Simultaneous detection of drug-resistant mutations in Mycobacterium tuberculosis and determining their role through in silico docking

2020 ◽  
Vol 20 ◽  
Author(s):  
Somanna Nachappa ◽  
Sumana Neelambike ◽  
Ahmad Sarikhani ◽  
Nallur Ramachandra
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
In Silico ◽  
Sequence Data ◽  
Simultaneous Detection ◽  
Fluoroquinolone Resistance ◽  
Drug Resistant ◽  
In Silico Docking

: A molecular method for diagnosis of drug-resistant Tuberculosis is Multiplex allele-specific PCR (MAS-PCR), which is more time-efficient. Also, understanding the role of mutations when translated to protein, in causing resistance helps better drug designing. Aims: To study MAS-PCR in the detection of drug resistance in comparison to DNA sequencing, and understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis. Methods: Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted four genes, iniA for the drug Ethambutol, rpsL and rrs for Streptomycin, and gyrA for Fluoroquinolone resistance, respectively. Further, the sequence data was analysed and modelled to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking. Results: We identified drug-resistant mutations in four out of 95 isolates with one of them carrying a mutation at codon iniA501, two at gyrA94, and one for both iniA501 and gyrA94 using MAS-PCR. DNA sequencing confirmed drug-resistant mutations in only two isolates, whereas two others had mutation adjacent to the target allele. Molecular docking showed Estimated Free Energy of Binding (ΔG) being higher for Fluoroquinolone binding with GyrA D94V mutant. Both, wild and mutant IniA interact with EMB but had no significant effect on binding energy. Conclusions: DNA sequencing-based drug resistance detection of TB is more accurate than MAS-PCR. Understanding the role of mutations in influencing the drug-protein interaction will help in designing effective drug alternatives.

scholarly journals Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS6110 Clustering Rather than Ethambutol Resistance

2005 ◽  
Vol 49 (9) ◽  
pp. 3794-3802 ◽  
Author(s):  
Manzour Hernando Hazbón ◽  
Miriam Bobadilla del Valle ◽  
Marta Inírida Guerrero ◽  
Mandira Varma-Basil ◽  
Ingrid Filliol ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Odds Ratio ◽  
Univariate Analysis ◽  
Drug Susceptibility ◽  
Drug Resistant ◽  
Development Of Resistance ◽  
Resistance Patterns ◽  
Novel Association

ABSTRACT Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

scholarly journals Detection of Second Line Drug Resistance among Drug Resistant Mycobacterium Tuberculosis Isolates in Botswana

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 208 ◽  
Author(s):  
Tuelo Mogashoa ◽  
Pinkie Melamu ◽  
Brigitta Derendinger ◽  
Serej D. Ley ◽  
Elizabeth M. Streicher ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Fluoroquinolone Resistance ◽  
Reference Laboratory ◽  
Drug Resistant ◽  
Second Line ◽  
Mdr Tb ◽  
Line Drug

The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.

scholarly journals Drug-sensitivity test and analysis of drug-resistant mutations in Mycobacterium tuberculosis isolates from Kashgar, China

2021 ◽  
Vol 19 ◽  
pp. 205873922110414
Author(s):  
Zhongchen Ma ◽  
Tianhao Sun ◽  
Xinyu Bai ◽  
Xiang Ji ◽  
Qian Zhang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
High Sensitivity ◽  
Drug Susceptibility ◽  
Resistance Rate ◽  
Clinical Isolates ◽  
Multi Drug Resistance ◽  
Drug Resistant ◽  
Resistance Rates

