ABSTRACT
Background & objectives: Ethambutol (EMB) resistance, thought to be occurring due to mutations in embB gene of Mycobacterium tuberculosis on the rise is a cause of grave concern. The present study was planned to investigate the presence of EMB resistance in M. tuberculosis isolates and to look for prevalent mutations in embB gene. Methods: A total of 591(283 from new and 308 from previously treated cases) sputum samples from the same number of pulmonary tuberculosis cases were cultured. Isolates were tested by 1 per cent proportion method for resistance to isoniazid, rifampicin streptomycin and ethambutol. Minimum inhibitory concentration (MIC) of EMB was measured by absolute concentration method. Ten randomly selected isolates were subjected to single strand conformational polymorphism (SSCP) and direct DNA sequencing to look for mutation in 364 bp segments of embB gene. Results: Of 353 isolates of M. tuberculosis from 591 sputum samples, 62 (17.58%) were resistant to EMB, of which, 16 (25.8%) showed initial resistance and 46 (74.2%) acquired. Mono resistance to EMB was rare. Only two isolates showed resistance to EMB alone. From 62 EMB resistant isolates, 88.7 per cent (55) were resistant to INH, 82.2 per cent (51) to rifampicin and 61.2 per cent (38) were resistant to streptomycin. Co-resistance to isoniazid and rifampicin (multidrug resistant, MDR-TB) with EMB resistance was seen in 41(66.1%) isolates. High level of EMB resistance was seen in 16.5 per cent isolates. SSCP showed altered mobility in 8 of 10 isolates tested. Among the 8 mutants, 4 had known mutations at codon Met 306 being replaced by Val/ Leu. The second most frequent mutation encountered was at codon Phe 287 being replaced by Val, Cys or Leu (novel mutations). Sequence analysis revealed 10 novel mutations in codon 221, 225, 227, 271, 272, 281, 282, 287, 293 and 294 within embB gene. Interpretation & conclusions: Presence of high frequency of EMB resistance, occurrence of high level EMB resistance, co-existence of MDR-TB with EMB resistance and novel mutations in emb B gene of M. tuberculosis clinical isolates reported highlight the need to work on larger samples to identify the diagnostic marker of EMB resistance in mycobacteria.
ABSTRACT
BACKGROUND: Ethambutol (EMB) is one of important first-line drug in the treatment of tuberculosis. Molecular techniques to detect embB gene mutations have been considered as an method to define the EMB resistance. We investigated the mutation rate within embB gene among EMB resistant strains using reverse hybridization techniques. METHODS: We made 11 probes that had wild or mutated sequences containing codons 306, 406, or 497 within embB gene respectively. These probes were reverse-hybridized with PCR products amplified from embB gene which were isolated from 149 ethambutol resistant strains and 50 pan-susceptible strains. RESULTS: Out of 149 ethambutol resistant strains, one hundred (67.1%) had mutation at least one base at codon 306, 406, or 497 in embB gene. Mutation at codon 306, 406, 497 were demonstrated in 75 (50.3%), 16 (10.7%), and 13 strains (8.7%) respectively. There were four strains that showed multi-mutation at codon 306 and codon 406 simultaneously. A high proportion (8.1%) had single mutation at codon 406. There was no mutation observed in embB gene among 50 pan-susceptible strains. CONCLUSION: Reverse hybridization will be useful technique for detection of gene mutation correlated to ethambutol resistance.
Subject(s)
Codon , Ethambutol , Genotype , Mutation Rate , Mycobacterium tuberculosis , Mycobacterium , Polymerase Chain Reaction , TuberculosisABSTRACT
Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y322C, D331Y double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.