Effects of efflux transporter genes on susceptibility of Escherichia coli to tigecycline (GAR-936)

Abstract

The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 microM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters.

FIG. 1.

Induction of the Tet(B) protein by tigecycline and tetracyclines. E. coli W3104/pLGT2 [tet(B)] was grown to an OD₅₃₀ of 0.4, and then the indicated concentrations of drugs were added for induction. After 2 h, the cells were harvested and membrane fractions were prepared. Ten micrograms of membrane proteins was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Tet(B) was visualized by Western blotting, as described in Materials and Methods.

FIG. 2.

Effect of tigecycine on Tet(B)-mediated proton translocation and AcrAB-mediated ethidium bromide transport. (a) Effect of tigecycline on proton translocation measured as quinacrine fluorescence in membrane vesicles of E. coli W3104/pLGT2 [tet(B)]. Solid line, tetracycline (TC) at 10 μM; dotted line, tigecycline (Tige) at 10 μM. Note that tigecycline quenched quinacrine fluorescence. (b) Effect of tigecycline on tetracycline efflux-coupled proton translocation in membrane vesicles of E. coli W3104/pLGT2 [tet(B)]. Tetracycline was used at 10 μM; and tigecycline was used at 0, 10, and 25 μM (from top to bottom). (c) Inhibition of ethidium bromide transport by tigecycline in E. coli KAM3/pAc8 (acrAB) whole cells. The tigecycline concentrations (top to bottom) were 200, 100, 50, 25, 12.5, and 0 μM. Glucose (0.2%) was added to start the efflux for ethidium bromide-preloaded and energy-depleted cells.