A Listeria Test Method Requiring Only a 12-Hour Enrichment in Leafy Green Matrices
Erica Miller 1, Rafael Davila 2, Alexa Grace Baldwin 3, and Christopher Crowe 2
Eurofins Microbiology Laboratories, Inc.: 1 Louisville, KY; 2 Salinas, CA; 3 Pathotrak, College Park, MD
ABSTRACT
Introduction
Recent outbreaks of Listeria monocytogenes in leafy greens have highlighted the need for Listeria product testing for these matrices. Because Listeria is a slow growing pathogen, commercial test methods take up to 24 hours or more, causing supply chain difficulties and issues getting product on the market with sufficient shelf life.
Purpose
To validate enrichment concentration as a method for shortening enrichment times to 12 hours for Listeria testing in leafy greens.
Methods
Twenty samples (125g) of romaine lettuce were inoculated with L. monocytogenes at 0.38 CFU/sample, a level designed to result in recovery from only a fraction of the samples. An additional five samples were inoculated at a level one log higher, and five samples were used as uninoculated controls. All samples were enriched in 250mL of pre warmed BACGro ULTRA™ Listeria Broth (BULB, Gold Standard Diagnostics) and incubated at 37°C for 12 hours. Following incubation, enrichments were concentrated using the Pathotrak™ concentration system. Concentrated samples were tested for both L. monocytogenes and Listeria spp. using BACGene real time PCR kits according to manufacturer’s instructions. Six additional produce matrices broccoli, green cabbage, iceberg lettuce, kale, spinach, and Mediterranean salad mix were also verified by inoculation of seven replicates with <10 CFU/sample and enriching and testing samples as described above.
Results
The romaine samples inoculated at a fractional level with L. monocytogenes demonstrated detection by both L. monocytogenes and Listeria spp. PCR in 7/20 replicates. All five romaine samples inoculated at a higher level were positive by both PCR kits, and all negative controls showed no detection. For
the remaining six matrices, all replicates showed detection of both L. monocytogenes and Listeria spp. All PCR results that were culturally confirmed agreed with cultural confirmation.
Significance
These data demonstrate a method to reduce Listeria enrichment times to 12 hours using enrichment concentration prior to PCR.
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