Molecular Methods for Campylobacter and Arcobacter Detection Author Abu-Halaweh, Marwan Published 2005 Thesis Type Thesis (PhD Doctorate) School School of Biomolecular and Biomedical Sciences DOI https://doi.org/10.25904/1912/2978 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/367268 Griffith Research Online https://research-repository.griffith.edu.au Molecular Methods for Campylobacter and Arcobacter Detection Marwan Abu-Halaweh, MSc. (Hons) A Thesis submitted in fulfillment of the requirements of the degree of Doctor of Philosophy in the School of Biomolecular and Biomedical Science, Faculty of, Griffith University, Nathan Campus, Queensland, Australia. January 2005 I Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. Marwan Abu-Halaweh I Acknowledgments First of all, thanks is to be directed to GOD. I thank Associate Professor Bharat Patel for the opportunity to work with him. His advice, support and assurance gave me enthusiasm and confidence during my research. I appreciate the advice, assistance and friendship of Dr Ben Mijts, Dr Mark Spanevello, Dr David Innes, Dr Sungwan Kanso, Dr Lyle McMillen, Mr Peter Bain and all members of our lab throughout the course of my research in Professor Patel’s laboratory. I acknowledge Dr John Bates, Miss Trudy and the Bacteriology Division of the Queensland Health Scientific Services and the staff for their help and assistance on site. I gratefully acknowledge the financial support from Queensland Health Scientific Services and Griffith University. Also I gratefully acknowledge the editors at WordsRU and Blayse Research for the proofreading of this thesis. All my friends who continued asking “So, how far to go now?” are mentioned for their ongoing companionship. Finally, I thank my family (especially my father and mother) for their continued interest, praying for me and encouraging me in all that I have done and will do, and my wife for her support during this time. II Publications and Proceedings Arising from this Thesis Research Paper Abu-Halaweh M, Bates J, Patel BKC: Rapid detection and differentiation of pathogenic Campylobacter jejuni and Campylobacter coli by real-time PCR. Res Microbiol 2005, 156(1):107-114. This paper generated from chapter 3 and chapter 4. Conference Presentations (supported by a grant for conference support by Griffith University) ¾ Abu-Halaweh M., Bates J. and Patel B.K.C. (2004). Rapid detection and identification of Campylobacter jejuni and Campylobacter coli directly from chicken samples by real-time PCR Bahrain: The first GCC Genetic conference - Bahrain, 5-7 October 2003. This presentation generated from chapter 3. ¾ Abu-Halaweh M., Bates J. and Patel B.K.C. (2004). Rapid detection and identification of Campylobacter and Arcobacter species by real-time PCR. European Meeting on Molecular Diagnostics - Kurhaus Hotel The Hague / Scheveningen – Netherlands - 16th & 17th October 2003. This presentation generated from chapter 6. ¾ Abu-Halaweh M., Bates J. and Patel B.K.C. (2004). Rapid detection of Campylobacter species using Ligase Detection Reaction (LDR). European Meeting on Molecular Diagnostics - Kurhaus Hotel The Hague / Scheveningen – Netherlands - 16th & 17th October 2003. This presentation generated from chapter 9. III Publications in Preparation ¾ Rapid detection and identification of Arcobacter species by real-time PCR. ¾ Rapid detection and identification of C. coli and C. jejuni and C. lari from other Campylobacter species by Ligase Chain Reaction. ¾ A real-time PCR multiplexed for rapid detection and identification of C. coli and C. jejuni from other Campylobacter species. IV Abstract Twenty species and six subspecies of the genera Arcobacter and Campylobacter have been described to date. All are Gram-negative, microaerophilic, curved, spiral or S- shaped cells, and are members of the order Campylobacterales, class Epsilonproteobacteria phylum Proteobacteria. Though most members are pathogenic, C. jejuni, C. coli and A. butzleri are the most frequently isolated species from patients suffering from gastrointestinal illness. The current methods for their detection, identification, and differentiation are cumbersome, time consuming and lack specificity. DNA based molecular techniques including real-time Polymerase Chain Reaction (PCR) and Fingerprinting methods Terminal Restriction Fragments Length Polymorphism (T-RFLP) and Ligase Detection Reaction (LDR) have been used in this project to develop rapid detection and identification methods for Campylobacter and Arcobacter species. Five real-time PCR methods were developed which include: (a) rapid detection and identification of Campylobacter species using real-time PCR adjacent hybridisation probes, (b) rapid identification of C. jejuni using SYBR Green I, (c) rapid detection and differentiation of Arcobacter species using adjacent hybridisation probes, (d) rapid detection and differentiation of