Immunomagnetic separation IMS of E' coli O157

1
Immunomagnetic separation (IMS) of E. coli O157
Objective - to develop an IMS procedure for
sensitive detection of E. coli O157
2
Principle of IMS

• IMS low volumes of sample ? low sensitivity of
  detection
• (e.g. 20 cfu/ml in PBS)

3
One way flow through IMS(1 way FTIMS)
-to increase the sensitivity ? increase the
volume processed and the conc. of magnetic
beads ?optimise the protocol
4
Modelling the IMS optimal parameters
Trajectories of particles to evaluate the size
of the system
The size of the system as function of various
parametrs field strength, Flow velocity, size of
particles
It is advantageous to use high and non-uniform
magnetic fields (650 kA/m) at moderate flow
velocities (2-2.5 cm/s) ?small IMS systems (3-5
cm length)
5
Recovery of magnetic beadsas function of field
strength(experimental)

• - 96 recovery from PBS at 640 kA/m
• 89 recovery from beef mince homogenate
• Flow rate (Q 7 ml/min)
• 93.5 from 1 ml by using Standard Dynal IMS

6
(4 ways FTIMS)
7
Magnetic field pattern
8
Recovery of magnetic beads- experimental device-
9
Recovery of magnetic beads as function of flow
rate

• - 98 recovery in PBS
• 97 recovery in beef
• mince homogenate
• 93.5 recovery 1ml Standard Dynal
• (flow rate Q 7 ml/min)

10
Recovery of E. coli O157 from PBS
250 ml FTIMS 3300 cfu/ml -0.02 ml magnetic
beads -30 min mixing 150 rev/min
1ml Dynal 3300 cfu/ml
FTIMS works OK with high concentrations of E.
coli O157 At low concentrations of bugs ?low
sensitivity ?optimisation
11
Sensitivity at low concentrations of E. coli
O157 in PBS
1ml Standard Dynal At 95 -recovery Sensitivity
20 cfu/ml Confidence interval12-30 cfu/ml
50 ml FTIMS 0.05 ml beads 30 min mixing 300
rev/min At 95 -recovery Sensitivity 1.7
cfu/ml Confidence interval0.8-3 cfu/ml
50 ml PBS sol. FTIMS 12 fold better sensitivity
than 1ml St. Dynal
12
Number of E. coli O157 recovered from PBS
Between 70 to 113 fold more colonies are detected
by using FTIMS system than 1ml Standard Dynal
system decimal dillution (-6 and -5)
13
Recovery of E. coli O157 from beef mince
homogenates (sensitivity)
1ml Standard Dynal At 95 -recovery Sensitivity
0.60 cfu/g Confidence interval0.12-1.12 cfu/g
50 ml FTIMS 0.05 ml beads 30 min mixing 150
rev/min At 95 -recovery Sensitivity 0.14
cfu/g Confidence interval0.08-0.2 cfu/g
50 ml FTIMS (4 hrs incub.) 4.3 fold better
sensitivity than 1ml St. Dynal (6 hrs incub.).
FTIMS can detect low level contaminants (gt3-4
cfu/sample) 1 ml Dynal system (sensitive at gt15
cfu/sample )
14
Number of E. coli O157 recovered from beef mince
homogenates
Between 82 fold more colonies are detected by
using the 50 ml FTIMS system than 1ml Standard
Dynal system concentration (0.3 cfu/g)
15
Future work

• Further improvements are required to enable
  routine
• application in microbiology laboratories (e.g.
  sterilisation
• between samples)
• Testing the system for
• the recovery of E. coli O157 from other sample
• matrixes (clinical, soil, fecal and
  environmental samples )
• the concentration of other pathogenic
  micro-organisms
• (Salmonella, Listeria, Cryptosporidium,
  Campylobacter
• and Vibrio spp)
• -negative separation