Unified Sampling Across European Food Processing Environments Reveals Complex Genetic and Contextual Factors Underlying Listeria monocytogenes Persistence

Listeria (L.) monocytogenes is a major foodborne pathogen that can persist in food processing environments for months or even years, posing a risk for product contamination and listeriosis outbreaks. Despite routine environmental monitoring and studies investigating the factors underlying persistence, genetic and phenotypic traits do not fully explain why some strains are able to persist. In this study, 1,538 environmental swabs across Europe (Austria, Greece, and Spain) were collected to assess distribution and persistence. Overall, 22% of samples tested positive, particularly on non-food contact surfaces and in post-cleaning samples. We sequenced 316 isolates representing 18 sequence types (STs) and 69 cgMLST types. Based on repeated isolation (3x) and a SNP difference of ≤10 across the whole genome, 177 isolates were classified as persistent, supplemented by 37 historical persistent isolates. Persistent strains appeared in all facility types. Pangenome-wide analysis revealed a set of 2,232 accessory genes, of which 354 genes were significantly associated with persistent isolates. Persistent isolates were enriched in genes related to genome maintenance, regulatory functions, stress response, and mobile genetic elements, while genes involved in metabolism, nutrient processing, and intracellular trafficking were depleted. Virulence and environmental stress tolerance gene profiles were driven more by ST type rather than persistence classification. The high detection rate in this study demonstrates that genomic flexibility makes it challenging to control persistent strains under highly dynamic and heterogeneous conditions present across food production environments. In summary, our findings underscore the need to integrate genomic, ecological and environmental data to predict and control persistence.