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  • Title: Antiseptic quaternary ammonium compound tolerance by gram-negative bacteria can be rapidly detected using an impermeant fluorescent dye-based assay.
    Author: Gregorchuk BSJ, Reimer SL, Beniac DR, Hiebert SL, Booth TF, Wuzinski M, Funk BE, Milner KA, Cartwright NH, Doucet AN, Mulvey MR, Khajehpour M, Zhanel GG, Bay DC.
    Journal: Sci Rep; 2020 Nov 25; 10(1):20543. PubMed ID: 33239659.
    Abstract:
    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.
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