Novel Bacterial Phylotypes in Endodontic Infections

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Novel Bacterial Phylotypes in Endodontic Infections
2005
J.F. Siqueira, Jr.1,2,*, I.N. RŸ¡as1, C.D. Cunha2, and A.S. Rosado2
Journal of Dental Research

© 2005 International and American Associations for Dental Research

1 Department of Endodontics, Est?cio de S? University, Rio de Janeiro, Brazil; and
2 Institute of Microbiobiology Prof. Paulo de G€es, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil;

* corresponding authorÌs address, R. Herotides de Oliveira 61/601, IcaraÃ, Niter€i, RJ, Brazil 24230-230; siqueira@estacio.br

ABSTRACT

Although molecular studies have revealed potential oral pathogens among the phyla Spirochaetes and Deferribacteres, their occurrence in endodontic infections has not been consistently investigated. In this study, we devised a nested PCR-DGGE approach to survey samples from infected root canals for the presence of members of these two phyla, and to examine their diversity. The primers used also amplified DNA from Atopobium species. Eight of 10 cases showed bands representative of the target bacterial groups. DGGE profiles revealed a mean number of 6.5 intense and faint bands. No single band occurred in all profiles. Sequences from intense bands excised from the gel showed similarities to species/phylotypes of all target groupsÛFlexistipes species (Deferribacteres phylum), uncharacterized spirochetes, and Atopobium species. Analysis of these data indicates that uncultivated Spirochaetes and Deferribacteres phylotypes are frequent members of the endodontic microbiota and may be potential pathogens involved with the etiology of periradicular diseases.


KEY WORDS: endodontic microbiology Ô 16S rDNA Ô molecular biology

INTRODUCTION

Although bacterial species or phylotypes detected in the oral cavity fall within 11 different phyla (Paster et al., 2001, 2002), representatives from only 6 of those have been reported in endodontic infections to date, namely, Bacteroidetes, Spirochaetes, Firmicutes, Actinobacteria, Fusobacteria, and Proteobacteria (Siqueira, 2003). Even so, the possibility exists that the diversity within some of these phyla has not been thoroughly explored in endodontic infections. For instance, 8 of the 10 cultivable oral Treponema species have been detected in endodontic infections by molecular methods (Baumgartner et al., 2003; RŸ¡as et al., 2003; Siqueira and RŸ¡as, 2003 Siqueira and RŸ¡as, 2004). However, the diversity of spirochetes in the oral cavity has been reported to be far greater than expected, and estimates suggest that about 80% of the oral treponemes remain uncultivable (Dewhirst et al., 2000; Paster et al., 2001). Consequently, it is possible that other spirochetes may occur in infected root canals. Moreover, it is possible that species belonging to the other phyla occurring in the oral cavity can also infect root canals. Members of the Deferribacteres phylum have been recently suggested to be involved with periodontal diseases (Paster et al., 2001; Kumar et al., 2003). These bacteria have not been found in endodontic infections, and this may be because they are actually absent, or they are difficult to isolate and/or identify by cultivation procedures.

It has been postulated that a comprehensive description of microbial communities inhabiting different habitats requires the use of techniques that sidestep cultivation (Hugenholtz et al., 1998). Genetic fingerprinting techniques represent a powerful tool for the investigation of the structure of microbial communities in diverse ecosystems and can be used for microbial identification. A commonly used strategy for the fingerprinting of complex bacterial communities consists of 16S rDNA-based PCR, followed by product analysis by denaturing gradient gel electrophoresis (DGGE) (Muyzer, 1999). The DGGE approach is based on electrophoresis of PCR products in polyacrylamide gels containing a linearly increasing gradient of DNA denaturants (Muyzer, 1999). In DGGE, DNA fragments of the same length, but with different base-pair sequences, can be separated. A single-base change in a sequence can be resolved (Fischer and Lerman, 1983), which provides PCR-DGGE with a great potential to identify closely related species based on 16S rDNA sequence divergence. In addition to being applied to the broad-range analysis of complex microbial communities (Muyzer and Smalla, 1998), the method can also be used to detect specific bacterial groups by altering the primer targets or by using a nested PCR (nPCR) approach (Ercolini, 2004).

We undertook the present study to investigate the occurrence of members of the phyla Spirochaetes and Deferribacteres in endodontic infections, and to examine their diversity through a culture-independent approach. Representatives of these phyla have been associated with periodontal diseases, and the possibility exists that these phyla also contain potential endodontic pathogens.

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