The potential pathogenicity of Dolosigranulum pigrum in multiple sclerosis, and the occurrence of the organism in the upper respiratory tract by Francis Mark Jorge MSc CSci FIBMS CBiol MSB A portfolio of research and development in a professional context Submitted in partial fulfilment of the Degree of Professional Doctorate in Biomedical Science School of Pharmacy and Biomedical Sciences Faculty of Science University of Portsmouth May 2014 Abstract The bacterial toxins hypothesis in multiple sclerosis postulates that bacterial toxins from the human nasopharynx access the central nervous system and are implicated in the disease. Dolosigranulum pigrum was originally found in acute multiple sclerosis tissues. This study seeks to determine if the organism is found in human nasal tracts and explores the relationship between Dolosigranulum pigrum infection and multiple sclerosis by measuring antibody to the bacterium in multiple sclerosis patients and matched controls. Eighty eight clinical specimens were cultured onto blood agar and analysed using rabbit anti-Dolosigranulum pigrum sera, and fluorescein isothiocyanate. None tested positive for Dolosigranulum pigrum. Dolosigranulum pigrum was looked for using a novel polymerase chain reaction test. Thirty samples tested positive, 17 male, 13 female, with an age range from 8 days to 74 years. Twelve were sent for sequencing, seven matched Dolosigranulum pigrum, two showed mixed amplicons and three produced signals not matching Dolosigranulum pigrum. Sixty five multiple sclerosis sera and matched controls were tested for anti- Dolosigranulum pigrum using a novel enzyme linked immunosorbant assay. The multiple sclerosis group showed raised antibodies, significantly different to the controls, p ≤ 0.001. Fifteen of the multiple sclerosis sera and controls were Western blotted. There were no common bands when compared with rabbit sera containing antibodies to Dolosigranulum pigrum. This study shows that Dolosigranulum pigrum is found in the nasal tract. The elevated antibodies were not found to be anti-Dolosigranulum pigrum but their presence may be suggestive of bacterial products leaking from the nasal tract into the central nervous system. Further research into bacteria in the nasal tract is indicated. Introduction of markers into the nasal passages with subsequent tracking into the central nervous system by magnetic resonance imaging could provide evidence of a route by which bacterial molecules enter the central nervous system „behind‟ the blood brain barrier. ii Table of Contents Abstract ii Table of Contents iii List of Tables vi List of Figures vii Abbreviations viii Acknowledgements x Dissemination xi Declaration xii Dedication xiii Foreword 1 Chapter 1: Introduction and literature review 1.1 History of multiple sclerosis 3 1.1.1 Naming and classifying the disease 3 1.1.2 Clinical features and pathology 4 1.1.3 Epidemiology 6 1.1.3.1 Genetics and racial prevalence 6 1.1.3.2 Geographical prevalence and migration 7 1.1.3.3 Epidemic multiple sclerosis 9 1.1.4 Prospective aetiologies for multiple sclerosis 10 1.1.4.1 Autoimmune hypothesis 10 1.1.4.2 Viral hypothesis 10 1.1.4.3 Heterogeneity hypothesis 11 1.1.4.4 Spirochaetal hypothesis 12 1.1.4.5 Chlamydial hypothesis 13 1.2 Bacteria, sinusitis and multiple sclerosis 13 1.2.1 Oligoclonal bands 13 1.2.2 Multiple sclerosis, optic neuritis and sinusitis 14 1.2.3 Evidence for a link between sinusitis and multiple sclerosis 17 1.2.4 Bacterial infections and multiple sclerosis 19 1.2.5 Possible mechanisms for a bacterial aetiology 22 1.3 The bacterial toxins hypothesis 25 1.3.1 Association of bacterial toxins with sinusitis and multiple 25 sclerosis 1.3.2 The blood brain barrier 25 1.3.3 Evidence for bacterial toxins in multiple sclerosis 28 1.4 Dolosigranulum pigrum 30 1.4.1 Initial isolation and identification 30 1.4.2 Characterization and naming of Dolosigranulum pigrum 33 1.4.3 Production of anti-Dolosigranulum pigrum in rabbits 35 1.4.4 Experiments using rabbit antisera 36 1.4.5 Epidemiology of Dolosigranulum pigrum 38 1.4.6 Association of Dolosigranulum pigrum to multiple 40 sclerosis 1.5 Aims and objectives of the study 41 iii Chapter 2: Methods 2.1 Flow charts showing methodology for the two aims of the 43 project 45 2.2 Specimens used for the project 2.2.1 Ethical approval 45 2.2.2 Specimens used for culture, fluorescence microscopy and 45 polymerase chain reaction 2.2.3 Specimens used for enzyme linked immunosorbent assay 45 and Western blotting 2.3 Culture for Dolosigranulum pigrum 46 2.4 Fluorescein isothiocyante method to detect 47 Dolosigranulum pigrum 2.5 DNA extraction method 47 2.5.1 Preparation of naso-pharyngeal