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

Dairy products are linked to half of the reported listeriosis outbreaks. Most dairy-related outbreaks are associated with products made of unpasteurized milk, enriched in hypervirulent clonal complexes from lineage I. Here, using a Listeria monocytogenes lineage I strain responsible for an outbreak related to cheese as a reference, we first analyzed Listeria innocua, Listeria valentina, and Listeria ivanovii as surrogates for growth in UHT and raw milk at refrigeration temperature. We determined the extent to which virulence factors, such as the bacteriocin listeriolysin S encoded in LIPI-3 or those controlled by PrfA, contribute to L. monocytogenes growth in UHT and raw milk. Finally, we analyzed the impact of milk contamination with all these strains on the composition of the native raw milk microbiota. Our data showed that L. innocua, L. valentina and L. ivanovii are non-reliable surrogates for L. monocytogenes in UHT and raw milk. L. monocytogenes overexpressing LIPI-3 exhibited similar growth parameters in UHT and raw milk to those of the isogenic parental strain. However, overexpression of the PrfA regulon resulted in a fitness cost to L. monocytogenes in UHT and raw milk. The growth and composition of the naturally occurring microbial communities in raw milk were independent of the contaminating Listeria spp., as well as the overexpression of LIPI-3 or PrfA. Altogether, these findings may aid in selecting more suitable surrogates for food challenge tests and contribute to understanding the relevance of milk contamination with strains that overexpress PrfA or LIPI-3, ultimately helping to reduce dairy product contamination.