Introduction In recent years, drug-resistant Mycobacterium tuberculosis strains have gradually become widespread. Most drug resistance is related to specific mutations. We investigated M. tuberculosis drug resistance in the Kashgar area, China. Methods The drug-susceptibility test was conducted to clinical isolates of M. tuberculosis. Genomic-sequencing technology was used for the drug-resistant strains and the significance of DNA sequencing as a rapid aid for drug-resistance detection and the diagnosis method was evaluated. Results The resistance rates of clinical isolates to rifampicin (RFP), isoniazid (INH), streptomycin (SM), ethambutol (EMB), and ofloxacin (OFX) were, respectively, 4.4%, 12.3%, 8.8%, 2.6%, and 3.5%. The single- and multi-drug resistance rates were, respectively, 80.0% and 20.0%. The resistance genes RopB, katG, InhA, RpsL, rrs, gyrA, and embB displayed codon mutations, while InhA was mutated in its promoter region. Kappa scores, evaluating the consistency between DNA sequencing and the resistance ratio methods for the detection of isolates’ resistance to RFP, INH, SM, OFX, and EMB, were 1, 0.955, 0.721, 0.796, and 1, respectively. Conclusion The resistance rate of INH and SM is relatively high in the Kashgar area. Detection of mutations in RopB, katG, InhA, RpsL, rrs, gyrA, and embB by DNA sequencing can predict drug resistance of M. tuberculosis strains with high sensitivity and specificity, and can be used for diagnosis.

scholarly journals Analysis of drug resistance and mutation profiles in Mycobacterium tuberculosis isolates in a surveillance site in Beijing, China

2021 ◽  
Vol 49 (1) ◽  
pp. 030006052098493
Author(s):  
Jie Zhang ◽  
Yixuan Ren ◽  
Liping Pan ◽  
Junli Yi ◽  
Tong Guan ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Drug Testing ◽  
Multidrug Resistant ◽  
High Rate ◽  
Drug Resistant ◽  
Surveillance Site ◽  
Mdr Tb ◽  
Previously Treated Patients ◽  
Beijing China

Objective This study analyzed drug resistance and mutations profiles in Mycobacterium tuberculosis isolates in a surveillance site in Huairou District, Beijing, China. Methods The proportion method was used to assess drug resistance profiles for four first-line and seven second-line anti-tuberculosis (TB) drugs. Molecular line probe assays were used for the rapid detection of resistance to rifampicin (RIF) and isoniazid (INH). Results Among 235 strains of M. tuberculosis, 79 (33.6%) isolates were resistant to one or more drugs. The isolates included 18 monoresistant (7.7%), 19 polyresistant (8.1%), 28 RIF-resistant (11.9%), 24 multidrug-resistant (MDR) (10.2%), 7 pre-extensively drug-resistant (XDR, 3.0%), and 2 XDR strains (0.9%). A higher rate of MDR-TB was detected among previously treated patients than among patients with newly diagnosed TB (34.5% vs. 6.8%). The majority (62.5%) of RIF-resistant isolates exhibited a mutation at S531L in the DNA-dependent RNA polymerase gene. Meanwhile, 62.9% of INH-resistant isolates carried a mutation at S315T1 in the katG gene. Conclusion Our results confirmed the high rate of drug-resistant TB, especially MDR-TB, in Huairou District, Beijing, China. Therefore, detailed drug testing is crucial in the evaluation of MDR-TB treatment.

embB Gene association with Ethambutol drug Resistance in Mycobacterium Tuberculosis Patients

2021 ◽  
Vol 15 (12) ◽  
pp. 3273-3276
Author(s):  
Sana Hafeez ◽  
Haleema Sajid ◽  
Farouk Qamar Malik ◽  
Imran Ali Zaidi ◽  
Sobia Niaz ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Codon Position ◽  
Mutational Analysis ◽  
Transmission Rate ◽  
Drug Resistant ◽  
Tuberculosis Patients ◽  
Two Samples ◽  
Suspected Tuberculosis ◽  
Embb Gene

