Transcript Rapid detction and Identification of Campylobacter and

Rapid detection and Identification of
Campylobacter and Arcobacter species
Marwan Abu-Halaweh
Phylogeny
• Campylobacter and Arcobacter are microaerophiles and member of
the order Campylobacterales , Class Epsilonproteobacteria phylum
Protobacteria
• Natural inhabitants of intestinal tracts of poultry and worm blooded
domestic
animals
10%
Campylobacter helveticus (ATCC 12470T)
Campylobacter upsaliensis (CCUG14913T)
Campylobacter lari (NCTC 11352T)
Campylobacter jejuni subsp. jejuni (CCUG 11248T)
Campylobacter jejuni subsp. doylei (CCUG 24567T)
Campylobacter coli (NCTC11366T)
Campylobacter lanienae (NCTC 1300 T)
Campylobacter hyointestinalis subsp. lawsonii (CCUG 34538T)
)
Campylobacter
hyointestinalis subsp. hyointestinalis (NCTC11608T)
Campylobacter hyointestinalis (ATCC 35217T)
Campylobacter fetus subsp. Venerealis (ATCC 19438T)
Campylobacter fetus subsp. Fetus (CIP 5396T)
Campylobacter mucosalis (CCUG 6822)
Campylobacter concisus ( ATCC 33237T)
Campylobacter curvus (ATCC 35224T)
Campylobacter rectus (ATCC 19438T)
Campylobacter showae (CCUG 11641)
Campylobacter hominis (NCTC 13146)
Campylobacter gracilis (ATCC 33236T)
Campylobacter sputorum subsp. bubulus (ATCC 33491)
Campylobacter sputorum subsp. sputorum (ATCC 35980 T)
Arcobacter nitrofigilis (CCUG 15893)
Arcobacter butzleri (CCUG10373)
Arcobacter cryaerophilus (CCUG 17801T)
Arcobacter skirrowii (CCUG 10374T)
Escherichia coli
Campylobacter history
• In 1886 Esherich observed organism resembling
Campylobacters in stool samples of children with
diarrhoea
• In 1913 Campylobacter were mis-identified as Vibrio.
• King In 1957 reported that a thermophilic Vibrio-like
bacterium associated with human acute enteritis.
• In 1973 Campylobacter have been identified as new genus
by Veron and Chatelain.
• C. jejuni the first species to be identified
Campylobacter Phenotypic Characterization
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Members are
Thermotolerant bacteria.
Curved, spiral or S-shaped, Gram-ve
Cells measure 1.5-6.0 m x 0.9 m
Motile by monopolar flagella
Microaerophilic
Grow aerobically or anaerobically between 15-42 oC.
Intolerate to freezing and drying
Campylobacter Molecular characterization
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Genome size is 1.6-1.7 Mb, except C. upsaliensis 2.0 Mb
GC contents around 30%
Multiple copies of 16S rDNA have been described
Campylobacter jejuni 81116 genome had been sequenced.
LPS as most G-ve bacteria is the pathogenic factor.
• LPS had been sequenced and express in E. coli
Campylobacter Infection
• Campylobacter associated with human and animal diseases
• incubation periods of illness vary from 1 day to seven
days.
• Guillain-Barré Syndrome(GBS)
• AIDS and Immunocomprmised patients are at high risk of
Campylobacter infection.
• Most of the Campylobacter related illness caused by C.
jejuni.
Epidemiology of gastrointestinal pathogens
Campylobacter is the most
common bacterial disease
Causing 46% of diarrhoeal
illness reported by Centre
for Disease control and
prevention (CDC)/USA
Arcobacter phenotypic characteristics
Members are:
• Gram -ve, curved or slightly curved, S-shaped, or helical
• Non spore former
• Motile by monopolar flagella
• Microaerophilic
•
Intolerate to freezing and drying..
Arcobacter Infection
• Arcobacter Associated with animal disease (Abortion,
Diarrhoea and mastitis)
• Arcobacter isolated from patient with diarrhoea.
Campylobacter And Arcobacter Detection methods
• Selective media, Biochemical test, DNA-DNA
hybridization, gene sequencing, PCR and Maldi mass
spectrometry.
• The appearance of colonies in Campylobacter selective
media at 42 º C indicate the presence of Campylobacter.
• Biochemical test such as catalase and cytochrome oxidase
tests which are positive for C. jejuni, C. coli and C. lari.
