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Gene sequencing for routine verification of pyrazinamide resistance in Mycobacterium tuberculosis: a role for pncA but not rpsA.

ABSTRACT: Pyrazinamide (PZA) is an important component of first-line therapy for the treatment of tuberculosis. Here, we evaluate targeted gene sequencing as a supplement to phenotypic PZA susceptibility testing of Mycobacterium tuberculosis. Routine sequencing of pncA, but not rpsA, is effective for verification of PZA susceptibility results.

SUBMITTER: Alexander DC

PROVIDER: S-EPMC3486241 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Gene sequencing for routine verification of pyrazinamide resistance in Mycobacterium tuberculosis: a role for pncA but not rpsA.

Alexander David C DC Ma Jennifer H JH Guthrie Jennifer L JL Blair Joanne J Chedore Pam P Jamieson Frances B FB

Journal of clinical microbiology 20120815 11

Pyrazinamide (PZA) is an important component of first-line therapy for the treatment of tuberculosis. Here, we evaluate targeted gene sequencing as a supplement to phenotypic PZA susceptibility testing of Mycobacterium tuberculosis. Routine sequencing of pncA, but not rpsA, is effective for verification of PZA susceptibility results. ...[more]

PMID: 22895038

Similar Datasets

Role of pncA and rpsA gene sequencing in detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates from southern China.

Project description:We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in pyrazinamide-resistant strains determined by the Bactec MGIT 960 system. The 3' end of rpsA might be added as the target of molecular detection of pyrazinamide susceptibility.

| S-EPMC3911430 | biostudies-literature

Pyrazinamide Resistance and pncA Mutation Profiles in Multidrug Resistant Mycobacterium Tuberculosis.

Project description:PurposePyrazinamide (PZA) is a critical component of standardized chemotherapy for tuberculosis (TB) and is recommended for the treatment of multidrug-resistant (MDR) TB. We aimed to characterize mutations in pncA of M. tuberculosis and evaluate their diagnostic accuracy for PZA susceptibility in China. We also combined genotypic methods with phenotypic susceptibility testing and pyrazinamidase (PZAse) activity to confirm PZA-resistant M. tuberculosis isolates.ResultsAn evaluation of 82 MDR M. tuberculosis strains revealed that 28.0% (23/82) were phenotypically resistant to 100 mg/L PZA and 15.9% (13/82) showed resistance to 300 mg/L PZA. Mutations in pncA were detected at 33 unique sites, and the majority were point mutations. No evident mutation hotspots or mutations affecting multiple amino acids were found, but the association between pncA mutations and PZA resistance was significant under 100 and 300 mg/L. The sensitivity of pncA mutation detection for predicting PZA susceptibility was 82.6% (19/23), and the specificity was 61.0% (36/59), based on 100 mg/L PZA, whereas the sensitivity was 84.6% (11/13) and the specificity was 55.1% (38/69), based on 300 mg/L PZA. All mutations identified in the highly PZA-resistant (300 mg/L) strains had an 80% loss relative to PZAse activity. No evident PZAse activity loss was observed in one synonymous mutation strain and the loss exceed 60% in all other strains.ConclusionThe association between pncA mutation and PZA resistance was significant. Relatively, the molecular method have shown better reliability than the phenotypic method for the detection of PZA resistance. This provides a theoretical basis for the clinical diagnosis of drug-resistant TB.

| S-EPMC9440668 | biostudies-literature

Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms.

Project description:A gene (pncA) with mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis complex members was characterized in 67 pyrazinamide-resistant and 51 pyrazinamide-susceptible isolates recovered from diverse geographic localities and anatomic sites and typed by IS6110 profiling. All pyrazinamide-susceptible organisms had identical pncA alleles. In striking contrast, 72% of the 67 resistant organisms had pncA mutations that altered the primary amino acid sequence of pyrazinamidase. A total of 17 previously undescribed mutations were found, including upstream mutations, missense changes, nucleotide insertions and deletions, and termination mutations. The mutations were arrayed along virtually the entire length of the gene. These data are further evidence that most drug resistance in M. tuberculosis is due to simple mutations occurring in chromosomally encoded genes rather than to acquisition of resistance genes by horizontal transfer events.

| S-EPMC163764 | biostudies-other

Rapid sequencing of the Mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility.

Project description:We developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.

| S-EPMC4313251 | biostudies-literature

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.

