## 4 Discussion  
PZA, as an important first-line anti-TB drug, has unique sterilizing activity in the therapeutic regimens against persistent Mtb [3; 13]. Considering its crucial role in treatment therapies, accurate detection of PZA resistance is vital for the effective inclusion of PZA in treatment regimens [12; 31]. Consistent with previous studies, the rate of PZA resistance (58.4%) in our study is comparable to those reported in Hunan (60.4%) [32], Ningbo (59.1%) [33], and Myanmar (58.9%) [34]. These rates exceed those documented in South Korea (31.5%) [35], Beijing (38.3%) [36], Peru (47.7%) [37], and Ireland (55.0%) [30]. While these figures are based on clinical isolates collected from central TB hospitals or organizations and may not fully represent the general population-level prevalence, they nonetheless provide valuable insight into the regional burden of PZA resistance. Such comparative data contribute to a better understanding of resistance trends and highlight the urgent need for rapid and reliable detection methods to strengthen MDR-TB management strategies. PZA resistance is more frequently observed in re-treated TB cases [11], underscoring the necessity for precise susceptibility testing, especially given the critical role of PZA in MDR-TB regimens. However, routine phenotypic testing remains challenging due to the requirement for an acidic environment in culture-based assays [38]. As genetic mutations are major contributors to drug resistance in Mtb, most studies on molecular diagnostics have been based on the detection of _pncA_ mutations for PZA susceptibility testing [28]. In this study, 83.8% of the PZA\({}^{\text{R}}\) Mtb isolates had genetic mutations in pncA or its upstream region. Mutations leading to the loss of PZase activity are the major mechanisms leading to PZA resistance. Globally, the prevalence of _pncA_ mutations among PZA\({}^{\text{R}}\) Mtb isolates varies significantly: 41.2% in Nepal [39], 63.6% in Fujian [40], 74.0% in Ukraine [41], 81.9-97.4% in Chongqing [42; 43], 89.5% in Slovenia [44], and 95.9% in Hunan [32]. Furthermore, the dominance of the Beijing genotype (74.1%) among PZAR isolates in our study mirrors findings from other studies reporting a higher frequency of MDR- and XDR-TB outbreaks caused by Beijing family strains [36; 45]. These comparative data highlight the widespread involvement of _pncA_ mutations in PZA resistance across different geographic regions and lineages. Such insights are important for global surveillance and the development of robust molecular diagnostics that can detect PZA resistance regardless of lineage background. Importantly, a subset of PZA\({}^{\text{R}}\) Mtb isolates in our study lacked mutations in _pncA_ and other conventional resistance-associated genes (_rpsA_, _panD_, and _Rv2783c_), posing a significant diagnostic challenge for _pncA_-based molecular tests. To validate these findings, repeated phenotypic susceptibility testing using a reduced inoculum [29; 30] and PZase activity assays confirmed true PZA resistance, as our results remained consistent. Interestingly, the WGS analysis of these five PZA\({}^{\text{R}}\) Mtb isolates identified novel non-synonymous mutations in _clpC1_, encoding an ATP-dependent chaperone ATPase crucial for protein degradation. ClpC1 belongs to the class II AAA+ family of proteins, comprising one N-terminal and two nucleotide-binding domains (D1 and D2), with the D2 domain linked to a critical C-terminal extension involved in oligomerization and substrate recognition [46; 47]. The N-terminal region of ClpC1 has been recognized as a promising target site for various anti-persister drugs [48]. We identified mutations (Gly58 Ser and Val63 Ala) in the N-terminal domain of the ClpC1 in our PZA\({}^{\text{R}}\) clinical strains (Figure 4), different from those detected at the distinct codons in the same region, causing resistance to lassomycin (Gln17 Arg, Arg21 Ser, Pro79 Thr) [49], rufomycin (Val13, His77, and Phe80) [50], and ecumicin (Leu92 Ser/Phe or Leu69 Pro) [51].