• Further biochemical test to identify the species (eg.
Hippurarte hydrolysis)
Campylobacter and Arcobacter biochemical
detection problems
• Isolation and Identification of Campylobacter is time
consuming require up to 4 days.
• Biochemical test depend on biochemical pathways and
their disruption can lead to product failure leading to false
result.
Advantage of Molecular technique detection
• Denis et al observed that biochemical test provide only
34% efficiency compared to 100% with the PCR.
• PCR tests have been developed for the detection and
identification directly from pathological and food sample.
Aim and advantage of this project
• 67 biochemical test and molecular technique have been
devised for Campylobacter identification
• Not all these test are suitable for routine testing in
microbiological laboratory.
• Project aim is to Develop a new rapid, easy to use,
sensitive, accurate and low cost molecular methods for the
detection, identification and quantitation of Campylobacter
and Arcobacter species from enriched and isolated culture
or directly from environmental and /or clinical sample.
Choice of Targets and Sensitivity
DNA
RNA
TOTAL DNA:
•Mol%G+C
•Restriction Patterns ( RFLP,
PFGE)
•Genome size
•DNA homol ogy
DNA SEGMENTS:
• PCR based fingerprinting
(ribotypi ng, AR DDRA, R APD,
AFLP, AP-PCR , rep-PCR)
•DNA probes
•DNA sequencing
• rRNA sequenci ng
23
•LMW RNA profi les
S
S
16
5S
tRNA
Plasmid DNA
DNA
•Ele ctrophoretic patterns of total
cellular or cell envel ope proteins
(1D or 2D)
PROTEINS
mRNA
•Multienzyme pa tte rns (multilocus
enzyme ele ctrophoresis)
CHEMOTAXONOMIC MARKERS
•Cellular fatty acids (FAME)
•Mycoli c acids
•Polar li pids
•Quinones
•Polyamine s
•Cell wal l compounds
•Exopolysacchar ides
EXPRESSED FEATUR ES
•Morphology
•Physiology (Biol og, API, …)
•Enzymol olgy ( APIzyme)
•Se rology (monoclona l, polyclona l)
R estri cti on F ragm ent Le ng th Po ly morp hi sm (RF LP )
Lo w f re qu enc y restri cti on f rag men t a na ly sis (P FG E)
P hag e an d ba cteri oci n typ i ng
Se rol og ic al te chn i que s
R ib otyp i ng
D N A amp li f i cati on (A FL P, AP -PC R, RA PD )
Zy mog rams (mu l til oc us en zyme s)
To tal c el lu l ar prote in el ectro pho reti c pa tte rns
D N A ho mol og y
M ol % G+ C
D N A amp li f i cati on (A R DR A )
tD NA -P CR
C hem otax on omi c mark ers
C el lu la r f a tty aci d f i nge rpri nti ng (FAM E)
rD N A / rRN A s equ en ci ng
D N A pro bes
D N A seq ue nci ng
H ig hth rou gpu t assa ys (M i croa rrays, Ca nti le ver ar rays)
St
ra
i
Tec hn iq ue
s
ly
s
ci e
mi
nu
a
F
Ge Spe
n
Moleculare Techniques
Real Time PCR
• What is REAL TIME PCR?
• Continues monitoring of fluorescent signals derived from
fluorescent resonance energy transfer (FRET).
• FRET PCR (ABI PRISM and the LightCyclerTM) was
described in 1996. Since then, there have been major
innovations in both probe technology and instrumentation
design.
Fluorophore probe innovations
Real Time PCR instrumentation innovations
Real Time PCR instruments used in this project
LightCycler
iCycler
Fluroprobe mechanism
Hybrdization
probe
Taqman probe
Project Outline
Real time PCR
T-RFLP
Adjacent hybridization probes
One tube assay
Multiplex Chapter
Two tube assay
C. coli
Arcobacter (Chapter 5)
Adjacent probes
C. jejuni
Other
Campyloba
cter
Campylobacter (Chapter 3)
Adjacent probes
C. coli and C. jejuni
A. butzleri
A. skirrowii
A. nitrofigilis
SYBR Green I
(Chapter 4)
Multi FAM probe (Chapter 6)
C. coli
C.jejuni
Campylobacter Sequencing
Campylobacter coli and C. jejuni detection
Normalized fluorescence
60
1
2
3
4
5
6
50
C. jejuni
40
683 bp
30
C. coli
20
primer
dimer
10
43
40
37
34
31
28
25
22
19
16
13
7
10
4
1
0
Cycle number
Increase in flouresence during specifciehybridisation of probes Jejuni-coli FITC and Universal- CY5 to the target site in the 16S
rDNA of (--) C. jejuni, (--) C. coli suring PCR as measured in the
LightCycler. Purified DNA was prepared using CTAB method. (-×-)
C. hyointestinalis, (--) C. upsaliansis, (--) E. coli and (-+-) No
template were used as negative controls.
