Correlation between GyrA substitutions and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium tuberculosis.

The newer fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of tuberculosis (TB) treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, the MICs for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs of >1 µg/ml and MXF MICs of >0.5 µg/ml. The presence of mutations within the gyrA quinolone resistance-determining regions (QRDR) correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific gyrA mutation present. Substitutions Ala90Val, Asp94Ala, and Asp94Tyr resulted in low-level MXF resistance (MICs were >0.5 but ≤2 µg/ml), while other mutations led to MXF MICs of >2 µg/ml. Based on these results, a critical concentration of 1 µg/ml is suggested for LVX and 0.5 µg/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 µg/ml.

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis.

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis.

The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)