katG Ser315Thr Mutation Research Articles

Whole-genome sequence analysis and comparisons between drug-resistance mutations and minimum inhibitory concentrations of Mycobacterium tuberculosis isolates causing M/XDR-TB

Drug resistance (DR) remains a major challenge for tuberculosis (TB) control. Whole-genome sequencing (WGS) provides the highest genetic resolution for genotypic drug-susceptibility tests (DST). We compared DST profiles of 60 Mycobacterium tuberculosis isolates which were drug resistant according to agar proportion tests (one poly DR-TB, 34 multidrug-resistant TB and 25 extensively drug-resistant TB). We additionally performed minimum inhibitory concentration (MIC) tests using Sensititre MYCOTBI plates (MYCOTB) and a WGS-based DST. Agreement between WGS-based DST and MYCOTB was high for all drugs except ethambutol (65%) and ethionamide (62%). Isolates harboring the -15 c/t inhA promoter mutation had a significantly lower MIC for isoniazid than did isolates with the katG Ser315Thr mutation (p < 0.001). Similar patterns were seen for ethambutol (embB Gly406Asp vs. embB Met306Ile), streptomycin (gid Gly73Ala vs. rpsL Lys43Arg), moxifloxacin (gyrA Ala90Val vs. gyrA Asp94Gly) and rifabutin (rpoB Asp435Phe/Tyr/Val vs. rpoB Ser450Leu). For genotypic heteroresistance, isolates with lower proportion of mapped read tended to has lower MIC of anti-TB drugs than those with higher proportion. These results emphasize the high applicability of WGS for determination of DR-TB and the association of particular mutations with MIC levels.

Evolution of virulent genotypes and an emerging threat of multidrug resistant tuberculosis in Bamako, Mali

Background: Bamako, Mali, has a well-structured hierarchy for tuberculosis (TB) case management. However, in recent years Bamako has been faced with an emerging threat from multidrug resistant TB (MDR-TB). Here we present insights into the genomic epidemiology of TB and the evolutionary mechanisms driving the emergence of MDR-TB in Bamako. Methods & Materials: Isolates recovered from tuberculosis patients from local reference centers and the University Teaching Hospital at Point G, in Bamako, Mali between 2006 and 2012 (n = 208), were tested for antimicrobial susceptibility at the MRC Unit The Gambia. A subset of 76 isolates were analysed using whole genome sequencing. A time dated phylogenetic tree was reconstructed using BEAST. Lineage and resistance conferring mutations were inferred using PhyResSe. Results: Patients included 21 females and 55 males aged between 3 to 78 years, among whom 12(16%) were infected by MDR-TB. Most patients 61(80%) were new cases and among 15 retreatment cases 9(60%) were MDR-TB. The phylogeny was reconstructed from 8508 variant core genome sites. The dominant lineage was the Euro-American super lineage, lineage 4. Within lineage 4, the Cameroon genotype was the most prevalent genotype (n = 20, 26%) followed by the Ghana genotype (n = 16, 21%). Cameroon genotype isolates diverged from a common recent ancestor ∼161 years ago to form three clusters, one of which emerged ∼22 years ago and is likely to be involved in on-going transmission. Seven Ghana genotype isolates were MDR-TB representing over half all MDR-TB in this dataset (7/12). Ghana genotype isolates were more likely to cause MDR-TB than other genotypes after controlling for treatment status (OR = 5.6, p-value = 0.043). The MDR-TB Ghana genotype isolates formed a clade that diverged approximately 30 years ago, in which the katGSer315Thr mutation was conserved. Other Euro-American genotypes included the six LAM, two H37Rv-like and one Uganda. Four patients were infected with closely related Beijing strains and five patients were infected with non-MDR Mycobacterium africanum2. Conclusion: The Cameroon and Ghana genotypes are endemic to West Africa. Their association with on-going spread and MDR-TB in Bamako respectively is a major concern. The rise of Beijing genotype in Bamako is worrying given its high transmissibility and virulence.

Isoniazid resistance levels of Mycobacterium tuberculosis can largely be predicted by high-confidence resistance-conferring mutations

The majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15–20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA.

