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Screening System Development for the rapid detection of cationic antimicrobial resistance mechanisms

Andy (Injun) Seo

University of Manitoba Max Rady College of Medicine

Introduction: Cationic antimicrobials (CAs) are positively charged compounds used in hospitals, food industries, and commercial products that work by disrupting bacterial membranes. The inappropriate and increased use of these compounds has contributed to the development of resistance to CA antiseptics/biocides in Gram-negative bacteria. These mechanisms often confer cross-resistance to clinically relevant antibiotics, adding to the alarming rate of antibiotic resistance development. Clinical surveillance for cationic antimicrobial disinfectants are not performed due to the tedious nature of the methods and a lack of standardized drug concentrations. Objective: To develop a rapid fluorescent-based assay to screen for membrane-based cationic antimicrobial resistance in Gram-negative bacteria Methods: In the Rapid Fluorescent Dye Membrane Intactness Assay (RFDMIA), we measured the uptake of an impermeant fluorescent dye by Gram-negative bacterial cells in the presence of increasing concentrations of commonly used CAs (colistin, cetrimide, tobramycin). The fluorescent emissions were compared to detect significant differences between CA susceptible and resistant strains at a p-value of <0.05. Results: The RFDMIA method developed was able to distinguish between the fluorescent emissions of resistant and susceptible E. coli strains for colistin and cetrimide, two well-known CAs (p-value<0.05). Tobramycin was unable to elicit fluorescent dye emissions from tested E. coli strains, suggesting the aminoglycoside was unable to disrupt the bacterial membrane to allow dye entry into the cell. Conclusions: The RFDMIA method, after further optimization and standardization, could serve as an important tool to guide policies regarding usage of CA in various industries.

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