Identification of nontuberculous mycobacteria using commercial DNA probes and gene sequencing

Abstract

Diagnosis of NonTuberculous Mycobacteria (NMT) infection frequently runs into difficulties regarding a precise definition of the strains. The use of molecular assays is the technique of choice for the identification of species, but commercial methods recognize only a limited number of species. Aim of this study was molecular identification of NonTuberculous Mycobacteria (NTM) in clinical specimens using commercial methods and automated sequencing. We analyzed 6192 clinical specimens for the isolation of Mycobacteria. One hundred and twelve strains of NTM were previously analyzed with GenoType Mycobacteria CM/AS kit and then with entire 16S rDNA and partial hsp65 sequencing. 100/112 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (n°25), M. xenopi (n°24), M. fortuitum (n°15), M. avium (n°12), M. intracellulare (n°7), M. chelonae (n°6), M. malmoense (n°4), M. peregrinum (n°3), M. mucogenicum (n°1), M. kansasii (n°1), M. abscessus (n°1), M. heckeshornense (n°1).Twelve unidentified strains were subjected to the entire 16S rDNA gene sequencing and nine were identified as M. arupense (n°7), M. avium complex (n°1), M. kumamotonense (n°1).Three unidentified strains were subjected to partial hsp65 gene sequencing and one was identified as M. arupense. Conclusions. Direct sequencing of entire 16SrDNA gene and of partial hsp65 gene appears to be a useful tool for the study of strains that are not identifiable by commercial methods.This new approach, applied to clinical diagnostic, allows also recognition of unusual strains or new species.

Keywords

• Nontuberculous mycobacteria, GenoType Mycobacteria CM/AS, 16S rDNA, hsp65, sequencing