Food Protection Trends - May/June 2018 - 179

O45, O121, O111, and O145), commonly known as the
"big six," as adulterants in non-intact beef products in
1994 and 2012, respectively (27). Subsequently, FSIS
implemented routine testing for the regulated STEC
serogroups, particularly in ground beef and other non-intact beef products (26, 27). Although use of molecular
methods such as PCR is very common for STEC detection, isolation of viable colonies is frequently carried out
using conventional techniques and is required to confirm
positive results.
The ability to isolate presumptive STEC colonies is
perhaps the primary challenge with STEC methodology,
particularly when low numbers of microorganisms are
present or when high concentrations of background
microflora exist in the sample. One of the techniques
most commonly used to facilitate STEC isolation is IMS,
a method regarded as highly effective, even when used
with samples having a high background microflora (7).
Hence, the technique is part of the FSIS STEC detection
and isolation protocol once potential positive results are
obtained via PCR (28). IMS recovers target cells from
the enrichment cultures using paramagnetic beads coated
with polyclonal antibodies specific for STEC serogroups;
the bead size ranges from 1 to 4 μm in diameter, with an
average of 2.5 μm. Beads are mixed with the enrichment,
incubated to allow binding to the cell-surface antigen, and
subjected to repeated rinsing to remove non-specifically
attached cells. An antibody-antigen complex is formed,
and a magnetic field is used to capture and recover target
cells (21). Magnetic beads labeled with antibodies for
O157 and the big six non-O157 STEC are commercially
available (1, 24). Because IMS efficiency may vary from
serogroup to serogroup, the focus of this study is to
evaluate differences in cell recovery for each serogroup.
The IMS method is efficient for bacterial cell recovery
when the appropriate enrichment conditions are provided to the microorganisms (8). Chapman et al. (1994)
indicated that IMS is more sensitive than direct culture
on selective agar and increases about 100-fold the ability
to recover E. coli O157 from artificially inoculated bovine
fecal samples (6). Despite this, the technique works more
efficiently when higher bacterial loads of the microorganisms are present in the sample (11); hence, magnetic bead
manufacturers recommend that a concentration greater
than 100 cells per ml be present for STEC detection. This
implies that lower bacterial concentrations in the sample
may lead to false negative results.
The goals of this study were to evaluate the differences
in capture efficiency of the magnetic beads for each of
the regulated STEC serogroups (O157 and the big six
non-O157), and to evaluate if the detection limits of IMS
per serogroup is affected by modifying the bead volume
used to perform the technique.

MATERIALS AND METHODS
Bacterial strains
Strains used for this study originated from human and
animal sources (Table 1). Strains were maintained frozen
at -80°C in tryptic soy broth (TSB - EMD Millipore
Chemicals; Darmstadt, Germany) with 10% glycerol. A 10
µl aliquot was transferred from the stock culture into a 9-ml
TSB tube and incubated for 24 h at 37°C; a subsequent
transfer of 10 µl into fresh TSB was performed.
Immunomagnetic separation
Antibody-coated magnetic microspheres were used to
perform IMS. For STEC serotypes O157, O26, O103, O111
and O145, commercially available paramagnetic beads were
obtained from one manufacturer (Dynabeads®, Invitrogen;
Carlsbad, CA), while paramagnetic beads for serotypes O121
and O45 were obtained from a different supplier (Abraxis;
Warminster, PA). IMS was conducted using an automated
system (BeadRetriever™. Invitrogen; Carlsbad, CA) that
captures the target microorganisms bound to antibody-coated
magnetic beads by utilizing a pre-programmed magnetic bead
processor. Instructions provided by the manufacturer of the
bead retriever were followed, and the same procedure was
applied to all serogroups as recommended by the bead suppliers.
After the IMS process was completed, 50 µl of the bacteriabead complex was plated onto tryptic soy agar (TSA. EMD
Millipore Chemicals; Darmstadt, Germany) or mRainbow
Agar (mRBA, Rainbow agar USDA recipe, MLG appendix
1.08), and incubated for 24 h at 37°C. Recovered colonies were
always confirmed using latex agglutination tests specific to each
O group, (Oxoid Ltd., Hants, UK) for the STEC O157, O26,
O103, O111 and O145, and Abraxis (Warminster, PA) for O121
and O45). Total STEC concentration (CFU/ml) recovered
after IMS from each sample was estimated by enumeration of
colonies grown on the respective solid media used.
Sample preparation to conduct IMS
Cultures in enrichment broth
For each STEC serogroup (O157, O26, O103, O111,
O145, O121, and O45), five-strain bacterial cocktails were
prepared separately by combining 2-ml aliquots of each strain
and homogenizing with a vortex mixer for about 5 sec. Serial
dilutions were prepared in 9-ml buffered peptone water
(BPW. EMD Millipore Chemicals; Darmstadt, Germany)
to achieve the desired final culture concentrations of 1.0,
2.0, 3.0, 4.0, or 5.0 log CFU/ml. Bacterial concentrations in
the sample subjected to IMS were confirmed during each
repetition by plating on TSA and incubating for 24 h at 37°C.
Colonies were enumerated and reported as CFU/ml. Log
CFU/ml conversion was done as needed.
Ground beef
A five-strain cocktail of E. coli O157:H7 was prepared as
previously described. The bacterial cocktail was centrifuged

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