Quaternary ammonium compounds (QACs) are widely used antiseptics and disinfectants with growing concerns about their contribution to antimicrobial resistance. The lack of standardized testing methods hinders understanding of QAC tolerance. Current methods, such as agar dilution and broth microdilution, are laborious and time-consuming (24-48 hours). This research aimed to develop a rapid and sensitive assay to overcome this limitation. The study focused on developing a rapid fluorescent dye-based membrane integrity assay (RFDMIA) to discriminate between QAC-susceptible and QAC-tolerant Gram-negative bacteria. The hypothesis was that QAC-susceptible bacteria would show increased membrane permeability and thus increased fluorescent dye (propidium iodide, PI) emission compared to QAC-tolerant isolates. The assay's speed and sensitivity were compared to traditional antimicrobial susceptibility testing (AST) methods. The widespread use of QACs in various settings, from healthcare to household products, necessitates a better understanding of their role in driving antimicrobial resistance. The high volume of QAC usage far surpasses that of therapeutic antibiotics, making this a critical area of investigation. This study aimed to fill a significant knowledge gap in standardized testing for QAC susceptibility by developing a rapid and sensitive assay.

Existing literature highlights the diverse applications of QACs as antiseptics, disinfectants, and industrial surfactants. Benzalkonium chloride (BZK) and cetrimide (CET) are among the most prevalent QACs. QACs act by disrupting bacterial membranes, leading to protein denaturation and increased reactive oxygen/nitrogen species. Previous research indicates that several Gram-negative bacteria, including Enterobacterales and Pseudomonadales, exhibit intrinsic or acquired tolerance to high QAC concentrations. Prolonged exposure to sub-lethal concentrations can lead to increased QAC tolerance, cross-tolerance to other biocides, and even cross-resistance to antibiotics. The absence of standardized QAC susceptibility testing is a significant obstacle. EUCAST and CLSI guidelines do not cover QACs, lacking defined breakpoint concentrations for AST. This lack of standardization necessitates the development of new, robust, and rapid methods for determining QAC tolerance.

The researchers developed a rapid fluorescent dye-based membrane impermeant assay (RFDMIA) to assess QAC susceptibility. RFDMIA utilizes a membrane-impermeant fluorescent dye, propidium iodide (PI), which only enters cells with compromised membrane integrity. Bacterial suspensions were exposed to increasing concentrations of QACs (BZK and CET), and changes in PI fluorescence emission (EM) were monitored over 30 minutes in a 96-well plate reader. The assay's performance was evaluated by comparing RFDMIA results with MIC values obtained from standard broth microdilution AST. The study used *E. coli* K-12 BW25113 isolates (unadapted, BZK-adapted, and CET-adapted) as primary models, with additional testing performed on *A. baumannii*, *P. aeruginosa*, *S. sonnei*, and *K. pneumoniae* isolates. Different cell preparation methods (stationary phase, mid-log phase, and agar plate colonies) were compared to determine optimal conditions. Scanning electron microscopy (SEM) was used to visualize cell morphology changes in response to QAC exposure. A second impermeant dye, SYTOX Blue, was also tested as an alternative to PI. Statistical analyses, including Student's t-tests and Mann-Whitney U tests, were used to assess significant differences in fluorescence emission and cell morphology.

RFDMIA effectively discriminated between QAC-susceptible and -tolerant *E. coli* isolates. The assay accurately predicted MIC values for BZK and CET in *E. coli*, with the lowest QAC concentration inducing a significant increase in PI fluorescence corresponding to the MIC. Stationary-phase cultures yielded the most reliable RFDMIA results, with lower error compared to mid-log or colony preparations. SEM analysis of QAC-adapted *E. coli* revealed altered cell morphologies, suggesting a potential QAC dependence for CET-adapted isolates, where sub-inhibitory CET concentrations were needed to maintain cell integrity. RFDMIA accurately predicted QAC susceptibility in *A. baumannii*, but not *P. aeruginosa*, likely due to the intrinsic fluorescence of *P. aeruginosa*. The assay also discriminated between QAC-susceptible and -tolerant isolates of *S. sonnei* and *K. pneumoniae*. High QAC concentrations (above the critical micelle concentration) reduced RFDMIA detection accuracy due to QAC-membrane interactions and potential fluorescence quenching effects. The 30-minute MBC values determined by spot plating often coincided with the maximum PI fluorescence in the RFDMIA, providing a more accurate measure of cell viability in the context of the rapid assay.

RFDMIA offers a significant advance over traditional methods for assessing QAC susceptibility. Its speed and sensitivity make it suitable for high-throughput screening. The findings highlight the importance of cell growth phase in assay performance, with stationary-phase cultures optimal for reliable results. The observed QAC dependence in adapted isolates underscores the complex interplay between QAC exposure and bacterial cell physiology. The inability to accurately determine QAC susceptibility in *P. aeruginosa* due to its intrinsic fluorescence emphasizes a limitation of the current RFDMIA protocol. Further development might include exploring alternative dyes with different spectral properties. Overall, the results support the application of RFDMIA as a valuable tool for studying QAC tolerance and for developing strategies to combat antimicrobial resistance.

This study successfully developed RFDMIA, a rapid and sensitive method for detecting QAC susceptibility in Gram-negative bacteria. The assay’s efficiency, especially using stationary-phase cultures, makes it suitable for high-throughput screening. While limitations exist regarding intrinsically fluorescent bacteria such as *P. aeruginosa*, RFDMIA shows promise for advancing our understanding of QAC tolerance and antimicrobial resistance. Future research could explore the use of alternative dyes, optimize the assay for a broader range of QACs and bacterial species, and investigate the molecular mechanisms underlying QAC dependence in adapted isolates.

The assay's inability to accurately predict QAC susceptibility in *P. aeruginosa* due to its intrinsic fluorescence represents a limitation. High QAC concentrations above the CMC also compromised detection accuracy. The use of laboratory-adapted isolates, rather than naturally occurring resistant strains, might limit the generalizability of findings. The 30-minute exposure time of RFDMIA differs from the longer incubation periods of standard AST, potentially affecting direct MIC value comparisons. Further research is needed to determine if longer exposure times would more accurately predict MIC values from RFU values in QAC-adapted strains.