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Role in quinolone resistance of the new DNA gyrase mutations, T73A+A83E in GyrA and R411H in GyrB, found in clinical MDR and XDR strains of M. tuberculosis

Abstract number: P1949

Matrat S., Sougakoff W., Jarlier V., Cambau E., Aubry A.

Objectives: Rapid detection of fluoroquinolone (FQ) resistance in multidrug-resistance tuberculosis (MDR-TB) is crucial since ultraresistant TB (XDR-TB, defined as resistant to rifamipicin, isoniazid, FQ, aminosides) is emerging as warned by WHO. Indeed, XDR-TB, which represents 10% of the MDR-TB, are nearly untreatable. Molecular detection of mutations in the DNA gyrase (sole target of FQ in M. tuberculosis) could allow the rapid detection of resistance to FQ in M. tuberculosis. However, all new mutations found in clinical strains should be evaluated for their implication in FQ resistance. In this work, we investigated the role of two new mutations (T73A+A83E in GyrA and R411H in GyrB) found in DNA gyrases of MDR and XDR clinical strains in resistance to FQ in M. tuberculosis.

Methods: Four mutant genes, reproducing the mutations (T73A+A83E in GyrA and R411H in GyrB) found in clinical strains, as well as the two single mutations (T73A or A83E), were produced by site-directed mutagenesis of the wild type gyrA or gyrB genes previously cloned in pET vectors. WT and mutant GyrA and GyrB subunits were overexpressed in E. coli, purified and used to reconstitute highly active gyrase complex. Enzyme inhibition (concentration of drug required to inhibit the supercoiling activity of the enzyme by 50% = IC50) were determined for moxifloxacin, gatifloxacin, ofloxacin and garenoxacin.

Results: IC50s of FQ were identical for the WT enzyme and the mutant gyrase carrying the GyrB R411H substitution, demonstrating that this mutation is not implicated per se in FQ resistance. Interestingly, gyrase complexes bearing GyrA T73A were hypersusceptible to FQ as previously shown (IC50s at least 2 fold lower than the WT IC50s), whereas those bearing GyrA A83E were highly resistant to FQ (IC50s 15 to 25 fold higher than the WT IC50s), an effect that was attenuated for enzymes bearing both mutations (IC50s 3 to 4 fold higher than the WT IC50s).

Conclusion: We demonstrated unequivocally (a) the implication of the single mutation A83E and the double mutation T73A+A83E in GyrA in resistance to FQ in M. tuberculosis., and (b) the non implication of the mutation R411H in GyrB in FQ resistance in M. tuberculosis. These results underlies (a) the importance of demonstrating the role of each new DNA gyrase mutation in FQ resistance, and (b) the occurrence of mutations leading to hypersusceptibility and attenuating the level of resistance of associated mutations.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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