Immunomagnetic separation (IMS) for Shiga toxin-producing E.

coli

REAGENTS and SUPPLIES: all sterile or autoclaved

Reference Toxin-producing Escherichia coli Target Strains:
O26
O111
O145
O103
O157:H7

Reference Non-target E. coli Strain:

E. coli K12, E. coli 25922, E. coli DH1, E. coli DH5, E. coli HB101.

Growth Medium: Gram-negative (GN) broth

Growth Agar Plate: CT-SMAC (Sorbitol MacConkey agar supplemented with Cefixime and
Tellurite), MIC=WSBM (washed sheep blood), SMAC or CHROMagar for
ID.
Luria-Bertani agar plate (LB agar) for titer (per liter, 10 g bacto-tryptone,
5 g bacto-yeast extract, 5 g NaCl, stir to until mixed, adjust to pH7.0,
bacto-agar 15 g per liter).

Top Agar: Luria-Bertani broth, bacto-agar 6.5 g per liter.

Phosphate Buffered Saline + 0.05% Tween-20 (PBST, Sigma P3563)

Pipets, tips, flasks, the usual glasswares and plastic wares.

Dynabeads (Invitrogen): Paramagnetic beads conjugated with specific antibody, stored at 4°C
with a shelf life 6 months only because of the antibody conjugates.
Anti-E. coli O157 (Invitrogen #71004, 5 ml, $566.00)
EPEC/VTEC O26 (Invitrogen #71013, 2 ml, $308.00)
EPEC/VTEC O111 (Invitrogen #71009, 2 ml, $308.00)
EPEC/VTEC O145 (Invitrogen #71007, 2 ml, $308.00)
EPEC/VTEC O103 (Invitrogen #71011, 2 ml, $308.00)

Table 1 Colony Colony Colony Colony

Appearance on Appearance on Appearance on Appearance on
CT-SMAC Plate SMAC Plate CHROMagar® MIC/WSBM Plate
O157 Non-O157 O157 Plate
O157 Colorless, acidic Colorless, acidic Mauve-black ppt hemolysis
O26 red red Purple magenta Strong hemolysis
O111 red red Mauve-black ppt hemolysis
O145 red red Purple magenta hemolysis
O103 Red, slow growth Red, concave No growth Weak hemolysis
 *Sorbitol MacConkey agar supplemented with Cefixime and Tellurite (CT-SMAC), sorbitol
MacConkey agar (SMAC), CHROMagar® O157-selective (from Invitrogen), MIC agar EHEC
selective. Non-O157 on CHROMagar blue.

Pathatrix: sample tubes, elution/wash tubes, capture phase kits, extra lids.

BeadRetriever: tube strips, tip combs (need autoclave.)

Determination of Overnight Growth Bacterial Titer:

Day 1: E. coli or other bacterial reference strains, start with ATCC vials or 15% glycerol stocks
saved in -70°C freezer, streak out for single colony on an appropriate agar plate.
Incubate overnight at 37°C.
Day 2: Pick up a single colony, inoculate into 2 ml of GN growth medium in glass culture tube
with screw cap, and incubate overnight in a shaking, 37°C incubator. The overnight
culture should have reached plateau and a titer ~10 8.
Day3: Do serial dilutions using 8 culture tubes each containing 0.9 ml of GN growth medium.
Transfer 0.1 ml of the overnight culture to tube 1, mix well. Change to a clean pipet,
transfer 0.1 ml from tube 1 to tube 2, mix well. Repeat for the next 6 tubes.

Add 3 X 0.1 ml of dilutes from tube 4 to tube 7 into sterile 5 ml round-bottom Falcon
tubes (VWR 60819-728). Pipet in 3.5 ml molten (42-50°C) LB top agar per tube, snap on
cap tight, invert to mix, open cap and pour onto pre-labeled and pre-warmed LB agar
plates, 12 in total. Swirl the plate to ensure even distribution of bacteria and top agar.
Let plates sit on bench to until top agar set. Incubate inverted overnight at 37°C.

Day 4: Count colonies from at least 2 X 3 of the serial dilution plates. Factor in the dilution
factors and calculate average for the overnight bacterial culture titer at cfu/ml.

