Transfer of embB Codon 306 Mutations into Clinical Mycobacterium tuberculosis Strains Alters Susceptibility to Ethambutol, Isoniazid, and Rifampin

Abstract

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMBresistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 g/ml to 7, 7, 8.5, and 14 g/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 g/ml or 28 g/ml, respectively, to 3 g/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.

Frequency of mutational changes in the embB among the ethambutol-resistant strains of Mycobacterium tuberculosis in Iran

“…Patients infected with embB306 mutants should be carefully monitored for treatment failure and the possible emergence of MDR [28]. In an investigation between EMB resistance and strain genotype, we found Beijing and CAS genotypes to be resistant to EMB.…”

Tuberculosis ethambutol resistance: Concordance between phenotypic and genotypic test results

“…In a recent study, Safi et al demonstrated that transferring embB306 point mutations into EMB susceptible clinical Mycobacterium tuberculosis strain 210 increased the EMB MIC. 9 However, if clinical isolates are considered, the percentage of EMB resistant strains which show mutations in embB306 ranges from 30 to 68%. 10,11 Additionally, a number of recent investigations described the occurrence of embB306 mutations in EMB susceptible strains, [12][13][14] and several studies reporting an association with broad drug resistance rather than with EMB resistance alone.…”

Why Phenotypic Drug Susceptibility Testing of Mycobacterium tuberculosis to First-Line Drugs is not Sufficient for Proper Management of Drug-Resistant and Multidrug-Resistant tuberculosis?

“…Mutations in embCAB operon lead to ethambutol resistance but only modestly (3-8 fold) increase its MIC while highlevel resistance develops later due to acquisition of additional mutations either in embCAB operon or in other genes [29,39,40]. Mutations in embB gene are more common and mostly occur at codons 306, 406 and 497 [41][42][43].…”

“…Phenotypic DST methods for ethambutol often report false susceptibility of M. tuberculosis. The radiometric BACTEC 460TB system (now discontinued) was more accurate compared to the current MGIT 960 system, particularly for isolates containing embB mutations [29, [39][40][41][42][43]. Recent studies have shown that patients infected with embB mutants should be considered as having ethambutol-resistant TB even if the isolates appear to be ethambutol-susceptible by phenotypic DST methods to avoid evolution of secondary mutations and selection of fully drug-resistant strains [22,39,40].…”

“…The radiometric BACTEC 460TB system (now discontinued) was more accurate compared to the current MGIT 960 system, particularly for isolates containing embB mutations [29, [39][40][41][42][43]. Recent studies have shown that patients infected with embB mutants should be considered as having ethambutol-resistant TB even if the isolates appear to be ethambutol-susceptible by phenotypic DST methods to avoid evolution of secondary mutations and selection of fully drug-resistant strains [22,39,40]. False susceptibility to ethambutol is not critical for the treatment of drug-susceptible TB, however, it is detrimental for successful treatment of MDR-TB as drug regimens for this disease entity should not include ineffective first-line drugs [5,7,10,44].…”