Deletion of lfrA (XZL1675) increased the susceptibility to ciprofloxacin

and ethambutol (Table 2), which suggests that LfrA might contribute to the intrinsic resistance of M. smegmatis to these drugs, as already reported by other studies [15]. Moreover, the LfrA mutant also showed increased susceptibility to EtBr, thioridazine and verapamil (Table 1). Table 2 Effect of efflux inhibitors on the MICs of antibiotics for Ro 61-8048 clinical trial wild-type and mutant PSI-7977 chemical structure strains of M. smegmatis MICs (mg/L)     M. smegmatis strains Antibiotic/EPI mc 2 155 (wild-type) SMR5 (mc 2 155 STR r ) MN01 (SMR5 Δ mspA Belnacasan ) ML10 (SMR5 Δ mspA Δ mspC ) XZL1675 (mc 2 155 Δ lfrA ) XZL1720 (mc 2 155 Δ lfrR )   No EPI 0.5 0.5 0.5 0.5 0.5 0.5 AMK CPZ 0.125 0.125 0.125 0.25 0.063 0.063   TZ 0.063 0.063 0.125 0.25 0.063 0.063   VP 0.125 0.125 0.125

0.25 0.125 0.125   No EPI 0.25 0.25 0.25 0.25 0.125 0.125 CIP CPZ 0.063 0.063 0.063 0.063 0.063 0.063   TZ 0.063 0.063 0.063 0.063 0.032 0.032   VP 0.063 0.063 0.063 0.063 0.063 0.063   No EPI 2 2 8 8 2 2 CLT CPZ 0.25 0.25 0.5 1 0.25 0.25   TZ 0.25 0.25 1 1 0.25 0.25   VP 0.5 0.5 0.5 1 0.5 0.5   No EPI 1 1 1 1 0.5 1 EMB CPZ 1 1 1 1 0.5 1   TZ 1 1 1 1 0.5 1   VP 1 1 1 1 0.5 1   No EPI 32 32 64 64 32 32 ERY CPZ 4 4 8 8 4 4   TZ 4 4 16 16 4 4   VP 8 8 8 8 8 8   No EPI 4 4 8 8 0.5 0.5 RIF CPZ 1 1 2 2 0.125 0.125   TZ 2 2 4 4 0.125 0.125   VP 2 2 4 4 0.125 0.25   No EPI 0.5 >256 >256 >256 0.5 0.5 STR CPZ 0.125 >256 >256 >256 0.032 0.063   TZ 0.125 >256 >256 >256 0.125 0.25   VP 0.25 >256 >256 >256 0.25 0.125 AMK, amikacin; CIP, ciprofloxacin; CLT, clarithromycin; CPZ,

chlorpromazine; EMB, ethambutol; EPI, efflux pump either inhibitor; ERY, erythromycin; RIF, rifampicin; STR, streptomycin; TZ, thioridazine; VP, verapamil. Data in bold type represents significant (at least 4-fold) reduction of the MIC produced by the presence of an efflux inhibitor. Relatively to the effect of the efflux inhibitors on the MICs of the tested antibiotics, there is an overall reduction of the MICs, with the exception of ethambutol, in all of the studied strains. The fact that the effect of these inhibitors is not dependent of a given genotype suggests that these compounds have a wide range of activity against efflux and are not specific of a particular efflux pump. Some of the results obtained in this study are at variance with those reported by others.