scholarly journals Acetylation of isoniazid - a novel mechanism of isoniazid resistance in Mycobacterium tuberculosis

2020 ◽  
Author(s):  
K. B. Arun ◽  
Aravind Madhavan ◽  
Billu Abraham ◽  
M. Balaji ◽  
K. C. Sivakumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Isonicotinic Acid ◽  
Active Form ◽  
Drug Susceptibility ◽  
Cell Wall Biosynthesis ◽  
First Line ◽  
Isoniazid Resistance ◽  
Acetyl Group ◽  
Resistant Strains ◽  
Susceptibility Assay

AbstractIsoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a pro-drug which is converted into its active form by the intracellular KatG enzyme of Mycobacterium tuberculosis. The activated drug hinders cell wall biosynthesis by inhibiting InhA protein. INH resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze transfer of acetyl group to INH from acetyl CoA. HPLC and LC-MS analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. Drug susceptibility assay and confocal analysis showed that M. smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at minimum inhibitory concentrations that inhibited wildtype strains. In addition, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity.

scholarly journals Acetylation of Isoniazid Is a Novel Mechanism of Isoniazid Resistance in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e00456-20
Author(s):  
K. B. Arun ◽  
Aravind Madhavan ◽  
Billu Abraham ◽  
M. Balaji ◽  
K. C. Sivakumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Chromatography ◽  
High Performance ◽  
Isonicotinic Acid ◽  
Drug Susceptibility ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Mutant Proteins ◽  
Acetyl Group ◽  
Resistant Strains

ABSTRACTIsoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a prodrug which is activated by the intracellular KatG enzyme of Mycobacterium tuberculosis. The activated drug hinders cell wall biosynthesis by inhibiting the InhA protein. INH-resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH-resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study, we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze the transfer of the acetyl group to INH from acetyl coenzyme A (acetyl-CoA). High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. A drug susceptibility assay and confocal analysis showed that Mycobacterium smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Mutant proteins of Rv2170 failed to acetylate INH. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at MICs that inhibited wild-type strains. Besides, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity, although this needs to be tested in INH-resistant clinical strains.

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.

scholarly journals Black Cumin (Nigella sativa) Against Mycobacterium tuberculosis Strain H37RV And MDR-TB

Elkawnie ◽  
2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Mashuri Masri ◽  
Cut Muthiadin ◽  
Masita Masita ◽  
Tri Cahyanto ◽  
Lianah Lianah ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistance ◽  
Microscopic Observation ◽  
Nigella Sativa ◽  
Drug Susceptibility ◽  
Tuberculosis Strain ◽  
Black Cumin ◽  
Mdr Tb ◽  
Susceptibility Assay ◽  
Strain H37rv

Abstract: Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis. 10 million people suffer from TB Every year. Although TB is a preventable and treatable disease, 1.5 million people die every year due to TB. Alternative treatments continue to be pursued, and treatment with the latest TB drugs that are continuously being encouraged. Black cumin (Nigella sativa) seed contains essential oils with active compounds such as thymohydroquinone, Oleoresins, flavonoids, alkaloids, saponins, tannins, and terpenoids that act as antibacterial drugs. This study aims to determine the sensitivity of  N. sativa seed extract in inhibiting the growth of  M. tuberculosis strain H37RV and MDR-TB (Multidrug Resistance-TB). This research using Microscopic-Observation and Drug-Susceptibility Assay (MODS) method. Extraction of N. sativa was carried out by the maceration method using 70% methanol as a solvent. The results showed that the M. tuberculosis strain H37RV and MDR-TB were sensitive to N. sativa extract at concentrations of 5 and 10% but resistant to N. sativa extract at concentrations of 1 and 3%.Abstrak: Tuberkulosis (TB) adalah penyakit menular yang disebabkan oleh Bakteri Mycobacterium tuberculosis. Penyakit ini menimbulkan dampak kematian yang cukup mengkhawatirkan.  Penyakit tersebut dapat dicegah dan diobati. Salah satu sumber pengobatannya menggunakan biji jintan hitam (Nigella sativa) yang mengandung minyak atsiri dengan senyawa aktif seperti timohidrokuinon, oleoresin, flavonoid, alkaloid, saponin, tanin, dan terpenoid yang berfungsi sebagai obat antibakteri. Penelitian ini bertujuan untuk mengetahui sensitivitas ekstrak biji N. sativa dalam menghambat pertumbuhan M. tuberculosis strain H37RV and MDR-TB (Multidrug-Resistance-TB). Penelitian ini menggunakan metode Microscopic-Observation and Drug-Susceptibility Assay (MODS). Ekstraksi N. sativa dilakukan dengan metode maserasi menggunakan pelarut metanol 70%. Hasil yang diperoleh menunjukkan bahwa bakteri M. tuberculosis strain H37RV dan TB-MDR, kedua  strain tsb sensitif terhadap ekstrak N. sativa konsentrasi 5 dan 10%,  tetapi resisten terhadap  ekstrak N. sativa konsentrasi 1 dan 3%.

