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

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.67.1.469-472.2001 ◽

2001 ◽

Vol 67 (1) ◽

pp. 469-472 ◽

Cited By ~ 175

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.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.38.10.3623-3630.2000 ◽

2000 ◽

Vol 38 (10) ◽

pp. 3623-3630 ◽

Cited By ~ 581

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.

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