The small molecule IITR08027 restores the antibacterial activity of fluoroquinolones against multidrug-resistant Acinetobacter baumannii by efflux inhibition

- A small-molecule library was screened for inhibitors of the AbeM efflux pump.
- IITR08027 reduces the MIC of fluoroquinolones in resistant bacteria.
- IITR08027 inhibits the proton gradient but is non-toxic per se.
- IITR08027 could be used in combination therapy with fluoroquinolones.

Efflux pumps are one of the major determinants of multiple drug resistance. In this study, AbeM, a multidrug efflux pump in Acinetobacter baumannii, was cloned into a multicopy plasmid (pUC18) and was transformed into an efflux-deficient mutant of Escherichia coli (KAM32). The elevated resistance profile of the recombinant E. coli for ciprofloxacin was utilised to screen a small-molecule library of 8000 molecules to identify IITR08027, a small molecule that is not inhibitory on its own but that could potentiate the activity of ciprofloxacin. When used in combination against A. baumannii, the molecule improves the killing efficiency of ciprofloxacin, extends its post-antibiotic effect and causes a decrease in frequency of resistant mutant selection with the antibiotic. Quinacrine-based fluorescence quenching and membrane depolarisation assays revealed that IITR08027 functions as a proton gradient inhibitor. This extends the activity of IITR08027 against other H+-driven efflux pumps such as AbeS. MTT assay against HeLa and HEK293 cells suggested that the molecule is non-toxic at its minimum effective concentration. We propose that, in combination with fluoroquinolones (ciprofloxacin and norfloxacin), IITR08027 may be effective against multidrug-resistant A. baumannii expressing AbeM or other efflux pumps that use the proton gradient as an efflux mechanism.