scholarly journals Molecular Analysis of Codon 548 in therpoBGene Involved in Mycobacterium tuberculosis Resistance to Rifampin

2014 ◽  
Vol 59 (3) ◽  
pp. 1542-1548 ◽  
Author(s):  
Yu-Tze Horng ◽  
Wen-Yih Jeng ◽  
Yih-Yuan Chen ◽  
Che-Hung Liu ◽  
Horng-Yunn Dou ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Resistance ◽  
Drug Susceptibility ◽  
Content Type ◽  
Stable Hydrogen Bond ◽  
Resistance Patterns ◽  
Resistant Strains ◽  
Rifampin Resistance ◽  
Susceptibility Tests ◽  
Dna Complex

ABSTRACTMostMycobacterium tuberculosisrifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the generpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinantMycobacterium smegmatisandM. tuberculosisisolates carrying mutatedrpoB(Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of therpoBgene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

Spoligotyping and Drug Sensitivity of Mycobacterium tuberculosis isolated from Pulmonary Tuberculosis Patients in the Arsi Zone of South Eastern Ethiopia

2019 ◽  
Author(s):  
Belete Haile Nega ◽  
Ketema Tafess ◽  
Aboma Zewude ◽  
Bazezew Yenew ◽  
Gilman SIU ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Sensitivity ◽  
Scientific Information ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Strain Identification ◽  
First Line ◽  
Eastern Ethiopia ◽  
Arsi Zone ◽  
Mycobacterium Africanum

Abstract Background Tuberculosis (TB) is one of the leading disease causing morbidity and mortality in different zones of Ethiopia including the Arsi Zone. However, little or no scientific information is available on the strains of Mycobacterium tuberculosis and their drug sensitivity profiles in this Zone. This study was conducted to identify the strains of M. tuberculosis and evaluate their drug sensitivity profiles. Methodology A total of 111 clinical isolates of M. tuberculosis from patients with pulmonary TB in the Arsi Zone were used for this study. The region of difference 9 (RD 9)-based polymerase chain reaction (PCR)and spoligotyping methods were used for speciation and strain identification of Mycobacterium tuberculosis respectively.The spoligotyping patterns were compared with the international SpolDB4 (SITVIT) and Run TB-Lineage used for the identification of lineages. The phenotypic drug susceptibility patterns were confirmed by BD BactecMGIT 960 SIRE test and GenoType MTBDRplus line probe assays were used for the detection the drug resistance-conferring mutations of the isolates. Result The spoligotype patterns of 83% (92/111) of the isolates were interpretable and 56 different patterns were identified. Twenty-two of these patterns were shared types while the remaining 34 were orphans. The predominant shared types were spoligotype international type (SIT) 149 and SIT53, each consisting of 12 and 11 isolates, respectively. The lineages identified were Euro-American, East-African-Indian, Mycobacterium-africanum, and Indo-Oceanic in descending order. Phenotypically, 17.2% of the 64 tested isolates were resistant to any of the four first-line drugs while 3.1% of them were multi-drug resistant (MDR). Higher (6.2%) monoresistance was observed to Streptomycin followed by Isoniazid (3.1%) while no resistance was observed either to Rifampicin or to Ethambutol. Genotypically, five (5.4%) isolates were resistant to Isoniazid and mutated at codon S315T1 of katG. On the other hand, only 1.1% of the isolates was resistant to Rifampicin and mutated at codon S531L of rpoB gene. Conclusion The proportion of orphan strains isolated in this study was high, which could suggest the presence of new strains in the Zone. Moreover, the study showed relatively high percentage of mono-resistance to any four first-line drugs warranting for the need to strengthen the control efforts.

scholarly journals Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of pncA PCR Sequencing

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Kingsley King-Gee Tam ◽  
Kenneth Siu-Sing Leung ◽  
Gilman Kit-Hang Siu ◽  
Kwok-Chiu Chang ◽  
Samson Sai-Yin Wong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Detection ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Activity Data ◽  
Content Type ◽  
Treatment Regimens ◽  
Mdr Tb ◽  
Resistant Strains ◽  
Respiratory Specimens

ABSTRACT An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtime MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

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