Phenotypic Characterization of pncA Mutants of Mycobacterium tuberculosis

Abstract

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were >100 g/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a threecodon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 g/ml, 3 had the same mutation (Thr473Ala) and 18 had the wild-type sequence. For the three Thr473Ala mutants PZA MICs were 12.5 g/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 g of PZA per ml and the third was resistant to 800 g of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to >100 g of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr473Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to >100 g of PZA per ml.

Tuberculose resistente: revisão molecular

“…20,22 Mais de 70% dos isolados de M. tuberculosis PZA resistentes (PZA R ) apresentaram mutações no gene pncA, que codifica a enzima pirazinamidase, a qual converte o fármaco PZA em sua forma ativa. 32 Recentemente, Morlock et al 26 (2000) identificaram inúmeras mutações no gene pncA em linhagens de M. tuberculosis, que podem servir como um indicador de resistência à PZA. Várias mutações encontradas estão associadas a uma pirazinamidase ineficiente.…”

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

“…The genetic basis for PZA resistance involves mutation within the pncA gene, which encodes Pzase activity (20,28). Although cases of PZA-resistant M. tuberculosis isolates with no pncA mutations have been reported, mutations of pncA and its putative promoter remain the major mechanism of PZA resistance (15,20).…”

Wayne’s Assay: A Screening Method for Indirect Detection of Pyrazinamide Resistance in Mycobacterium Tuberculosis Clinical Isolates

“…Thus, finding mutations on the pncA gene makes the rapid detection of PZA-resistant MTB possible [1]. The identification of mutations within the pncA gene has been proposed as a surrogate marker for PZA resistance in MTB [8]. Unfortunately, DST to PZA is still not sufficiently standardized to help guide therapy.…”