Background: Tuberculosis (TB) is fatal and life threatening infectious disease. The transmission rate of tuberculosis is very high. Various drugs are used as treatment for TB. Recently it has been observed that one of the most important factor for fast TB spread is development of anti-TB drug resistant mycobacterium tuberculosis (MTB). Various combination of drugs like isoniazid (INH), rifampicin (RIF), Streptomycin(SM), pyrazinamide (PZA) or ethambutol (EMB) are in global use for TB treatment. Improper usage of these drugs makes the person prone to develop anti-TB drug resistant tuberculosis. Aim: To evaluate association of embB gene with ethambutol resistance in Mycobacterium Tuberculosis. Methods: 104 Specimens of sputum from suspected tuberculosis patients were processed for inoculation in Lowenstein J Medium after it has been decontaminated properly. Kit method by using QIAamp DNA Mini kit was utilized for extraction of DNA. Then region from base 6953 to 10249 of embB gene was amplified through PCR and then followed by sequencing with the aid of softwares blast2seq and ClustalW2. Three primer sets were utilized to amplify embB gene. Ethambutol (EMB) Resistant MTB specimens were processed to study mutation in embB gene. Results: Out of the total 104 sputum specimens, 14 samples were found to have ethambutol resistance. These 14 samples were then processed for mutational analysis. DNA sequence analysis of these 14 samples confirmed embB gene mutation in 10 samples. Mutational analysis revealed that 08 samples showed mutation at codon 306 and two samples showed mutation at 319 codon. The reported mutation Methionine →Isoleucine was seen in 07 samples with ATG codon replaced by ATA codon at codon position 306. One sample showed mutation as Methionine →Isoleucine with ATG codon replaced by ATC codon at codon position 306. Two samples showed mutation as Tyrosine →Serine with TAT codon replaced by TCT at 319 codon position in embB gene. Conclusion: This study concludes that mutation of certain genes particularly point mutation of embB gene at codon 306 and 319 is associated with drug resistance of ethambutol in ethambutol resistant mycobacterium tuberculosis patients. Keywords: Ethambutol, embB gene, Mycobacterium tuberculosis.

scholarly journals Genotypes of Mycobacterium tuberculosis isolates circulating in Shaanxi Province, China

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242971
Author(s):  
Yan Li ◽  
Yu Pang ◽  
Tianhua Zhang ◽  
Xiaoping Xian ◽  
Jian Yang ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Multidrug Resistant ◽  
Shaanxi Province ◽  
Drug Resistant ◽  
Beijing Family ◽  
Tuberculosis Patients ◽  
Smear Positive Pulmonary Tuberculosis ◽  
Discriminatory Index

Objectives The prevalence of drug-resistant TB in Shaanxi Province is higher than other areas. This study was aimed to investigate the genetic diversity and epidemiology of Mycobacterium tuberculosis clinical strains in Shaanxi Province, China. Methods From January to December 2016, a total of 298 Mycobacterium tuberculosis clinical isolates from smear-positive pulmonary tuberculosis patients were genotyped by Mcspoligotyping and 15-locus VNTR. Results We found that the Beijing family strains was the most prominent family(81.54%, 243/298). Other family strains included T family(9.06%, 27/298), U family(0.67%, 2/298), LAM9 family(0.34%, 1/298) and Manu family(0.34%, 1/298). The rates of multidrug-resistant (MDR) M.Tuberculosis, age, type of case and education between Beijing and non-Beijing family strains were not statistically different, while the distribution in the three different regions among these was statistically significant. VNTR results showed that strains were classified into 280 genotypes, and 33 (11.07%) strains could be grouped into 14 clusters. 11 of the 15-VNTR loci were highly or moderately discriminative according to the Hunter-Gaston discriminatory index. Conclusions We concluded that the Beijing family genotype was the most prevalent genotype and 15-locus VNTR typing might be suitable for genotyping of M. tuberculosis in Shaanxi Province. There was less association between Beijing family genotypes and drug resistance in our study area.

scholarly journals Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1990-1996 ◽  
Author(s):  
Alix Pantel ◽  
Stéphanie Petrella ◽  
Nicolas Veziris ◽  
Florence Brossier ◽  
Sylvaine Bastian ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hot Spot ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Content Type ◽  
Highly Active ◽  
Definition Of

ABSTRACTFluoroquinolone (FQ) resistance is emerging inMycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target inM. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified inM. tuberculosisclinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

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