Systematic Analysis of Pyrazinamide-Resistant Spontaneous Mutants and Clinical Isolates of Mycobacterium tuberculosis

Abstract

ABSTRACTPyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistentMycobacterium tuberculosisbacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZAr) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10−5bacilliin vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZArphenotype correlated with mutations in thepncAgene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZArmutants did not have apncAmutation, strongly indicating the presence of at least one other mechanism(s) of PZAr.