Pyrazinamide Resistance In Mycobacterium Tuberculosis Research Articles

Non-pncA Gene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

Pyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB). pncA gene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-S TB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-type pncA gene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy: panD or rpsA mutations identified by whole-genome sequencing (WGS) (n = 7), heteroresistance (n = 8), and mixed populations with Mycobacterium avium (n = 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-R clinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymous pncA mutation (Thr87Met), whereas a nonsynonymous rpsA mutation was found in 17 isolates. None of these isolates carried a nonsynonymous panD mutation, while all 15 of the MDRPZA-R isolates harbored a nonsynonymous pncA mutation. Our findings indicate that it is necessary to consider the occurrence of panD mutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymous rpsA mutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role of rpsA in PZA resistance.

Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.

ABSTRACTPreviously, we showed that mutations in Mycobacterium tuberculosis panD, involved in coenzyme A biosynthesis, cause resistance against pyrazinoic acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against pyrazinoic acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.

Molecular analysis of pyrazinamide resistance in Mycobacterium tuberculosis in Vietnam highlights the high rate of pyrazinamide resistance-associated mutations in clinical isolates

Pyrazinamide (PZA) is a key antibiotic in current anti-tuberculosis regimens. Although the WHO has stressed the urgent need to obtain data on PZA resistance, in high tuberculosis burden countries, little is known about the level of PZA resistance, the genetic basis of such resistance or its link with Mycobacterium tuberculosis families. In this context, this study assessed PZA resistance through the molecular analysis of 260 Vietnamese M. tuberculosis isolates. First-line drug susceptibility testing, pncA gene sequencing, spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing were performed. Overall, the pncA mutation frequency was 38.1% (99 out of 260 isolates) but was higher than 72% (89 out of 123 isolates) in multidrug and quadruple-drug resistant isolates. Many different pncA mutations (71 types) were detected, of which 55 have been previously described and 50 were linked to PZA resistance. Among the 16 novel mutations, 14 are likely to be linked to PZA resistance because of their mutation types or codon positions. Genotype analysis revealed that PZA resistance can emerge in any M. tuberculosis cluster or family, although the mutation frequency was the highest in Beijing family isolates (47.7%, 62 out of 130 isolates). These data highlight the high rate of PZA resistance-associated mutations in M. tuberculosis clinical isolates in Vietnam and bring into question the use of PZA for current and future treatment regimens of multidrug-resistant tuberculosis without PZA resistance testing.Emerging Microbes & Infections (2017) 6, e86; doi:10.1038/emi.2017.73; published online 11 October 2017

Mutation in clpC1 encoding an ATP-dependent ATPase involved in protein degradation is associated with pyrazinamide resistance in Mycobacterium tuberculosis

Emerging Microbes & Infections (2017) 6, e8; doi:10.1038/emi.2017.1; published online 15 February 2017

Pncagenesequence analysis for pyrazinamide resistance inmycobacterium tuberculosis from High-incidence setting

Pncagenesequence analysis for pyrazinamide resistance inmycobacterium tuberculosis from High-incidence setting

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Pyrazinamide resistance in Mycobacterium tuberculosis fails to bite?

In contrast to most other antimycobacterial drugs where--particularly in multidrug-resistant (MDR) strains--a limited number of resistance mutations dominate, pyrazinamide (PZA) resistance associated mutations remain highly diverse with limited clustering. This apparent lack of evolutionary selection for successful PZA resistance mechanisms deserves attention. A clear understanding of the epidemiology of PZA resistance acquisition and spread would be expected to result in important insights into how PZA might be better exploited in treatment regimens to minimize the amplification of Mycobacterium tuberculosis (MTB) drug resistance. We propose that PZA resistance typically induces a fitness cost that impairs MTB transmission. This would explain the lack of extensive clustering for PZA-resistant mutants. Our hypothesis also leads to a series of testable predictions which we outline that could confirm or refute our ideas.

