bhunia talk 1 multi pathogen screening - Center for Food Safety ...

Funding period: Jan 2003-Feb 2006
Multipathogen screening and /or
confirmation via microarray detection
Arun Bhunia, Mark Morgan, Shu-I Tu
Center for Food Safety and Engineering,
Purdue University
ERRC, Wyndmoor, PA
Primary objective – to develop microarray
based platforms to detect three major
pathogens (Listeria, Salmonella, E. coli)
simultaneously
Focus
1. Nucleic acid –based platform – Applegate (PI)
2. Antibody- based platform – Bhunia (PI)
Team members: BK Hahm (PD), Priya Banada (PD),
Viswaprakash Nanduri (PD), Tao Geng (PD), Amanda
Lathrop (GS), Hyochin Kim (GS), Andrew Gehring (USDA-
ARS, ERRC)
CFSE Annual Review – Oct 12-13, 2006
1
2
Immunomicroarray for multipathogens
Essentials
• Specific antibodies and immune reaction (Objective 1
& 1b)
• Platforms: Fiber optic and 96-well/slide (Objective 2)
• Sample preparation strategy-PEDD (Objective 3)
• Selective growth media (Objective 4)
Objective 1: Antibody and antibody
reaction
a. Antibody Development: Genomic and
proteomic approaches in antibody
development for Listeria, E. coli and
Salmonella.
b. Antigen Expression: Effect of stress
responses (media and environments) on
antigen expression and immunoassays
3
4
Objective 1a: Genomic and proteomic approaches in
antibody development for Listeria, E. coli and
Salmonella
• Search for species-specific
surface exposed amino
acid sequence
• Determine hydropathy
profiles, surface
localization, and antigenic
properties
• Synthesize candidate
peptide (15-20 amino
acid) antigen
• Immunize rabbits and test
for antibody production
Strategies
5’-TCC-CGT-ACT-GAC-ATT-CTC-3’
N’- S - R - T - D - I - L -C’
N’
C’
5
Antibody
E. coli-specific antibodies
Anti-E. coli
O157:H7 PAb
2F11 MAb
ST3229 PAb
ST3230 PAb
ST3236 PAb
ST3238 PAb
Anti-Salmonella
PAb
Anti-Salmonella
CSA-1 PAb
Target
antigen
Anti-E. coli
O157:H7 PAb
LPS
Anti-E. coli PAb Whole cells
Intimin
Salmonella-specific antibodies
LPS
32 kDa
32 kDa
205 kDa
25 kDa
Multiple
proteins
Multiple
proteins
Antibody List
Pathogen
Antibody
specificity
E. coli O157:H7
E. coli
E. coli O157:H7
Sal. Enteritidis
Sal. Typhimurium
Sal. Typhimurium
Sal. Typhimurium
Sal. Typhimurium
Sal. Enteritidis
Sal. Typhimurium
Listeria–specific antibodies
C11E9 MAb
EM-7G1 MAb
EM-6E11 MAb
P66 PAb
MAb-EM10
Anti-Listeria
PAb
Lm404 PAb
Lm407 PAb
LmC639 PAb
Target
antigen
66 kDa
66 kDa
97 and 43 kDa
66 kDa
104 kDa
68, 62, 58, 50, 43
and 30 kDa
63 kDa InlB
52 kDa
90 kDa ActA
Pathogen
specificity
L. mono, L.
innocua
L. mono
Listeria spp
Listeria spp
Listeria spp
Listeria spp
L. mono
Listeria spp
L. mono
6
1

Objective 1b: Effect of stress responses
on antigen expression and immunoassays
Motivation: As biosensors are becoming increasingly
sensitive, a slight reductions in immune reactions due to
“cellular or environmental factors” will result in “false” results
Effect of selective enrichment media on InlB and
ActA expression for L. mono –specific antibodies
BHI
V7 (1/2a)
Scott A (4b)
MurrayB (4ab)
ATCC 19114 (4a)
SLCC 2479 (3c)
ATCC 7644 (1/2c)
F4233 (1/2b)
ATCC 2540 (3b)
ATCC 19117 (4d)
ATCC 19118 (4e)
ATCC 19116 (4c)
SLCC 2482 (7)
SLCC 2373 (3a)
BHI
V7 (1/2a)
Scott A (4b)
MurrayB (4ab)
ATCC 19114 (4a)
SLCC 2479 (3c)
ATCC 7644 (1/2c)
F4233 (1/2b)
ATCC 2540 (3b)
ATCC 19117 (4d)
ATCC 19118 (4e)
ATCC 19116 (4c)
SLCC 2482 (7)
SLCC 2373 (3a)
• Effect of selective enrichment broths on InlB,
ActA and several unknown antigens in L.
