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Abstract
Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis.
Highlights
Endodontic failures correspond with a persistence of periradicular lesions [1,2]
Our study aimed to investigate the microbial flora of treated root canals associated with apical periodontitis combining cultural methods with the cultureindependent approach
The density of microorganisms ranged from 16103 colony forming units (CFU)/ml to 6.86104 CFU/ml for aerobic cultivation and from 16103 CFU/ml to 2.46104 CFU/ml for anaerobic cultivation
Summary
Endodontic failures correspond with a persistence of periradicular lesions [1,2]. To conserve the tooth a revision of the endodontic treatment becomes necessary, because otherwise persistent microorganisms or secondary infections mainly caused by insufficient coronal restoration can lead to loss of the tooth. Only three culture-independent studies using the 16S-rDNA cloning technique have been done to analyze the microbial diversity of secondary endodontic infections [8,16,17]. PCR and cloning analysis will lead to some bias due to the detection of DNA from dead cells, differential DNA-extraction or preferential DNA amplification [18]. This might cause an overestimation of the role of certain species that can reach the root canal but might not actively grow there [19]
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