This study developed a rapid fluorescent dye-based membrane impermeant assay (RFDMIA) to detect quaternary ammonium compound (QAC) tolerance in Gram-negative bacteria. RFDMIA uses propidium iodide to measure membrane permeability changes in response to increasing QAC concentrations. The assay successfully discriminated between QAC-susceptible and -tolerant *E. coli* phenotypes and predicted benzalkonium and cetrimide tolerance in most species tested, except for intrinsically fluorescent *P. aeruginosa*. RFDMIA results closely correlated with minimum inhibitory concentration (MIC) values from broth microdilution assays. Scanning electron microscopy suggested a cetrimide dependence in CET-adapted *E. coli*, requiring sub-inhibitory concentrations to maintain cell integrity. RFDMIA offers a rapid and sensitive method for detecting QAC susceptibility and membrane perturbations in Gram-negative bacteria.

Branden S. J. Gregorchuk, Shelby L. Reimer, Daniel R. Beniac, Shannon L. Hiebert, Timothy F. Booth, Michelle Wuzinski, Brielle E. Funk, Kieran A. Milner, Nicola H. Cartwright, Ali N. Doucet, Michael R. Mulvey, Mazdak Khajehpour, George G. Zhanel, Denice C. Bay