Pyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan. We performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations. A total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n = 4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C > T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%). Mutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.

Pyrazinamide (PZA) is one of the essential anti-mycobacterium drugs, active against non-replicating Mycobacterium tuberculosis (MTB) isolates. PZA is converted into its active state, called pyrazinoic acid (POA), by action of pncA encoding pyrazinamidase (PZase). In the majority of PZA-resistance isolates, pncA harbored mutations in the coding region. In our recent report, we detected a number of novel variants in PZA-resistance (PZA^R) MTB isolates, whose resistance mechanisms were yet to be determined. Here we performed several analyses to unveil the PZA^R mechanism of R123P, T76P, G150A, and H71R mutants (MTs) through molecular dynamics (MD) simulations. In brief, culture positive MTB isolates were subjected to PZA susceptibility tests using the WHO recommended concentration of PZA (100 μg ml⁻¹). The PZA^R samples were screened for mutations in pncA along sensitive isolates through polymerase chain reactions and sequencing. A large number of variants (GeneBank accession no. MH461111), including R123P, T76P, G150A, and H71R, have been spotted in more than 70% of isolates. However, the mechanism of PZA^R for mutants (MTs) R123P, T76P, G150A, and H71R was unknown. For the MTs and native PZase structures (WT), thermodynamic properties were compared using molecular dynamics simulations for 100 ns. The MTs structural activity was compared to the WT. Folding effect and pocket volume variations have been detected when comparing between WT and MTs. Geometric matching further confirmed the effect of R123P, T76P, G150A, and H71R mutations on PZase dynamics, making them vulnerable for activating the pro-drug into POA. This study offers a better understanding for management of PZA^R TB. The results may be used as alternative diagnostic tools to infer PZA resistance at a structural dynamics level.

Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health issue in Pakistan. Isoniazid is a first-line pro-drug that requires activation through an enzyme called catalase peroxidase, but is subject to widespread resistance, driven by mutations in katG and inhA genes and other loci with compensatory effects (e.g., ahpC). Here, we used whole genome sequencing data from 51 M. tuberculosis isolates collected from Khyber Pakhtunkhwa province (years 2016-2019; all isoniazid phenotypically resistant) to investigate the genetic diversity of mutations in isoniazid candidate genes. The most common mutations underlying resistance were katG S315T (37/51), fabG1 -15C>T (13/51; inhA promoter), and inhA -154G>A (7/51). Other less common mutations (n < 5) were also identified in katG (R128Q, V1A, W505*, A109T, D311G) and candidate compensatory genes ahpC (-54C>T, -51G>A) and oxyS (M249T). Using DynaMut2 software, the mutants exhibited various degrees of stability and flexibility on protein structures, with some katG mutations leading to a decrease in KatG protein flexibility. Overall, the characterisation of circulating isoniazid resistant-linked mutations will assist in drug resistant TB management and control activities in a highly endemic area of Pakistan.