scholarly journals Molecular Genetics of para-Aminosalicylic Acid Resistance in Clinical Isolates and Spontaneous Mutants of Mycobacterium tuberculosis

2009 ◽  
Vol 53 (5) ◽  
pp. 2100-2109 ◽  
Author(s):  
Vanessa Mathys ◽  
René Wintjens ◽  
Philippe Lefevre ◽  
Julie Bertout ◽  
Amit Singhal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Acid Resistance ◽  
Clinical Isolates ◽  
Alternative Mechanism ◽  
Aminosalicylic Acid ◽  
Coding Region ◽  
First Line ◽  
Marked Contrast ◽  
Dilution Assay ◽  
Spontaneous Mutants

ABSTRACT The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

scholarly journals Para-aminosalicylic acid increases the susceptibility to isoniazid in clinical isolates of Mycobacterium tuberculosis

2019 ◽  
Vol Volume 12 ◽  
pp. 825-829 ◽  
Author(s):  
Tingting Zhang ◽  
Guanglu Jiang ◽  
Shu’an Wen ◽  
Fengmin Huo ◽  
Fen Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Isolates ◽  
Aminosalicylic Acid

scholarly journals Binding Pocket Alterations in Dihydrofolate Synthase Confer Resistance topara-Aminosalicylic Acid in Clinical Isolates of Mycobacterium tuberculosis

2013 ◽  
Vol 58 (3) ◽  
pp. 1479-1487 ◽  
Author(s):  
Fei Zhao ◽  
Xu-De Wang ◽  
Luke N. Erber ◽  
Ming Luo ◽  
Ai-zhen Guo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Binding Pocket ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Missense Mutations ◽  
Aminosalicylic Acid ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Mechanistic Basis

ABSTRACTThe mechanistic basis for the resistance ofMycobacterium tuberculosistopara-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursorpara-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance inM. tuberculosisstrain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates ofM. tuberculosisandMycobacterium bovis. From a panel of 85 multidrug-resistantM. tuberculosisclinical isolates, 5 were found to harbor mutations in thefolCgene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy offolCfully restored PAS susceptibility infolCmutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics forM. tuberculosisclinical isolates and for further defining the mode of action of this important tuberculosis drug.

scholarly journals Thr202Ala in thyA Is a Marker for the Latin American Mediterranean Lineage of the Mycobacterium tuberculosis Complex Rather than Para-Aminosalicylic Acid Resistance

2010 ◽  
Vol 54 (11) ◽  
pp. 4794-4798 ◽  
Author(s):  
Silke Feuerriegel ◽  
Claudio Köser ◽  
Leona Trübe ◽  
John Archer ◽  
Sabine Rüsch Gerdes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latin American ◽  
Acid Resistance ◽  
Multidrug Resistant ◽  
Nucleotide Polymorphisms ◽  
Aminosalicylic Acid ◽  
Phenotypic Resistance ◽  
Clear Cut ◽  
Development Of Resistance ◽  
Phylogenetic Lineages

ABSTRACT Single nucleotide polymorphisms (SNPs) involved in the development of resistance represent powerful markers for the rapid detection of first- and second-line resistance in clinical Mycobacterium tuberculosis complex (MTBC) isolates. However, the association between particular mutations and phenotypic resistance is not always clear-cut, and phylogenetic SNPs have been misclassified as resistance markers in the past. In the present study, we investigated the utility of a specific polymorphism in thyA (Thr202Ala) as a marker for resistance to para-aminosalicyclic acid (PAS). Sixty-three PAS-susceptible MTBC strains comprising all major phylogenetic lineages, reference strain H37Rv, and 135 multidrug-resistant (MDR) strains from Germany (comprising 8 PAS-resistant isolates) were investigated for the presence of Thr202Ala. In both strain collections, the Thr202Ala SNP was found exclusively in strains of the Latin American Mediterranean (LAM) lineage irrespective of PAS resistance. Furthermore, PAS MICs (0.5 mg/liter) for selected LAM strains (all containing the SNP) and non-LAM strains (not containing the SNP), as well as the results of growth curve analyses performed in liquid 7H9 medium in the presence of increasing PAS concentrations (0 to 2.0 mg/liter), were identical. In conclusion, our data demonstrate that the Thr202Ala polymorphism in thyA is not a valid marker for PAS resistance but, instead, represents a phylogenetic marker for the LAM lineage of the M. tuberculosis complex. These findings challenge some of the previous understanding of PAS resistance and, as a consequence, warrant further in-depth investigations of the genetic variation in PAS-resistant clinical isolates and spontaneous mutants.

