The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates

Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZA^R) primarily related to <i>pncA</i> mutations. However, discordance between phenotypic resistance and conventional <i>pncA</i>-based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among <i>Mycobacterium tuberculosis</i> (Mtb) clinical isolates. Fifty-three Mtb isolates from Guangzhou Chest Hospital were tested for PZA resistance using the BACTEC MGIT 960 system and PZase activity assay. Thirty-one phenotypically PZA^R strains were genetically assessed by Sanger sequencing of PZA^R-associated customary genes. Five <i>pncA</i>-wild-type PZA^R strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZA^R strains harbored mutations in <i>pncA</i> and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZA^R strains lacked <i>pncA</i> mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating <i>clpC1</i> mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% (<i>pncA</i> alone) to 100%. This is the first report from southern China that identifies novel <i>clpC1</i> mutations in wild-type <i>pncA</i> PZA^R Mtb isolates. Our findings underscore the limitations of <i>pncA</i>-targeted diagnostics and support the integration of WGS and <i>clpC1</i> analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes.