P3-08 Simultaneous Immunomagnetic Separation (IMS) of Five Escherichia coli STEC O-groups with Subsequent Differentiation Using Modified Rainbow Agar

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Alexandra Calle, Texas Tech University, Lubbock, TX
Matthew Sellers, Texas Tech University, Lubbock, TX
Guy H. Loneragan, Texas Tech University, Lubbock, TX
Mindy M. Brashears, Texas Tech University, Lubbock, TX
Introduction: The USDA FSIS regulation establishes 7 STEC strains to be adulterants in non-intact beef products. The FSIS testing protocols for STECs rely mostly on the use of PCR-based methods. Unfortunately, they are not the most practical approach, especially when isolation and enumeration of the microorganisms is required.

Purpose: To 1) determine whether magnetic beads available for five Escherichia coli O groups (O157, O26, O145, O111, and O103) can be combined in a single IMS assay, and 2) evaluate if O group differentiation is possible using a chromogenic agar.

Methods: A cocktail of the five O groups was prepared by incubating each strain in tryptic soy broth for ca. 18 h at 37°C, combining the strains in equal aliquots and performing serial dilutions to achieve final culture concentrations of ca. 3 and 4 log CFU/ml. Each concentration of cocktail was subjected to IMS using two levels (20 and 10ml of magnetic beads with beads of the five O groups combined for a single IMS. Recovered cells were plated on modified Rainbow agar and incubated for ca. 24 h at 37°C. Colonies were confirmed using latex agglutination and counted to evaluate the ability to recover all O groups simultaneously on one plate. 

Results: Statistical analysis revealed no significant difference (P > 0.005) between the two volumes of beads at any of the bacterial concentrations tested.  Difference between the cells recovered by each microorganism was found (P< 0.05); however, serogroups O157, O26, O145, and O103 were recovered as expected based on IMS detection limits, and O111 did not grow at expected concentrations.

Significance: This study revealed that a simultaneous IMS could be conducted to recover E. coli O157, O26, O145, and O103 at once using a selective media that allows for O group differentiation. Because the amount of beads can be reduced and less time is required this could be considered as a cost and time efficient alternative to isolation of STECs one strain at a time.