The Susceptibility of katG Ser315Thr Mycobacterium tuberculosis mutant Against Anti Tuberculosis Drugs

Mycobacterium tuberculosis resistant strains is a matter of great concern for TB control Program since nocure for some multi drug resistance TB (MDR-TB) cases. These strains could spread around the world, makea great challenges for control measures. Mycobacterium tuberculosis strains resistance to anti tuberculosis drugsis mainly caused by the alteration in several genes encoding the molecular targets. Mutation of katG at codon315 especially Ser315Thr are responsible for INH resistance in a large proportion of TB cases. The aim of thisstudy is to know the frequency of of katG Ser315Thr of M. tuberculosis mutant and the pattern of resistance toAnti TB drugs. The study design was observational laboratoric. Eighty fi ve M. tuberculosis INH resistant clinicalisolates were screened for mutation of katGSer315Thr by using PCR-RFLP and DNA sequence analysis.Theresults showed that katG Ser 315Thr were identifi ed in 23 (27,05%) of INH resistance isolates. Frequency ofMDR among the katG Ser315Thr mutants almost double than the non mutant. The result suggested that thethe katG Ser315Thr mutation may play important role in the development of MDR-TB.

Fitness Costs of Drug Resistance Mutations in Multidrug-Resistant Mycobacterium tuberculosis: A Household-Based Case-Control Study.

The projected long-term prevalence of multidrug-resistant (MDR) tuberculosis depends upon the relative fitness of MDR Mycobacterium tuberculosis strains, compared with non-MDR strains. While many experimental models have tested the in vitro or in vivo fitness costs of various drug resistance mutations, fewer epidemiologic studies have attempted to validate these experimental findings. We performed a case-control study comparing drug resistance-associated mutations from MDR M. tuberculosis strains causing multiple cases in a household to matched MDR strains without evidence of secondary household cases. Eighty-eight multiple-case and 88 single-case household MDR strains were analyzed for 10 specific drug resistance-associated polymorphisms previously associated with fitness effects. We found that the isoniazid-resistant katG Ser315Thr mutation occurred more than twice as frequently in multiple-case households than in single-case households (odds ratio [OR], 2.39; 95% confidence interval [CI], 1.21-4.70), corroborating previous experimental findings. However, strains carrying both the katG Ser315Thr mutation and the rpsL Lys43Arg mutation were less likely to be found in multiple-case households (OR, 0.09; 95% CI, .01-.73), suggesting a negative epistatic interaction which contrasts previous findings. The case-control design presents a useful approach for assessing in vivo fitness effects of drug resistance mutations.

Analysis of mutational characteristics of the drug-resistant gene katG in multi-drug resistant Mycobacterium tuberculosis L-form among patients with pneumoconiosis complicated with tuberculosis

The aim of the present study was to investigate the mutational characteristics of drug‑resistant genetic mutations in the katG gene to isoniazid (INH) in multi‑drug resistant Mycobacterium tuberculosis (MTB) L‑form among patients with pneumoconiosis complicated with tuberculosis (TB), in order to reduce the occurrence of drug resistance in patients, and gain further insight into the mechanisms underlying drug resistance in MDR‑TB L‑form. A total of 114 clinically isolated strains of MTB L‑forms were collected. The MDR‑TB L‑forms were identified using a conventional antimicrobial susceptibility test (AST). The DNA genomes were extracted, the target genes were amplified by polymerase chain reaction technology and the hotspot mutational regions in the katG gene were analyzed by direct sequencing. The results of AST analysis demonstrated that there were 31 strains of MDR‑TB L‑forms in 114 clinical isolates. The mutation rate of katG was 61.29% (19/31) in INH‑resistant isolates, mainly concentrated in codon 315 (Ser315Thr, 48.39% and Ser315Asn, 9.68%) and 431 (Ala431Val, 3.23%). Base substitutions were identified, however, no multisite mutations were found. No mutations in katG were identified in 10 INH‑sensitive strains that were randomly selected. INH‑resistance was more severe in MDR‑TB L‑form isolates among patients with pneumoconiosis complicated with TB. The substitution of highly conserved amino acids encoded by the katG gene resulted in the molecular mechanisms responsible for INH resistance in MDR‑TB L‑form isolates. It was also verified that the katG gene was in diversiform. The katG Ser315Thr mutation is one of the main causes of resistance to INH in MDR‑TB L-form isolates.