monocytogenes
• Effect of environmental stresses on antibody
based detection of E. coli O157:H7, S. enterica
and L. monocytogenes
7
LB
BLEB
UVM No expression observed
FB
anti-InlB PAb (Lm404)
Lathrop et al 2006 (Submitted)
LB
BLEB
UVM
FB
anti-ActA PAb (LmC369)
8
RT-PCR ELISA
OD490
OD490
M
F4244 (4b)
F4244-ActA
2
1.5
1
0.5
0
LB BHI BLEB UVM FB
2
F4244-InlB
1.5
1
0.5
0
LB BHI BLEB UVM FB
actA inlB 16S
LB
BHI
BLEB
UVM
FB
LB
BHI
BLEB
UVM
FB
LB
BHI
BLEB
UVM
FB
No temp ctrl
M
~90 bp
OD490
OD490
M
F4233 (1/2b)
1.5
F4233-ActA
1
0.5
0
LB BHI BLEB UVM FB
2 F4233-InlB
1.5
1
0.5
0
LB BHI BLEB UVM FB
actA inlB 16S
LB
BHI
BLEB
UVM
FB
LB
BHI
BLEB
UVM
FB
LB
BHI
BLEB
UVM
FB
9
M
Transmission electron microscopic (TEM) analysis of InlB
and ActA expression in different media
LB
BLEB
PAb
Lm404
PAb
LmC369
(a) InlB
(c) ActA
(b) InlB
(d) ActA
InlB
ActA
10
Effect of enrichment broth on antigen
expression for a Listeria specific PAb
Protein Identity
Calculated
Gene name
molecular mass
Putative LPXTG-motif cell wall
65 kDa
anchor domain protein
Lmo 0610 62,841.1 kDa
58 kDa
Flavocytochrome C Fumarate
Reductase chain A homolog
Lmo 0355 54,462.4 kDa
eno
50 kDa Enolase 46,472.4 kDa
(Lmo 2455)
45 kDa
Glyceraldehyde 3-phosphate gap
36,286.2 kDa
dehydrogenase (Lmo 2459)
35 kDa
Putative phosphosugar binding protein
Hypothetical regulator
Lmo 0041 30,113.7 kDa
Effect of environmental stresses on antibody
based detection of E. coli O157:H7, S. enterica
and L. monocytogenes
Stress conditions investigated
• Heat 45 o C; Cold (4 o C); Acid (pH 5.0); Salt
(NaCl- 5.5%), oxidative stress (H 2 O 2 ;15 mM,
ethanol (5%)
Duration
• Short-term (3h)
• Long term (>6 h)
Geng et al. 2006. J. Food Prot. 69(8): 1879-1886
Hahm and Bhunia. 2006. J. Appl. Microbiol. 100:1017-1027
Lm PAb 11
12
2

Western blot analysis of antigen expression with
long-term combined stress
60 kDa
36 kDa
30 kDa
25 kDa
_
_
_
_
Sal. Enteritidis PAb (our lab)
37°C, no stress
12°C, pH 5.5,
3.5% NaCl
4°C, pH 5.5,
3.5% NaCl
6 24 120 6 24 120 6 24 120 h
68 kDa
62 kDa
58 kDa
50 kDa
43 kDa
30 kDa
L. monocytogenes PAb (our lab)
_
_
_
_
_
_
37°C, no stress
12°C, pH 5.5,
3.5% NaCl
6 24 120 6 24 120
4°C, pH 5.5,
3.5% NaCl
6 24
120 h
ELISA490+SD
2.5
2.0
1.5
1.0
0.5
0.0
Examination of antibody reactions with bacteria
grown in hotdogs in the presence of stress
E.coliO 157:H7
PAb (KPL)
ELISA analysis
Sal.C SA-1 PAb
(KPL)
37`C,no stress
Sal.Enteritidis
PAb (ourlab)
12`C,pH 5.5,NaCl3.5%
L.
monocytogenes
PAb (ourlab)
60 kDa
_
36 kDa _
30 kDa _
25 kDa
_
Western blot analysis
Control
Stressed
Control
→
E. coli
O157:H
7 PAb
(KPL)
Stressed
→
Control
→
Stressed
Sal. Sal.
