This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 3 Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.Drug resistance is a major threat for the control of tuberculosis (TB). Multidrug-resistant (MDR) Mycobacterium tuberculosis strains resistant to at least rifampin (rifampicin) and isoniazid and extensively drug resistant TB caused by M. tuberculosis strains that in addition to being MDR are resistant to any fluoroquinolone and to at least one of three injectable second-line drugs, amikacin, kanamycin, or capreomycin, have now been reported worldwide (25).Ofloxacin, ciprofloxacin, and levofloxacin have been used as second-line drugs for the treatment of drug-resistant TB or in patients with intolerance to one of the first-line drugs (24). A new generation of fluoroquinolones is under clinical evaluation, and these are being proposed as first-line regimens for shortening the duration of treatment (2, 12, 17). Moxifloxacin and gatifloxacin are promising drugs undergoing phase III trials (27), having previously shown favorable pharmacokinetic activity and low MICs against M. tuberculosis (5, 9, 14).The main target of fluoroquinolones in M. tuberculosis is the DNA gyrase, encoded by gyrA and gyrB (26). Mutations in two short regions known as "quinolone resistance-determining regions" have been associated with fluoroquinolone resistance in M. tuberculosis (20). Mutations in Ala-90 and Asp-94 of gyrA have been the most commonly found (1, 7, 16). We looked for mutations gyrA and gyrB in a panel of fluoroquinolone-resistant M. tuberculosis strains and evaluated the possible correlation between gene mutations and resistance to ofloxacin, moxifloxacin, and gatifloxacin based on their MICs.A total of 41 M. tuberculosis clinical isolates were studied. Twenty-six strains were MDR. Fresh subcultures were prepared on Löwenstein-Jensen medium and kept at 37°C for no longer than 4 weeks. Moxifloxacin (Bayer, Brussels, Belgium), gatifloxacin (Lupin Ltd., India), and ofloxacin (Sigma-Aldrich, Bornem, Belgium) stock solutions were prepared at 1 mg/ml in 0.1 N NaOH, filter sterilized, and stored at Ϫ20°C for less than 2 months. DNA was obtained from a loopful of culture resuspended in 200 l of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), heat inactivated at 100°C for 10 min, and centrifuged at 10,000 ϫ g at 4°C for 20 min. Two microliters of supernatant was used for amplification of the gyrA and gyrB genes using the primers (oligo 101) GyraseB-sense (5Ј-TAAGAGCGCCACC GACATCGGTGGATTG-3Ј) (positions 1404 to 1431 of gyrB) and (oligo 102) GyraseA-reverse (5Ј-GATGAAATCGAC...