Ethambutol-resistant Isolates Research Articles

The Mutations on embA, embB and embC Gene Region in Ethambutol-Resistant and Susceptible Clinical Mycobacterium tuberculosis Complex Isolates

Ethambutol (EMB) is one of the first-line drugs used in the standard combination therapy for tuberculosis (TB) caused by Mycobacterium tuberculosis complex (MTC), and resistance to drugs that play a key role in treatment is increasing worldwide. Mutations in the embCAB operon that have been confirmed to be associated with resistance are responsible for EMB resistance. In this study, it was aimed to determine the frequency and patterns of mutations in embA, embB and embC gene regions in clinical MTC isolates found to be phenotypically resistant and susceptible to EMB. A total of 64 MTC isolates, 44 of resistant to EMB and 20 of susceptible to EMB, isoniazid, rifampicin, and streptomycin by conventional phenotypic drug susceptibility test, were included in the study. Following the DNA isolation, embA, embB and embC gene regions associated with EMB resistance were amplified with specific primer sequences. The PCR products were cycle sequenced using the Bigdye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA) and electrophoretically separated on the ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). Mutated gene regions were identified by aligning sequence analysis data in multiple sequence analysis programs. In the study, genomic mutations in the embCAB operon were detected in 68.2% (30/44) of the EMB resistant isolates. Mutations in the embB gene region were detected in 66% (29/44) of the resistant isolates, 76% (22/29) of these mutations were at codon 306 and the most common mutation patterns in this codon were determined as ATG→GTG (M306V; 58.6%; 17/29), ATG→ATA, ATC or ATT (M306I; 17.2%; 5/29). Other mutations in the embB gene region were determined as Y334H (3.4%; 1/29), D354A (6.9%; 2/29), E378A (3.4%; 1/29), G406C (3.4%; 1/29), M423I (3.4%; 1/29) and E521A (3.4%; 1/29). Of the 44 EMB-resistant isolates, mutations were detected in one (2.3%) of the isolate in the embA gene region (L330L) and in two (4.5%) of the isolates in the embC gene region (T270I in one isolate and T270I and E305E in the other isolate). Of the phenotypically EMB susceptible isolates, mutation was detected in only one (5%) of the isolates in the embA gene region (E180G). In our study, it was determined that mutations frequently occur in codon 306 of the embB gene in EMB-resistant MTC isolates and this mutation has a potential role in the development of EMB resistance. However, it was concluded that the absence of mutations does not exclude phenotypic EMB resistance. Our results will shed light on the molecular epidemiology of embCAB operon mutations that cause EMB resistance in our country.

Mycobacterium tuberculosis Complex Mutations in Drug Resistant Clinical Isolates from Southwest Mexico

Mutations in target genes have been described in Mycobacterium tuberculosis Complex (MTBc) drug resistant isolates worldwide. In Mexico, not enough information has been reported in this concern. The aim of this study was to characterize mutations related to resistance to first line drugs in MTBc isolates from Oaxaca, Mexico. MTBc isolates were identified in clinical samples from Tuberculosis (TB) patients. Susceptibility to isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide was tested through nitrate reductase assay. PCR based analysis and sequencing were employed to characterize mutations in katG, inhA, rpoB, embB, rrs, rpsL and pncA genes. Mutations in katG and the promoter of the mabA-inhA operon were found in isoniazid resistant isolates. Sequence analysis of Rifampin Resistance-Determining Region in the rpoB gene showed novel mutations along this region besides mutations at codons 516, 526 and 531. Polymorphisms at codon 306 embB gene were found in ethambutol resistant isolates. Frequent mutations associated to resistance to streptomycin were characterized in rrs and/or rpsL genes. pncA analysis showed variable number of mutations in resistant and susceptible pyrazinamide isolates. Most frequent mutations related to resistance to first line antituberculous drugs were identified in phenotypically resistant MTBc isolates. New mutations were characterized in rpoB, rrs and rpsL genes.

