Whole-genome sequencing reveals clonal connectivity of Listeria monocytogenes across hygienically separated processing lines within a fresh-cut produce facility prior to sanitation
The presence of Listeria monocytogenes in fresh-cut fruit and vegetable processing environments remains a major challenge for food safety management. Control in these settings relies heavily on hygienic architecture (physical separation between processing lines, zoning, and personnel and footwear barriers) under the implicit assumption that such compartmentalization limits microbiological connectivity between compartments. Whether physically separated lines within a single facility harbour genomically independent populations of L. monocytogenes has rarely been tested at high resolution. Here we tested whether two physically and hygienically separated processing lines within a single fresh-cut facility (cut-fruit and cut-lettuce) harbour genomically independent populations of L. monocytogenes. Whole-genome sequencing was performed on 99 isolates collected across three pre-sanitation sampling campaigns, and high-quality assemblies were analysed using MLST, cgMLST, high-resolution SNP (hqSNP) approaches. The population was dominated by two sequence types, ST155 (80%) and ST6 (19%), both showing limited intra-lineage diversity consistent with closely related clonal populations. Hygienic compartmentalization did not produce genomically independent populations: within each lineage, processing line explained only a minor fraction of the genomic variation, between-line distances were comparable to within-line distances, and near-identical genotypes were recovered from both lines. This connectivity took two forms: a pervasive distribution of ST155 across the facility, with boot-washer and floor isolates clustering across lines, consistent with footwear-mediated transfer; and a focal, drain-associated connection of ST6 between the two lines. Core virulence was conserved and no acquired antimicrobial resistance was detected. These findings indicate that hygienic compartmentalization alone does not guarantee microbiological independence, and that environmental monitoring in multi-line plants should be designed to detect connectivity between compartments, not only persistence within them, with sampling targeted at shared personnel routes and footwear, mobile equipment, and drainage systems.
The presence of Listeria monocytogenes in fresh-cut fruit and vegetable processing environments remains a major challenge for food safety management. Control in these settings relies heavily on hygienic architecture (physical separation between processing lines, zoning, and personnel and footwear barriers) under the implicit assumption that such compartmentalization limits microbiological connectivity between compartments. Whether physically separated lines within a single facility harbour genomically independent populations of L. monocytogenes has rarely been tested at high resolution. Here we tested whether two physically and hygienically separated processing lines within a single fresh-cut facility (cut-fruit and cut-lettuce) harbour genomically independent populations of L. monocytogenes. Whole-genome sequencing was performed on 99 isolates collected across three pre-sanitation sampling campaigns, and high-quality assemblies were analysed using MLST, cgMLST, high-resolution SNP (hqSNP) approaches. The population was dominated by two sequence types, ST155 (80%) and ST6 (19%), both showing limited intra-lineage diversity consistent with closely related clonal populations. Hygienic compartmentalization did not produce genomically independent populations: within each lineage, processing line explained only a minor fraction of the genomic variation, between-line distances were comparable to within-line distances, and near-identical genotypes were recovered from both lines. This connectivity took two forms: a pervasive distribution of ST155 across the facility, with boot-washer and floor isolates clustering across lines, consistent with footwear-mediated transfer; and a focal, drain-associated connection of ST6 between the two lines. Core virulence was conserved and no acquired antimicrobial resistance was detected. These findings indicate that hygienic compartmentalization alone does not guarantee microbiological independence, and that environmental monitoring in multi-line plants should be designed to detect connectivity between compartments, not only persistence within them, with sampling targeted at shared personnel routes and footwear, mobile equipment, and drainage systems.
Centro de Edafologia y Biologia Aplicada del Segura