Mark A. Munson

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% CO2 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.

Molecular and Cultural Analysis of the Microflora Associated with Endodontic Infections

Cultural studies have indicated that a subset of the oral microflora is responsible for endodontic infections. Approximately 50% of oral bacteria are unculturable, so it is likely that currently unknown bacteria are present in such infections. In this study, cultural and molecular analyses were performed on the microflora in aspirate samples collected from 5 infected root canals. 16S rDNA sequences from 261 isolates and 624 clones were identified by comparison with database sequences. Sixty-five taxa were identified, of which 26 were found by the molecular method alone. A mean of 20.2 taxa was found in each sample. A new species of Dialister was the only organism present in all 5 samples. Twenty-seven novel taxa were detected, 18 of which belonged to the phylum Firmicutes and 8 to Bacteroidetes. Culture-independent, molecular analysis has revealed a more diverse microflora associated with endodontic infections than that revealed by cultural methods alone.

Characterisation of Eubacterium-like strains isolated from oral infections

The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests. As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent. In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis. Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A. rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E. sulci (8), E. yurii subsp. yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M. vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13). The remaining 14 isolates did not correspond to existing species. This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods. This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined.

Shuttleworthia satelles gen. nov., sp. nov., isolated from the human oral cavity

Nine strains of anaerobic, non-spore-forming, gram-positive bacilli, isolated from the human oral cavity and provisionally identified as belonging to the genus Eubacterium, were subjected to a comprehensive range of phenotypic and genetic tests. Biochemically, they were found to comprise a homogeneous group, and phylogenetic analysis of their 16S rRNA sequences indicated that they constitute a unique branch within the Clostridium-Bacillus subphylum of the phylum Firmicutes. All of the isolates displayed an unusual colonial morphology after extended incubation. This resembled a contaminated culture in that small, secondary colonies were seen to arise around and from within the primary colony form, and a third, independent, colony type was also seen. However, inspection of the colonies by Gram-staining and scanning electron microscopy together with protein profile analysis and 16S rRNA gene sequence comparison of the two independent colony types revealed that only a single organism was present. A new genus, Shuttleworthia, and the species Shuttleworthia satelles gen. nov., sp. nov., are proposed. The cells are saccharolytic, and acetate, butyrate and lactate are produced as end products of glucose fermentation. Aesculin is hydrolysed and indole is produced. The G+C content of the DNA of the type strain is 51 mol%. The type strain is strain DSM 14600T (= CCUG 45864T = VPI D143K-13T).

Eubacterium minutum is an earlier synonym of Eubacterium tardum and has priority

The recently proposed species Eubacterium minutum and Eubacterium tardum appeared to be similar from their published descriptions. The aim of this study was to perform phenotypic and genetic analyses of strains of both species to clarify their taxonomic position. The type strains of E. minutum and E. tardum exhibited identical biochemical and protein profiles and their 16S rRNA gene sequences displayed 99.9% similarity. The G + C content of the DNA of both strains was estimated at 45 mol%. It is concluded that E. minutum and E. tardum are synonyms; E. minutum has priority. An emended description of E. minutum is given.