The relationship between embb306 and embb406 mutations and ethambutol resistant in Mycobacterium tuberculosis isolated from patiens in west of Iran.

Background: Mutations in embB gene have been reported in ethambutol (EMB) resistance Mycobacterium tuberculosis (M. tuberculosis) isolates. The aim of this study was survey on embB 306 and 406 EMB resistant M. tuberculosis isolated from patients in West of Iran (2014-2015). Methods: Fifty strains of M. tuberculosis from patients with pulmonary tuberculosis (TB) were considered. Drug susceptibility using proportional method was performed. Polymerase chain reaction (PCR) -DNA sequencing was applied for mutation in embB 306 and 406 codon detection. Data were analyzed in SPSS 16 software using descriptive statistics and Fisher's exact test (p<0.05). Results: In this study 7 (14%) M. tuberculosis isolates were resistant to EMB. 6 (85.71%) and 1 (14.28%) resistant isolates had embB 306 and 304 codon mutations, respectively. Between embB306 mutations and resistance to EMB and MDR isolates had a significant relationship (p<0.001). Conclusion: The data indicated that embB 306 mutations have effect on EMB resistant. Detection of EMB resistant and these mutations prominent for antibiotic prescription.

Detecting Ethambutol Resistance in Mycobacterium tuberculosis Isolates in China: A Comparison Between Phenotypic Drug Susceptibility Testing Methods and DNA Sequencing of embAB.

With the increasing incidence of drug-resistant tuberculosis (DR-TB), determining a rapid and accurate drug susceptibility testing (DST) method to identify ethambutol (EMB) resistance in Mycobacterium tuberculosis has become essential for patient management in China. Herein, we evaluated the correlation between three phenotypic DST methods, namely, proportion method (PM), MGIT 960 system, and microplate alamar Blue assay (MABA), and DNA sequencing of embAB in 118 M. tuberculosis isolates from China. When the results of the phenotypic DST methods were compared with those of DNA sequencing, the overall agreement and kappa values of the PM, MGIT 960 system, and MABA were 81.4% and 0.61, 77.1% and 0.55, and 84.7% and 0.67, respectively. The agreement for EMB resistance between MABA and PM was significantly higher than that between the MGIT 960 system and PM (P = 0.02). Moreover, among the isolates with detectable embAB mutations, 97.2% (70/72 isolates) harbored mutations in embB. The analysis of embB mutations predicted EMB resistance with 81.3% sensitivity, 86.8% specificity, and 83.1% accuracy. Thus, MABA may be a better phenotypic DST method for detecting EMB resistance. DNA sequencing of embB may be useful for the early identification of EMB resistance and the consequent optimization of the treatment regimen.

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.

Association of ubiA mutations and high-level of ethambutol resistance among Mycobacterium tuberculosis Thai clinical isolates.

Ethambutol (EMB) is the first-line antituberculosis drug and a potential supplementary agent for a treatment regimen of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). It has long been known that mutations in embCAB operon, encoding EMB target, arabinosyltransferase, confer resistance to EMB. Recently, ubiA was additionally reported to be specifically associated with high-level EMB resistance in Mycobacterium tuberculosis. However, such information on ubiA is very limited. This study aimed to investigate correlations between mutations in ubiA and phenotypic EMB resistance among EMB-resistant (EMBR) M. tuberculosis Thai clinical isolates. Minimum inhibitory concentration (MIC) level of EMB and ubiA sequences were determined and analyzed. Of 68 EMBR-MDR isolates, 8.9% harbored mutations in ubiA. However, 10.0% and 46.6% of EMB-sensitive (EMBS)-MDR and pan-susceptible isolates also had ubiA mutations detected, respectively. Most nonsynonymous mutations, L31P, A35S, and V55M were only found in the EMBR-MDR isolates except E149D which was also found in EMBS-MDR and pan-susceptible isolates. A further phylogenetic analysis based on spoligotyping and IS6110-RFLP illustrated that E149D was in fact associated to EAI-families rather than EMB resistance. By excluding synonymous mutations and the E149D, we found a high correlation between ubiA mutations and high-level of EMB resistance with 100.0% specificity. In conclusion, despite its rare occurrence, mutations in ubiA can potentially be a marker for a detection of high level of EMB resistance at least in the MDR M. tuberculosis background.

ubiA (Rv3806c) encoding DPPR synthase involved in cell wall synthesis is associated with ethambutol resistance in Mycobacterium tuberculosis.

OBJECTIVES: Ethambutol (EMB) is a frontline antituberculosis drug for the treatment of tuberculosis (TB). The embB gene is responsible for EMB resistance in only about 50-60% clinical isolates of Mycobacterium tuberculosis, and the mechanism of resistance in the remaining EMB-resistant strains is not clear. We assessed the role of the ubiA gene encoding 5-phospho-α-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase, UbiA) involved in decaprenylphosphoryl-d-arabinose (DPA) pathway for cell wall synthesis in EMB resistance. METHODS: Antimicrobial susceptibility testing was performed using broth colorimetric method or an agar proportion method to compare the MICs of recombinant M. tuberculosis strains that overexpressed the wild type or mutant ubiA gene. PCR and DNA sequencing were used to detect ubiA mutations in EMB-resistant M. tuberculosis clinical isolates with or without embB mutations. ubiA mRNA expression levels were measured by qRT-PCR. Enzymatic activity of UbiA in different M. tuberculosis strains was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RESULTS: Overexpression of the wild type M. tuberculosis ubiA led to increased resistance to EMB in M. tuberculosis. In addition, novel ubiA mutations were found in some EMB-resistant XDR-TB isolates without common embB mutations. Topological modeling analysis showed that the mutation sites in ubiA were mainly localized in the sixth transmembrane domain of the UbiA protein. Moreover, mutated ubiA when overexpressed led to higher level of EMB resistance and increased DPA levels, compared to wild type ubiA in M. tuberculosis. CONCLUSIONS: Our results indicate that ubiA is involved in EMB resistance in M. tuberculosis and that ubiA mutations that caused elevated DPA levels may be responsible for EMB resistance. The essentiality of UbiA, its involvement in EMB resistance, and lack of human homologs make UbiA an attractive target for new drug development.