CSA-1 Enteriti
PAb dis PAb
(KPL) (our lab)
Control
Stressed
68 kDa
62 kDa
58 kDa
50 kDa
43 kDa
30 kDa
L.
monocytogenes
PAb (our lab)
_
_
_
_
_
_
13
14
Summary: ELISA response to environmental stress
Antibody
E. coli O157:H7 PAb
(KPL)
Sal . CSA-1 PAb
(KPL)
Sal . Enteritidis PAb
L. monocytogenes PAb
ELISA response
Stress
(Compared to
control -37C)
4°C 16% ↓
Short-term (3 h)
45°C 12% ↓
5.5% NaCl 15% ↓
Long-term and combined 4°C up to 6 h 13% ↓
(pH 5.5, 3.5% NaCl) 12°C up to 6 h 33% ↓
Short-term (3 h) 4°C 34% ↓
Long-term and combined
(pH 5.5, 3.5% NaCl)
Short-term (3 h)
Long-term and combined
(pH 5.5, 3.5% NaCl)
Short-term (3 h)
Long-term and combined
(pH 5.5, 3.5% NaCl)
4°C or 12°C up to 120
h
13-40% ↓
4°C 48% ↓
45°C 37% ↓
5.5% NaCl 30% ↓
15 mM H2O2 20% ↓
4°C up to 120 h 32% ↓
pH 5.5
44% ↑
5.5% NaCl 100% ↑
5% ethanol 53% ↑
45°C 53% ↑
4°C up to 120 h 40% ↓
4°C or 12°C up to 24 h 8-30% ↑
15
Objective 2: Fiber optic sensor for food
pathogens
• Listeria monocytogenes
• E. coli O157:H7
• Salmonella Enteritidis
16
Fiber optic sensor for pathogen detection
Detector
FO based detection of L. monocytogenes from
inoculated meat samples – after 20 h-enrichment
F
Fluorescent
molecules
Laser detector
Fiber
laser
Evanescent wave
Signal (pA
2500
2000
1500
1000
Bologna
Hotdog
(1) (2) (3)
500
top middle bottom
0
10^1 inoculum 10^2 inoculum 10^3 inoculum neg control pos control
17
Geng et al. 2004 Appl. Environ. Microbiol. 70:6138-6146
18
3

L. monocytogenes detection using RAPTOR
Detection of cells with fiber optic sensor grown in
BHI or EC at 2 h intervals
Continous detection of E. coli O157:H7 grown in BHI, EC or GB at 2 h interval
Concentration of E. coli grown in BHI
1.0E+08
3000
Concentration of E. coli grown in EC
10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 5 10 6 10 7 10 8 10 9 Concentration of E. coli grown in GB w/ EC
1.0E+07
250
250
LM PAb (0.01mg/ml)
400
400
2500
L. monocytogenes
1.0E+06
LM PAb (0.02mg/ml)
200
LM P66 (0.01mg/ml)
200
Antibody devoid
350 E. fecalis
350
LM P66 (0.02mg/ml)
2000
1.0E+05
L. rhamnosus
150
150
300
300
1.0E+04
1500
250
250
100
100
1.0E+03
200
200
1000
50
50
1.0E+02
150
150
500
0
0
1.0E+01
100
100
-50
-50
50
50
0
1.0E+00
0 2 4 6 8
0
0
-100
-100
Time (hours)
10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10
10 5 10 6 10 7 10 8 10 9
Listeria monocytogenes,cfu/ml
CONCENTRATION OF BACTERIA, cfu/ml
PBS Hotdog
Geng et al 2006. Sensors 6:796-807
19
20
Nanduri et al 2006. Sensors 6:808-822
Signal,pA
coupon
RAPTOR
SIGNAL,pA
Signals (pA)
4000
Signals from E. coli grown in BHI
1.0E+10
Signals from E. coli grown in EC
1.0E+09
3500
Signals from E. coli grown in GB w/ EC
E. coli concentration
(CFU/ml)
Summary: Fiber optic sensor for food
pathogens
Objective 3. Sample preparation strategy
for use with microarray platforms
Pathogen
Listeria
monocytogenes
E. coli O157: H7
SLT
Salmonella
Detection limit Detection
time
10 3 cfu/ml
23 h
10 4 -10 5 cfu/ml meat
10 3 cfu/ml meat
0.5 ug/ml
10 4 cfu/ml
4-6 h
Publications
•Geng et al. 2004. AEM. 70:
6138
•Nanduri et al 2006. Sensors,
6:808
•Kim et al. 2006. Key Eng.
