Pyrazinamidase activity of Mycobacterium tuberculosis—A test of sensitivity to pyrazinamide

Trivedi, Sunil; Desai, Sharav

doi:10.1016/0041-3879(87)90058-4

Cited by 44 publications

(31 citation statements)

References 3 publications

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“…PZA is a prodrug that requires activation to its toxic form, pyrazinoic acid, a process mediated by pyrazinamidase (Pzase), an enzyme produced by mycobacterial species (29). The correlation between PZA resistance and Pzase activity is supported by the demonstration of loss of this activity in resistant isolates (18,32).…”

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confidence: 90%

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

Bastola

Morlock

et al. 2004

J Clin Microbiol

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The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing highperformance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.

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confidence: 90%

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

Bastola

Morlock

et al. 2004

J Clin Microbiol

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“…So, the PZA susceptibility test now depends on the pyrazinamidase test because of the strong correlation of reduced pyrazinamidase activity with the PZA resistance phenotype (4,13,22). However, the pyrazinamidase assay method has shortcomings; e.g., it takes at least 3 weeks to prepare a sufficient amount of bacilli and it also takes 4 days for the pyrazinamidase assay.…”

Section: Fig 2 Activities Of In Vitro-synthesized Pyrazinamidasementioning

confidence: 99%

Rapid Detection of Pyrazinamide-Resistant Mycobacterium tuberculosis by a PCR-Based In Vitro System

Suzuki¹,

Suzuki²,

Tamaru³

et al. 2002

J Clin Microbiol

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Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5 ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.Tuberculosis (TB) caused by members of Mycobacterium tuberculosis complex is one of the common human diseases, causing 3,000,000 deaths per year worldwide (23). While the disease is associated with economic impoverishment, TB is on the rise in many industrialized nations. The spread of TB is due to immigration, the emergence of drug-resistant strains (24), and the AIDS epidemics. The increasing number of drug-resistant M. tuberculosis strains has made the rapid identification of susceptibility clinically important because those patients suffering from these strains require specialized antibiotic treatment.Recently, a number of genetic changes leading to rifampin, isoniazide, pyrazinamide (PZA), streptomycin, and kanamycin resistances have been characterized. Nearly 95% of rifampinresistant M. tuberculosis strains carry mutations in a certain region of the beta subunit of the RNA polymerase encoded by the rpoB gene (20, 21). The mutations correlated to isoniazide resistance were found on the katG (25), inhA (1), and ahpC/oxyR (26) genes. Ethambutol-resistant M. tuberculosis strains carried mutations on a certain part of the gene coding for alabinosyl transferase (2, 17). Mutations on the rrs and rpsL genes coding for the 16S ribosomal RNA and ribosome protein S12, respectively, confer streptomycin resistance (8, 11). A part of the kanamycin-resistant phenotype can be detected by identifying the mutations on the rrs gene (19). Missense mutations in the DNA gyrase gene (gyrA) have been reported to be associated with increased levels of resistance to fluoroquinolones (5).PZA is one of the most important drugs for anti-TB shortcourse chemotherapy. PZA is converted to pyrazinoic acid by mediation of pyr...

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“…Alternatively, PZA susceptibility has also been determined by detecting PZase activity of the cultured M. tuberculosis (11,12) as PZase activity is lost in PZA-resistant isolates (13). However, these conventional culture-based methods require up to 3 months and exhibit high discordance rates among laboratories (14).…”

Section: Introductionmentioning

confidence: 99%

Characterization ofpncAMutations of Pyrazinamide-ResistantMycobacterium tuberculosisin Korea

Lee

Jung

2001

J Korean Med Sci

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Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.

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Pyrazinamidase activity of Mycobacterium tuberculosis—A test of sensitivity to pyrazinamide

Cited by 44 publications

References 3 publications

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

Rapid Detection of Pyrazinamide-Resistant Mycobacterium tuberculosis by a PCR-Based In Vitro System

Characterization ofpncAMutations of Pyrazinamide-ResistantMycobacterium tuberculosisin Korea

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