Agarose gel electrophoresis of PCR products from
C. coli (Lane 1), C. jejuni (Lane 2), and C.
hyointestinalis (Lane 3), C. upsaliensis (Lane 4), E.
coli (Lane 5) and no DNA template control (Lane
6). Only C. coli and C. jejuni but none of the others
produce the amplicons of 683 bp as expected.
DNA melting curve
C. jejuni
C. coli
C. hyointestinalis
C. upsaliansis
E.coli
No-template
DNA quantification
70
Normalized fluoresence
60
0.000192 0.00192 0.0192 19.2 0.192
50
40
30
681 bp
20
10
Cycle number
Real-time detection of C. jejuni CTAB-purified
DNA at different concentrations. 1920 ng (-),192 ng (-19.2 ng (--), 1.92 ng (-+-), 0.192
ng (-×-), 0.0192 ng (--), 0.00192 ng (-ž
-) and
0.000192 ng (--)
43
40
37
34
31
28
25
22
19
16
13
10
7
4
1
0
Primer
dimer
1.92
192
1920 ng
Colony serial dilution
16
14
Flouresence
12
10
8
6
4
2
Cycle Number
Real time PCR of different dilution from one colony. The numbers of cells in each
dilution was determined by plating the dilutions onto BA plates. 50000 cells (--), 5000
cells (-×-), 500 cells (--), 125 cells (--), 50 cells (-о-) and Negative (--)
46
43
40
37
34
31
28
25
22
19
16
13
7
10
-2
4
1
0
50
100
45
90
Normailised flourescence
40
35
30
25
20
15
10
5
80
70
60
50
40
30
20
10
Cycle number
Real time PCR of different culture incubation
time of C coli 24 hours incubation (--), 8 hours
(--), 6 hours (--), 4 hours (--), 2 hours (-•
-)
and 0hours(--)
Real time PCR of different culture incubation time
of C jejuni 24 hours incubation (--), 8 hours (--),
6 hours (--), 4 hours (--), 2 hours (-•
-) and 0hours(-)
46
43
40
37
34
31
28
25
22
19
16
13
7
4
0
10
Cycle number
46
43
40
37
34
31
28
25
22
19
16
13
10
7
4
1
0
1
normailzed flourescence
Different growth phase
Hippuricase gene (HipO)
• HipO code for hippuricase enzyme.
• Catalyses the hydrolysis of N-benzoyleglycin (Hippuric
acid) to glycine and benzoic acid.
• HipO gene present only in C. jejuni but not in any other
Campylobacter spp.
Hippuricase detection using the LightCycler
40
1
2
3
4
5
35
Fluorescence
30
25
20
15
270 bp
10
primer
dimer
5
29
27
25
23
21
19
17
15
13
11
9
7
5
3
1
0
Cycle Number
Real time SYBR Green 1 assay with CTAB-purified
DNA from C. jejuni ATCC 940565(-  -), C. coli
NCTC 11366 (--), C. upsaliensis strain 99M126 (--)
C. hyointestinalis strain 99M2318 (-  -) for the
detection of hippuricase gene (hipO).
Agarose gel electrophoresis of the PCR products
from Real Time SYBR Green 1 assay showing the
expected 270 bp specific product for C. jejuni
(Lane 1), and much lower sized non-specific
products from C. coli (Lane 2), C. hyointestinalis
(Lane 3), C. upsaliensis (Lane 4). and No
Template (Lane 5)
Melting curve of the HipO PCR product
A
C. jejuni
B
C. coli
C. hyointestinalios
C. upsaliansis
Hippuricase detection using the iCycler
C .jejuni
C. coli
C. hyointestinalis
C. upsaliansis
1
C. upsaliansis
C. hyointestinalis
C. coli
C. jejuni
2
3
4
292 bp
Primer
dimer
Arcobacter detection
Normalized fluorescence
25
1
20
2
3
4
5
6
15
10
315 bp
5
primer
demer
39
37
35
33
31
29
27
25
23
21
19
17
15
13
9
11
7
5
3
1
0
Cycle number
The increase in fluorescence during specific
hybridisation of probes probe Butz, probe Skir-Cry
and Universal CY5 to the target site in the 16S
rDNA of A. butzleri (--), A. skirrowii (--), A.
nitrofigilis (-+-), C. coli (--), C. jejuni (--) and
no template (--) suring PCR as measured in the
LightCycler. Purified DNA was prepared using
CTAB method. (--) C. jejuni, (-●-) C. coli. No
templates (-+-) were used as negative controls.
