Pyrazinamide-resistant mycobacterium tuberculosis isolates from Khyber Pakhtunkhwa and rpsA mutations

Pyrazinamide (PZA) is a key first-line antibiotic used for the short-course treatment of drug-sensitive and multidrug-resistant (MDR) isolates of tuberculosis. PZA exhibits potent action against semi-dormant bacilli in acidic environments. However, mutations that occur in target genes may cause technical difficulties in the diagnosis of PZA resistance during drug susceptibility testing. The objective of the current study is to identify mutations in pncAWT rpsA and rpsAWT panD genes among PZA-resistant isolates of Mycobacterium tuberculosis (MTB) circulating in the Pashtun dominant region, Khyber Pakhtunkhwa, Pakistan. We selected 18 PZA-resistant pncAWT strains from the Provincial Tuberculosis Reference Laboratory (PTRL) Khyber Pakhtunkhwa to investigate mutations in the coding region of rpsA and panD genes. The experiments were repeated for drug susceptibility testing using MGIT 960 automated system. In addition, eighteen PZA-resistant rpsA genes along with 5 susceptible strains and one H37Rv strain were sequenced. All 18 isolates were PZA-resistant. The majority of these isolates exhibited multidrug resistance (MDR) (13/18). We identified 14 non-synonymous and one synonymous mutation in the coding region of rpsA in 11 strains. All mutations were scattered throughout the gene and not reported previously. Further, we did not identify any mutation in 7 rpsAWT panD genes. Mutations in rpsA but not in panD occur in PZA-resistant pncAWT MTB isolates circulating in Khyber Pakhtunkhwa, Pakistan.