Materials
•Geng et al. 2006. Sensors,
6:796
•Tu et al. 2006. SPIE
•Morgan et al. 2006. Key
Eng. Materials
21
• Pathogen enrichment
detection device
(PEDD)
• Self-contained, easyto-use
device to
deliver particle–free
and pH adjusted
bacterial suspension
22
Performance evaluation of
PEDD with food sample
Growth of pathogens spiked onto food sample
in PEDD
Pathogen
Food
sample
Media
Growth Detection
10
E. coli O157:H7 Sal. Typhimurium L. monocytogenes
12
10
E. coli O157:H7
Ground
beef
mEC +
novobiocin
CT-SMAC
Log CFU/m
8
6
4
Log CFU/m
10
8
6
4
Log CFU/m
8
6
4
Salmonella
Typhimurium
Egg
RV
XLD
2
0
0 4 8 12 16 20 24
Time (h)
2
0
0 4 8 12 16 20 24
Time (h)
2
0
0 4 8 12 16 20 24
Time (h)
Listeria
monocytogenes
Hotdog
BLEB
MOX
PEDD 1 CFU/ml PEDD 10 CFU/ml PEDD 100 CFU/ml
Bag 1 CFU/ml Bag 10 CFU/ml Bag 100 CFU/ml
23
24
4

Lateral flow
immunoassay of L.
monocytogenes
retrieved from
PEDD
PEDD
0 1 10 100 CFU/g
PEDD
BAG
E. coli O157:H7
0 1 10 100 CFU/g
Salmonella Typhimurium
0 1 10 100 CFU/g
8 h incubation
PEDD
BAG
BAG
8 h incubation
25
10 h incubation
26
After
Before
PCR tube
PCR analysis E. coli O157:H7 culture before
or after passing through the cartridge
8 h 10 h 12 h
after before after before after before
M 0 1 10 100 0 1 10 100 0 1 10 100 0 1 10 100 0 1 10 100 0 1 10 100
M : 100 bp DNA ladder
0, 1, 10, 100: CFU/ml of inoculation
PCR mix
27
eaeA
stx2
stx1
Objective 4: Formulation of selective
universal enrichment broth for
Salmonella, E. coli and Listeria (SEL)
• Media is called “SEL – Salmonella, E. coli
and Listeria
• Base – Buffered Listeria Enrichment Broth
(BLEB) with acriflavin, cyclohexamide,
fosfomycin, nalidixic acid)
• Media performance will be addressed in our
next project.
Hyochin Kim 2006
28
Multipathogen Screening
Fiber optic
Sensor
Test sample
Enrichment in PEDD using universal
selective enrichment broth (eg., SEL media)
Microarray
ELISA
29
Need work!
Conclusions
• Specific antibodies are available for
immunomicroarray assay
• Stress and media affect antibody reaction either by
affecting transcription, translation or translocation
• Data with physiologically stressed bacterial cells would
be helpful in formulating assay procedure for optimum
signal
• Fiber optic sensor data are promising and multiplex
assay could be developed using the automated system-
RAPTOR
• PEDD system removes particles and inhibitors from
food and should be useful for detection by biosensor
platforms
30
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