Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

Pyrazinamide (PZA) is a critical component of first- and second-line treatments of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate Mycobacterium bovis BCG mutants resistant to pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA resistant strains. Rather than finding mutations in the proposed candidate targets fatty acid synthase I and ribosomal protein S1, we found resistance conferring mutations in two pathways: missense mutations in aspartate decarboxylase panD, involved in the synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations in the vitro nonessential polyketide synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM). Probing for cross resistance to two structural analogs of POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM- but not the CoA-related mechanism of action with POA. We demonstrated that POA depletes CoA in wild-type bacteria, which is prevented by mutations in panD. Sequencing 10 POA-resistant Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least 2 distinct mechanisms of resistance to the drug. The emergence of resistance through the loss of a virulence factor in vitro may explain the lack of clear molecular patterns in PZA-resistant clinical isolates, other than mutations in the prodrug-converting enzyme. The apparent interference of POA with virulence pathways may contribute to the drug’s excellent in vivo efficacy compared to its modest in vitro potency.

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“…47 We also observed the same when we looked into sequences of XDR strains from Pakistan that were pncA wild type and resistant to PZA. 48 …”

Section: Discussionmentioning

confidence: 99%

Pyrazinamide Resistance Is Caused by Two Distinct Mechanisms: Prevention of Coenzyme A Depletion and Loss of Virulence Factor Synthesis

et al. 2016

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“…An isolate was considered resistant to a given drug when growth of 1% or more as compared with the control was observed in drug‐containing medium. MTB H 37 Rv wild‐type strain (ATCC 27294) was used as a control for drug susceptibility testing (WHO ; Ali et al, ).…”

Section: Methodsmentioning

confidence: 99%

“…MTB H 37 Rv wild-type strain (ATCC 27294) was used as a control for drug susceptibility testing (WHO 2009;Ali et al, 2015).…”

Section: Drug Susceptibility Testing (Dst)mentioning

confidence: 99%

Clonality and genetic profiles of drug‐resistant Mycobacterium tuberculosis in the Eastern Cape Province, South Africa

et al. 2019

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In this study, we investigated the diversity of drug‐resistant Mycobacterium tuberculosis isolates from families who own cattle in the Eastern Cape Province of South Africa using spoligotyping and mycobacterial interspersed repetitive‐unit‐variable number tandem repeat (MIRU‐VNTR) typing. The Mycobacterium tuberculosis was investigated using MIRU‐VNTR and the Mycobacterium tuberculosis families were evaluated using spoligotyping. Spoligotyping grouped 91% of the isolates into seven clusters, while 9% of the deoxyribonucleic acid (DNA) from TB isolates were unclustered from a total of 154 DNA used. Previously described shared types were observed in 89.6% of the isolates, with the Beijing family, SIT1, the principal genotype in the province, while the families T, SIT53 and X1, SIT1329 were the least detected genotypes. MIRU‐VNTR grouped 81% of the isolates in 23 clusters while 19% were unclustered. A combination of the VNTR and spoligotyping grouped 79% of the isolates into 23 clusters with 21% unclustered. The low level of diversity and the clonal spread of drug‐resistant Mycobacterium tuberculosis isolates advocate that the spread of TB in this study may be instigated by the clonal spread of Beijing genotype. The results from this study provide vital information about the lack of TB control and distribution of Mycobacterium tuberculosis complex strain types in the Eastern Cape Province of South Africa.

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“…Our study followed primer patterns from one related study in Hong Kong that could predict 96.8% specificity and 64.2% sensitivity for kanamycin from rrs on gene marker 1401 [11]. showed 78% of kanamycin resistance correlated to SNPs in rrs at nt1401 [28] and concluded that the commercial diagnostic tool determining the aminoglycoside drug resistance target at rrs nt 1401 could detect 78% of kanamycin resistance in XDR-TB isolates [28]. One related study in Thailand showed that the designed rrs primer using 3 positions and 1680 PCR product size could predict 100% specificity and 72.4% sensitivity of kanamycin resistant M/XDR-TB [29].…”

Section: Kanamycin Resistancementioning

confidence: 91%

Correlations between Gene Resistant Markers and Second-Line Anti-TB Drug Resistance in Pre-XDR and XDR-TB Patients

Jaksuwan¹,

Tharavichikul²,

Chuchottaworn³

et al. 2017

JTR

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Background: Extensively drug resistant tuberculosis (XDR-TB) is a serious problem in public health and XDR-TB patients usually develop from multi-drug resistance tuberculosis (MDR-TB) and pre-XDR-TB. The rapid molecular test for drug susceptibility testing (DST) can be used for early detection to prevent XDR-TB. Methods: We examined 34 clinical Mycobacterium tuberculosis (M. tuberculosis) isolates from MDR/XDR-TB patients in the upper north of Thailand that were identified with drug susceptibility profiles by indirect agar proportion method from 2005-2012. Our study investigated the genetic mutations in gyrA for ofloxacin resistance and rrs for kanamycin resistance. The genetic mutations and drug susceptibility test results were analyzed using the exact test. Results: The majority of the ofloxacin resistance was detected in gyrA 21, gyrA 70, gyrA 87, gyrA 102, gyrA 162, and gyrA 187 were at 0%, 12.5%, 37.5%, 0%, 50.0% and 25.0% sensitivity, respectively, and at 96.2, 96.2%, 20.1%, 96.2%, 57.7% and 61.5% specificity, respectively. Kanamycin resistance was found in rrs 512, rrs 241, rrs 223, rrs 414 and rrs 408 at 16.7%, 0%, 0%, 16.7% and 16.7% sensitivity, respectively, and at 96.4%, 92.9%, 82.1%, 82.1% and 71.4% specificity, respectively. This study found no significant correlation between gyrA mutations and ofloxacin resistance and also no correlation between the rrs gene and kanamycin resistance. Conclusion: These primer sequences and PCR products in our study such as gyrA and rrs might be unsuitable to detect ofloxacin and kanamycin resistance in the upper How to cite this paper:

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Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

Cited by 66 publications

References 66 publications

Pyrazinamide Resistance Is Caused by Two Distinct Mechanisms: Prevention of Coenzyme A Depletion and Loss of Virulence Factor Synthesis

Pyrazinamide Resistance Is Caused by Two Distinct Mechanisms: Prevention of Coenzyme A Depletion and Loss of Virulence Factor Synthesis

Clonality and genetic profiles of drug‐resistant Mycobacterium tuberculosis in the Eastern Cape Province, South Africa

Correlations between Gene Resistant Markers and Second-Line Anti-TB Drug Resistance in Pre-XDR and XDR-TB Patients

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