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

Antimicrobial resistance is a massive threat, but it can take decades to develop a new antibiotic. That time could be drastically 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 prioritise 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 E. coli and A. baylyi. We then chose Rhodamine 123 to screen a commercial library of chemical compounds. Using flow cytometry, several drugs present in the Prestwick library were observed to have cytotoxic effects 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. 3. Impact statementThe discovery of novel antimicrobials is essential to build resilience against infectious diseases. Understanding the mechanism of action of known as well as novel compounds is equally crucial. Achieving this understanding is a long and complex process that is usually addressed on an individual basis for an antibiotic, or a class of antibiotics. Using high-throughput pipelines such as those possible with flow cytometry can help to shorten this process. We present a flow cytometry-based approach capable of determining if a given molecule has rapid antimicrobial effects that involve increasing the permeability of the cell membrane. The latter is a property that usually makes a chemical a very effective antimicrobial. 4. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.