Abstract: To enhance analytical throughput and enable rapid assessment of bacterial susceptibility to disinfectants, developing an accelerated evaluation protocol is critically required,a new method for rapid phenotypic assessment of Lm's BAC tolerance was established based on SYBR Green I (SG) and propidium iodide (PI) double staining-the SG-PI dual-staining methodology. Results showed that the BAC tolerance of 104 Lm isolates from swine slaughterhouse samples was assessed using the dilution method. Analysis revealed 11 BAC-tolerant and 93 BAC-sensitive isolates, establishing these findings as the reference standard for validating the newly developed methodology. Firstly, the SG-PI dual-staining methodology was optimized, and the optimal concentration combination of the double staining was finally determined to be 20× SG + 5 μmol/L PI, and the optimal double staining incubation time was 5 min. Subsequently, the optimal conditions of the SG-PI dual-staining methodology were applied to Lm suspensions with different percentages of viable bacteria (P), and the fluorescence intensity ratio (F) between SG and PI was calculated. The functional relationship between the F value and the P value was determined to be F=0.004 996P+0.303 6, R²=0.997 6. The quantitative detection model formula for the relative cell activity of Lm derived from the relationship was RCA=[(F-0.303 6)/0.004 996]×100%. Finally, through exposure experiments, the optimal exposure concentration of BAC was determined to be 16 mg/L. The SG-PI double-staining method developed in this study exhibited 100% sensitivity for detecting BAC-tolerant strains, demonstrating superior accuracy compared to traditional dilution methods. The SG-PI dual-staining technique offered simplicity, rapidity, and precision, having been effectively applied in assessing bacterial antibiotic resistance.

Key words: Listeria monocytogenes, benzalkonium bromide(BAC), tolerance assessment, SYBR Green I (SG), propidium iodide (PI)