New mutation at the position 81 in GyrA in clinical strains of Mycobacterium tuberculosis resistant to quinolone: report of two clinical cases and functional analysis of DNA gyrase mutant enzymes
Abstract number: o57
Matrat S., Veziris N., Jarlier V., Cambau E., Truffot-Pernot C., Camuset J., Bouvet E., Aubry A.
Fluoroquinolone resistance is emerging in M. tuberculosis (Mtb) with increasing drug use. DNA gyrase is the sole target of quinolone in Mtb (lack of topoisomerase IV). Mutations in the heterotetramer GyrA2GyrB2 are associated with resistance to quinolones. We report two cases of infection with multidrug resistant tuberculosis (MDR-TB) strains carrying a new G81A mutation in GyrA. We also investigated the role of the amino acid at this position in the resistance to quinolones in Mtb.
Two mutant genes were produced by site-directed mutagenesis of the wild type gyrA gene from Mtb strain H37Rv: gyrA g263c (as found in our clinical strains, see results) and gyrA g262t (as found by Takiff in an in vitro mutant selection, AAC, 1994). GyrA and GyrB subunits (WT and mutants GyrA G81A and GyrA G81C) were overexpressed in Escherichia coli, purified and used to reconstitute highly active gyrase complex. MICs and enzyme inhibition (concentration of drug required to inhibit the supercoiling activity of the enzyme by 50% = IC50) were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin and enoxacin.
Two men aged 24 and 45 years old, both with a previous story of TB, born in Algeria and Congo respectively, had MDR TB caused by Mtb strain carrying a G81A mutation in GyrA. One had cavitary pulmonary TB, was AFB positive and HIV negative. The other one had both pulmonary and extrapulmonary TB, was AFB positive and HIV positive. Both had been treated by fluoroquinolone within the months before the diagnosis of MDR-TB. MICs and IC50s of quinolones were 2 to 16 fold and 3 to 32 fold higher than for the WT for the GyrA G81A mutant and for the GyrA G81C mutant, respectively.
We demonstrated unequivocally that modifying the amino acid at position 81 (whatever G81A or G81C) in GyrA subunit of DNA gyrase lead to quinolone resistance in Mtb. This point contributes to clarify the nature of the drug-enzyme binding pocket in Mtb, which most likely include the aminoacid 81.
|Session name:||XXIst ISTH Congress|
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