Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacter ium tuberculosis isolates. PZase activity was determined by the method of W ayne (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 greater than or equal to 100 mu g/ml for 37 isolates, 34 of which had alte rations in the pncA gene, These mutations included missense substitutions f or 24 isolates, nonsense substitutions for 3 isolates, frameshifts by delet ion for 4 isolates, a three codon insertion for 1 isolate, and putative reg ulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 mu g/ml, 3 had the same mutation (Thr47-->Ala) and 18 had the wild-ty pe sequence. For the three Thr47-->Ala mutants PZA MICs were 12.5 mu g/ml b y the method of proportions on 7H10 agar; two of these were resistant to 10 0 mu g of PZA per ml and the third was resistant to 800 mu 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 tha t were resistant to greater than or equal to 100 mu g of PZA per ml or in 3 4 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47-->Ala mutation. This study demonstrates that mutations in t he pncA gene may serve as a reliable indicator of resistance to greater tha n or equal to 100 mu g of PZA per ml.