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%

Escherichia coli

Campylobacter curvus (ATCC 35224T)

Campylobacter coli (NCTC11366T)

Arcobacter nitrofigilis (CCUG 15893)

Campylobacter lanienae (NCTC 1300 T)

Campylobacter hyointestinalis (ATCC 35217T)

Arcobacter butzleri (CCUG10373)

Campylobacter lari (NCTC 11352T)

Campylobacter helveticus (ATCC 12470T)

Campylobacter upsaliensis (CCUG14913T)

Campylobacter jejuni subsp. jejuni (CCUG 11248T)Campylobacter jejuni subsp. doylei (CCUG 24567T)

Campylobacter hyointestinalis subsp. lawsonii (CCUG 34538T) )Campylobacter hyointestinalis subsp. hyointestinalis (NCTC11608T)

Campylobacter fetus subsp. Venerealis (ATCC 19438T)Campylobacter fetus subsp. Fetus (CIP 5396T)

Campylobacter mucosalis (CCUG 6822)Campylobacter concisus ( ATCC 33237T)

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 cryaerophilus (CCUG 17801T)Arcobacter skirrowii (CCUG 10374T)

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

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

• 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 SEGMENTS:• PCR based fingerprinting(ribotyping, ARDDRA, RAPD,AFLP, AP-PCR, rep-PCR)•DNA probes•DNA sequencing

TOTAL DNA:•Mol%G+C•Restriction Patterns (RFLP,PFGE)•Genome size•DNA homology

DNA

DNA

Plasmid DNA

RNA

• rRNA sequencing

•LMW RNA profiles

mRNAPROTEINS

•Electrophoretic patterns of totalcellular or cell envelope proteins(1D or 2D)

•Multienzyme patterns (multilocusenzyme electrophoresis)

CHEMOTAXONOMI C MARKERS EXPRESSED FEATURES

•Cellular fatty acids (FAME)•Mycolic acids•Polar lipids•Quinones•Polyamines•Cell wall compounds•Exopolysaccharides

•Morphology•Physiology (Biolog, API, …)•Enzymololgy (APIzyme)•Serology (monoclonal, polyclonal)

Moleculare Techniques

TechniqueR estri ction F ragm ent Le ng th Po ly morp hism (RF LP )

Lo w fre qu enc y restricti on frag men t a na ly sis (P FG E)

P hag e an d ba cteriocin typ ing

Se rolog ic al te chn ique s

R ib otyp ing

D NA amp lifi cation (A FL P, AP -PC R, RA PD )

Zy mog rams (mu ltil oc us en zyme s)

To tal c el lu lar prote in electro pho retic pa tte rns

D NA ho molog y

Mol% G+C

D NA amp lifi cation (A R DR A )

tD NA -P CR

C hem otax on omic mark ers

C el lu la r fa tty acid fi nge rprinting (FAM E)

rD NA / rRNA s equ en cing

D NA pro bes

D NA seq ue ncing

H ig hth rou gpu t assa ys (Microa rrays, Ca nti le ver ar rays)

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

Multi FAM probe (Chapter 6)

Multiplex Chapter

A. butzleri A. skirrowii A. nitrofigilis

C. jejuni

Adjacent probes

Real time PCR T-RFLP

C. coli and C. jejuni

C. coli C.jejuni

SYBR Green I (Chapter 4)

Campylobacter (Chapter 3)Arcobacter (Chapter 5)

Two tube assayOne tube assay

Adjacent probes

C. coli Other Campylobacter

Adjacent hybridization probes

Campylobacter Sequencing

Campylobacter coli and C. jejuni detection

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Cycle number

Normalized fluorescence C. jejuni

C. coli

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.

683 bp

primerdimer

1 2 3 4 5 6

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

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Cycle number

Nor

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flu

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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 (--) and �

0.000192 ng (--)

0.000192 0.00192 0.0192 19.2 0.192 1.92 192 1920 ng

681 bp

Primerdimer

Colony serial dilution

-2

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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 (--)

Different growth phase

Real time PCR of different culture incubation time of C coli 24 hours incubation (--), 8 hours (--), 6 hours (--), 4 hours (--), 2 hours (--) �and 0hours(--)

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Cycle number

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Cycle numberN

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Real time PCR of different culture incubation time of C jejuni 24 hours incubation (--), 8 hours (--), 6 hours (--), 4 hours (--), 2 hours (--) and �0hours(--)

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

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Cycle Number

Flu

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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).

270 bp

primer dimer

1 2 3 4 5

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

C. jejuni

C. coli

C. hyointestinalios

C. upsaliansis

A

B

Hippuricase detection using the iCycler

C .jejuni

C. coli

C. hyointestinalis

C. upsaliansis

C. upsaliansis

C. hyointestinalis

C. coli

C. jejuni

292 bpPrimer dimer

1 2 3 4

Arcobacter detection

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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.

315 bp

primerdemer

1 2 3 4 5 6

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. skirrowii

A. butzleri

A. nitrofigalis

A

B

Colony serial Dilution and Growth Time

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nor

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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 (- -)

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Cycle numberN

orm

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Flo

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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

-10

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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 (-□)

1 2 3 4 5 6 7 8 9 10

315 bp

Primer dimer

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

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Fluoresence

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 (--).

23.1….2…….3……4……5…….6

315 bp

Primerdimer

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

Arcobacter skirwii

A. butzleri

Campylobacter coli

C. jejuni

A. bultzeri

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