## 5 Conclusions  
In conclusion, this study aims to update the growing list of PZA-associated targets by reporting the new PZA resistance-associated mutations in ClpC1 of Mtb clinical isolates. To the best of our knowledge, this is the first report from southern China that identifies novel mutations (Gly58 Ser, Ala567 Val, and Pro796 Leu) in the ClpC1 of PZA\({}^{\text{R}}\) clinical isolates (MIC \(\geq\) 400 ug/mL) bearing wild-type _pncA_. Though Val63 Ala in ClpC1 was recently stated as a low-level resistance marker in Indo-Oceanie lineage isolates [62], in southern China, it was reported for the first time in the Beijing genotype of PZA\({}^{\text{R}}\) clinical isolates used in this study, which indicates that Val63 Ala substitution is not lineage-specific.  
Clinically, our findings advocate for the integration of long-read sequencing and parallel WGS into diagnostic workflows, particularly for isolates exhibiting phenotypic-genotypic discordance. Such approaches will not only enhance the sensitivity and accuracy of genome-based molecular diagnostics but also enable the early detection of elusive resistance mechanisms. The mechanistic investigations of our novel findings through mutagenesis in future studies will additionally pave the way for a better understanding of enigmatic resistance mechanisms and assist in the development of next-generation diagnostics to timely diagnose the elusive resistance in TB patients.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/microorganisms13061401/s1](https://www.mdpi.com/article/10.3390/microorganisms13061401/s1)]([https://www.mdpi.com/article/10.3390/microorganisms13061401/s1](https://www.mdpi.com/article/10.3390/microorganisms13061401/s1)), Table S1: DST profiles of Mtb clinical isolates.  
**Author Contributions:** Conceptualization, H.M.A.H. and T.Z.; Methodology, H.M.A.H. and C.F.; Software, Z.L., S.W. and H.L.A.; Validation, X.C., N.Z., J.H. and T.Z.; Formal analysis, H.M.A.H. and C.F.; Investigation, H.M.A.H., C.F., Y.G. and T.Z.; Resources, J.H.; Data curation, H.M.A.H., C.F. and Y.G.; Writing--original draft, H.M.A.H.; Writing--review & editing, H.M.A.H., N.Z. and T.Z.; Visualization, Z.L., S.W., X.C., H.L.A. and J.H.; Supervision, H.M.A.H. and T.Z.; Project administration, H.M.A.H. and T.Z.; Funding acquisition, H.M.A.H. and T.Z. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This work was supported by the National Key R&D Program of China (2021 YFA1300904), State Key Laboratory of Respiratory Diseases (SKLRD-Z-202301, SKLRD-Z-202412, SKLRD-OP-202324), Guangzhou Science and Technology Plan-Youth Doctoral \"Sail\" Project (2024A044273), President's International Fellowship Initiative-CAS (2023 VBC0015), National Foreign Expert Project--Foreign Young Talents Program (QN2022031002L), and Guangzhou Key Medical Disciplines (2025-2027). The funders had no role in study design, data collection, analysis, decision to publish, or manuscript preparation.  
**Institutional Review Board Statement:** This study was conducted in compliance with WHO guidelines and approved by the ethics committee of Guangzhou Chest Hospital (GZXK-2022-24, approved at 10 March 2022).  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.  
**Acknowledgments:** We acknowledge the Guangzhou Chest Hospital team for providing the clinical isolates and the Guangzhou National Laboratory team for reviewing our study and offering valuable suggestions.  
**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.