## 3 Results
### Identification of Novel Mutations in _clpC1_ of the Sequenced PZAR Strains Using WGS  
To resolve the observed discrepancy between phenotypic and genetic characteristics, WGS was performed on the five PZAR isolates exhibiting wild-type customary genes (_pncA_, _rpsA_, _panD_, and _Rv2783c_). Comparative genomic analyses against the reference strain Mtb H37 Rv (NC_000962.3) and genomic databases revealed that five PZAR Mtb clinical strains contained novel nonsynonymous mutations in the _clpC1_ gene, encoding an ATP-dependent ATPase involved in protein degradation.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Locus** & \begin{tabular}{c} **Nucleotide** \\ **Change** \\ \end{tabular} & **Codon Change** & **Amino Acid Change** & \
\begin{tabular}{c} **PZase** \\ **Activity** \\ \end{tabular} &
\begin{tabular}{c} **No. of** \\ **Isolates** \\ \end{tabular} \\ \hline \multirow{4}{*}{UFR} & A-11C & - & - & N & 1 \\  & A-11G & - & - & N & 2 \\  & T-122 & deletion & Frameshift & N & 1 \\  & C-114, A-11G & deletion + substitution & Frameshift + substitution & N & 1 \\ \hline \multirow{4}{*}{_pncA_ + UFR} & G-115C, T476G & CTG \(\rightarrow\) CGG & Leu159 Arg & N & 1 \\  & C-110G, T355G & TGG \(\rightarrow\) GGG & Trp119 Gly & N & 1 \\  & A403C & ACC \(\rightarrow\) CCC & Thr135 Pro & N & 1 \\  & A422C & CAG \(\rightarrow\) CCG & Gln141 Pro & N & 2 \\  & A-144 ins, T416C & GTG \(\rightarrow\) CGG & Val139 Ala & N & 1 \\  & C28T & CAG \(\rightarrow\) TAG & Gln10 Stop & N & 1 \\  & T80G & CTG \(\rightarrow\) CGG & Leu27 Arg & N & 1 \\ \hline \multirow{2}{*}{_pncA\({}^{c}\)_} & A128 & deletion & Frameshift & N & 1 \\  & G133T & GTG \(\rightarrow\) TTG & Val45 Leu & N & 1 \\  & A142T & AAG \(\rightarrow\) TAG & Lys48 Stop & N & 1 \\  & C161A & CCG \(\rightarrow\) CAG & Pro54 Gln & N & 1 \\  & C185T & CCG \(\rightarrow\) CTG & Pro62 Leu & N & 1 \\  & C189A & GAC \(\rightarrow\) GAA & Asp63 Glu & N & 1 \\  & G233A & GGC \(\rightarrow\) GAC & Gly78 Asp & N & 2 \\  & C282G & TTC \(\rightarrow\) TTG & Phe94 Leu & N & 1 \\  & A286G & AAG \(\rightarrow\) GAG & Lys96 Glu & N & 2 \\  & C299T & ACC \(\rightarrow\) ATC & Thr100 Ile & N & 1 \\  & G311T & AGC \(\rightarrow\) ATC & Ser104 Ile & N & 1 \\ \hline \hline \end{tabular}  
* UFR: mutations only in the upstream flanking region (UFR) of _pncA_. _pncA_ + UFR: strains with mutations both in UFR and _pncA_. _pncA_: mutations only in the coding region of _pncA_. N: negative.  
\end{table}
Table 2: Mutations in _pncA_ and its flanking regions in PZAR MöbiusClinical isolates.  
This resulted in a clinically significant condition in which approximately 16.1% of the PZAR Mtb isolates were initially misclassified as PZA-susceptible based on _pncA_-specific existing primer schemes or using conventional _pncA_-targeted molecular diagnostics alone [28], potentially leading to suboptimal treatment outcomes. Notably, the reconfirmation of these five clinical isolates through phenotypic susceptibility testing using a reduced inoculum as previously described [29; 30] and targeted gene resequencing by the [PERSON] method verified the PZA resistance (400 ug/mL) and the existence of novel mutations in _clpC1_, respectively. In _Mtb_, ClpC1 works with the proteolytic domains ClpP1 and ClpP2 of the Clp complex to degrade misfolded proteins, a process dependent on ATP hydrolysis by ClpC1, but protein degradation is diminished or entirely halted when lacking functional ClpC1. The in silico mutagenesis by PyMOL showed structural alteration associated with these amino acid substitutions in the ClpC1 protein (Figure 2).