BACTERIAL INVOLVEMENTS IN ULCERATIVE COLITIS: MOLECULAR AND MICROBIOLOGICAL STUDIES Samia Alkhalil A thesis submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy of the University of Portsmouth Institute of Biomedical and biomolecular Sciences School of Pharmacy and Biomedical Sciences October 2017 AUTHORS’ DECLARATION I declare that whilst registered as a candidate for the degree of Doctor of Philosophy at University of Portsmouth, I have not been registered as a candidate for any other research award. The results and conclusions embodied in this thesis are the work of the named candidate and have not been submitted for any other academic award. Samia Alkhalil I ABSTRACT Inflammatory bowel disease (IBD) is a series of disorders characterised by chronic intestinal inflammation, with the principal examples being Crohn’s Disease (CD) and ulcerative colitis (UC). A paradigm of these disorders is that the composition of the colon microbiota changes, with increases in bacterial numbers and a reduction in diversity, particularly within the Firmicutes. Sulfate reducing bacteria (SRB) are believed to be involved in the etiology of these disorders, because they produce hydrogen sulfide which may be a causative agent of epithelial inflammation, although little supportive evidence exists for this possibility. The purpose of this study was (1) to detect and compare the relative levels of gut bacterial populations among patients suffering from ulcerative colitis and healthy individuals using PCR-DGGE, sequence analysis and biochip technology; (2) develop a rapid detection method for SRBs and (3) determine the susceptibility of Desulfovibrio indonesiensis in biofilms to Manuka honey with and without antibiotic treatment. Mucosal biopsy DNA from 4 colitis patients and one healthy individual was used to amplify 16S rRNA fragments, which were separated either by DGGE or by molecular cloning prior to sequencing, and dissimilatory sulphite reductase (dsr) gene fragments, which were cloned and sequenced. The number of bands separated by DGGE varied from 3 to 12 for an individual. The profiles from the UC patients had a greater similarity than the one from the healthy individual. In total, 25 bands were excised for sequence analysis but only 4 produced usable sequences. The 16S rRNA gene library comprised 250 clones, the sequences of which showed great changes in diversity from the healthy individual to the UC patients. The sequences from the healthy individual represented members of the Bacteroidetes, Clostridiaceae, Ruminococcaceae, Enterobacteriaceae, Coriobacteriaceae and Lactobacillaceae, whereas those form the UC patients comprise only members of the Enterobacteriaceae. The library for the dsr gene comprised 30 clones, with sequences from the healthy individual representing members of the Desulfobacteraceae (45%), and the Desulfovibrionaceae (33%), with minor components from the Desulfohalobiaceae, Desulfomicrobiaceae and Desulfonatronaceae. Sequences from the UC patients were less diverse, being composed primarily of Desulfovibrionaceae sequences. II Oligonucleotide probes targeting SRB and other bacterial species found to be involved in UC were developed or selected from published sources. Cassettes with multiple probes were used to evaluate the hybridisation probes for inclusion in a biochip. Validated probes (23), with similar binding profiles, were then used to assess the levels of SRB and other bacteria associated with UC in healthy control and UC patient’s samples. The DNA from the healthy individual gave a strong hybridisation signal for Desulfovibrio piger, and weaker signals for Escherichia coli, Bacteroides fragilis, and a Clostridium species, whereas the signals Desulfobacter and Desulfovibrio gigas were present in all of the samples from the UC patients. Signals were also recorded for species of Fuscobacterium, which have been implicated in colitis. In this investigation, procedure for rapid detection and quantification of SRB was developed. The SRB growth was monitored in the presence and absence of Escherichia coli BP, the copy number of the dsrA and adenosine-5′-phosphosulfate reductase (aps) genes was detected in DNA and RNA purified from different ages of SRB culture using TaqMan qPCR. The result showed that in the presence of E.coli BP enhanced the growth of SRB and allowed the detection of SRB in low dilutions comparing with the growth of SRB only. E. coli and SRB are both implicated in colonic infections. They pose several mechanisms for immune evasion and antibiotic resistance, one of these being their ability to grow in a biofilm. The present investigation has highlighted that Manuka honey and antibiotic treatment might have a protective role against SRBs and enteric bacteria. However, mixed