1696Total-deletion mutation of pncA as a new mechanism of pyrazinamide resistance in Mycobacterium tuberculosis - The first report from Japan

1696Total-deletion mutation of pncA as a new mechanism of pyrazinamide resistance in Mycobacterium tuberculosis - The first report from Japan

PncAgene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis

summary Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system.

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity.

To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.

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

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

Association between genotypic and phenotypic pyrazinamide resistance in Mycobacterium tuberculosis

Background: Carbapenem-resistant Gram-negative bacteria (CR GNB) are subject of public health concerns and may be associatedwithworse outcomes. In this studyweaimed to assess 28-days survival of patientswith bacteraemia due toGramnegative species. Methods & Materials: Cross-sectional multicentre study was performed in patients with drug-resistant Gram-negative bacteraemia in Slovakia in November 2011January 2012. Antibiotic susceptibility testing was performed according to the European Committee on Antimicrobial Susceptibility Testing. Kaplan-Meier curves were used to assess 28-days survival and were analysed with Log rank test to compare survival of patientswith andwithout infection caused by CR GNB. Results: Altogether, 257 cases with drug-resistant GNB bacteraemia were identified (59,4% males; median age 62 years, IQR 50-75), of them 45 (17,5%)were due to CR GNB. Twenty-eight-days mortality, was significantly higher in group of CR GNB cases (42,2% vs. 25,5%, p=0,029), while in overall mortality there was no statistically significant difference (64,4% vs. 49,06%, p=0,086). Median time from positive blood culture to death was 9,5 days in CR GNB group, and 34,5 days in carbapenem-susceptible group (p=0,042). Also, significant difference in28-days survivalwasnoticed comparing patients with carbapenem susceptible and CR GNB infection (Log rank test p=0,033). Regarding the risk factors, polymicrobial infection (p=0,019), hospitalization in intensive care unit (p<0,001) andhospitalizationduring thewinter and/or spring (p=0,054),were associated with CR infection. Conclusion: In this study, carbapenem-resistance in GNB species causing bloodstream infection was associated with significantly worse outcome and decreased probability of survival. This is underscoring importance of careful control strategies to avoid spread of CR GNB among patients. (This study was partially supported with Trnava University Grant, No 7/TU/12).

Next-Generation Sequencing for Identifying Pyrazinamide Resistance in Mycobacterium tuberculosis

Next-Generation Sequencing for Identifying Pyrazinamide Resistance in Mycobacterium tuberculosis

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant Mycobacterium tuberculosis. Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M.tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M.tuberculosis samples for PZA resistance.

PncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M.tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.

Detection of pncA mutation associated with pyrazinamide resistance in Mycobacterium tuberculosis by high-resolution melting cure analysis

To evaluate the application of a high-resolution melting (HRM) curve analysis for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis (M. tuberculosis). Clinical isolates of M. tuberculosis were collected from Shenzhen from 2007 - 2009 and pncA mutations were characterized by sequence analysis. HRM analysis method was established and used to determine pncA gene mutation in 49 pncA mutant isolates and 78 wild type isolates. BCATEC MGIT 960 was used as the standard method to evaluate application of the HRM curve. Among 49 pncA mutant isolates, 48 isolates were characterized as mutant and 1 isolate with double point mutations misclassified as wild type by using HRM detection. In 78 wild type isolates, HRM found 6 mutant isolates. Using the BCATEC MGIT 960 to detect pyrazinamide drug susceptibility, 62 isolates were found resistant and 65 were susceptible. Among the 62 resistant strains by HRM results 53 strains were resistant, and among the 65 susceptible stains by HRM results 64 were susceptible. Compared to the results of BACTEC MGIT 960, the sensitivity and specificity of HRM were 85.5% (53/62) and 98.5% (64/65), respectively. The Youden index was found to be 0.840. The results showed a good correlation between HRM curve analysis and BACTEC MGIT 960. HRM curve analysis is a rapid and accurate method that can be used as a screening test for identification of resistance to pyrazinamide.