NOTE: Titer needs to be done for all 10 bacterial strains before proceeding to IMS, unless
overnight culture titer was done before.
Stool Matrix Preparation:
Multiple stool specimens that have tested negative for STX 1 and STX2 will be retained. All
specimens will be weighted and combined, mixed thoroughly with 10 volumes (w:v) of PBS (phosphate
buffered saline) to create one uniform stool matrix. The non-sterilized stool matrix will be aliquoted at 6
ml X 60 tubes plus 2 ml X 20 tubes and stored frozen at -80°C for use throughout the validation. Make
sure that 60/20 aliquots are stored before beginning the procedures (double of the minimum numbers
and double for 2/3 mixed beads selection). For the sterilized 1:10 stool matrix, 10 X 400 ml will be
needed. The 1:10 stool matrix means 1:10 dilution with GN broth and incubation overnight, followed by
autoclave. After autoclave, sterilized 1:10 stool matrix will be divided into 400 ml X 10 containers, and
stored frozen at -80°C. In total, 80 grams starting stool specimen will be needed.

Detection Level: E. coli O26, O111, O145, O103 and O157:H7.

The stool matrix for this section will be diluted 1:10 with GN broth, incubated overnight at 37°C
with shaking, then sterilized by autoclave. One 60 ml 1:10 stool matrix will be spiked with one (one
bacterium at a time, all will be done individually) of the above organisms at 10 6 cfu/ml. A dilution series
will be made ranging from 106 to 100 cfu/ml using the 1:10 stool matrix, and used immediately.

The 7 spiked 54 ml samples will be immediately analyzed the same day with and without IMS
(Pathatrix (50 ml), BeadRetriever (3 replicates of 1 ml), and direct plating (3 replicates of 10 l)). The
objective is to test with known quantities of E. coli targets, the lowest limit of recovery by IMS.

Pathatrix:
1. Read the Pathatrix manual ahead. Clean work surface with 70% EtOH.
2. Place 1 sample vessel and 1 elution/wash vessel in the 2-position tube holder, loosening
the lids.
3. Load 50 ml of one enriched sample into the sample vessel and load 35 ml PBS into the
elution/wash vessel, close the lids tightly. Add 50 l of well-suspended beads (OK to
vortex, those are tough polystyrene beads) into the spout on the lid of the sample
vessel.
4. Assemble the capture phase kit to the sample vessel and the elution/wash vessel and
then attach the assembled sample vessel, elution/wash vessel, and capture phase kit
 into a cartridge on the cartridge holder, start at the bottom and move upwards, push in
firmly. Test the magnet assembly. Engage the magnet. Load the assembled cartridge
into Pathatrix
5. Repeat for the next 4 samples, mind cross contamination and change glove in between.
6. START RUN by push the number botton, LED change from red to green. At the end of
run the LED will flash green.
7. One at a time, press the number bottom to initialize the draining step to the specific
cartridge (~1 min. LED red at the end of draining) Pull the cartridge out of Phathrax and
place on the cartridge rack.
8. Expel excess liquid by gently pressing the syringe up to the closed position (I found no
need). Remove the syringe from the cartridge and carefully pull out the rest of the
assembly, starting at the top and working downwards. Do not spill any liquid.
9. Remove the sample vessel, elution/wash vessel, and capture phase kit assembly and
place on the 2-position tube holder.
10. Remove lid from the elution/wash vessel and, leaving only the elution/wash vessel in
the 2-position tube holder, lift away the rest of the assembly and discard as biohazard
waste.
11. Place a clean lid on the elution/wash vessel and place it into the 5-position tube rack
with magnets, let it sit for 1 min to allow the capture of the beads at the bottom.
Remove all liquid from the elution vessel without disturbing the beads using a fine
transfer pipet. (It’s a good idea to finish with a fine gel loading tip to avoid losing beads,
200 ul flat gel tip, 0.17 mm, racks, sterile, USA Scientific 1022-2610) Remove the elution
vessel and place in a rack without magnet.
12. Add 150 l PBS to the beads and resuspend the beads.
13. Repeat for the next 4 cartridges. Then repeat for the next 2 enriched samples.
14. Out of the 150 l beads, 3 X 10 l are used to streak out single colonies per selected
plates, 4 selected plates per organism (table 1). Incubate at 37°C overnight and evaluate
the next day. The leftover 30 l will be stored frozen at -80°C for later PCR quantification
of STX1/STX2 genes.

BeadRetriever
1. Read the BeadRetriever manual ahead. Clean up work surface with 70% EtOH.
2. Insert the 15 labeled tube strips into tube strip tray (the slip ends face left, ridge on left
most tube that matches the notch on tube strip tray, 3 X 5 samples orientation below.)
3. Remove 3 tube strips for one organism to a tube rack 1 meter away (to avoid
contamination), add PBS as in figure below.
4. Add 10 l well suspended beads each into wells 1 and 2 per tube stripe. These are tough
polystyrene beads that can be vortexed.
5. Add samples as in figure below.
6. Return the 3 tube strips to the tube strip tray and proceed to the next 3 tube strips to
until finish.
7. Insert the 3 tip combs into the slots in the immobile comb tray (slip facing front).
8. Slide tube strip tray all the way in. Close both front and top lids of the BeadRetriever.
Power should be ON at this point. Make sure the program is at “EPEC/VTEC”. START and
wait for finish.
9. Out of the 150 l beads, 3 X 10 l are used to streak out single colonies per selected
plates, 4 selected agar plates per organism (table 1 above.) Incubate at 37°C overnight
 and evaluate the next day. The leftover 30 l will be stored frozen at -80°C for later PCR
quantification of STX1/STX2 genes.
10. Two more samples left, process it next.

