Sequencing of the pncA Gene in Members of the Mycobacterium tuberculosis Complex Has Important Diagnostic Applications: Identification of a Species-Specific pncA Mutation in “ Mycobacterium canettii ” and the Reliable and Rapid Predictor of Pyrazinamide Resistance

Abstract

Testing for susceptibility to pyrazinamide (PZA) and analysis of the pncA gene sequences of 423 Mycobacterium tuberculosis complex isolates have revealed a unique silent nucleotide substitution that enables the rapid identification of "M. canettii" (proposed name). Moreover, the lack of a defined mutation within the pncA gene strongly suggests that an alternative mechanism is responsible for PZA resistance. Our results indicate that DNA sequencing of the pncA gene has the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing of the M. tuberculosis complex. The Mycobacterium tuberculosis complex (MTB complex) consists of the closely related organisms M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. caprae, M. microti, M.pinnipedii, the dassie bacillus, and "M. canettii" (proposed name) (3,5,6,26). Although the members of the complex may differ in their epidemiologies, host spectra, geographic ranges, pathogenicities, and antituberculosis drug susceptibilities and although they display different phenotypic characteristics by conventional biochemical tests, they show high degrees of genetic homogeneity (10, 25).Conventional phenotypic methods for the identification of the members of the MTB complex are laborious and timeconsuming, and they require a large biomass (13). Thus, molecular methods (such as DNA sequencing of the oxyR, pncA, gyrB, or hsp65 gene; analysis of spacers between direct repeats in the direct repeat region; and deletion analysis of the regions of difference [RD]) provide a more rapid and accurate approach to the differentiation of the members of the MTB complex (8,9,16,23,24).The findings of a recent report that examined seven genes (katG, gyrB, gyrA, rpoB, hsp65, sodA, and the 16S rRNA gene) indicate that M. canettii represents the most ancient phyloge-

“ Mycobacterium canettii ” Isolated from a Human Immunodeficiency Virus-Positive Patient: First Case Recognized in the United States

“…Results of these assays revealed that the isolate carried the "Mycobacterium canettii"-specific C-to-T mutation in the hsp65 gene (8) and the A-to-G mutation at position 138 in the pncA gene (22). Deletion analysis identified the presence of RD1, RD4, RD9, RD10, and tuberculosis-specific deletion 1 as well as the absence of RD12, also characteristic for "M. canettii" (10).…”

“…Genetic analysis of the isolate was performed according to the following technologies: PCR-restriction fragment length polymorphism analysis of the hsp65 gene, mutation analysis of the pncA gene by automated DNA sequencing, PCR-based genomic deletion analysis (region of difference 1 [RD1], RD4, RD9, RD10, RD12, and M. tuberculosis-specific deletion 1), and spoligotyping (8,16,17,21,22). Results of these assays revealed that the isolate carried the "Mycobacterium canettii"-specific C-to-T mutation in the hsp65 gene (8) and the A-to-G mutation at position 138 in the pncA gene (22).…”

Comparative study of Mycobacterium tuberculosis and Mycobacterium bovis protein profiles

“…The complex of Mycobacterium tuberculosis (MTB), including M. tuberculosis, M. africanum, M. bovis, Bovis BCG, M. caprae, M. microti, M. pinnipedii, M. dassie bacillus and M. canettii, although shows different phenotype characteristics in their biochemical tests, but they have high similarity in genetic terms [4]. To differentiate complex members is essential to promote successful treatment, especially in areas where the disease is epidemic or exposure to human and animal is high [5].…”

Discovery of the New Target for Identification of Mycobacterium tuberculosis

“…The mtp-40 and CYP 141 genes are not present in all members of the Mycobacterium tuberculosis strains and, therefore, are not useful for differentiating Mycobacterium tuberculosis from Mycobacterium bovis (10,11). The gene for histone-like protein hupB of Mycobacterium tuberculosis has been identified as a target for differentiation of MTB complex and also polymorphisms in pncA and oxyR are good options for rapidly differentiate M. bovis from M. tuberculosis; thus, for rapid and reliable detection of Mycobacterium tuberculosis complex from clinical specimens, it is needed to study genes that are simply analyzed and are conserved (9)(10)(11)(12).…”