Rapid accumulation of fluorophores and fast kill identify drugs with bactericidal effects against Gram-negative bacteria

Antimicrobial resistance is a massive threat, but developing a new antibiotic can take decades. That time could be drasti-cally reduced if we were able to anticipate desirable properties of a chemical, such as its potential to target specific bacterial compartments. This would provide the opportunity to prioritize the development of molecules that target, for instance, the cell membrane, as this does not rely on transporters and usually results in a fast-acting bactericidal effect. We used flow cytometry and a set of fluorophores together with a group of antibiotics to discriminate between antimicrobials acting on cell membrane versus intracellularly against two Gram-negative bacteria, Escherichia coli and Acinetobacter baylyi. We then chose Rhodamine 123 as a fluorescent marker to screen a commercial library of chemical compounds. Using flow cytometry, several drugs present in the Prestwick library were observed to have cytotoxic effects at 1 µM final concentration towards E. coli. This was confirmed with growth inhibitory assays in both E. coli and A. baylyi for pantoprazole, theophylline and zoledronic acid. This represents an approach to the large-scale screening of small molecules with the potential to deliver fast-acting molecules that target cell membranes in Gram-negative bacteria.