Investigation of bacteriophages as potential sources of oral antimicrobials By: Mohammed I. Abud Al-Shaheed Al-Zubidi A thesis submitted for the degree of Doctor of Philosophy December 2017 The University of Sheffield Faculty of Medicine, Dentistry and Health School of Clinical Dentistry ii Abstract Bacteriophages are natural viruses that attack bacteria and are abundant in all environments, including air, water, and soil- following and co-existing with their hosts. Unlike antibiotics, bacteriophages are specific to their bacterial hosts without affecting other microflora. The use of bacteriophages and phage endolysin based therapies to kill pathogens without harming the majority of harmless bacteria has received growing attention during the past decade, especially in the era where bacterial resistance to antibiotic treatment is increasing. The aims of this study were to characterise a putative prophage (phiFNP1) residing in the genome of the periodontal pathogens Fusobacterium nucleatum polymorphum ATCC 10953, investigating the possibility of prophage induction, examining its presence in clinical plaque samples taken from patients with chronic periodontitis cases, and purify its putative lysis module genes to examine their potential antimicrobial activity. In addition, attempts will be made to catalogue phages that are presents in samples from the oral cavity of patients with chronic periodontitis and in wastewater, followed by isolation of lytic phages targeting endodontic and oral associated pathogens, characterisation of the isolated phages, evaluation of the efficiency of phage towards biofilm elimination, and established an animal model to study phage-bacterial interaction in order to develop them for treatments. The results revealed that phiFNP1 prophage are common in subgingival plaque of patients suffering from chronic periodontal disease but attempts to induce phiFNP1 using Mitomycin C were inconclusive, indicating that it might be defective. Bioinformatics revealed that the genome of phiFNP1 contained potential lysin genes, with one being cloned and purified successfully in soluble form, though its activity as antibacterial was not confirmed, and needs to be further explored as phage lytic enzymes against Fusobacterium have not been identified yet. Various phage like particles with different morphology were visualised by direct electron microscopy from oral and wastewater samples, which reflect the richness and diversity of bacteriophage within those samples. Several phages were isolated namely phiSHEF 2,3,4,5,6,7 which belong to the Siphoviridae family which are specific to Enterococcus faecalis and the full chromosome sequence comparisons for three of the isolated phages (phiSHEF 2,4 and 5) revealed that they are lytic in nature reflected by iii absence of genes associated with lysogenic cycle, therefore place them as suitable candidate for therapy. They exhibit genomic variations corresponded to their difference in phage-host range especially in the tail region. In addition, phiSHEF 2 was capable of complete biofilm eradication on abiotic surfaces and significantly reduced biofilms formed on the surfaces of tooth root slices. Most importantly phiSHEF 2 recovered a Zebrafish larvae from the deadly infection caused by an oral clinical isolate of E. faecalis, indicating the establishment of a successful animal model for testing the efficiency of phage therapy towards E. faecalis infection. Finally, Exopolysaccharide mutants of E. faecalis were not infected by three of our phiSHEF phages tested, indicating that E. faecalis exopolysaccharide capsule and proper bacterial membrane integrity play an important role during the initial stages of phage infection. In conclusion, bacteriophages exhibit an apparent superiority to fight hard to eradicate pathogens such as those associated with recalcitrant endodontic infections thus “bacteriophage based therapy” could represent an innovative alternative to control and eliminate oral infections caused by biofilm forming and antibiotic resistant pathogens. iv Acknowledgments Firstly, my sincerest gratitude and appreciation goes to my supervisors Dr. Graham Stafford, Prof. Andrew Rawlinson and Prof. Ian Douglas for their attention to details, endless motivation and never ending scientific knowledge. Their kindness, unlimited support, guidance and encouragement throughout this study have been invaluable. I have been blessed to learn from the supportive staff of the Oral and Maxillofacial Department and my PhD colleagues at Graham Stafford research group. My gratefulness also goes to my colleague Sahang Gul who has always supported me in clinical samples collection part of this study, and I will never forget his friendly fellowship and lovely moments that we spent together in