CHARACTERIZATION OF FLUOROQUINOLONE-RESISTANT MUTANT STRAINS OF MYCOBACTERIUM-TUBERCULOSIS SELECTED IN THE LABORATORY AND ISOLATED FROM PATIENTS

To examine the mechanism of resistance to fluoroquinolones in Mycobact erium tuberculosis, we selected spontaneous fluoroquinolone-resistant mutants from a susceptible strain, H37Rv, and studied the susceptibili ties of these mutants and two fluoroquinolone-resistant clinical isola tes (A-382, A-564) to various fluoroquinolones and to isoniazid and ri fampin. Furthermore, since mutations within the quinolone resistance-d etermining region of the structural gene encoding the A subunit of DNA gyrase are the most common mechanism of acquired resistance, we ampli fied this region by PCR and compared the nucleotide sequences of the f luoroquinolone-resistant strains with that of the susceptible strain. Fluoroquinolone-resistant mutants of H37Rv appeared at frequencies of 2 x 10(-6) to 1 x 10(-8), For three mutants selected on ciprofloxacin, ofloxacin, and sparfloxacin, respectively, and the two clinical isola tes, MICs of ciprofloxacin and ofloxacin were as high as 16 mu g/ml, a nd those of sparfloxacin were 3 to 8 mu g/ml. They displayed cross-res istance to all fluoroquinolones tested but not to isoniazid or rifampi n, Sparfloxacin and FQ-A (PD 127391-0002) were the most potent fluoroq uinolones. All of the fluoroquinolone-resistant strains (MICs, greater than or equal to 4 mu g/ml) had mutations in the quinolone resistance -determining region which led to substitution of the Asp residue at po sition 87 (Asp-87) by Asn or Ala or the substitution of Ala-83 by Val in the A subunit of DNA gyrase. Similar mutations have been noted in o ther bacterial species and recently in mycobacteria. The broad resista nce to fluoroquinolones that arose readily by point mutation in the la boratory and apparently during inadequate therapy, as was the case in the clinical isolates, may ultimately lead to serious restriction of t he use of these drugs in the treatment of tuberculosis.