Detection of Pyrazinamide Resisitance in Mycobacterium Tuberculosis by Sequencing of pncA Gene

Hwang, Jee Yoon; Kwak, Kyung Rok; Park, Hye Kyung; Lee, Ji Seok; Park, Sam Seok; Kim, Yun Seong; Lee, Jung Yoo; Chang, Chul Hun; Lee, Min Ki; Park, Soon Kew

Abstract

BACKGROUND
Examining the biological susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) in vitro is very difficult as PZA is inactive under normal culture conditions. The susceptibility test, an enzyme assay for Pzase activity, and a genetic test for pncA gene mutations, were performed in order to predict PZA resistance.
METHODS
28 cultured clinical isolates of Mycobacterium tuberculosis were tested. The biological susceptibility was performed by the absolute concentration method using Lowenstein-Jensen media. The PZase activity was tested by means of Wayne's method. A 710-bp region includes the entire open reading frame of pncA was amplified and sequenced.
RESULTS
All six strains with positive PZase activity exhibited no pncA mutations with one strain showing a false resistance in the biological susceptibility test. Among the 22 strains with no PZase activity, 21 exhibited showed pncA mutations. In the biological suscaptibility test, 20 strains were resistant, and one was susceptible, and the other failed to test. The mutation types varied with ten missense, one silent and one nonsense mutation 1 slipped-strand mispairing, and 6 frameshift mutations. Three strains had an adenine to guanine mutation at position - 11 upstream of the start codon.
CONCLUSION
The mutation at the pncA promotor region is frequent at -11 upstream position. Automatic sequencing of pncA is a useful tool for rapid and accurate detection of PZA resistant M.tuberculosis, and for demonstrating the epidemiological relatedness of the PZA-resistant M.tubersulosis strains.