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<sup>R</sup>) primarily related to <i>pncA</i> mutations. However, discordance between phenotypic resistance and conventional <i>pncA</i>-based molecular diagnostics challeng...
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2025-06-01
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| author | H. M. Adnan Hameed Cuiting Fang Zhiyong Liu Yamin Gao Shuai Wang Xinwen Chen Nanshan Zhong Htin Lin Aung Jinxing Hu Tianyu Zhang |
| author_facet | H. M. Adnan Hameed Cuiting Fang Zhiyong Liu Yamin Gao Shuai Wang Xinwen Chen Nanshan Zhong Htin Lin Aung Jinxing Hu Tianyu Zhang |
| author_sort | H. M. Adnan Hameed |
| collection | DOAJ |
| description | Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZA<sup>R</sup>) 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<sup>R</sup> strains were genetically assessed by Sanger sequencing of PZA<sup>R</sup>-associated customary genes. Five <i>pncA</i>-wild-type PZA<sup>R</sup> strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZA<sup>R</sup> strains harbored mutations in <i>pncA</i> and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZA<sup>R</sup> 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<sup>R</sup> 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. |
| format | Article |
| id | doaj-art-746bd9fdb2b6439298773415ea8b0668 |
| institution | Kabale University |
| issn | 2076-2607 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
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| series | Microorganisms |
| spelling | doaj-art-746bd9fdb2b6439298773415ea8b06682025-08-20T03:27:25ZengMDPI AGMicroorganisms2076-26072025-06-01136140110.3390/microorganisms13061401The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical IsolatesH. M. Adnan Hameed0Cuiting Fang1Zhiyong Liu2Yamin Gao3Shuai Wang4Xinwen Chen5Nanshan Zhong6Htin Lin Aung7Jinxing Hu8Tianyu Zhang9State 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, ChinaState 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, ChinaState 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, ChinaState 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, ChinaState 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, ChinaGuangzhou National Laboratory, Guangzhou 510005, ChinaGuangzhou National Laboratory, Guangzhou 510005, ChinaChina-New Zealand Joint Laboratory of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, ChinaState 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, ChinaState 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, ChinaPyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZA<sup>R</sup>) 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<sup>R</sup> strains were genetically assessed by Sanger sequencing of PZA<sup>R</sup>-associated customary genes. Five <i>pncA</i>-wild-type PZA<sup>R</sup> strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZA<sup>R</sup> strains harbored mutations in <i>pncA</i> and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZA<sup>R</sup> 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<sup>R</sup> 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.https://www.mdpi.com/2076-2607/13/6/1401<i>Mycobacterium tuberculosis</i>pyrazinamidedrug resistance<i>clpC1</i>clinical isolatesmolecular diagnosis |
| spellingShingle | H. M. Adnan Hameed Cuiting Fang Zhiyong Liu Yamin Gao Shuai Wang Xinwen Chen Nanshan Zhong Htin Lin Aung Jinxing Hu Tianyu Zhang The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates Microorganisms <i>Mycobacterium tuberculosis</i> pyrazinamide drug resistance <i>clpC1</i> clinical isolates molecular diagnosis |
| title | The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates |
| title_full | The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates |
| title_fullStr | The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates |
| title_full_unstemmed | The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates |
| title_short | The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates |
| title_sort | identification of novel mutations in atp dependent protease clpc1 assists in the molecular diagnosis of obscured pyrazinamide resistant tuberculosis clinical isolates |
| topic | <i>Mycobacterium tuberculosis</i> pyrazinamide drug resistance <i>clpC1</i> clinical isolates molecular diagnosis |
| url | https://www.mdpi.com/2076-2607/13/6/1401 |
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