Utilization of Pre-enrichment for Improved Sensitivity of an ELISA-based Detection System for Escherichia coli O157:H7

Introduction:  Along with Escherichia coliO157:H7, emerging non-O157 strains (O26, O103, O121, O145, O111, and O45) are now recognized by the Centers for Disease Control and Prevention as major contributors of severe foodborne illness. Recent outbreaks associated with emerging non-O157 strains have heightened the need for their enhanced surveillance. For field deployment, a rapid, robust, sensitive, and user-friendly portable assay for these emerging pathogens is much needed.

Purpose:  This study evaluated the utilization of a pre-enrichment step for increased sensitivity in an ELISA-based detection system, using commercially available antibodies against E. coli O157:H7 for rapid detection in foods in a portable format.

Methods:  Overnight bacterial cultures (n=10) were ten-fold serially diluted in pre-enrichment broth (Universal Pre-enrichment broth) and incubated for 6 h at 37°C prior to analysis with a standard sandwich ELISA. E. coli isolates comprised both O157:H7 isolates (n=4) and non-O157 isolates (n=6), that were evaluated in triplicate with 3 biological replicates. Lowest detection limit of the assay was determined.

Results:  After pre-enrichment, this ELISA assay showed increased sensitivity by two to four-fold from 5.0x10⁴ colony forming units (CFU)/ml to 3.0x10¹ CFU/ml for 2 E. coli O157:H7 isolates. Two E. coli O157:H7 isolates were detected at 4.5x10² CFU/ml and 3.80x10³ CFU/ml.  Three of the 5 non-O157:H7 isolates (O26:H11, O103:H11, and O45:H2) showed detection limits ranging from 3.2 x10² to 72.9 x10³ CFU/ml, and the other three isolates (O145, O111, and O12)  were not detected.

Significance:  These data suggest that pre-enrichment of samples prior to detection with an ELISA-based system is necessary for adequate/improved detection of E. coli O157:H7. Further evaluation and development of these assay components in a lateral-flow luminometer system is ongoing. This simplistic system has potential to further improve the detection sensitivity and specificity for E. coli O157:H7 and the emerging non-O157:H7 strains.