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A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study mode-of-action and resistance mechanisms in bacteria

Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate mode-of-action studies, we synthesized a fluorescent analogue of the quaternary ammonium compound benzalkonium chloride, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against E. coli, S. aureus and P. aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. Leveraging these findings and NBD-DDA’s fluorescent properties, we show that reduced cellular adsorption is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized in the cell envelope of E. coli, which is a primary target of BAC and other QACs. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study the mode-of-action and the resistance mechanisms of QACs in bacteria.