Comparison of IMS Platforms for detecting and recovering Escherichia coli O157 and Shigella flexneri in foods.

Two automated platforms using immunomagnetic separation technology were compared for detecting and recovering Escherichia coli O157 in ground beef and sprouts and Shigella flexneri in green onions. The foods were inoculated with <20 CFU/25 g and tested at 5 and 24 h postincubation. Immunomagnetic beads were mixed with food enrichments, processed through the Pathatrix Auto or KingFisher Flex, and tested by real-time PCR (qPCR) and recovery on selective agars. At 5 h, the Pathatrix Auto detected E. coli O157 in 90% and 60% of the ground beef and sprouts samples and S. flexneri in all of the green onion samples. It also recovered E. coli O157 in all the samples but could not recover S. flexneri in any of the green onion samples. In comparison, the KingFisher Flex detected E. coli O157 in 80% and 30% of the ground beef and sprouts samples and S. flexneri in all of the green onion samples. It also recovered E. coli O157 in 90% of the ground beef samples but none of the sprouts samples and S. flexneri in 20% of the green onion samples. At 24 h, both platforms detected and recovered the target bacteria in all of the samples.

Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods.

Shiga toxin-producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx(1)] and 2 [Stx(2)]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx(1) and/or Stx(2). The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with ≤2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to simultaneously detect additional foodborne pathogens and their toxic markers.