Apical Root Canal Microbiota as Determined by Reverse-capture Checkerboard Analysis of Cryogenically Ground Root Samples from Teeth with Apical Periodontitis

doi:10.1016/j.joen.2010.07.001

Journal of Endodontics

Volume 36, Issue 10, October 2010, Pages 1617-1621

https://doi.org/10.1016/j.joen.2010.07.001 Get rights and content

Abstract

Introduction

Bacteria located in the apical root canal system potentially participate in the pathogenesis of apical periodontitis. Detection and identification of apical bacteria can be compromised because of limitations in conventional sampling and identification procedures. This study identified several bacterial taxa in the apical and middle/coronal segments of primarily infected root canal system by using pulverized root segments and a culture-independent molecular method.

Methods

Seventeen extracted teeth with attached apical periodontitis lesions were sectioned to obtain 2 root fragments (apical and middle/coronal segments). Root fragments were cryogenically ground, and DNA was extracted from samples. After multiple displacement amplification, DNA from samples was used as template in a reverse-capture checkerboard hybridization assay targeting 28 bacterial taxa.

Results

Bacterial DNA was detected in all samples. The most prevalent taxa in the apical root canal system were Olsenella uli (76.5%), Prevotella baroniae (71%), Porphyromonas endodontalis (65%), Fusobacterium nucleatum (53%), and Tannerella forsythia (47%). O. uli, P. endodontalis, and Propionibacterium acnes were as frequently detected in apical samples as they were in middle/coronal samples. P. baroniae, T. forsythia, and F. nucleatum were found more frequently in the apical part of the canal as compared with matched coronal segments. Streptococcus species were more prevalent in middle/coronal samples. The median and mean of shared bacterial taxa between matched apical and middle/coronal segments were 27% and 41%, respectively.

Conclusions

Several candidate endodontic pathogens were very prevalent in the apical root canal system. The apical microbiota was usually complex and differed in species composition when compared with the microbiota of middle/coronal samples from the same tooth.

Section snippets

Specimen Collection and Processing

DNA extracts from samples collected for a previous investigation (5) were stored and available for reanalysis in this study. The examined material consisted of 17 extracted teeth randomly collected from 14 patients in the Oral Surgery Clinic, Estácio de Sá University, Rio de Janeiro. All teeth had necrotic pulps, radiographic evidence of apical periodontitis and extensive caries lesions, and were extracted for prosthetic reasons or by request of the patient. The study protocol was approved by

Results

Sterility control samples taken from the external root surface after disinfection yielded no PCR product. Negative PCR controls showed the predicted results. All apical and middle/coronal samples were positive for the presence of bacteria as determined by PCR with universal 16S rRNA gene primers.

All samples were also positive for bacteria in the reverse-capture checkerboard assay as revealed by the 2 universal probes. The results revealed that 27 and 20 taxon-specific probes tested reactive

Discussion

Only 2 studies have been published that use molecular methods to identify bacteria located in the apical root canal. One study (19) used a conventional species-specific PCR assay directed to 10 bacterial species, whereas the other (18) used the same reverse-capture checkerboard approach as used in the present study to detect 28 target species/phylotypes. The main difference between those studies and the present one was that a cryogenic grinding approach was used to pulverize root samples so the

Acknowledgments

This study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazilian Governmental Institutions. The authors are grateful to Marlei Gomes da Silva for technical assistance and Dr Rodrigo Soares de Moura Neto from the Institute for Research and Expertise in Forensic Genetics of the State Police Academy Sylvio Terra, Rio de Janeiro, for assistance with the

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Clinical ResearchApical Root Canal Microbiota as Determined by Reverse-capture Checkerboard Analysis of Cryogenically Ground Root Samples from Teeth with Apical Periodontitis

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Specimen Collection and Processing

Results

Discussion

Acknowledgments

J Endod

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

J Endod

J Endod

J Endod

J Endod

J Endod

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

J Endod

J Endod

J Endod

J Endod

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

Endodontic microbiology and treatment of infections

Reaction of periradicular tissues to root canal treatment: benefits and drawbacks

Endod Topics

Molecular analysis of endodontic infections

Significance of bacterial identification by molecular biology methods

Endod Topics

Clinical Research
Apical Root Canal Microbiota as Determined by Reverse-capture Checkerboard Analysis of Cryogenically Ground Root Samples from Teeth with Apical Periodontitis