bacterial population in biofilms exhibit greater resistance to both treatments, and this needs to be taken into account when devising a treatment based on ingestion of honey. The present investigation were conducted as a baseline study for determination the role of SRB in the pathogenesis of colitis through the use of novel methodology allowing for rapid detection and screening investigations into species and genera of interest present in colonic mucosa; that may allowed in further investigation to evaluate the bacterial role in UC through extensive studies in broad multinational cohort of patients suffering from this UC. III ACKNOWLEDGEMENTS First of all, all thanks and gratitude go to Almighty ALLAH for guiding and inspiring me during this research process. Secondly, I offer my sincerest gratitude to my parents who supported me during my study, without them and their help this mission would have been impossible. My deepest gratitude to my husband Tariq for his patience and support while I’m doing this research and to my sons, Mohammed, Abdulaziz and my daughter Monera with love to all of you. I would like to express my thanks to my first supervisor Dr. Vitaly Zinkevich for giving me the opportunity to give a great hand in this project, this thesis would not be achieved without his presence and support. I am sincerely grateful to my supervisor Dr. Julian Mitchell for his supervision, motivation for friendly guidance and enormous support and help during the stages of this thesis. Without his constructive feedback and tremendous support this thesis would not have seen the light of day. Thank you, Dr. Julian for all positive energy that you gave me. I would like to tank Dr. Irina Bogdarina for her continues help and also Dr. Nelly Sapojnikova who was always available for help and Dr. Tamar Kartvelishvili for the warm welcome and for their assistance during the stay in the University of Tbilisi. Thanks to my sponsor Ministry for Higher Education in Saudi Arabia for their financial and academic support represented by Saudi Arabian Cultural Bureau in London. Finally, I offer my regards and blessings to all of those who have supported me and given assistance to me in any respect during the completion of this thesis. IV LIST OF ABBREVIATIONS APS Ammonium persulphate APS Adenosine phosphosulfate ATCC American type culture collection ATP Adenosine triphosphate BLAST Basic Local Alignment Search Tool bp Base pairs cDNA Complementary DNA CD Crohn’s Disease Ct Cycle threshold Cy3 Cyanine 3 DGGE Denaturing gradient gel electrophoresis DIEA Diisopropylethyl amine DMF Anhydrous amine free N,N-dimethylformamide DNA Deoxyribonucleic acid dsDNA double-stranded DNA Dsr Dissimilatory sulphite reductase EDTA Ethyleno-diamino tetra-acetic acid FAM 6-carboxy-fluorescein, a blue fluorescent dye FISH Fluorescence in situ hybridization GIT Gastrointestinal Tract H2S Hydrogen sulphide IDT Integrated DNA Techenlogy IPTG Isopropyl-β-D-thiogalactopyranoside kb Kilobase pairs LB Lysogeny or Luria Broth MBC Minimum Bactericidal Concentration V MGO Methylglyoxal MIC Minimum Inhibitory Concentration mRNA Messenger RNA MW Molecular weight NA Avogadro’s number PCR Polymerase chain reaction PFGE Pulsed Filed gel Electrophoresis QPCR Quantitative PCR QS Quorum sensing RNA Ribonucleic acid rpm Revolutions per minute rRNA Ribosomal RNA RT Room Temperature sp. Species SRB Sulfate-reducing bacteria ssDNA single stranded DNA TAMRA Tetramethylrhodamine TEMED N,N,N’,N’Tetramethylethylenediamine Tm Melting temperature, the temperature at which a DNA double helix dissociates into single strands Tris-HCl Tris (hydroxylmethyl) aminomethane UC Ulcerative Colitis UV Ultra Violet light VM Vitamin Medium VMI Vitamin Medium with Iron VMR Vitamin Medium with Reduced iron X-Gal 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside %(v/v) Percentage volume/volume VI %(w/v) Percentage mass/volume VII TABLE OF CONTENTS AUTHORS’ DECLARATION…………………………………………….……………………………………………..I ABSTRACT .............................................................................................................................. II ACKNOWLEDGEMENTS .................................................................................................... IV LIST OF ABBREVIATIONS ................................................................................................... V TABLE OF CONTENTS ...................................................................................................... VIII LIST OF FIGURES ................................................................................................................ XII LIST OF TABLES ............................................................................................................. XVIII