Objective To investigate the distribution characteristics and transmission routes of Listeria monocytogenes contamination in the production and sales of enoki mushrooms, and to identify key contamination points, thereby providing scientific evidence for controlling pathogenic bacterial contamination in the enoki mushroom production chain.

Methods  A study was conducted on a specific enoki mushroom production enterprise and its commercial products. A total of 277 samples were collected from seven production stages and the sales stage for the isolation of L. monocytogenes. The minimum inhibitory concentration (MIC) of the isolated strains was determined for ampicillin, penicillin, tetracycline, meropenem, co-trimoxazole, and erythromycin using the minimum inhibitory concentration method. Drug susceptibility was assessed according to CLSI and EUCAST standards. The DNA of the strains was extracted and sequenced using a second-generation sequencing platform. The sequencing data were analyzed using a bioinformatics platform to predict drug resistance genes and virulence genes, and to determine the lineage, ST type, CC group, and serotype of the strains. Multilocus sequence analysis of core genomes was performed, and an evolutionary tree was constructed for molecular tracing.

Results A total of 54 Listeria monocytogenes strains were detected in the 277 samples, with a detection rate of 19.49%. Among these, the detection rate in the enoki mushroom production stage was 20.72% (46/222), while that in the commercial stage was 14.55% (8/55), showing no statistically significant difference. In the production process, the detection rates in the inoculation room (36.11%) and harvesting/packaging area (27.88%) were significantly higher than those in other production stages (e.g., inoculation room, sterilization area, and mushroom house, all 0%). Among the sample types, operators (29.03%), environment (25.71%), enoki mushrooms (22.73%), and tools/equipment (21.51%) showed relatively high detection rates but no statistically significant differences (P=0.182). Among the 30 sequenced strains, lineage Ⅱ (56.67%) predominated, with dominant clone groups being CC8 (50.00%) and CC5 (43.33%). Serotypes were concentrated in Ⅱa (17 strains) and Ⅱb (13 strains), while ST8 (15 strains) and ST1032 (13 strains) were consistently detected across multiple stages. Three highly virulent strains (DZ-25GSA110, DZ-25GSA111, DZ-25GSA105) were all isolated from the harvesting/packaging area, carrying significantly more virulence factors than other strains. Among the 30 strains, erythromycin resistance was the highest (26.67%), ampicillin sensitivity the highest (93.33%), and multidrug resistance the highest (16.67%). cgMLST revealed homologous strains of ST8 and ST1032 in both production and market stages, indicating contamination spread from production to end products.

Conclusion  L. monocytogenes contamination in enoki mushrooms showed no difference in detection rates between production and sales stages, with inoculation and harvesting/packaging being key control points. The ST8/CC8 clone group was the primary genotype for cross-stage transmission. Targeted disinfection and genomic monitoring are recommended.