Antiseptic quaternary ammonium compound tolerance by gram-negative bacteria can be rapidly detected using an impermeant fluorescent dye-based assay

Biocides such as quaternary ammonium compounds (QACs) are potentially important contributors towards bacterial antimicrobial resistance development; however, their contributions are unclear due to a lack of internationally recognized biocide testing standards. Methods to detect QAC tolerance are limited to laborious traditional antimicrobial susceptibility testing (AST) methods. Here, we developed a rapid fluorescent dye-based membrane impermeant assay (RFDMIA) to discriminate QAC susceptibility among Gram-negative Enterobacterales and Pseudomonadales species. RFDMIA uses a membrane impermeant fluorescent dye, propidium iodide, in a 30-min 96-well fluorescent microplate-based assay where cell suspensions are exposed to increasing QAC concentrations. Our results demonstrate that RFDMIA can discriminate between QAC-susceptible and QAC-adapted Escherichia coli tolerant phenotypes and predict benzalkonium and cetrimide tolerance in all species tested except for intrinsically fluorescent Pseudomonas aeruginosa. RFDMIA identified a close association to minimum inhibitory concentration values determined by broth microdilution AST and increasing fluorescent dye emission values. RFDMIA emission values and scanning electron microscopy results also suggest that CET-adapted E. coli isolates have a CET dependence, where cells require sub-inhibitory CET concentrations to maintain bacilliform cell integrity. Overall, this study generates a new, rapid, sensitive fluorescent assay capable of detecting QAC-susceptible Gram-negative bacteria phenotypes and cell membrane perturbations.

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