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...

Tuberculosis remains an important global public health problem, with an estimated prevalence of 14 million individuals with tuberculosis worldwide in 2007. Because antibiotic treatment is one of the main tools for tuberculosis control, knowledge of Mycobacterium tuberculosis drug resistance is an important component for the disease control strategy. Although several gene mutations in specific loci of the M. tuberculosis genome have been reported as the basis for drug resistance, additional resistance mechanisms are now believed to exist. Efflux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. Mycobacterium tuberculosis presents one of the largest numbers of putative drug efflux pumps compared with its genome size. Bioinformatics as well as direct and indirect evidence have established relationships among drug efflux with intrinsic or acquired resistance in M. tuberculosis. This minireview describes the current knowledge on drug efflux in M. tuberculosis.

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

We demonstrated that polymorphisms in the IL-8 gene was significantly associated with H. pylori infection. Furthermore, polymorphisms in the IL-8 and IL-10 genes were associated with an enhanced risk of peptic ulcer disease in H. pylori-positive patients.