Purification and inhibition by quinolones of DNA gyrases from Mycobacterium avium, Mycobacterium smegmatis and Mycobacterium fortuitum bv. peregrinum

Laboratoire de Recherche Moléculaire sur les Antibiotiques (LRMA), Université Pierre et Marie Curie (Paris VI), Faculté de Médecine Pitié-Salpêtrière, 91 Bd de l’Hôpital, 75634 Paris Cédex 13, France 1 LRMA, Faculté de Médecine, Broussais-Hôtel Dieu, 75005 Paris, France 2 Author for correspondence: Wladimir Sougakoff. Tel: +33 1 40 77 97 46. Fax: +33 1 45 82 75 77. e-mail: sougakof@ lmcp.jussieu.fr The DNA gyrases from Mycobacterium avium , Mycobacterium smegmatis and Mycobacterium fortuitum bv. peregrinum , which are species naturally resistant, moderately susceptible and susceptible to fluoroquinolones, respectively, were purified by affinity chromatography on novobiocin-Sepharose columns. The DNA gyrase inhibiting activities (IC 50 values) of classical quinolones and fluoroquinolones were determined from the purified enzymes and were compared to the corresponding antibacterial activities (MICs). Regarding M. fortuitum bv. peregrinum , which is nearly as susceptible as Escherichia coli , the corresponding MIC and IC 50 values of quinolones were significantly lower than those found for M. avium and M. smegmatis (e.g. for ofloxacin, MICs of 0·25 versus 32 and 1 µg ml -1 , and IC 50 values of 1 versus 8 and 6 µg ml -1 , respectively). Such a result could be related to the presence of Ser-83 in the quinolone-resistance-determining region of the gyrase A subunit of M. fortuitum bv. peregrinum , as found in wild-type E. coli , instead of Ala-83 in M. avium and M. smegmatis , as found in fluoroquinolone-resistant E. coli mutants. The IC 50 values of quinolones against the M. avium and M. smegmatis DNA gyrases were similar, while the corresponding MICs were 32-fold higher for M. avium when compared to M. smegmatis , suggesting that an additional mechanism, such as a low cell wall permeability or a drug efflux, could contribute to the low antibacterial potency of quinolones against M. avium . Keywords: Mycobacterium , DNA gyrase, quinolone inhibition assays, fluoroquinolone resistance Abbreviations: QRDR, quinolone-resistance-determining regions