Affiliation:
1. Department of Medical Microbiology, Royal Free and University College Medical School,1 and
2. Department of Medical Microbiology, St George's Hospital Medical School,2 London, United Kingdom
Abstract
ABSTRACT
Mycobacterium tuberculosis
converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the
pncA
gene, and mutations in the
pncA
gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of
Mycobacterium tuberculosis
suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The
pncA
gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal
pncA
gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The
pncA
gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.
Publisher
American Society for Microbiology
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