DETECTION OF TETRACYCLINES AND EFFLUX PUMP INHIBITORS

Screening assays for the detection of tetracyclines and inhibitors of tetracycline efflux pumps are described. The tetracycline assay is bas ed on the observation that the tetA(B) gene encoding the efflux pump o f transposon Tn10 is induced by tetracycline. The Escherichia coli str ain designed to detect tetracyclines contains a single copy of a tetA( B)-lacZ transcriptional fusion integrated into the chromosome and the tetR gene encoding the tetracycline repressor on a plasmid. The assay specifically detects tetracyclines of distinct structures, but not oth er classes of drugs. A strain capable of detecting inhibitors of the T etA(B) efflux pump contained the tetA(B)-lacZ fusion and, in addition, a tetA(B) structural gene lacking its transcriptional regulatory sign als which mediated resistance to only 5 mug of tetracycline per ml. Th is strain was more refractory to induction by tetracycline because of the action of the pump. Inhibitors were detected in two ways: (i) beta -galactosidase induction in the presence of 5 ng of tetracycline per m l, a subinducing concentration, and (ii) growth inhibition in the pres ence of 5 mug of tetracycline per ml. A strain designed to detect inhi bitors of the Tet(K) efflux pump from Staphylococcus aureus was constr ucted by substituting the tet(K) structural gene for the tetA(B) gene. Nocardamine and other siderophores were found to interfere with the a ction of tetracycline efflux pumps.