Enhanced Detection of Listeria spp. in Farmstead Cheese Processing Environments through Dual Primary Enrichment, PCR, and Molecular Subtyping

Abstract

The incidence and ecology of Listeria spp. in farmstead cheese processing environments were assessed through environmental sampling conducted in nine different plants over a 10-week period. Environmental samples (n = 705) were examined for the presence of Listeria spp. by using three detection/isolation protocols. The use of dual enrichment methods, which allowed for the recovery of injured Listeria spp. (mUSDA), identified more Listeria species–positive samples with higher sensitivity than the standard USDA method. The addition of PCR to the mUSDA method identified the most Listeria monocytogenes–positive samples, achieving greater sensitivity of detection while substantially reducing time. Overall, 7.5% of samples were positive for Listeria spp., yielding 710 isolates, 253 of which were subtyped by automated ribotyping to examine strain diversity within and between plants over time. The isolation of specific ribotypes did not appear to be affected by the enrichment protocol used. Fifteen (2.1%) samples yielded L. monocytogenes isolates differentiated almost equally into ribotypes of lineages I and II. Of most concern was the persistent and widespread contamination of a plant with L. monocytogenes DUP-1042B, a ribotype previously associated with multiple outbreaks of listeriosis. Our results suggest that the extent of contamination by Listeria spp., notably L. monocytogenes, in farmstead cheese plants is comparatively low, especially for those with on-site farms. The results of this study also identified points of control for use in designing more effective Listeria spp. control and monitoring programs with a focus on ribotypes of epidemiological significance.