Large-scale genome analysis of Listeria monocytogenes from Austrian food producers reveals differences in accessory genome dynamics and phage integration in persistent genomes

Abstract Background As the causative agent of listeriosis, Listeria ( L .) monocytogenes is an important food safety hazard and public health concern. The ability of some L. monocytogenes strains to establish in food processing environments poses the threat of contamination to food products. The repeated isolation of identical strains over time has been termed persistence, however the lack of consensus definition and standard approaches for characterizing and analyzing isolate genomes remain key challenges for understanding traits and/or mechanisms underlying persistence. In this study, we leverage the L. monocytogenes genome database generated by the Austrian National Reference Laboratory to investigate sequence type (ST)-level, population-level, and gene-level patterns associated with persistence. Results We applied a stringent and consistent genome-based approach to define isolate relatedness (≤ 10 single nucleotide polymorphism (SNP) differences across the whole genome against an internal reference for each ST/core genome multilocus ST complex type (CT)/company cluster, and at least three repeated isolations between 2018 and 2023) in combination with isolate metadata to contextualize persistence patterns. We classified 1,699 isolate genomes, revealing 979 persistent, 697 non-persistent, and 23 sporadic isolates across 18 Austrian food producers. We then investigated dynamics of gene gain and loss using a phylogenetically-informed approach, revealing that although gene gain and loss occurs across L. monocytogenes , persistent isolates demonstrate evidence of more stable integration of acquired elements. Finally, we analyzed the association between specific genes and persistence classification across the combined dataset and for selected STs. This revealed stress tolerance genes and phage elements to be enriched in persistent isolates, suggesting that persistence may be driven by adaptations in gene regulation and/or phage-mediated genomic diversification. Conclusions Our results demonstrate L. monocytogenes persistence as a widespread phenomenon across diverse sequence types and different food sectors. Additionally, the results highlight that although gene-level patterns are associated, no single marker gene or set of genes may fully explain the complex phenotype of persistence. However, consideration of pangenome dynamics supports the role of acquiring and maintaining genomic novelty in persistence. This study represents a model for future collaborations between government agencies, industry, and academia, which could improve our understanding of the complex phenomenon of persistence and support management of L. monocytogenes in food production.