Characterization of pncA and rpsA mutations in pyrazinamide-resistant M. tuberculosis

Abstract

Objective To investigate the characterizations and contributions of mutations in pncA and rpsA whole genes sequence in pyrazinamide(PZA) resistant Mycobacterium tuberculosis(MTB). Methods All of the 161 clinical strains of MTB collected from Guangzhou Chest Hospital during September 2010 and November 2012 were subjected to determine the susceptibilities to PZA by the MGIT 960 PZA system and to obtain pncA and rpsA whole gene sequences by DNA sequencing.Then the significant difference of pncA and rpsA mutation between the PZA resistant and susceptible isolates were analyzed by chi square test. Results The mutation frequency of 52 PZA resistant isolates was 84.6%(44/52), but the 109 PZA susceptible isolates had no mutations, which showed highly significant difference of pncA mutation frequency between the PZA resistant and the susceptible isolates (χ2=126.92, P=0.00).rpsA mutation was observed in 3 PZA resistant MTB isolates while only 1 PZA susceptible ones was found rpsA mutation, which exhibited no significant difference of rpsA mutation between the PZA resistant and the susceptible isolates (χ2=3.42, P=0.06).Thirty four types of pncA mutations were found in 44 PZA resistant isolates of MTB, including 12 novel mutations (L deletion at 27, E deletion at 91, E deletion at 111, Q deletion at 122, VVG deletions at 130 to 132, V deletion at 131, D8A, S18P, H57Y, F58S, E174G and M175R substitutions) and 1 promoter mutation at -11 nucleotide position with A to G.The identified mutations were randomly dispersed on the pncA whole gene sequence, but 9 isolates were observed with pncA mutations centralized in the region of G132-T142. Three PZA-resistant isolates，without pncA mutation, were observed with rpsA mutations of R474W, R474L, and E433D at C-terminus, and 1 PZA-susceptible strain showed mutation of Q162R in rpsA. Conclusions In the study, mutations in pncA gene is the major mechanism of PZA resistance and direct sequencing the pncA gene by PCR should help to rapidly identify PZA-resistant clinical strains of MTB.Furthermore, novel mutations of pncA in the study indicate the regional investigations are necessary for learning about the characteristics of pncA mutations in PZA-resistant strains of MTB.However, 3′ terminal of rpsA gene sequence may be added as the second PZA resistant relevant region for molecular identification of PZA susceptibility.（Chin J Lab Med,2014,37:285-289） Key words: Mycobacterium tuberculosis; Pyrazinamide; Mutation