Phenotypic and Molecular Characterization of Mycobacterium tuberculosis Isolates Resistant to both Isoniazid and Ethambutol

Abstract

In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 g/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 g/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 g/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 g/ml EMB (61 of 67 ‫؍‬ 91.0%) than in isolates either susceptible to EMB or resistant to 2.5 g/ml EMB (39 of 60 ‫؍‬ 65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 g/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 g/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52 ‫؍‬ 94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.

Feasibility of the GenoType MTBDR sl Assay for Fluoroquinolone, Amikacin-Capreomycin, and Ethambutol Resistance Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

“…Some authors also propose that embB codon 306 mutations are associated with resistance to other antituberculosis drugs and with MDR M. tuberculosis. This fact underlines a more general impact of this mutation on drug resistance (6,14). More extensive and detailed studies in different settings are needed to clarify this.…”

Performance Assessment of the GenoType MTBDR sl Test and DNA Sequencing for Detection of Second-Line and Ethambutol Drug Resistance among Patients Infected with Multidrug-Resistant Mycobacterium tuberculosis

“…Since 43.8% (71/162) of our EMB-resistant isolates exhibited WT patterns when using the GenoType MTBDRsl test alone, 9 isolates harbored mutations in the embA, embC, and embR genes. In addition, some reports have suggested that embB codon 306 mutations do not confer resistance to EMB but rather constitute a common polymorphism of the susceptibility to develop any type of drug resistance or MDR (9,18,22,27). Although those reports offer speculation on the biological role of this mutation (3), further studies are needed to identify actual mechanisms of EMB resistance.…”

Diagnostic Accuracy of a Molecular Drug Susceptibility Testing Method for the Antituberculosis Drug Ethambutol: a Systematic Review and Meta-Analysis

“…This depends on EMB resistance mechanisms. EMB resistance is regulated by both gene mutations and gene expression (51). embB 306, 406, and 497 were only shown to be associated with embB mutations and EMB resistance.…”

“…Although the embB 306 mutation is the mutant hot site in EMB-resistant strains (45-48), it was not enough to cover a single detection site for embB 306 (50,51). Other studies used multiple sites of the embB gene as detection sites when using molecular detection techniques (16,17,(26)(27)(28).…”