Gene mutations related to rifampin resistance of tuberculosis in northwest of Iran

Mycobacterium tuberculosis causes tuberculosis disease, is a communicable disease and multidrug-resistant or rifampicin-resistant M. tuberculosis continuous to be a public health threat worldwide. Therefore, the aim of this study was the assessment of the mutations profile of rifampin-resistant and MDR M. tuberculosis in the north-west of Iran. The specimens of suspicious individuals were collected and processed by direct microscopy and culture. Drug susceptibility testing was assessed using the conventional proportional method, and the rpoB genes of rifampin-resistant isolates were amplified and sequence analyses were performed. A total of 6294 individuals referred, 295 (4.68%) isolates of Mycobacterium spp. were isolated, which only 5 (1.69%) isolates were found resistant to rifampin. Three of the rifampin-resistant isolates were multidrug-resistant, originated from Azerbaijani patients, and two from Iran that were monoresistant to rifampin. Three different types of mutations were identified in the rpoB gene, involving four codons Gln510His, Asp516Val, His526Tyr and Ser531Leu. The most frequent codons affected by point mutation were codons 531 and 526. The katG315 and emb306 mutations were responsible for isoniazid- and ethambutol-resistant isolates. The results of this study revealed that the frequency of the mutations in the RRDR region of the rpoB gene were low, but most of the isolates were collected from patients originated from Rep. of Azerbaijan, which have a high potential for transmission of MDR tuberculosis. These results indicate the importance of policies to restrict infection distribution in the region by patients from neighbor countries. The rate of mutations in the isolates of the present study was lower than the rest of the world. • Rifampicin-resistant M. tuberculosis is a public health threat worldwide. • The frequencies of the mutations in the RRDR region of the rpoB gene were low. • Most of the resistance isolates were collected from patients originated from Azerbaijan. • Policies to restrict infection distribution in the regions are important.

Lack of association of novel mutation Asp397Gly in aftB gene with ethambutol resistance in clinical isolates of Mycobacterium tuberculosis

To discover additional genotypic indicators for ethambutol (EMB) resistant M. tuberculosis, we studied polymorphisms in arabinofuranosyl transferase encoding genes aftA (Rv3792), aftB (Rv3805) and aftC (Rv2673) in 38 EMB resistant and 34 EMB susceptible isolates from India and a repository established by the World Health Organization (WHO) Special Programme for Research and Training in Tropical Disease (TDR) by DNA sequencing. The results were correlated with the minimum inhibitory concentration (MIC) of EMB and mutations in embB (Rv3795). The most common non-synonymous polymorphism identified in aftB was Asp397Gly in 12/38 (31.6%) EMB resistant and 3/34 (8.8%) EMB susceptible isolates. Interestingly, 10/12 (83.3%) EMB resistant isolates with aftB Asp397Gly mutation also carried embB306, embB402 or embB497 mutations. Association of Asp397Gly polymorphism with EMB resistance was statistically significant (p 0.0216). However, overexpression of the mutant aftB in M. tuberculosis H37Rv did not exhibit any change in the MIC. Whole genome sequencing of a panel of Indian isolates and SNP cluster grouping (SCG) of TDR strains revealed an association between aftB mutation Asp397Gly and Beijing genotype or SCG2, a cluster group representing the Beijing genotype. To conclude, though aftBAsp397Gly mutation is not associated with EMB resistance, this mutation may be a phylogenetic marker for the Beijing clade.

Polymorphisms in Rv3806c (ubiA) and the upstream region of embA in relation to ethambutol resistance in clinical isolates of Mycobacterium tuberculosis from North India

Mutations at embB306 are the most prevalent polymorphisms associated with ethambutol (EMB) resistance, responsible for 40-60% of EMB resistant clinical cases of tuberculosis (TB). The present study analyzed additional mutations associated with EMB resistance in the embB, embC, embA and Rv3806c (ubiA) genes in 29 EMB resistant and 29 EMB susceptible clinical isolates of M. tuberculosis selected from 360 patients with TB. The entire ubiA gene, mutational hotspot regions of embB, embC, and upstream region of embA were screened for polymorphisms by DNA sequencing and the results correlated with minimum inhibitory concentrations (MIC) of EMB. The most common polymorphism identified in ubiA was at codon 149 (GAA to GAC), occurring in 5/29 (17.2%) resistant isolates and 7/29 (24%) susceptible isolates. Mutations in embB were most common at codon 306 (ATG to ATC/GTG), occurring only in EMB resistant isolates (20/29; 69%). Mutations in the upstream region of embA at -8, -11, -12 and -60 codons also occurred in EMB resistant strains (8/29; 27.5%) of which 6/8 (75%) were observed in isolates with EMB MIC ≥16μg/ml. Though no polymorphisms associated with EMB resistance were identified in ubiA, polymorphisms upstream to embA may contribute to high level EMB resistance.

Characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array

Ethambutol resistance has mostly been related to mutations in the embB gene. The objective of the present study was to characterize the embB gene in a collection of ethambutol-resistant and ethambutol-susceptible isolates of Mycobacterium tuberculosis complex (MTBC) from Barcelona, and to develop a DNA microarray for the rapid detection of embB mutations in our area. Fifty-three ethambutol-resistant and 702 ethambutol-susceptible isolates of MTBC were sequenced in internal 982-1495 bp fragments of the embB gene. In addition, a low-cost, low-density array was designed to include the embB codons identified as being most frequently mutated in our area (LD-EMB array). The global prevalence of embB mutations found among the ethambutol-resistant isolates was 77.4% (41/53). Substitutions in embB306 were the most common [53.7% (22/41)], followed by substitutions in embB406 [26.8% (11/41)]. The presence of mutations in embB406 was related to higher levels of ethambutol resistance and to multidrug resistance. Among unrelated isolates (from 24-locus MIRU-VNTR genotyping), the percentage of embB-mutated isolates was 72.9% (27/37)--59.3% (16/27) in embB306 and 25.9% (7/27) in embB406. None of the ethambutol-susceptible isolates studied showed a mutation in codon 306 or 406. The LD-EMB array showed 100% sensitivity and specificity in identifying the main embB substitutions in our area. Mutations at codons 306 and 406 of embB have a relevant role in resistance to ethambutol in our area. The LD-EMB array developed in this study would appear to be a good molecular test for rapid detection of ethambutol resistance.

Drug Resistance in Mycobacterium tuberculosis Isolates from Northeastern Sudan

The present study intended to determine the drug resistance patterns of Mycobacterium tuberculosis isolates among human new tuberculosis cases from northeastern Sudan using conventional and molecular techniques. Of 100 mycobacterial isolates tested by proportion method, 6% and 2% were identified, respectively, as multi drug resistanttuberculosis (MDR-TB) and non-multi drug resistant-rifampicin (non-MDR-RIF). A total of 8% was found resistant to rifampicin (RIF), 13% to isoniazid (INH), 34% to streptomycin (STM) and 12% to ethambutol (EMB). Mono-resistant were 0% for RIF, 1% INH, 18% STM and 3% ETH. The remaining 61% isolates were identified as sensitive. Resistance to pyrazinamide was evaluated conventionally for the first time in this country and was found as low as 1%. Molecularly, mutations of MDR-TB, RIF, INH, EMB and STM resistant isolates were detected in 66.7%, 87.5%, 76.9%, 66.7% and 17.6% of corresponding conventional findings, respectively. The genetic mutations were related to the following codons: rpoB 531 (71.4%) and rpoB 526 (28.6%) for RIF, katG 315 (90%) and mabA-inhA-15 (10%) for INH, embB 306 (66.7%) for EMB and rpsL (17.6%) for STM. The study showed that drug resistant tuberculosis increased steadily and provided

