Niclas Nordholt (Berlin / DE), Kate O'Hara (Berlin / DE), Ute Resch-Genger (Berlin / DE), Mark A.T. Blaskovich (Brisbane / AU), Bastian Rühle (Berlin / DE), Frank Schreiber (Berlin / DE)
Introduction: 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 that can make bacteria less susceptible to QACs, including phenotypic heterogeneity.
Goals: To develop a fluorescent quaternary ammonium compound and to use it to study the mechanisms underlying QAC susceptibility in single bacterial cells.
Methods: A fluorescent quaternary ammonium compound, N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl] azanium-iodide (NBD-DDA), was synthesized. Chemical structure and purity of NBD-DDA was confirmed by mass spectrometry and NMR spectroscopy. Antimicrobial activity against S. aureus, P. aeruginosa and E. coli was assessed by determination of minimum inhibitory concentrations and time-kill assays. Uptake, heterogeneity and efflux of NBD-DDA in wild-type and efflux-deficient mutants (ΔtolC) were determined, using flow cytometry and fluorometry. Sub-cellular localization of NBD-DDA was determined using confocal laser scanning microscopy.
Results: NBD-DDA was readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings. NBD-DDA retained antimicrobial activity comparable to the structurally similar, widely used QAC benzalkonium chloride (BAC). 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. NBD-DDA was preferentially localized to the cell envelope, which is a primary target of BAC and other QACs. Leveraging NBD-DDA"s fluorescent properties, it was demonstrated that reduced cellular accumulation underlies BAC tolerance in a BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC.
Conclusion: Overall, NBD-DDA"s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study mechanisms of QAC susceptibility and mode-of-action in bacteria, both on the level of populations and single-cells.