Copyright © 1993, American Society for Microbiology 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 based on the observation that the tetA4(B) gene encoding the efflux pump of transposon TnlO is induced by tetracycline. The Escherichia coli strain 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 other classes of drugs. A strain capable of detecting inhibitors of the TetA(B) efflux pump contained the tetA(B)-lacZ fusion and, in addition, a tetA(B) structural gene lacking its transcriptional regulatory signals which mediated resistance to only 5,g of tetracycline per ml. This strain was more refractory to induction by tetracycline because of the action of the pump. Inhibitors were detected in two ways: (i) 13-galactosidase induction in the presence of 5 ng of tetracycline per ml, a subinducing concentration, and (ii) growth inhibition in the presence of 5 jLg of tetracycline per ml. A strain designed to detect inhibitors of the Tet(K) effiux pump from Staphylococcus aureus was constructed by substituting the tet(K) structural gene for the tetA(B) gene. Nocardamine and other siderophores were found to interfere with the action of tetracycline efflux pumps. The emergence of tetracycline-resistant bacteria has ren-dered the tetracyclines less useful than when they were firs