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

H M Adnan Hameed; Cuiting Fang; Zhiyong Liu; Yamin Gao; Shuai Wang; Xinwen Chen; Nanshan Zhong; Htin Lin Aung; Jinxing Hu; Tianyu Zhang

doi:10.3390/microorganisms13061401

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

Microorganisms. 2025 Jun 16;13(6):1401. doi: 10.3390/microorganisms13061401.

Authors

H M Adnan Hameed^{1

2

3

4}, Cuiting Fang^{1

2

3

4}, Zhiyong Liu^{1

5}, Yamin Gao^{1

2

3

4}, Shuai Wang^{1

2

3

4}, Xinwen Chen⁵, Nanshan Zhong^{5

6}, Htin Lin Aung^{2

7}, Jinxing Hu¹, Tianyu Zhang^{1

2

3

4

5}

Affiliations

¹ State Key Laboratory of Respiratory Disease, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou Chest Hospital, Guangzhou 510530, China.
² China-New Zealand Joint Laboratory of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
³ Guangdong-Hong Kong-Macau Joint Laboratory of Respiratory Infectious Diseases, Guangzhou 510530, China.
⁴ University of Chinese Academy of Sciences, Beijing 100049, China.
⁵ Guangzhou National Laboratory, Guangzhou 510005, China.
⁶ State Key Laboratory of Respiratory Disease, National Clinical Research Centre for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China.
⁷ Department of Microbiology and Immunology, School of Biomedical Sciences, University of Otago, Dunedin 9016, New Zealand.

Abstract

Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZA^R) primarily related to pncA mutations. However, discordance between phenotypic resistance and conventional pncA-based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among Mycobacterium tuberculosis (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 pncA-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 pncA and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZA^R strains lacked pncA mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating clpC1 mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% (pncA alone) to 100%. This is the first report from southern China that identifies novel clpC1 mutations in wild-type pncA PZA^R Mtb isolates. Our findings underscore the limitations of pncA-targeted diagnostics and support the integration of WGS and clpC1 analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes.

Keywords: Mycobacterium tuberculosis; clinical isolates; clpC1; drug resistance; molecular diagnosis; pyrazinamide.

Abstract

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