Direct Plating: From the enriched samples, direct plating 3 X 10 l to one selected plates, 4
selected plates per organism. Incubate at 37°C overnight and evaluate the next day. Keep some
of the enriched samples for PCR later.

NOTE: Plates needed: 105X plates per selected plates per experiment. Start each experiment
with at least 350 ml PBS. Each experiment will process one bacterium enriched with one broth,
therefore expect at least 5 experiments.

POST NOTE 1: After the first experiment of “Detection Limit”, we decided to remove the highest
(106 CFU/ml) and the lowerest (1 CFU/ml) concentrations of the starting E. coli concentrations,
as no usable data can be obtained and not expected for subsequent “Detection Limit”
experiments.

Mixed Beads: Use BeadRetriever only, E. coli targets O26, O111, O145, O103 and O157:H7
This experiment can be done with only one target organism at one experiment. For example, for target
organism A, the beads (A, B, C, D, and E) combinations will be A, AB, AC, AD, AE, ABC, ABD, ABE, ACD,
ACE, ADE, ABCD, ABCE, ACDE, and ABCDE (14 total.) Prepare mixed bead combinations in excess for all
E. coli targets and keep at 4°C. The combinations must contain the target antibody, therefore different
for each target, but the redundant combinations need not be prepared each time. A diagram for the
sample arrangement in BeadRetriever is as below.

The 2 ml stool matrix will be spiked with one (one at a time for one experiment) target organism
3
at 10 cfu/ml, mixed and then added to 18 ml pre-warmed GN broth in a 100 ml flask. The flask will be
incubated overnight in a 37°C shaking incubator to obtain the enriched sample. Next day the enriched
sample will be analyzed with BeadRetriever and direct plating (3 replicates of 10 l). All specimens (14
BeadRetriever + one direct plating) 3 X 10 l will be plated (streak plate) per selected plates, 4 sets of
selected plates per organism. Plates will be incubated overnight at 37°C. Recovery for all mixed bead
combinations will be determined. Leftover enriched samples will be saved for Stx1/Stx2 PCR later.

BeadRetriever: Procedure same as above in Detection Level.

Direct Plating: Procedure same as above in Detection Level.

NOTE: Plates needed: 45X per selected agar plates per target organism (table 1 above). Start
each experiment with at least 50 ml PBS. There will be at least 5 experiments in 5 different days,
each day one organism, and the results should be analyzed for recovery and consistency, using A
(target bead for target organism) as baseline. If the 5 beads combination is not good for any of
the target organism, two combinations of 2 antibodies and 3 antibodies should be selected
based on the results. These two combinations should be used subsequently for for Inclusivity,
Exclusivity, and Competitor Strain tests with Pathatrix, triplicate BeadRetriever, and triplicate
direct plating. If the 5 beads combination is satisfactory for all 5 organisms, the 5 beads mix will
be used for Inclusivity, Exclusivity, and Competitor Strain tests.

If mixed beads is rejected based on this test, for Inclusivity, Exclusivity, and Competitor
Strain tests will be done in target organism against target bead.

Inclusivity: E. coli O26, O111, O145, O103 and O157:H7.

Six milliliter each stool matrix will be spiked with one (one at a time, all 5 strains will be done
individually in one experiment) of the above organisms at 10 3 cfu/ml. (Start from overnight cultures from
single colonies, calculate the volume for 10 3 cfu/ml X 6 ml, do a dilution if necessary.) The stool matrix
will be mixed and added to 54 ml pre-warmed GN broth in a 250 ml flask. The 5 flasks will be incubated
overnight in a 37°C shaking incubator and immediately analyzed the next morning with and without IMS
(Pathatrix (50 ml), BeadRetriever (3 replicates of 1 ml), and direct plating (3 replicates of 10 l)). All
specimens will be streak plated to 3X per selected plates, 3 selected plates per organism (see table 1
above.) Plates will be incubated overnight at 37°C. Recovery for all three methods will be determined.

Pathatrix: Procedure same as above in Detection Level.

