Whole-genome sequencing reveals clonal connectivity of Listeria monocytogenes across hygienically separated processing lines in a fresh-cut produce facility

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Listeria monocytogenes control in fresh-cut produce facilities relies heavily on hygienic zoning and physical segregation of processing lines, under the assumption that these measures limit microbiological connectivity between compartments. However, whether segregated lines within a single facility harbour genomically independent populations has rarely been assessed at high resolution. Here, we investigated the population structure and connectivity of L . monocytogenes in two physically and hygienically segregated processing lines (cut-fruit and cut-lettuce) within a fresh-cut produce facility. Whole-genome sequencing was performed on 100 isolates collected during three pre-sanitation sampling campaigns. High-quality assemblies were analysed using multilocus sequence typing (MLST), core-genome MLST (cgMLST) and high-quality SNP (hqSNP) approaches. The population was dominated by two sequence types, ST155 (81%) and ST6 (19%), both displaying limited intra-lineage diversity consistent with closely related clonal populations (median pairwise distances of 7 and 3 hqSNPs, respectively). Hygienic compartmentalization did not result in genomically independent populations. Within each lineage, processing line accounted for only a minor fraction of genomic variation, between-line genetic distances were comparable to within-line distances, and near-identical genotypes were recovered from both lines. Two distinct connectivity patterns were identified: a facility-wide distribution of ST155, with boot-washer and floor isolates clustering across lines, consistent with transfer through personnel-associated routes; and a more localized, drain-associated connectivity pattern involving ST6. Collectively, these findings demonstrate that hygienic segregation alone does not ensure microbiological independence between processing lines and highlight the importance of monitoring hygienic transition zones, including footwear sanitation stations, mobile equipment and drainage systems.