Project description:Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

| S-EPMC163747 | biostudies-other

Gibbs Free Energy Calculation of Mutation in PncA and RpsA Associated With Pyrazinamide Resistance.

Project description:A central approach for better understanding the forces involved in maintaining protein structures is to investigate the protein folding and thermodynamic properties. The effect of the folding process is often disturbed in mutated states. To explore the dynamic properties behind mutations, molecular dynamic (MD) simulations have been widely performed, especially in unveiling the mechanism of drug failure behind mutation. When comparing wild type (WT) and mutants (MTs), the structural changes along with solvation free energy (SFE), and Gibbs free energy (GFE) are calculated after the MD simulation, to measure the effect of mutations on protein structure. Pyrazinamide (PZA) is one of the first-line drugs, effective against latent Mycobacterium tuberculosis isolates, affecting the global TB control program 2030. Resistance to this drug emerges due to mutations in pncA and rpsA genes, encoding pyrazinamidase (PZase) and ribosomal protein S1 (RpsA) respectively. The question of how the GFE may be a measure of PZase and RpsA stabilities, has been addressed in the current review. The GFE and SFE of MTs have been compared with WT, which were already found to be PZA-resistant. WT structures attained a more stable state in comparison with MTs. The physiological effect of a mutation in PZase and RpsA may be due to the difference in energies. This difference between WT and MTs, depicted through GFE plots, might be useful in predicting the stability and PZA-resistance behind mutation. This study provides useful information for better management of drug resistance, to control the global TB problem.

| S-EPMC7160322 | biostudies-literature

Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan.

Project description:BACKGROUND:Pyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan. METHODS:We performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations. RESULTS:A total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n?=?4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C?>?T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%). CONCLUSION:Mutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.

| S-EPMC6364397 | biostudies-literature

Correlation between pyrazinamide activity and pncA mutations in Mycobacterium tuberculosis isolates in Taiwan.

Project description:A total of 76 clinical Mycobacterium tuberculosis isolates from Taiwan were tested for pyrazinamidase activity, pyrazinamide susceptibility, and pncA mutations. Frequency of resistance to PZA rose with increases in resistance to first-line drugs. Of 17 pyrazinamide-resistant strains, 7 (3 of which had not been previously described) possessed mutations in the pncA gene.

| S-EPMC253789 | biostudies-literature

Structural Dynamics Behind Clinical Mutants of PncA-Asp12Ala, Pro54Leu, and His57Pro of Mycobacterium tuberculosis Associated With Pyrazinamide Resistance.

Project description:Pyrazinamide (PZA) is one of the main FDA approved drugs to be used as the first line of defense against Mycobacterium Tuberculosis (MTB). It is activated into pyrazinoic acid (POA) via MTB's pncA gene-encoded pyrazinamidase (PZase). Mutations are most commonly responsible for PZA-resistance in nearly 70% of the resistant samples. In the present work, MTB positive samples were chosen for PZA drug susceptibility testing (DST) against critical concentration (100 ug/ml) of PZA. The resistant samples were subjected to pncA sequencing. As a result, 36 various mutations have been observed in the PZA resistant samples, uploaded to the NCBI (GeneBank accession no. MH461111). Here we report the mechanism of PZA resistance behind the three mutants (MTs), Asp12Ala, Pro54Leu, and His57Pro in comparison with the wild type (WT) through molecular dynamics simulation to unveil how these mutations affect the overall conformational stability. The post-simulation analyses revealed notable deviations as compared to the WT structure. Molecular docking studies of PZA with MTs and WT, pocket volume inspection and overall shape complementarity analysis confirmed the deleterious nature of these mutations and gave an insight into the mechanism behind PZA-resistance. These analyses provide vital information regarding MTB drug resistance and could be extremely useful in therapy management and overcoming its global burden.

| S-EPMC6914729 | biostudies-literature

Association between pncA gene mutations, pyrazinamidase activity, and pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

Project description:We determined MICs for, confirmed the presence of pncA mutations in, and performed pyrazinamidase testing on colonies (subclones) obtained from seven isolates that exhibited differential pyrazinamide (PZA) susceptibility. Six of the seven strains were found to exhibit characteristics resulting from the mixture of strains possessing different properties. In addition, our analysis revealed large pncA-spanning deletions (1,565 bp, 4,475 bp, and 6,258 bp) in three strains that showed high PZA resistance.

| S-EPMC4136016 | biostudies-literature

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