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J Med Microbiol 55 (2006), 101-107; DOI: 10.1099/jmm.0.46212-0
© 2006 Society for General Microbiology
ISSN 0022-2615

Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries

Daniel Saito1, Renato de Toledo Leonardo2, Jorge Luiz Mazza Rodrigues3, Siu Mui Tsai3, José Francisco Höfling1 and Reginaldo Bruno Gonçalves1

1 Department of Oral Diagnosis, Dental School of Piracicaba, State University of Campinas (UNICAMP), Av. Limeira 90, Piracicaba, 13414-903 São Paulo, Brazil

2 Department of Restorative Dentistry, Dental School of Araraquara, State University of São Paulo, Araraquara, São Paulo, Brazil

3 Laboratory of Cell and Molecular Biology, Center of Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, São Paulo, Brazil

Correspondence
Reginaldo Bruno Gonçalves
Reginald{at}fop.unicamp.br

Received 27 June 2005
Accepted 8 September 2005

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65·2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10·9 %), Spirochaetes (4·3 %), Bacteroidetes (6·5 %), Actinobacteria (2·2 %) and Deferribacteres (2·2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.

The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of uncultured Clostridiales bacterium clone AG_D03, uncultured Clostridiaceae bacterium clone AG_G04, uncultured Streptococcaceae bacterium clone AF_F05 and uncultured Mogibacterium sp. clone AF_H06 are AY821867, AY821868, AY821869 and AY821870, respectively.

PCR results with reference bacteria are available as supplementary material in JMM Online.




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