Gene mutations analysis in resistant Mycobacterium tuberculosis isolates

Objective To investigate the relationship between the phenotypes and the patterns of genetic mutations in the corresponding resistance genes (rpoB, katG, inhA, ahpC, rrs, rpsL, embB and gyrA) in resistant Mycobacterium tuberculosis (MTB) isolates. Methods Rifampicin-resistant gene (rpoB), isoniazid-resistant genes (katG, inhA, ahpC), streptomycin-resistant genes (rrs, rpsL), ethambutol-resistant gene (embB) and quinolinone-resistant gene (gyrA) were amplified by PCR with sequence-specific primers, then mutants screened by single-stranded conformation polymorphism (SSCP) were sequenced. Results rpoB mutation with predominant Ser450Trp pattern was 94. 9% (56/59) in 59 rifampicin-resistant isolates;katG mutation rate was 38. 9% (35/90) and the main pattern was Ser315Thr, but only 3 inhA mutants and no ahpC mutation were determined in 90 isoniazid-resistant isolates;gyrA mutation with main Asp94Gly then Ala90Val pattern was 82.4% (28/34) in 34 quinolinone-resistant isolates;the total mutation rate was 77.4% in 31 streptomycin-resistant isolates, of which 15 isolates mutated in rrs with main pattern A514C or A1041G, 10 isolates mutated in rpsL Lys88Arg;and embB mutation with main Met306Val accounted for 19.4% (6/31) in 31 ethambutol-resistant isolates. Conclusions The results showed that resistance of resistant MTB may be complicated, and DNA sequencing-based mutation analysis could efficiently detect the molecular makers such as rpoB, katG, gyrA, rrs, rpsL and embB in resistant MTB isolates. Meanwhile, it is notable that the rpoB mutation pattern in our isolates is different from previous report, further effort are needed to confirm the characteristics. The spectrum of potential resistance-related mutations in MTB clinical isolates may lay substantial foundation for the rapid molecular diagnosis and rational use of drug to MTB patients. Key words: Mycobacterium tuberculosis; Drug resistance, bacterial; Polymerase chain reaction; Mutation; Sequence analysis,DNA

PROGRESSION OF CHRONIC PULMONARY TUBERCULOSIS IN MICE INTRAVENOUSLY INFECTED WITH ETHAMBUTOL RESISTANT MYCOBACTERIUM TUBERCULOSIS

PROGRESSION OF CHRONIC PULMONARY TUBERCULOSIS IN MICE INTRAVENOUSLY INFECTED WITH ETHAMBUTOL RESISTANT MYCOBACTERIUM TUBERCULOSIS

Progression of chronic pulmonary tuberculosis in mice intravenously infected with ethambutol resistantMycobacterium tuberculosis

Ethambutol (EMB) is an important first line drug, however little information on its molecular mechanism of resistance and pathogenicity of resistant isolates is available. Present work was designed to study virulence of the EMB resistant M. tuberculosis strains and the host responses in-vivo on infection of EMB resistant M. tuberculosis using Balb/c mouse model of infection. Three groups of Balb/c mice (female, age 4-6 wk; 21 mice in each group) were infected intravenously with 106 CFU of M. tuberculosis H37Rv and two EMB resistant clinical isolates. Age and sex matched control animals were mock inoculated with Middlebrook 7H9 broth alone. At 10, 20, 30, 40, 50, 60, and 70 days post-infection three animals from each group were sacrificed by cervical dislocation and lung tissue was collected for further analysis. Infection with EMB resistant M. tuberculosis led to progressive and chronic disease with significantly high bacillary load (p=0.02). Massive infiltration and exacerbated lung pathology with increased expression of IFN-gamma and TNF-alpha was observed in lungs of mice infected with EMB resistant strains. The present study suggests that infection with EMB resistant M. tuberculosis leads to chronic infection with subsequent loss of lung function, bacterial persistence with elevated expression of TNF-alpha resulting in increased lung pathology. These findings highlight that EMB resistant M. tuberculosis regulates host immune response differentially and its pathogenicity is different from drug sensitive strains of M. tuberculosis.

Molecular approaches to tuberculosis.

Molecular approaches to tuberculosis.

Evaluation of the MB/BacT Mycobacterium Detection System for Susceptibility Testing of Mycobacterium tuberculosis

The MB/BacT mycobacterium detection system was evaluated for its performance in the susceptibility testing of Mycobacterium tuberculosis. Eighty-three M. tuberculosis isolates were processed. Results for all isoniazid-, rifampin- and streptomycin-susceptible, isoniazid-resistant, and rifampin-resistant M. tuberculosis isolates with the MB/BacT system agreed 100% with those obtained by the agar proportion method. The agreements between the two methods for streptomycin- and ethambutol-resistant isolates were 96.4 and 90.4%, respectively. The susceptibility test results were obtained in 7 days, on average. These data demonstrate that the MB/BacT system is an accurate, nonradiometric method for rapid susceptibility testing of M. tuberculosis.

Acute alcohol intake: Relationship between neurophysiological parameters and performance tasks

Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia.A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping.Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001).Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies.