid comparison of the community structure and diversity of different ecosyst...

Application of substrate utilization patterns and terminal restriction fragment length polymorphism analysis to characterize the oral bacterial community of healthy subjects and patients with periodontitis

“…The present results indicated that the TRFLP patterns derived from patients with periodontitis were significantly different from the patterns of periodontally healthy subjects. The data confirm findings from previous studies, which reported that the saliva from patients with periodontitis contains a special bacterial diversity (9,19). Notably, the present TRFLP results also suggested that the age of the subjects had a significant effect on the oral bacterial flora.…”

“…The TRFLP patterns allow the rapid comparison of the community composition among patients with periodontitis, and can show host-specific relatively stable oral bacterial flora (9). The present results indicated that the TRFLP patterns derived from patients with periodontitis were significantly different from the patterns of periodontally healthy subjects.…”

“…In the study of the oral bacterial community, saliva is considered to be the most suitable sample, as it is composed of a variety of bacteria from different oral sites (9). Although TRFLP analysis has been used for evaluating the oral bacterial community in a few studies (7,9), the TRFLP method has not been compared with other microbial ecology techniques.…”

“…Although TRFLP analysis has been used for evaluating the oral bacterial community in a few studies (7,9), the TRFLP method has not been compared with other microbial ecology techniques. The aim of the present study, therefore, was to measure colony-forming units (CFUs) in the unstimulated saliva samples of patients with chronic periodontitis and healthy subjects.…”

Comparison among Fecal Secondary Bile Acid Levels, Fecal Microbiota and Clostridium scindens Cell Numbers in Japanese

“…When the target of PCR is 16S ribosomal DNA, the terminal restriction fragments pattern reflects the taxonomic diversity of the bacteria in the sample. T-RFLP is a rapid and efficient approach for successfully obtaining profiles of the various members of a complex microbial community, for example, human feces (10,25), chicken cecal samples (6), pig colon (19), rat feces (15), oral bacterial flora (24), soil (20,21), and marine sediments (28). The present study is the first to our knowledge to analyze 40 fecal samples using T-RFLP and to compare them using a dendrogram.…”