Influence of the evaluation method on the assessment of the susceptibility of Listeria monocytogenes to quaternary ammonium compounds

Rapid and reliable methods are needed to evaluate Listeria monocytogenes resistance to disinfectants. The purpose of this study was to evaluate and compare the susceptibility of L. monocytogenes isolates from food-processing environments to quaternary ammonium compounds (QACs) using agar diffusion methods and suspension and surface challenge tests. L. monocytogenes strains isolated from frozen vegetables– (n = 23), broccoli- (1), cheese- (n = 3), and avocado- (n = 1) processing environments, and ATTC 19115 were used. Strains were characterized for antibiotic susceptibility and screened for bcr ABC and qac H resistance genes. The susceptibility to QACs (50, 200, 500, and 1000 ppm) was evaluated using agar diffusion methods. Growth inhibition zones were measured, and strains were categorized as resistant, intermediate, and sensitive to QACs (500 ppm). Strains were inoculated (10 log CFU/mL) in a QAC suspension (200 ppm/30 s) or on stainless steel (8.4 log CFU/cm 2 ) and exposed to QACs (200 ppm/30 s and 2 min, 500 ppm/30 s and 2 min) and surviving microbial population was quantified. Strain characterization revealed heterogeneous antimicrobial and gene-resistance profiles among L. monocytogenes isolates. The agar well diffusion method showed higher sensitivity than the disc diffusion method (p > 0.05). Based on the agar well diffusion method, isolates were classified as sensitive (≥ 21 mm), intermediate (16 mm ≤zone< 21 mm), or resistant (<16 mm). Regardless of the strain, the suspension method achieved complete inactivation of the microbial population (>9 log CFU/mL), whereas bacterial reductions oscillated from 0.9 to 4.8 log CFU/cm 2 on stainless steel. A negative correlation (ρ= −0.4) was observed between the agar diffusion methods and the stainless steel surface assay. Agar diffusion methods can be used as rapid and reliable screening tools to assess the QAC susceptibility of L. monocytogenes isolates from food-production environments. The proposed classification based on inhibition zone diameters provides a practical and reproducible approach to interpret susceptibility data in the absence of standardized cut-offs for disinfectants. The stainless steel surface assay remains essential for evaluating disinfection effectiveness in food-processing environments. • Well agar diffusion method showed higher sensitivity than the disk diffusion assay. • K-means clustering classified QACs susceptibility statistically. • Most L. monocytogenes isolates resisted QACs at 500 ppm. • bcr ABC gene predominated in L. monocytogenes isolates. • Agar diffusion is a rapid tool to monitor QACs susceptibility changes.