ABSTRACT
The purpose of the present study was to analyse mutations in the gyrA and gyrB genes of Mycobacterium tuberculosis and define the possible correlation between these mutations and resistance to levofloxacin (LVX), moxifloxacin (MFX) and gatifloxacin (GAT), based on their MICs. One hundred and forty-two M. tuberculosis clinical isolates were collected from pulmonary tuberculosis patients in the Moscow region. All M. tuberculosis strains were tested for drug susceptibility to rifampicin and isoniazid using the BACTEC MGIT 960 System and to ofloxacin (OFX) using the absolute concentration method on solid Lowenstein-Jensen slants. All in all, 68 strains were selected at random (38 strains were resistant and 30 were susceptible to OFX) for further analysis using the TB-BIOCHIP-2 test system and DNA sequence analysis. The MICs of LVX, MFX and GAT for selected strains were determined using the BACTEC MGIT 960 System. Mutations in the gyrA gene were observed in 36 out of 38 (94.7 %) OFX-resistant M. tuberculosis strains. Asn538Asp and Asp500His substitutions in the gyrB gene only were found in two (5.3 %) strains. Twenty-nine out of 30 OFX-sensitive M. tuberculosis strains had no mutations in either gene. One (3.3 %) OFX-sensitive M. tuberculosis strain carried an Arg485His substitution in gyrB. The results of our investigation showed that there is no clear correlation between the type of mutation in the genes gyrA and gyrB, and the MIC levels of LVX, MFX and GAT for resistant strains. Mutations in gyrA and Asn538Asp, and Asp500His substitutions in gyrB were associated with cross-resistance of M. tuberculosis to fluoroquinolones. The substitution Arg485His in gyrB does not confer resistance to LVX, MFX and GAT in M. tuberculosis.
Subject(s)
Antitubercular Agents/pharmacology , DNA Gyrase/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Aza Compounds/pharmacology , DNA Gyrase/metabolism , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Gatifloxacin , Gene Expression Regulation, Bacterial , Humans , Levofloxacin , Microbial Sensitivity Tests , Moxifloxacin , Mutation , Ofloxacin/pharmacology , Quinolines/pharmacology , Russia/epidemiology , Tuberculosis, Pulmonary/epidemiology , Tuberculosis, Pulmonary/microbiologyABSTRACT
OBJECTIVES: Kanamycin is an important second-line drug used to treat multidrug-resistant (MDR) tuberculosis (TB). Molecular analysis of the rrs gene seems to be not enough to identify every case of kanamycin resistance. In the present study we evaluated the incidence of eis mutations in kanamycin-resistant Mycobacterium tuberculosis isolates. METHODS: We analysed 70 MDR M. tuberculosis clinical isolates. All isolates were screened for rrs and eis mutations using single-strand conformation polymorphism and sequencing. Phenotypic drug susceptibility testing was performed using Bactec MGIT 960 and the absolute concentration method on Lowenstein-Jensen medium. RESULTS: eis mutations were found in 10 isolates. The most prevalent mutations were the A1401G substitution in the rrs gene and the C14T substitution in the eis promoter region. CONCLUSIONS: Our study shows that the eis promoter region is a useful molecular marker of kanamycin resistance in the Moscow region. Complex analysis of rrs and eis mutations will significantly reduce the time to diagnose kanamycin resistance in TB patients, compared with phenotypic drug resistance testing.
