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

• Published in: Scientific reports Vol. 10; no. 1; p. 20543
• Main Authors: Gregorchuk, Branden S. J., Reimer, Shelby L., Beniac, Daniel R., Hiebert, Shannon L., Booth, Timothy F., Wuzinski, Michelle, Funk, Brielle E., Milner, Kieran A., Cartwright, Nicola H., Doucet, Ali N., Mulvey, Michael R., Khajehpour, Mazdak, Zhanel, George G., Bay, Denice C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25-11-2020; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647; 631/326; 631/45; 692/308; 704/172; Ammonium; Anti-Bacterial Agents - pharmacology; Anti-Infective Agents - pharmacology; Anti-Infective Agents, Local - pharmacology; Antimicrobial agents; Antimicrobial resistance; Bacteria; Bacteria - metabolism; Bacterial Proteins - metabolism; Benzalkonium Compounds - pharmacology; Biocides; Cell membranes; Cell suspensions; Disinfectants - pharmacology; Drug Resistance, Bacterial - genetics; Dyes; E coli; Emissions; Escherichia coli; Fluorescent Dyes - pharmacology; Fluorescent indicators; Gram-negative bacteria; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - metabolism; Humanities and Social Sciences; Iodides; Microbial Sensitivity Tests - methods; Minimum inhibitory concentration; multidisciplinary; Phenotypes; Propidium iodide; Quaternary ammonium compounds; Quaternary Ammonium Compounds - pharmacology; Scanning electron microscopy; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-77446-8

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
r
apid
f
luorescent
d
ye-based
m
embrane
i
mpermeant
a
ssay (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.