Arcobacter species and the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. hyointestinalis, C. insulaenigrae, C lanienae) using melting temperature (Tm) of adjacent hybridisation probes, and (e) a one tube real-time PCR multiplex for the rapid detection and identification of Campylobacter species, C. coli and C. jejuni using a TaqMan Probe, in an iCycler iQTM (BioRad, USA) and Light CyclerTM (Idaho Technology, USA). The real-time PCR methods for a and b, was a two-tube assay which detected, identified and differentiated C. coli, C. lari and C. jejuni from other members of the family Campylobacteraceae. The first tube assay was based on the principle of Fluorescence V Resonance Energy Transfer (FRET) in which the FRET signal from the hybridisation of two adjacent fluoroprobes, probe Cy5+1046 and a specific downstream 6-FAM probe to the target site within the 16S rRNA gene of the 681 base pair amplicons produced during PCR with primers F2 and Cam Reverse, was continuously measured in a LightCyclerTM (Idaho Technology, USA) enabling the differentiation of C. coli, C. lari and C. jejuni from all other Campylobacter species. Following detection of these species, a second tube assay was used to differentiate C. jejuni from C. coli and C. lari based on the continuous monitoring of an increase in fluorescence in a LightCyclerTM due to the binding of the intercalating dye SYBR Green I to DNA amplicons, produced by primers Hip-2214F and Hip-2474 targeting the hippuricase (hipO) gene known to be present in the C. jejuni genome but not in C. coli, C. lari and other Campylobacter species. The subsequent temperature dependent dissociation of the strands to produce a ο ο specific Tm of 85±0.5 C confirmed the presence of C. jejuni, whereas a Tm of 56 C indicated the presence of non-specific primer dimers. This two tube assay was successfully used to identify and differentiate 176 cultures isolated from animals, humans, plants and birds as C. coli and C. lari group (77 isolates), C. jejuni (88 isolates), and other Campylobacter species (11 isolates). Furthermore, this assay was used to identify and differentiate 30-enrichment cultures initiated from chicken samples as C. jejuni and C. coli and C. lari group (26) and C. jejuni (18). In addition, more than one Campylobacter species could also be detected in the same enrichment culture. Method c was a single tube real-time PCR, which employed adjacent hybridisation probes to differentiate pathogenic Arcobacter species using the FRET principle. In this assay, two specific 6-FAM labelled probes, probe Butz specific for A. butzleri and probe Skir-Cry specific for A. skirrowii and A. cryaerophilius, and an adjacent universal Cy5 labelled probe were used for the simultaneous detection, identification and differentiation of A. butzleri from A. skirrowii and A. cryaerophilius. The binding of VI either probe Butz or probe Skir-Cry in conjunction with the probe Cy5+1046 to their respective 16S rRNA target regions within the 317 base pair amplicons produced by primers F2 and R5, resulted in an increase in fluorescence during real-time PCR. The subsequent temperature dependent dissociation of the strands to produce specific Tms based on nucleotide heterogeneity of the probe to the target binding sites enabled the o differentiation of the three Arcobacter species: a Tm of 67 C due to dissociation of o o probe Butz confirmed the presence of A. butzleri, and a Tm of 63 C and 65 C due to the dissociation of probe Skir-Cry differentiated A. skirrowii and A. nitrofigilis respectively. The method successfully identified all the 22 Arcobacter isolates obtained from humans and birds as A. butzleri (20 isolates), A. skirrowii (1 isolate), and A. nitrofigilis (1 isolate). The assay also successfully detected A. butzleri in 18 out of 30-enrichment cultures initiated from chicken samples and 9 as A. skirrowii and/or A. cryaerophilus. In addition, more than one Arcobacter species was also be detected in the same enrichment culture. Method d was a new, complex, single tube assay, termed multi FAM adjacent hybridisation real-time PCR assay. It has been developed for the simultaneous detection and differentiation of the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. hyointestinalis, C. insulaenigrae, and C. lanienae), A. butzleri, and A. skirrowii. The binding of 6-FAM labelled probes (probe Jejuni-coli, probe Butz, and probe Skir-Cry) in conjunction with the universal Cy5 labelled probe to their specific target sites within the amplicon produced an increase in fluorescence signal, which was measured in a LightCyclerTM. The subsequent dissociation of the probes to produce o specific Tms identified and differentiated the targeted species: a Tm of 67 C identified A. o o butzleri, a Tm of 63 C identified A. skirrowii and a Tm of 65 C identified the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. o hyointestinalis, C.