the department. I would like especially to thank my colleague Magdalena Widziolek who assists me in establishment of the animal model of my study. I would like to thank Alison Barber and Claire Vallance- Owen in the Charles Clifford Dental Hospital. They have been there to support me when we recruited patients and helped collect data for this study. My PhD journey would have been much more difficult without the amazing technical staff who are second to none. Particularly, technical expertise from Mrs. Sue Newton and the supportive role of Mr. Jason Heath and Mrs. Brenka McCabe has been indispensable regarding methodological problem solving and pleasant discussions. I would also like to express my appreciation for all the advice, encouragement and funny moments I received throughout my PhD time from all the lovely friends I got to know throughout my study. My gratitude and love goes to my family especially my mother for supporting me spiritually throughout my life and giving me all the advice and encouragement I needed throughout my study. Ultimately, my life would be impossible including this PhD study without the support of my lovely wife, Shaimaa. Her patience, endless support, and continuous encouragement have been invaluable and greatly appreciated. For this, I like to dedicate this work to her and my gorgeous daughters, Rawan and Rama. Last but not the least, I would like to express my gratitude for the Ministry of Higher Education and Scientific Research in Iraq for granting me this fantastic opportunity to finish my PhD study and excel academically, and I recognise that this research would not have been possible without it. v Abbreviations ® Trade mark α Alpha β Beta γ Gamma μ Micro Ap Ampicillin ATCC American Type Culture Centre bp Base pairs BHI Brain heart infusion BSA Bovine serum albumin °C Centigrade CO2 Carbon dioxide DNA Deoxyribonucleic acid dsDNA Double stranded deoxyribonucleotide DTT Dithiothreitol EDTA Ethylenediamine tetra-acetic acid FA Fastidious anaerobe agar G Gram g Gravity GST Glutathione S-transferase His-tag Histidine-tag h hour IL Interluekin IPTG Isopropyl β-D-1-thiogalactopyranoside kbp kilo base pairs kDa kilo Daltons L litre LB Luria-Bertani LPS Lipopolysaccharide ng nanogram μg Microgram μl Microlitre ml Millilitre min minute mM Mmillimolar MOI Multiplicity of Infection M Molar MW Molecular weight N2 Nitrogen Ni-NTA Nickel-nitriloacetic acid nm Nanometre OD Optical density PBS Phosphate buffered saline PCR Polymerase chain reaction % Percentage PFU Plaque forming units phi Phage ppm part per million psi Pounds per square inch (pressure) vi qPCR Quantitative polymerase chain reaction RNA Ribonucleic acid rpm Revolutions per minute s seconds SD Standard deviation SDS Sodium dodecyl sulphate SDS-PAGE SDS-polyacrylamide gel electrophoresis ssRNA Single stranded ribonucleotide ssDNA Single stranded deoxyribonucleotide TAE Tris-acetate-EDTA TEM Transmission electron microscope TEMED Tetramethylethylenediamine tRNA Transfer RNA TNF Tumour necrosis factor TSB Triptone soya broth UV Ultraviolet light V Volt vii Contents Abstract ............................................................................................................................ iii Acknowledgments ............................................................................................................. v Abbreviations ................................................................................................................... vi List of Figures ................................................................................................................. xii List of Tables ................................................................................................................. xvi Chapter 1: Literature Review ............................................................................................ 1 1.1 Introduction ............................................................................................................. 2 1.2 Role of oral microbiome in health .......................................................................... 6 1.3 Bacteriophage.......................................................................................................... 6 1.3.1 Definition of bacteriophage ............................................................................. 6 1.3.2 History of bacteriophage discovery ................................................................. 8 1.3.3 Classification of bacteriophage ........................................................................ 9 1.3.4 Lytic phages towards orally derived bacteria ................................................ 14 1.3.4.1 E. faecalis lytic phages...............................................................................