Molecular characteristics of pyrazinamide resistance in Mycobacterium tuberculosis strains

Abstract

Objective To investigate the correlation between the mutation of pncA gene and the susceptibility to pyrazinamide (PZA) in Mycobacterium tuberculosis complex (MTBC) strains and to analyze the mutation of panD and rpsA genes in wild type isolates without pncA gene mutation. Methods The susceptibilities of 108 MTBC strains to first-line drugs including PZA were detected by using the MGIT 960 TB system. PCR was performed to amplify the 16S rDNA and pncA, panD and rpsA genes. The PCR products were analyzed by DNA sequencing analysis. Results Among the 78 multidrug-resistant MTBC strains, 47 isolates (60%) were resistant to PZA. Four out of 30 (13%) strains that were sensitive to ethambutol, isoniazid, rifampicin and streptomycin (EIRS) were resistant to PZA. The drug-resistant MTBC strains showed higher resistance rate to PZA than that of the EIRS sensitive strains. There were 49 (96%) PZA-resistant isolates and 4 (7%) PZA-sensitive isolates occurred pncA gene mutation. Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation, especially the His57Asp substitution. The pncA gene mutations centralized in the regions of 160-169, 203-289, 309-396 and 413-467. Seven novel mutation sites of pncA gene were observed including T175C, C188A, G insertion at 68, AGC insertion at 235, C insertion at 339, CC insertion at 392 and GT deletion at 395. The mutation sites founded in the genomes of PZA-sensitive strains were different from those of the PZA-resistant strains. No mutation of the pncA gene and the upstream regulatory sequence was found in two PZA-resistant strains, NJ44 and NJ108. The sequence analysis of panD and rpsA gene showed that the NJ108 strain had panD gene mutation at G419A, but no mutation was detected in the NJ44 strain. Conclusion The multidrug-resistant MTBC strains showed higher resistance rate to PZA. The pncA gene mutation was common in PZA-resistant MTB strains and the panD gene mutation was also worthy of attention. Key words: Mycobacterium tuberculosis; Pyrazinamide; Drug resistance; Molecular characteristic