Optimization of a validated labor-efficient culture-based protocol to decrease false positives in Listeria monocytogenes detection

The ISO 11290:2017 method for detecting and enumerating Listeria monocytogenes is detailed but labor-intensive. To reduce labor, modifications such as using fewer selective media or confirmation tests are often implemented. To ensure modified protocols are as precise as the official ISO 11290:2017, they must (or should) be validated against this standard according to ISO 16140. This resulted in numerous validated alternative methods. However, false positives still occur, making it crucial to optimize these labor-efficient protocols. This study aimed to optimize an existing validated labor-efficient culture-based (LECB) protocol consisting of three steps: initial detection on a selective medium, followed by purification of the isolate, and confirmation of the isolate. Screening was conducted on eight false-positive and 13 true-positive L. monocytogenes strains using five selective media and four confirmation tests to identify appropriate culture media and confirmation tests to develop a reliable, optimized LECB protocol. Only RAPID’ L. mono distinguished false from true positives and was incorporated into the optimized protocol, along with the hemolysis test. The optimized LECB protocol was tested in parallel with the original LECB protocol using three stock cultures and 66 fresh presumptive L. monocytogenes and Listeria spp. isolates. Annotated genomes of the eight false-positive and 13 true-positive isolates were screened for genes linked to agar and test selectivity. Quantitative PCR was also evaluated as an alternative confirmation method. This study demonstrates an approach for optimizing validated protocols, although full ISO 16140 validation remains necessary.