Dataset Information

Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

ABSTRACT: OBJECTIVES:In addition to several molecular methods and in particular 16S rDNA analysis, the application of a more discriminatory genetic marker, i.e., 16S-23S internal transcribed spacer gene sequence has had a great impact on identification and classification of mycobacteria. In the current study we aimed to apply this sequencing power to conclusive identification of some Iranian clinical strains of mycobacteria. MATERIALS AND METHODS:The test strains consisted of nineteen mycobacterial isolates which were initially identified by the use of conventional phenotypic techniques and molecular methods and subjected to further definitive identification using the 16S-23S internal transcribed spacer gene sequencing. RESULTS:Out of 19 studied strains, 7 isolates were found to be rapidly growing and 12 isolates as slowly growing mycobacteria. With the exception of one isolate, i.e., the isolate HNTM87, which yielded a distinct ITS sequence incomparable with all previously identified mycobacteria, the remaining isolates produced the sequences similar to the established mycobacteria and were clearly identified and differentiated from closely related taxa. A phylogenetic tree based on maximum parsimony analysis of 16S-23S internal transcribed spacer gene sequences constructed showing the relatedness of Iranian clinical isolates with the closely related type species of mycobacteria. CONCLUSION:This study showed that the 16S-23S internal transcribed spacer gene of the genus Mycobacterium exhibits a high variation which is of value for discriminating closely related taxa and could be used independently or in combination with 16S rDNA sequencing to delineate the true identity of rare mycobacterial species.

SUBMITTER: Shojaei H

PROVIDER: S-EPMC3586870 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

Shojaei Hasan H Abodolrazagh Hashemi H Heidarieh Parvin P Pourahmad Fazel F Daei Naser Daei Naser DN

Iranian journal of basic medical sciences 20120101 1

<h4>Objectives</h4>In addition to several molecular methods and in particular 16S rDNA analysis, the application of a more discriminatory genetic marker, i.e., 16S-23S internal transcribed spacer gene sequence has had a great impact on identification and classification of mycobacteria. In the current study we aimed to apply this sequencing power to conclusive identification of some Iranian clinical strains of mycobacteria.<h4>Materials and methods</h4>The test strains consisted of nineteen mycob ...[more]

PMID: 23493089

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Project description:A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

Dataset Information

Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

Publications

Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

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