There is substantial need for the rapid and efficient detection and quantification of Escherichia coli and other fecal coliforms in shellfish intended for human consumption. Outbreaks of diarrhea, hemorrhagic colitis, and Hemolytic Uremic Syndrome (HUS) in humans have been associated with E. coli, most commonly the O157:H7 strain due to its production of pathogenic Shiga-toxins. The current FDA-approved most probable number (MPN) method used for quantifying pathogenic E. coli in shellfish is outdated and can take up to four days to complete. In this project, two triplex quantitative Polymerase Chain Reaction (qPCR) assays were developed using previously published primers and probes. These assays were intended to be combined with the overnight enrichment from the standard FDA MPN method to streamline the detection and quantification of the pathogenic O157:H7 E. coli strain.
This qPCR-MPN method was first tested with shellfish collected from both contaminated and uncontaminated sites on Cape Cod, MA; however, no O157:H7 was identified by qPCR or by plating on O157:H7-specific MacConkey Agar Medium with Sorbitol. To troubleshoot and validate the assays, a spiking experiment using known amounts of O157:H7 E. coli cultures and MPN-qPCR analysis was conducted as follows (Fig. 1). Sets of 10 oysters were sterilely shucked and homogenized. A 1:10 dilution series was created, then used to spike oyster homogenates before inoculation of the 10-mL lauryl sulfate tryptose broth (LST) tubes. Following an overnight enrichment, gas-positive LST tubes were presumed positive for coliforms and used to inoculate 8-mL E. coli media (EC) tubes to confirm the presence of E. coli. Following another overnight enrichment, gas-positive EC tubes were presumed positive for fecal coliforms. DNA was extracted from both LST and EC positive tubes and run in the newly designed qPCR assays. Positive results from qPCR were compared with MPN positive results with the goal of eliminating the EC media step, saving up to two days.