BeadRetriever: Procedure same as above in Detection Level.
Direct Plating: Procedure same as above in Detection Level.

NOTE: Plates needed: 21 X per selected agar plates per organism, 5 organisms per day. Start
each experiment with at least 250 ml PBS. The inclusivity tests should be done at least 3 times in
3 different days and the results should be analyzed for recovery and consistency.

This is “if 5 beads combination is selected” or “no mixed beads” is selected. If “2/3 bead
combinations is selected”, 2 rounds of Pathatrix and BeadRetriever will be done in one day for
the 5 organisms, the stool matrix, overnight enrichment cultures, and all reagents and plates will
be doubled.

Exclusivity: 5 additional non-target E.coli strains E. coli K12, E. coli 25922, E. coli DH1, E. coli DH5, E.
coli HB101.
Six milliliter each stool matrix will be spiked with one (one at a time, all 5 strains will be done
individually and in one experiment) of the above organisms at 10 3 cfu/ml. (Start from overnight cultures
from single colonies, calculate the volume for 10 3 cfu/ml X 6 ml, do a dilution if necessary.) The 6 ml
stool matrix will be added to 54 ml of pre-warmed GN broth in a 250 ml flask. The 5 flasks will be
incubated overnight in a shaking 37°C incubator and immediately analyzed the next morning with and
without IMS (Pathatrix (50 ml), BeadRetriever (3 replicates of 1 ml), and direct plating (3 replicates of 10
l)). All specimens will be streak plated to 3X per selected plates, 3 selected plates per organism (see
table 1 above.) Plates will be incubated overnight at 37°C. Recovery for all three methods will be
determined. Save remaining bead samples for PCR later and log the samples to excel sheet.

Pathatrix: Procedure same as above in Detection Level.

BeadRetriever: Procedure same as above in Detection Level.
Direct Plating: Procedure same as above in Detection Level.

NOTE: Plates needed: 21 X per selected agar plates per organism, 5 organisms per day. Start
each experiment with at least 250 ml PBS. The exclusivity experiment should be done 3 times in
3 different days for reproducibility and the results should be analyzed for recovery and
consistency.

This is “if 5 beads combination is selected” or “no mixed beads” is selected. If “2/3 bead
combinations is selected”, 2 rounds of Pathatrix and BeadRetriever will be done in one day for
the 5 organisms, the stool matrix, overnight enrichment cultures, and all reagents and plates will
be doubled.

Competitor Strain: E.coli O157:H7, E.coli 25922

The 6 ml stool matrix will be spiked with one (one at a time, all will be done individually in one
experiment) target organism at 103 cfu/ml and another non-target, for example E.coli 25922, at 104
cfu/ml. The spiked stool matrix will be added to 54 ml pre-warmed GN broth in a 250 ml flask. The flasks
will be incubated overnight in a 37°C shaking incubator. The next morning all 5 enriched samples will be
analyzed with and without IMS (Pathatrix (50 ml), BeadRetriever (3 replicates of 1 ml), and direct plating
(3 replicates of 10 l)). All specimens will be plated (spread plates) 3X 10 l per selected plate, 3
selected plates per organism (see table 1 above.) Recovery for all three methods will be determined. In
addition the ratio of suspect organisms to target organisms will be noted. The competitor strains
selected for each target organism are selected based on morphological differences on SMAC media (see
table 2 below.)

Pathatrix: Procedure same as above in Detection Level.

BeadRetriever: Procedure same as above in Detection Level.
Direct Plating: Procedure same as above in Detection Level.

NOTE: Plates needed: 21 X per selected agar plates per organism + competitor strain
combination, 5 organism + competitor strain combinations per day, and 3 selected agar plates
per combination. Start each experiment with at least 250 ml PBS. The competitor strain test
should be done 3 times in 3 different days for reproducibility and the results should be analyzed
for recovery and consistency.

This is “if 5 beads combination is selected” or “no mixed beads” is selected. If “2/3 bead
combinations is selected”, 2 rounds of Pathatrix and BeadRetriever will be done in one day for
the 5 organisms, the stool matrix, overnight enrichment cultures, and all reagents and plates will
be doubled.

Table 2
Target Strain Competitor Strain
1 E. coli O26 E.coli 25922
2 E. coli O111
3 E. coli O145
4 E. coli O103
5 E. coli O157:H7

 Implementation plan: Implementation at the WAPHL

At the completion of the validation plan the WAPHL will select the method with the best performance
for implementation (if direct plating out-performs IMS methods no changes to current methods will be
made). After method selection is complete, the WAPHL will write the appropriate protocols and train
staff for immediate implementation.