Non- pncA Gene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

Pyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S ) tuberculosis (TB).pncA gene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-S TB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncA gene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panD orrpsA mutations identified by whole-genome sequencing (WGS) (n = 7), heteroresistance (n = 8), and mixed populations withMycobacterium avium (n = 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-R clinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncA mutation (Thr87Met), whereas a nonsynonymousrpsA mutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanD mutation, while all 15 of the MDRPZA-R isolates harbored a nonsynonymouspncA mutation. Our findings indicate that it is necessary to consider the occurrence ofpanD mutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsA mutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsA in PZA resistance.