Hybrid Genome Sequencing and Comparative Analysis of Three Novel Listeria monocytogenes Strains: Insights into Lineage Diversity, Virulence, Antibiotic Resistance, and Defense Systems.

Listeria monocytogenes is a major foodborne pathogen, responsible for severe listeriosis outbreaks associated with contaminated foods. This study reports the comparative genomic analysis of three novel L. monocytogenes strains C5, A2D9 and A2D10, obtained from dairy and clinical sources. Hybrid genome sequencing with Oxford-Nanopore and Illumina technologies provided high-quality complete chromosomes. Phylogenomic analysis revealed a highly conserved core genome alongside accessory genome diversity. Strain C5 belonged to sequence type ST2, while A2D9 and A2D10 were assigned to ST155 and ST1, respectively. All strains exhibited close genomic relatedness to isolates from dairy animals and/or the dairy environment. Functional analysis identified conserved metabolic functions across all genomes. A total of 40 virulence genes were detected, including the LIPI-1 island in all strains and the LIPI-3 operon exclusively in A2D10, indicating a potential hypervirulent phenotype consistent with its ST1 background and the associated fatal clinical outcome. All strains exhibited similar antimicrobial resistance profiles typical of L. monocytogenes and diverse defense systems. The newly sequenced strains provide a valuable resource for functional analyses of the mechanisms underlying adaptation of L. monocytogenes to diverse environments.