2. Rapid detection and Identification of Campylobacter and Arcobacter species Marwan Abu-Halaweh

3. 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

4. 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

5. 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 o C. Intolerate to freezing and drying

6. 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

7. 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.

8. 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

9. 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..

10. Arcobacter Infection Arcobacter Associated with animal disease (Abortion, Diarrhoea and mastitis) Arcobacter isolated from patient with diarrhoea.

11. 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)

12. 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.

13. 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.

14. 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.

15. Choice of Targets and Sensitivity

16. Moleculare Techniques

17. 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 LightCycler TM ) was described in 1996. Since then, there have been major innovations in both probe technology and instrumentation design.

18. Fluorophore probe innovations

19. Real Time PCR instrumentation innovations

20. Real Time PCR instruments used in this project LightCycler iCycler

21. Fluroprobe mechanism Hybrdization probe Taqman probe

22. 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. coliC.jejuni SYBR Green I (Chapter 4) Campylobacter ( Chapter 3) Arcobacter ( Chapter 5) Two tube assayOne tube assay Adjacent probes C. coli Other Campyloba cter Adjacent hybridization probes

23. Campylobacter Sequencing

24. Campylobacter coli and C. jejuni detection 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.

25. DNA melting curve

26. DNA quantification 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 (-  -) 681 bp Primer dimer

27. Colony serial dilution 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 (-  -)

28. 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(-  -) Real time PCR of different culture incubation time of C jejuni 24 hours incubation (-  -), 8 hours (- -), 6 hours (-  -), 4 hours (- -), 2 hours (--) and 0hours(-  -)

29. 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.

30. Hippuricase detection using the LightCycler 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)

31. Melting curve of the HipO PCR product A B

32. Hippuricase detection using the iCycler 292 bp Primer dimer 1 2 3 4

33. Arcobacter detection 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).

34. DNA melting curve A B

35. Colony serial Dilution and Growth Time 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 byLightCycler TM were: (-  -) 0 hours, (- -) 2 hours, (- -) 4 hours, (-  -) 6 hours, (-  ○-) 8 hours, and (-  -□) 24 hours

36. Arcobacter DNA Quantification 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 (-  □) 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)

37. Multi-FAM detection to differentiate between C. coli, c. jejuni, A. butzleri and A. skirrowii 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 (- -). 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)

38. DNA melting curve

39. 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