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Pyrazinamide (PZA) is widely used to treat drug-sensitive or multidrug resistance tuberculosis. However, conventional PZA susceptibility tests of clinical isolates are rather difficult because of the requirement of acid pH. Since resistance to pyrazinamide is primary mediated by mutation of  pncA , an alternative way of PZA susceptibility test is to analyze the pyrazinamidase activities of  Mycobacterium tuberculosis  clinical isolates. Therefore, a database containing the full spectrum of  pncA  mutations along with pyrazinamidase activities will be beneficial. To characterize mutations of  pncA  in  M. tuberculosis  from Chongqing, China, the  pncA  gene was sequenced and analyzed in 465 clinical isolates. A total of 124 types of mutations were identified in 424 drug-resistant isolates, while no mutation was identified in the 31 pan-susceptible isolates. Ninety-four of the 124 mutations had previously been reported, and 30 new mutations were identified. Based on reported literatures, 294 isolates could be predicted resistant to pyrazinamide. Furthermore, pyrazinamidase activities of the 30 new mutations were tested using the  Escherichia coli pncA  gene knockout strain. The results showed that 24 of these new mutations (28 isolates) led to loss of pyrazinamidase activity and six (8 isolates) of them did not. Taken together, 322 isolates with  pncA  mutations could be predicted to be PZA resistant among the 424 drug-resistant isolates tested. Analysis of  pncA  mutations and their effects on pyrazinamidase activity will not only enrich our knowledge of comprehensive  pncA  mutations related with PZA resistance but also facilitate rapid molecular diagnosis of pyrazinamide resistance in  M. tuberculosis .

Characterization of pncA Mutations and Prediction of PZA Resistance in Mycobacterium tuberculosis Clinical Isolates From Chongqing, China