The tripartite efflux pump acrAB-tolC is an important mediator of antibiotic resistance in Escherichia and Salmonella. While expression of this pump is tightly regulated in wildtypes, antibiotics can induce an over-expression of the pump’s genes, especially acrB. Because this pump acts on a broad range of antibiotics, fluorescent dyes, and other chemicals, its over-expression grants multiple drug resistances.
Several natural and synthetic compounds are known to act as efflux pump inhibitors (EPI), which can revert efflux-based antibiotic resistance. In order to screen new substances for EPI activity, we developed a simple assay using Salmonella over-expressing acrB.
Over the course of several weeks, Salmonella typhimurium SL1344 was gradually adapted to grow in doubling concentrations of enrofloxacin (a common veterinary antibiotic), eventually growing at 32 mg/L (initial MIC was 0.06 mg/L). The adapted strain (named enro+) was found to be resistant against various antibiotics and detergents, even after several passages in antibiotic-free medium and showed an increase in acrB expression with RT-qPCR. When enro+ was incubated with the known EPI phenylalanine arginyl ß-naphthylamide (PAßN), MIC values of all tested substances dropped back to levels comparable to the unadapted parent strain, showing that the acquired resistances were reversible by inhibiting efflux activity.
A simple agar assay was established to screen substances for EPI activity: Enro+ was streaked on Mueller-Hinton agar plates containing 1 mg/L of the fluorescent dye acridine orange (AO) and discs loaded with PAßN or test samples were placed on the agar. The plates were incubated over-night and evaluated under a UV-lamp. Enro+ showed no fluorescence at this concertation of AO, due to its efflux activity, but samples with EPI activity yielded a zone of fluorescent colonies around the discs.
The assay presented can be used to screen substances with EPI activity. Promising substances will be investigated further.