Please refer to the following articles:

Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

J. Clin. Microbiol. March 2000 vol. 38 no. 3 1094-1104                                    

Andreas Roth1,*,

Udo Reischl2,

Anna Streubel1,

Ludmila Naumann2,

Reiner M. Kroppenstedt3,

Marion Habicht1,

Marga Fischer1, and

Harald Mauch1

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 inMycobacterium 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 aviumcomplex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, orAvaII). 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.

Utility of high-performance liquid chromatography analysis of mycolic acids and partial 16S rRNA gene sequencing for routine identification of Mycobacterium spp. in a national reference laboratory

Diagnostic Microbiology and Infectious Disease

Volume 67, Issue 2, June 2010, Pages 143–152

High-performance liquid chromatography analysis of mycolic acids and partial gene sequencing for the first 500-bp 5′ end of the 16S rRNA gene were used singularly and in combination to evaluate the final identification of species. Examination of 200 cultures revealed 100 strains of slowly growing mycobacteria (SGM), 91 strains of rapidly growing mycobacteria (RGM), and 9 strains of other genera. SGM were discriminated in complexes with both methods for 56 strains, composed primarily of the Mycobacterium spp.: Mycobacterium avium, Mycobacterium terrae, and Mycobacterium simiae–Mycobacterium lentiflavum. For RGM, 73 strains were associated with complexes designated as Mycobacterium abscessus–Mycobacterium chelonae, Mycobacterium fortuitum–Mycobacterium peregrinum, and Mycobacterium mucogenicum–Mycobacterium phocaicum. Consistent identification of all the isolates differentiated to single species within the Mycobacterium genus was not possible with either test method. Sequencing results often distinguished complexes containing fewer species, and combining the results from each method increased the confidence of identifying the correct species.

Different molecular methods for the identification of rarely isolated non-tuberculous mycobacteria and description of new hsp65 restriction fragment length polymorphism patterns

Molecular and Cellular Probes

Volume 18, Issue 1, February 2004, Pages 59–65

Analysis of heat shock protein 65 (hsp65) gene restriction fragment length polymorphism (RFLP) is done frequently to identify non-tuberculous mycobacteria (NTM) on a genetic basis. Here we report the results of analysing the hsp65 patterns of some rarely isolated NTM for which patterns have not been published before (Mycobacterium bohemicum, Mycobacterium hassiacum, Mycobacterium heckeshornense, Mycobacterium monacense, and Mycobacterium triplex). Furthermore new hsp65-variants for Mycobacterium interjectum (type II), Mycobacterium mucogenicum (type V), Mycobacterium gordonae (type VIII) and Mycobacterium paraffinicum (perhaps synonymous to Mycobacterium scrofulaceum) are described. All species were characterised by hsp65-RFLP, sequencing a 441-bp fragment of the hsp65 gene and sequencing the hypervariable region of the 16S rDNA. Additional data for less frequently isolated mycobacteria are provided and the hitherto described data for the Mycobacterium gordonae complex are summarised. Although the hsp65-RFLP analysis turned out to be a useful method a number of restraints (lack of standardisation, slight variability in fragment length) limits its broader use. Reliable identification of NTM needs, however, more than one molecular method. Identification results obtained by applying different methods yielded conflicting results. Confusion may be caused by older data base entries which are not updated and not longer reflect the actual systematic and taxonomy of the genus Mycobacterium.

Dear Samir,

sometimes it happens that it's difficult to identify NTMs as there are many species not studied yet. It might be a cluster with such kind of profile somewhere inbetween of rapid and slow growers especially if you combine several DNA loci.

I'm afraid there is no single algorythm to identify all NTMs. There are many methods, including hsp65-RFLP or biochemical analyses, and it will be interesting to combine all the results to see whether those results will give clear identification. Probably not. So you will discover a new species :)

Did you try to dig www.ezbiocloud.net ?

Thank you Dr Artem, yes i tried ezbiocloud.net, its results are also different. Whether it is a new species, i cannot say at present.. time will say more. But it seems it is falling in Mycobacterium avium complex. Thank you for your observations. I may need further your suggestions.

Thank you Dr Amar, I have gone through your articles and as it is in  Mycobacterium avium complex group and with your suggestions i have initiated work  on ITS 16s-23sRNA  also for this isolate . Thank you for your kind observations. I may need further your suggestions.

Very pleased, be happy to assist in the future.

Best,

A

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