Understanding Non-Confirmable Presumptives in Environmental Listeria Testing
Erica Miller 1, Emily Schmitt 2, Andrzej A. Benkowski 2, Alex Angel 1, Luke Anderson 1, Dustin DeLoach 1, Qinwei Lu 1, Daniel DeMarco 1, J. David Legan 2, and Christopher Crowe 3
Eurofins Microbiology Laboratories, Inc.: 1 Louisville, KY; 2 Madison, WI, 3 Salinas, CA
ABSTRACT
Introduction
Molecular methods have revolutionized pathogen testing, but samples that test presumptive positive are typically confirmed using cultural methods. As the sensitivity of molecular methods improve, the frequency of presumptive results that fail to culturally confirm has increased, though the factors underlying these lower confirmation rates are poorly defined or quantified.
Purpose
This study was designed to determine the main cause(s) of presumptive Listeria spp. environmental samples that fail to confirm by culture.
Methods
A total of 634 environmental sponges submitted over a 5 month period were tested for Listeria spp. using commercial real-time PCR and end-point assays. DNase treatment was employed prior to cell lysis to eliminate free DNA and reduce the possibility of false positives from dead cell detection. All presumptive positive samples were confirmed by the FDA-BAM culture method. Samples that did not confirm using the standard BAM method were subjected to further analysis, including a secondary enrichment, additional plating media and increased plating volumes. Enrichments were also sequenced to verify if Listeria was present.
Results
A total of 66 samples were presumptive for Listeria spp. by one or both of the PCR platforms used. Of these 66 presumptive samples, 15 were unable to be confirmed by standard culture methods. The addition of secondary enrichments, plating on different selective and differential agars, and increased plating volumes all failed to recover Listeria in any of these 15 samples. Whole genome sequencing of the enrichments demonstrated that Listeria DNA was present in all 15 samples. The use of Dnase prior to lysing each of these samples, suggests that viable Listeria was present in the samples, despite the cultural methods failing to recover the organism.
Significance
These data suggest that the results of molecular methods for Listeria spp. testing may be more reliable than cultural methods.
View the poster
Questions on how this information is applicable to your food process?
