P3-61 Development of an Automated Multiplexed Immunomagnetic Separation System for Isolating Shiga Toxin-producing Escherichia coli

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Laurie Clotilde, U.S. Food and Drug Administration, Alameda, CA
Nicole Herbold, California State University, Hayward, CA
Andrew Lin, U.S. Food and Drug Administration, San Francisco, CA
Clay Bernard, U.S. Department of Agriculture-ARS, Albany, CA
Alexandra Salvador, U.S. Department of Agriculture-ARS, Albany, CA
Carol Lauzon, California State University, Hayward, CA
Mark Muldoon, Romer Lab Technologies, Inc., Newark, DE
Yichun Xu, Romer Lab Technologies, Inc., Newark, DE
John Mark Carter, U.S. Department of Agriculture-ARS, Albany, CA
Introduction: In recent years, non-O157 Shiga toxin-producing Escherichia coli (STEC) have become an emerging problem. 

Purpose: Efforts have been devoted to facilitating and speeding their detection; however, their isolation from high background microbiota foods remains problematic.  To solve this problem, immunomagnetic separation (IMS) is commonly used. 

Methods: Here we describe the development of an automated multiplexed IMS assay capable of isolating E. coli O26, O45, O103, O111, O121, O145 and O157 and E. coli expressing intimin using the KingFisher Flex. 

Results: This platform presents many advantages: 1) high-throughput setup (up to 96 samples simultaneously); 2) programmable (number of washes, temperature, and mixing speed); and 3) automation.  The multiplexed format will save time, labor, reagents, and test sample.  The specificity of the serogroup-specific antibodies used was tested against 240 STEC isolates belonging to 29 serogroups.  Except for 4 strains belonging to the O111 (3) and O157 (1) serogroups, nearly all strains (98.3%) were correctly identified.  Of the 240 strains tested, 161 were genotyped for the attaching-and-effacing gene (eae) and expression of the corresponding intimin was confirmed using HeLa cells.  A total of 78 strains carried eae with 62 expressing intimin and 2 expressing type 1 fimbria, while 83 did not carry the eae with 8 expressing type 1 fimbria and 54 expressed some other adhesin(s).  A total of 7 intimin antibodies were also developed and tested against 12 of those strains. Of these, 4 intimin antibodies showed promising results (> 90% specificity) for inclusion in our assay. 

Significance: Future directions include testing those intimin antibodies against more STEC strains, including other serogroup-specific antibodies, and testing this assay in foods.  Our assay will provide meaningful data for releasing safer foods, thus minimizing annual cost and numbers of recalls, enhancing public health, and allowing utilization of the same standard protocol throughout regulatory agencies in a more user-friendly manner.