Agarose gel electrophoresis of PCR
products from no DNA template (Lane 1),
A. butzleri (Lane 2), A. skirrowii (Lane 3),
A. nitrofigilis (Lane 4), C. coli (Lane 5),
and C. jejuni (Lane 6).
DNA melting curve
A
A. skirrowii
B
A. butzleri
A. nitrofigalis
Colony serial Dilution and Growth Time
40
Normalized Flouresence
25
20
15
10
5
-5
43
40
37
34
31
28
25
22
19
35
30
25
20
15
10
5
46
43
40
37
34
31
28
25
22
19
16
13
10
7
4
0
Cycle number
1
16
13
10
7
4
0
1
normalized fluorescence
30
Cycle number
Real time PCR of different dilution from one
colony of A. butzleri. The numbers of cells in
each dilution was determined by plating the
dilutions onto BA plates. 50000 cells (--),
5000 cells (--), 500 cells (--), 125 cells (--)
and Negative (- -)
culture incubation time required by Arcobacter
species before it could be detected byLightCyclerTM
were: (--) 0 hours, (--) 2 hours, (--) 4 hours, (--)
6 hours, (-○-) 8 hours, and (--□) 24 hours
Arcobacter DNA Quantification
90
1
normalized fluorescence
80
2
3
4
5
6
7
8
9
10
70
60
50
40
315 bp
30
Primer
dimer
20
10
43
40
37
34
31
28
25
22
19
16
13
7
10
-10
4
1
0
Cycle number
Real-time detection of A. butzleri CTAB-purified DNA at
different concentrations. 113 ng (--), 11.3 ng (--), 1.13
ng (--), 0.113 ng (--), 0.0113 ng (-+-), 0.00113 ng (--),
and 0.000113 ng (-□)
Agarsoe gel electrophoresis of
different DNA concentration 113
ng ((Lane 1), 11.3 ng (Lane 2),
1.13 ng (Lane 3), 0.113 ng (Lane
4), 0.0113 ng (Lane 5), 0.00113
ng (Lane 6), 0.000113ng (Lane 7),
No template (Lane 8)
Multi-FAM detection to differentiate between
C. coli, c. jejuni, A. butzleri and A. skirrowii
180
23
.1….2…….3……4……5…….6
160
140
Fluo
rese 120
nce
100
315 bp
80
60
40
20
0
1
4
7
10
13
16
19
22
25
28
31
34
37
40
Cycle Number
Real time detection of Campylobacter species and
Arcobacter species using probe Skir-Cry, probe Butz
and probe Jejuni-coli, C. jejuni (00M2260) (--), C.
coli (P287/96) (--), A. butzleri (ATCC1248) (-*-), A.
skirrowii (ATCC 12713) (-^-), A butzleri (99M3958) (-) and NO template (--).
43
Primer
dimer
Agarose gel electrophoresis of PCR products
from no DNA template (Lane 1), A. butzleri
(ATCC1248) (Lane 2), A. skirrowii (ATCC 12713)
(Lane 3), A butzleri (99M3958) (Lane 4), C. coli
(P287/96) (Lane 5), and C. jejuni (00M2260) (Lane
6)
DNA melting curve
Campylobacter coli
C. jejuni
A. bultzeri
Arcobacter skirwii
A. butzleri
No template
Conclusion and Future Direction
• Rapid Methods for both C. coli and C. jejuni have been
developed.
• Distinguish between the closely related species C. coli and
C. jejuni.
• Time required for Campylobacter and Arcobacter
detection reduced tow minutes instead of days
• Application of the methods to environmental and food
samples.
• PCR multiplex for rapid detection and identification in one
step
• T-RFLP for rapid detection and identification of
Campylobacter and Arcobacter.
• Determine the pathogenisity gene that present in C. coli
and A. butzleri