Molecular Analysis of the Microflora Associated with Dental Caries

Abstract

Molecular techniques have revealed many novel, presumed unculturable, taxa in oral infections. The aim of this study was to characterize the bacterial community of the middle and advancing front of carious dental lesions by cultural and molecular analyses. Samples were collected with a hand excavator from five teeth with carious lesions involving dentine. Samples were cultured on blood agar and Rogosa agar incubated in air plus 5% CO 2 and on fastidious anaerobe agar anaerobically. DNA was also extracted directly from the samples and 16S rRNA genes were amplified by PCR with universal primers. PCR products were singularized by cloning, and the cloned inserts and cultured isolates were identified by 16S rRNA gene sequence analysis. We identified 95 taxa among the 496 isolates and 1,577 clones sequenced; 44 taxa were detected by the molecular method alone; 31 taxa were previously undescribed. Only three taxa, Streptococcus mutans, Rothia dentocariosa, and an unnamed Propionibacterium sp., were found in all five samples. The predominant taxa by anaerobic cultivation were the novel Propionibacterium sp. (18%), Olsenella profusa (14%), and Lactobacillus rhamnosus (8%). The predominant taxa in the molecular analysis were Streptococcus mutans (16%), Lactobacillus gasseri/johnsonii (13%), and Lactobacillus rhamnosus (8%). There was no significant difference between the compositions of the microflora in the middle and advancing front samples (P < 0.05, Wilcoxon matched pairs, signed ranks test). In conclusion, combined cultural and molecular analyses have shown that a diverse bacterial community is found in dentinal caries and that numerous novel taxa are present.

Olsenella umbonata sp. nov., a microaerotolerant anaerobic lactic acid bacterium from the sheep rumen and pig jejunum, and emended descriptions of Olsenella, Olsenella uli and Olsenella profusa

“…Isolates of O. uli and O. profusa are regularly recovered from disease sites in the human mouth (Munson et al, 2002(Munson et al, , 2004Hooper et al, 2006) and sometimes from blood of humans with local oral or gastrointestinal infections (Lau et al, 2004;Bahrani-Mougeot et al, 2008). Olsenellae are found in the healthy (Ozutsumi et al, 2005;Cho et al, 2006;Hernandez et al, 2008) and acidotic (Tajima et al, 2000) bovine rumen.…”

Culture clash: challenging the dogma of microbial diversity

“…OTU and culture group numbers are presented here as reported in the original paper. The seven habitats shown are (1) endodontic pathogenic oral biofilms (Munson et al, 2002); (2) exodontic oral biofilms (Munson et al, 2004); (3) Hawaiian lakes and Lō'ihi (Donachie et al, 2004a); (4) rhizoplane (Kaiser et al, 2001); (5) bird feathers (Shawkey et al, 2005); (6) ocean (Eilers et al, 2000); (7) hypersaline salterns (Maturrano et al, 2006). molecular methods alone can detect 'unculturable' (i.e., previously uncultivated) bacteria.…”

Propionibacterium acidifaciens sp. nov., isolated from the human mouth

“…In a combined cultural and molecular analysis of the microbiota in dentinal caries, we found that an as-yet unnamed taxon was the dominant Propionibacterium taxon present and was one of only three taxa at species level to be found in every lesion (Munson et al, 2004). The aim of the present study was to perform a genetic and phenotypic characterization of three strains that are representative of this unnamed Propionibacterium taxon.…”

“…Strains C3M_31 T , C2A_34 and C5A_34 were isolated from the dentine of carious lesions in three subjects, as described previously (Munson et al, 2004). Strains were grown at 37 u C on fastidious anaerobe agar (FAA; LabM) supplemented with 5 % horse blood in an anaerobic workstation (Don Whitley Scientific) under anaerobic conditions (80 % N 2 /10 % H 2 /10 % CO 2 ).…”