Tracing of persistent Listeria monocytogenes contamination in ewe's milk farm

Ewe's milk farm production is permanently associated with the risk of contamination by pathogenic bacteria, including Listeria monocytogenes. In the present study, the prevalence and diversity of L. monocytogenes strains repeatedly isolated from tank ewe's milk and the milking environment on a farm in Slovakia during a prolonged period were investigated to identify the source of potentially persistent contamination. A total of 140 samples along the milk production chain were collected during an 18-month period. From all these samples, 45 samples were found L. monocytogenes positive with 90.3% positivity of tank milk samples (28 positive samples from 31 analysed). Pulsed-field gel electrophoresis profiling resulted in strain discrimination into six profiles with one pulsotype (NS1) corresponding to MLST-ST14 being predominant. A total of 17 proportionally selected L. monocytogenes isolates, including 11 NS1/ST14 isolates, were subjected to whole genome sequencing. Resulted data were used to compare the genomes diversity and to confirm the persistent contamination when <10 allelic differences threshold in cgMLST analysis was applied. The source of persistent contamination was localized inside the milking apparatus, probably in shelters that were very difficult to clean. Despite great efforts, the ewe's milk contamination could not be eliminated during the reporting period. Significance and impact of the study Listeria monocytogenes is a serious pathogenic contaminant of the food-processing environment with a potential to colonize food-production facilities. In this study, molecular analysis was used to help a ewe's milk producer combat long-term contamination of milk by these bacteria. Pulsed-field gel electrophoreses profiling of L. monocytogenes isolates as well as analysis of whole genome sequencing (WGS) data revealed persistent contamination by a single strain. The study demonstrated that progressive molecular methods, in particularWGS, can help to improve L. monocytogenes control programmes in the food production chain to prevent the contamination of food products.