Paper Strip-Based Sensor for Detection of Antimicrobial Resistant Listeria monocytogenes in Milk

Abstract

To develop a rapid, selective, and field-deployable paper strip assay for the detection of Listeria monocytogenes and targeted screening of resistance to Penicillin G and Ampicillin in milk. A novel Listeria Selective Enrichment Medium (LSEM) was optimized to enhance the recovery of L. monocytogenes in milk while reducing interference from selected competing microorganisms evaluated in this study. The assay employed β-D-glucosidase and enzyme-substrate reactions for presumptive and species-specific detection, producing characteristic colour changes for Listeria spp. The developed paper strip detected L. monocytogenes within 7 h in broth and 9 h (with enrichment for 24 h at 1 CFU/25 mL) in spiked sterilized milk. The total detection time of the developed assay was approximately 33 h (24 h enrichment + 9 h strip-based confirmation). In the present study, antimicrobial resistance assessment was limited to two representative β-lactam antibiotics, Penicillin G and Ampicillin. Therefore, the assay should currently be considered a targeted screening platform rather than a universal antimicrobial susceptibility test. Resistant strains showed colour development in the presence of the respective antibiotic, whereas susceptible strains remained colourless. Validation with 70 milk samples confirmed one raw milk sample positive for L. monocytogenes, which was further verified using ISO 11290–1:2017 and disc diffusion methods. The developed assay provides a rapid, selective, cost-effective, and user-friendly platform for simultaneous detection of L. monocytogenes and targeted susceptibility screening to Penicillin G and Ampicillin in milk.