Characterization of pncA gene mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Mexico

Numerous studies have linked mutations in the pncA gene with resistance to pyrazinamide (Z) in Mycobacterium tuberculosis. However, variations in these mutations are specific to the country of origin of the isolate. The aim of this study was to characterize changes in pncA gene sequence in isolates of M. tuberculosis with resistance to Z, from patients in Mexico.M. tuberculosis isolates were recovered from individuals suspected of carrying drug resistant tuberculosis and respective susceptibility tests were developed. In isolates with resistance to pyrazinamide the pncA gene and its promoter were analyzed by capillary sequencing.From 127 drug-resistant isolates collected, 42 (33%) were resistant to pyrazinamide. The pncA sequences showed 26 changes in 34 (81%) isolates: 18 SNPs (n=26, 62%), four insertions (n=4, 9.5%) and four deletions (n=4, 9.5%). Absence of modifications was observed in eight (19%) sequences/isolates. The most frequent changes were the mutations L120P (n=7) and K96R (n=4). Twelve changes found are reported for the first time.This is the first description of pncA gene modifications in pyrazinamide resistant isolates originating in Mexico. We conclude that the diversity of changes in pncA indicates the presence of a noteworthy variety of pyrazinamide resistant strains occurring in the area.

Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug susceptible and multidrug-resistant tuberculosis (MDR-TB). Resistance to PZA is most commonly associated with mutations in the pncA gene encoding nicotinamidase/pyrazinamidase which converts the prodrug PZA to the active form pyrazinoic acid (POA). RpsA (ribosomal protein S1) involved in trans-translation was recently shown to be a target of PZA and mutations in RpsA are found in some PZA-resistant TB strains. However, some other PZA-resistant strains lack mutations in either pncA or rpsA. To identify potential new mechanisms of PZA resistance, we isolated 174 in vitro mutants of M. tuberculosis H37Rv resistant to PZA to search for resistant isolates that do not have pncA or rpsA mutations. DNA sequencing revealed that 169 of the 174 (97.1%) PZA-resistant mutants had pncA mutations but 5 mutants lacked pncA or rpsA mutations. Whole genome sequencing analyses revealed that the 5 PZA-resistant mutants had different mutations all occurring in the same gene panD encoding aspartate decarboxylase, which is involved in synthesis of β-alanine that is a precursor for pantothenate and co-enzyme A biosynthesis. panD mutations were identified in naturally PZA-resistant Mycobacterium canetti strain and a PZA-resistant MDR-TB clinical isolate. Future studies are needed to address the role of panD mutations in PZA resistance and confirm PanD as a new target of PZA.Emerging Microbes & Infections (2013) 2, e35; doi:10.1038/emi.2013.38

In silico Analysis of Novel Mutation ala102pro Targeting pncA Gene of M. Tuberculosis

This study reports on the structural and functional basis of pyrazinamide (PZA) resistance conferred by a novel mutation Ala102Pro in pncA gene as sequenced from a PZA resistant Mycobacterium tuberculosis strain. Molecular modeling studies of Wild Type (WT) and Mutant Type (MT) of Pyrazinamidase (PZase) showed the mutation at Ala102Pro does not impact on the conformation of the protein. However, the docking studies infer that MT has a higher inhibitory constant (Ki-990.0m) compared to WT (Ki-822.42m), which is indicative of drug resistance in MT. Furthermore, molecular interaction studies also reveal that WT forms 4 hydrogen bonds involved in PZA-WT Inhibitory interactions, whereas, in case of MT, it formed 5 hydrogen bonds with PZA. However, Ala102 in WT was found to be less fluctuating and more stable in Molecular dynamics Simulation when compared to Pro102 in MT which was highly fluctuating and unstable. This implies that Ala102 shall be a key residue involved in PZA inhibitory interactions. Moreover, MT does not show hydrogen bonding with PZA with Pro102 and also deviating in terms of PZA binding pose in comparison with WT. Hence, the observed deviations in terms of MT-PZA interactions shall be attributed to the drug resistance conferred.

An improved method for detection of pyrazinamide resistance in Mycobacterium tuberculosis

An improved method for detection of pyrazinamide resistance in Mycobacterium tuberculosis