KatG (SER 315 THR) Gene Mutation in Isoniazid Resistant Mycobacterium Tuberculosis

BACKGROUND Isoniazid (INH) together with Rifampicin (RFP) forms the cornerstone of a short chemotherapy course for tuberculosis (TB) treatment. Mutation at codon 315 of katG gene is most prevalent in isoniazid resistant Mycobacterium tuberculosis (MTB) and is high in area with high TB incidence. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) has been found to be a reliable and effective tool for the identification of the specific gene alteration. The objective of this study was to screen Ser315Thr mutation of KatG gene of INH resistant MTB strain by PCR-RFLP technique. Altogether 37 INHr MTB isolates obtained from German Nepal Tuberculosis Project (GENETUP) Kathmandu Nepal was included in the study. Deoxyribonucleic Acid (DNA) extraction was performed according to protocol of SORPOCLEAN from the culture isolates. Amplification of the fragment with katG codon 315 was performed in a Biometra Thermocycler using primers. The amplified fragment was cleaved with MspI. The restriction fragments obtained were electrophoresed in a 2% agarose gel and were visualized using transilluminator. The katG Ser315Thr mutation was observed in 23 (62.2%) out of 37 INH resistant isolates. The drug susceptibility profile of INHr MTB isolates showed all isolates to be resistant to INH and RFP whereas 26 and 27 MTB isolates were resistant to Ethambutol (EMB) and Streptomycin (S) respectively. Seventeen (17) patients were harbouring katG gene mutated strain among Ethambutol and Streptomycin resistant cases. The study identified high prevalence of Ser315Thr mutation in katG. The isolates harbouring this mutation were also simultaneously resistant to RFP. Ser315Th could be a potential genetic marker for predicting MDR-TB.

Molecular characterization of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from the USA

Drug-resistant tuberculosis poses a significant problem for treatment. The mechanisms of resistance to the front-line drug isoniazid (INH) are complex and can be mediated by katG, inhA and other unknown genes. To identify the percentage of INH-resistant strains with no katG or inhA mutation, this study characterized a panel of 28 clinical isolates of Mycobacterium tuberculosis and five mutants derived from H37Rv resistant to INH. Seventeen of 33 resistant strains (51 %) had katG mutations with 12 of the 17 strains having the most common KatG Ser315Thr mutation. Three of the 17 strains with the KatG 315 mutation had an additional mutation in the inhA promoter and were resistant to a high level of INH. Seventeen of the 33 INH-resistant strains (51 %) had inhA mutations. The most common inhA promoter mutation was -15C-->T and was present in 13 of the 17 inhA mutations. This promoter mutation occurred alone without katG mutations and was associated with a low level of INH and ethionamide resistance. However, other inhA mutations were associated with katG mutations. No mutations were found in the ndh gene. Three of 33 strains (9 %) had no mutations in katG, inhA or ndh, indicating that their resistance was due to a new mechanism of resistance. Detection of the KatG Ser315Thr mutation and the -15C-->T inhA mutation accounted for 76 % (25/33) of the INH-resistant strains and should be useful for rapid detection of INH-resistant strains by molecular tests.

Detection of katG Ser315Thr substitution in respiratory specimens from patients with isoniazid-resistant Mycobacterium tuberculosis using PCR-RFLP.

Mutations in the katG locus of catalase peroxidase in Mycobacterium tuberculosis (MTB) account for major isoniazid (INH) resistance. In the South China region, a collection of 906 respiratory specimens and 142 MTB isolates was used to evaluate the sensitivity and specificity of a PCR-RFLP method for the detection of INH resistance-associated mutations. Except for four catalase-negative MTB isolates, katG PCR for a 620-bp amplicon was successful for all purified MTB isolates. For respiratory specimens, diagnostic sensitivity and specificity of katG PCR was 85 and 100 %. Subsequent RFLP of the katG amplicons by MspI digestion identified that 51 % of INH-resistant MTB were associated with the Thr315 phenotype, and that codon 463 was a polymorphic site with no linkage to INH resistance. The Arg463 wild-type MTB isolates predominant in the Western world were replaced by isolates carrying Leu463 in the South China region. RFLP patterns of katG amplicons from respiratory specimens were identical to those of the corresponding MTB cultured colonies. This method has potential application for rapid diagnosis of INH resistance due to katG Ser315Thr mutation.