Identification of a Mycobacterium tuberculosis Strain with Stable, Low-Level Resistance to Isoniazid

Abstract

We have identified a potential quality control strain of Mycobacterium tuberculosis to monitor isoniazid susceptibility testing. This strain (strain A) has a stable phenotypic low-level resistance to isoniazid, has a mutation of C (؊15) 3 T in the inhA promoter region, and gave consistent susceptibility test results in 141 laboratories.Among the primary drugs used to perform susceptibility testing of Mycobacterium tuberculosis, isoniazid (INH) is often used in two concentrations, frequently showing low-level resistance (to 0.1 or 0.2 g/ml) and being fully susceptible to higher INH concentrations. A low-level resistance quality control (QC) strain for monitoring the accuracy of susceptibility testing, especially with low INH concentrations, has not yet been identified (6). The available highly resistant strain H37Rv is not recommended as a QC strain, since it is unable to detect efficiency of low-level resistance and does not provide information to discriminate the quality of test media, drug strength, or technical performance in the testing process. It is also believed that low-level INH resistance is often not stable during long-term storage, subculturing, and freezing-thawing of the cultures. This study was conducted to identify a potential QC strain with stable low-level resistance to INH which is stable on several subculturing and freezing cycles.Three low-level INH-monoresistant M. tuberculosis clinical isolates (A, B, and C) were selected from the CDC M. tuberculosis Drug Susceptibility Testing Performance Evaluation Program (PEP) and subcultured on Middlebrook 7H10 agar to obtain isolated colonies. To purify one low-level INH-resistant isolate from each subculture, the selected colonies were tested for MICs with twofold dilutions of INH, starting with 0.05 g/ml, in 7H12 BACTEC broth and by agar dilution method. The selected low-level INH-resistant subcultures from each strain were inoculated into Middlebrook 7H9 broth and then subcultured on Lowenstein-Jensen slants. These cultures were sent to two laboratories for confirmation by MIC testing using agar dilution and broth dilution (7H12 BACTEC broth) methods (1, 2). The MICs for the three strains ranged from 0.1 to 0.4 g/ml in BACTEC 7H12 broth and from 0.2 to 0.8 g/ml by the agar dilution method. The MIC for the susceptible control strain H37Rv tested at the same time was 0.1 g/ml, and the MIC for the resistant control strain ATCC 35822 was Ն1.0 g/ml by the agar dilution method (7H10 Middlebrook agar).Strains A, B, C, and D (a fully susceptible strain) were sequenced for detecting genes associated with INH resistance. Three laboratories performed studies on the stability of lowlevel INH resistance with all four cultures. Susceptibility testing was performed using a bacterial suspension made from a freshly grown culture. The bacterial suspension was then frozen at Ϫ70 Ϯ 10°C, thawed a few days later, subcultured again, and tested for susceptibility to INH. This cycle of freezing, thawing, subculturing, and testing was repeated three times for each culture...

Performance of Tuberculosis Drug Susceptibility Testing in U.S. Laboratories from 1994 to 2008

“…There may be significant regional differences in the prevalence of low-level INH-resistant strains, with this level of resistance rare in some parts of Europe (S. Ruesch Gerdes, personal communication). Many studies evaluating performance of specific methods or interlaboratory agreement include only strains with high-level INH resistance (18) (18).…”

Genotypic and phenotypic characteristics of tunisian isoniazid-resistant Mycobacterium tuberculosis strains

“…Nucleotide substitution in region flanking a presumed ribosomal binding site (RBS) located in the upstream region of the mabA-inhA operon is thought to increase the target levels, thereby causing resistance by a drug titration mechanism . The point mutation -15C T mabA-inhA promoter region was the most frequently involved in INH-resistance (Madison et al, 2004).…”

Altered NADH/NAD + Ratio Mediates Coresistance to Isoniazid and Ethionamide in Mycobacteria

“…In contrast, overexpressed inhA alleles or alleles causing amino acid substitutions within the structural gene have been shown to confer INH resistance in a dominant fashion, i.e., conferring INH resistance when the mutant alleles replace or complement the wild-type gene (1,26,51). Mutations in INH r clinical isolates of M. tuberculosis have been mapped to the promoter region or the structural gene of inhA (1,2,17,22,27,31,38,41,43). The dominant nature of these mutations was consistent with the hypothesis that inhA encodes the target of INH (1,23,26).…”