scholarly journals Detection of Mycobacterium tuberculosis pncA Mutations by the Nipro Genoscholar PZA-TB II Assay Compared to Conventional Sequencing

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Melisa J. Willby ◽  
Maria Wijkander ◽  
Joshua Havumaki ◽  
Kartee Johnson ◽  
Jim Werngren ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Confidence Interval ◽  
Coding Region ◽  
First Line ◽  
Content Type ◽  
Treatment Regimens ◽  
Molecular Tests ◽  
Initiation Of Therapy ◽  
Probe Assay ◽  
Detection Of Mutations

ABSTRACT Pyrazinamide (PZA) is a standard component of first-line treatment regimens for Mycobacterium tuberculosis and is included in treatment regimens for drug-resistant M. tuberculosis whenever possible. Therefore, it is imperative that susceptibility to PZA be assessed reliably prior to the initiation of therapy. Currently available growth-based PZA susceptibility tests are time-consuming, and results can be inconsistent. Molecular tests have been developed for most first-line antituberculosis drugs; however, a commercial molecular test is not yet available for rapid detection of PZA resistance. Recently, a line probe assay, the Nipro Genoscholar PZA-TB II assay, was developed for the detection of mutations within the pncA gene, including the promoter region, that are likely to lead to PZA resistance. The sensitivity and specificity of this assay were evaluated by two independent laboratories, using a combined total of 249 strains with mutations in pncA or its promoter and 21 strains with wild-type pncA. Overall, the assay showed good sensitivity (93.2% [95% confidence interval, 89.3 to 95.8%]) and moderate specificity (91.2% [95% confidence interval, 77.0 to 97.0%]) for the identification of M. tuberculosis strains predicted to be resistant to PZA on the basis of the presence of mutations (excluding known PZA-susceptible mutations) in the pncA coding region or promoter. The assay shows promise for the molecular prediction of PZA resistance.

scholarly journals Revisiting Activation of and Mechanism of Resistance to Compound IQG-607 in Mycobacterium tuberculosis

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Bruno L. Abbadi ◽  
Anne D. Villela ◽  
Valnês S. Rodrigues-Junior ◽  
Fernanda T. Subtil ◽  
Pedro F. Dalberto ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metal Complex ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Isolates ◽  
Whole Genome ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Resistant Strains ◽  
Spontaneous Mutants

ABSTRACT IQG-607 is a metal complex previously reported as a promising anti-tuberculosis (TB) drug against isoniazid (INH)-resistant strains of Mycobacterium tuberculosis. Unexpectedly, we found that INH-resistant clinical isolates were resistant to IQG-607. Spontaneous mutants resistant to IQG-607 were subjected to whole-genome sequencing, and all sequenced colonies carried alterations in the katG gene. The katG(S315T) mutation was sufficient to confer resistance to IQG-607 in both MIC assays and inside macrophages. Moreover, overexpression of the InhA(S94A) protein caused IQG-607's resistance.

scholarly journals Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

2011 ◽  
Vol 56 (3) ◽  
pp. 1253-1257 ◽  
Author(s):  
J. Werngren ◽  
E. Sturegård ◽  
P. Juréen ◽  
K. Ängeby ◽  
S. Hoffner ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Wild Type ◽  
First Line ◽  
Content Type ◽  
Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

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