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Title: SUBTYPING LISTERIA MONOCYTOGENES FROM BULK TANK MILK USING AUTOMATED REPETITIVE ELEMENT-BASED PCR

Author
item Van Kessel, Jo Ann
item Karns, Jeffrey
item Gorski, Lisa
item Perdue, Michael

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2005
Publication Date: 12/1/2005
Citation: Van Kessel, J.S., Karns, J.S., Gorski, L.A., Perdue, M.L. 2006. Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR. Journal of Food Protection. 6:2707-2712.

Interpretive Summary: Listeriosis is a food-borne disease that is caused by the bacterium, Listeria monocytogenes. This disease affects approximately 2500 people in the US each year, and, although the case load appears unremarkable, the hospitalization rate for listeriosis sufferers is 92% and the fatality rate is 20%. Although pasteurization is effective at killing L. monocytogenes, dairy products have been implicated in several listeriosis outbreaks. Rapid methods that can readily differentiate between strains of bacteria are needed to identify and track the strains responsible for infection. In a previous study we isolated L. monocytogenes from raw bulk tank milk. L. monocytogenes isolates have traditionally been sub-classified into serogroups based on their cell surface structure. Serotyping is a time consuming, expensive process and the resulting groups are often too broad to enable epidemiological tracking of organisms. Several DNA-based methodologies are available that have been shown to be effective at differentiating between bacterial strains. Our goal in this study was to examine the utility of one of these systems, an automated repetitive element-based PCR (rep-PCR), for serotype grouping of our milk-derived L. monocytogenes isolates, and to determine if specific regional relationships could be identified. Using this system, we compared the DNA fingerprints of 65 L. monocytogenes isolates. Our results indicate that the automated rep-PCR method could not discriminate between two main serogroups (serotypes 1/2b and 4b), but this method has the potential for discriminating between L. monocytogenes 1/2a isolates. The automated rep-PCR used in this study may be useful for differentiating L. monocytogenes isolates within the serogroup 1/2a and for distinguishing these isolates from those of other serogroups.

Technical Abstract: A total of 65 L. monocytogenes strains from raw milk were analyzed using an automated repetitive element-based PCR system to examine the utility of this system for serotype grouping and to determine if specific regional relationships could be identified. Results of the similarity analysis of the L. monocytogenes isolates revealed three primary clusters of isolates. Isolates in Cluster 1 represented serogroups 1/2a, 1/2b, 4b, 3b, and 4c. Cluster 2 exclusively contained serogroup 1/2a isolates, although two 1/2a isolates were also found in each of Clusters 1 and 3. Cluster 3 contained only 4 isolates from serogroups 1/2a (2), 1/2b (1), and 4c (1). Clusters 1 and 2 were separated at a relative similarity of 86% and these two clusters had <65% similarity with Cluster 3. Cluster 3 isolates were shown to be more similar to Cluster 1 and 2 isolates than to L. ivanovii and L. seeligeri, and less similar to Cluster 1 and 2 isolates than L. welshimeri, L. grayi, and L. innocua. When rep-PCR fingerprints of the L. monocytogenes 1/2a isolates were compared, there was no apparent regional grouping. However, discrimination between isolates suggests that this method might have utility in tracking L. monocytogenes 1/2a. This would be useful for tracking isolates across regions or within smaller ecological niches. It appears that the automated rep-PCR method used could not discriminate between serotypes 1/2b and 4b, but this method has the potential for discriminating between L. monocytogenes 1/2a isolates and may be useful for differentiating 1/2a isolates from other serovars and for tracking isolates within this serotype.