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

2006 ◽  
Vol 55 (1) ◽  
pp. 101-107 ◽  
Author(s):  
Daniel Saito ◽  
Renato de Toledo Leonardo ◽  
Jorge Luiz Mazza Rodrigues ◽  
Siu Mui Tsai ◽  
José Francisco Höfling ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rdna ◽  
Significant Proportion ◽  
Oral Bacteria ◽  
Clone Libraries ◽  
Root Canals ◽  
Gram Positive Bacteria ◽  
Identification Of Bacteria ◽  
Endodontic Infections ◽  
Rdna Clone

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.

Identification of bacteria associated with periapical abscesses of primary teeth by sequence analysis of 16S rDNA clone libraries

2020 ◽  
Vol 141 ◽  
pp. 103954 ◽  
Author(s):  
Wenwen Zhang ◽  
Yuanyuan Chen ◽  
Qing Shi ◽  
Benxiang Hou ◽  
Qiubo Yang
Keyword(s):  
Sequence Analysis ◽  
16S Rdna ◽  
Primary Teeth ◽  
Clone Libraries ◽  
Identification Of Bacteria ◽  
Rdna Clone

scholarly journals Identification of microorganisms in persistent/secondary endodontic infections with respect to clinical and radiographic findings: bacterial culture and molecular detection

2019 ◽  
Author(s):  
Nazanin Zargar ◽  
Mahmoud Amin Marashi ◽  
Hengameh Ashraf ◽  
Rene Hakopian ◽  
Peyman Beigi
Keyword(s):  
16S Rdna ◽  
Bacterial Species ◽  
Significant Proportion ◽  
Clinical Signs ◽  
Oral Bacteria ◽  
16S Rdna Gene ◽  
Radiographic Findings ◽  
Rdna Gene ◽  
Endodontic Infections ◽  
Iranian Patients

Background and Objectives: Bacterial agents are commonly accepted as the main etiology of endodontic infections. A significant proportion of oral bacteria cannot be cultured using existing methods. Since diversity and abundance of bacterial species are different in different populations, the present study was aimed to identify effective microorganisms in persistent endodontic infections in Iranian patients based on culture and molecular biology methods using sequence analysis of 16S rDNA gene. Materials and Methods: Thirty patients with previous failure of endodontic treatment were enrolled in the study. After isolation and disinfection of the tooth surrounding area with 3% sodium hypochlorite and 30% hydrogen peroxide, sampling from the root canals was carried out using two sterile Hedstrom files and two sterile paper points, and then the specimens were transferred to the microbiology laboratory in thioglycolate transport medium so that they undergo aerobic-anaerobic culture, PCR, and 16S rDNA gene sequencing. Results: Of 30 patients (15 women and 15 men), 15 patients had radiographic lesions smaller than 5 mm and other 15 pa- tients had radiographic lesions larger than 5 mm. The mean age of patients was 40.20 ± 13.76 years. A total of 26 patients were asymptomatic. Only four patients had clinical signs such as pain and percussion sensitivity and Tannerella forsythia was the most common bacterium found in this group of patients. 13 bacterial species were found in 11 different genus, one virus strain and one fungus strain. From 30 studied specimens, Enterococcus faecalis was the most common microorganism with prevalence rate of 63.63%. Conclusion: This study showed the type and prevalence of effective bacteria in secondary/persistent endodontic infections in Iranian patients. E. faecalis is the most commonly found microorganism in Iranian patients

scholarly journals Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

2001 ◽  
Vol 67 (1) ◽  
pp. 469-472 ◽  
Author(s):  
Arjen G. C. L. Speksnijder ◽  
George A. Kowalchuk ◽  
Sander De Jong ◽  
Elizabeth Kline ◽  
John R. Stephen ◽  
...  
Keyword(s):  
16S Rdna ◽  
Sequence Variation ◽  
Hot Spot ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Pcr Cloning ◽  
Clone Libraries ◽  
High Degree ◽  
Cloning Experiment ◽  
Rdna Clone

ABSTRACT A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

scholarly journals 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

2000 ◽  
Vol 38 (10) ◽  
pp. 3623-3630 ◽  
Author(s):  
Michel Drancourt ◽  
Claude Bollet ◽  
Antoine Carlioz ◽  
Rolland Martelin ◽  
Jean-Pierre Gayral ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rdna ◽  
Similarity Score ◽  
16S Rdna Sequencing ◽  
16S Rdna Sequence ◽  
16S Rdna Sequence Analysis ◽  
Rdna Sequences ◽  
Rdna Sequencing ◽  
16S Rdna Sequences ◽  
Identification Of Bacteria

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter andPantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries

1997 ◽  
Vol 6 (5) ◽  
pp. 475-482 ◽  
Author(s):  
D. P. Chandler ◽  
J. K. Fredrickson ◽  
F. J. Brockman
Keyword(s):  
16S Rdna ◽  
Clone Libraries ◽  
Total Community ◽  
Rdna Clone

Molecular and Cultural Analysis of the Microflora Associated with Endodontic Infections

2002 ◽  
Vol 81 (11) ◽  
pp. 761-766 ◽  
Author(s):  
M.A. Munson ◽  
T. Pitt-Ford ◽  
B. Chong ◽  
A. Weightman ◽  
W.G. Wade
Keyword(s):  
Oral Bacteria ◽  
Cultural Analysis ◽  
Root Canals ◽  
Cultural Methods ◽  
Rdna Sequences ◽  
Infected Root ◽  
16S Rdna Sequences ◽  
Culture Independent ◽  
Novel Taxa ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document