Impact of virulence factors overexpression on Listeria monocytogenes F2365 epidemic strain fitness and the limitations of surrogate species in UHT and raw milk

Identifying reliable surrogates for Listeria monocytogenes is critical to safely model its behavior in dairy environments. Many dairy-related listeriosis outbreaks are linked to unpasteurized milk products enriched in hypervirulent L. monocytogenes from lineage I. We used an outbreak-associated lineage I strain to evaluate Listeria innocua, Listeria valentina, and Listeria ivanovii as potential surrogates in UHT and raw milk at 4 °C. We also assessed how overexpression of key virulence factors, including LIPI-3 and the PrfA regulon, influenced growth. While LIPI-3 overexpression had no significant impact, PrfA overexpression reduced fitness in both matrices. None of the other Listeria species tested accurately replicated the L. monocytogenes wild type strain growth patterns. Additionally, the native raw milk microbiota remained largely unaffected by the presence of any Listeria strain. Our findings emphasize the limitations of commonly used surrogates and underline the importance of selecting appropriate models for food challenge studies, especially in complex dairy matrices.