aspirates, mucous and 47 swabs 2.5.2 DNA extraction 48 2.6 Polymerase chain reaction method 49 2.6.1 Primers 49 2.6.2 Master mix formulation 51 2.6.3 Electrophoretic separation of polymerase chain reaction 51 products 2.6.4 Purification of products for sequencing 52 2.7 Development of an enzyme linked immunosorbent assay 53 method for testing for Dolosigranulum pigrum antibody in serum 2.7.1 Preparation of microtitre trays 53 2.7.2 Establishing a methodology for an enzyme linked 54 immunosorbent assay test for anti- Dolosigranulum pigrum 2.7.3 Multiple sclerosis and matched control sera for 55 Dolosigranulum pigrum antibody screen 2.7.4 Enzyme linked immunosorbent assay method to detect 56 Dolosigranulum pigrum antibody in human sera 2.8 Western blotting 58 2.8.1 Gel preparation 58 2.8.2 Electrophoresis and Western blotting 59 Chapter 3: Results 3.1 Samples tested by culture, fluorescein isothiocyante and 62 polymerase chain reaction for Dolosigranulum pigrum 3.1.1 Fluorescein isothiocyanate and culture results for 62 Dolosigranulum pigrum 3.1.2 Polymerase chain reaction results for Dolosigranulum 65 pigrum 3.2 Dolosigranulum pigrum enzyme linked immunosorbent 76 assay results 3.3 Western blotting of Dolosigranulum pigrum against 79 multiple sclerosis sera and matched control sera using R6 sera Chapter 4: Discussion 4.1 Comparison of culture methods and fluorescein 81 isothiocyanate techniques with polymerase chain reaction iv for the study of Dolosigranulum pigrum in multiple sclerosis 4.2 Sequence analysis of polymerase chain reaction products 88 4.3 Detection of Dolosigranulum pigrum 95 4.4 Enzyme linked immunosorbent assay and Western blotting 96 4.5 Future work to validate the bacterial toxins hypothesis 100 4.6 Conclusions 103 Chapter 5: Reflections on the Professional Doctorate 105 References 111 Appendix A: Ethics, research grants and awards A.1 Ethics 132 A.2 Research grants and awards 136 Appendix B: Reagents and materials B.1 Reagents for DNA extraction using PureLink DNA kits 139 B.2 Electrophoresis reagents and materials 139 B.3 Gene ruler DNA ladder 140 B.4 Dolosigranulum pigrum enzyme linked immunosorbent 140 assay reagents and materials B.5 Western blotting reagents 141 Appendix C: Companies 144 Appendix D: Original identification of Dolosigranulum 145 pigrum Appendix E: Preparation of Dolosigranulum pigrum 148 antisera Appendix F: UPR16 149 v List of Tables Table 1.1 Sources, clinical diagnoses, and demographic information 35 on 27 strains of Dolosigranulum pigrum Table 1.2 Reported clinical isolates of Dolosigranulum pigrum 40 since LaClaire and Facklam‟s study (2000) study Table 2.1 Titration of R6 and anti-rabbit IgG peroxidase conjugate 55 to find ideal concentrations Table 2.2 Detection of antibodies to brain heart infusion in multiple 57 sclerosis and control sera Table 2.3 Multiple sclerosis and matched control sera used for 61 Western blotting Table 3.1 Comparison of polymerase chain reaction results with 70 nasal specimen culture results showing patient demographics and type of specimen Table 3.1 Comparison of polymerase chain reaction results with 71 continued throat specimen culture results showing patient demographics and type of specimen Table 3.1 Comparison of polymerase chain reaction results with 72 continued naso-pharyngeal aspirate specimen culture results showing patient demographics and type of specimen Table 3.1 Comparison of polymerase chain reaction results with 73 continued miscellaneous specimen culture results showing patient demographics and type of specimen Table 3.2 Summary of DNA sequence results 75 Table 3.3 Sequence identification of BLAST data produced from 76 sequences A10, A12 and B25 Table 3.4 Anti-Dolosigranulum pigrum optical density enzyme 77 linked immunosorbent assay results of multiple sclerosis sera Table 3.5 Anti-Dolosigranulum pigrum optical density enzyme 78 linked immunosorbent assay results of control sera (age and sex matched with multiple sclerosis sera Table 4.1 Reactions of miscellaneous catalase-negative Gram 83 positive cocci in basic phenotypic tests vi List of Figures Figure 1.1 Charcot‟s pathological triad 4 Figure 1.2 A representation of the four distinct courses of multiple 5 sclerosis Figure 1.3 Worldwide distribution of multiple sclerosis as of 1994 9 Figure 1.4 Median sagittal section through sella turcica 16 Figure 1.5 Localised defect in the wall of the optic canal 16 Figure 1.6 Immunocytochemistry of acute multiple sclerosis tissues 30 Figure 1.7 Dolosigranulum pigrum on aerobic Columbia blood 32 agar showing α haemolysis after 5 days incubation Figure 1.8 Dolosigranulum pigrum on aerobic Columbia blood 32 agar showing β haemolysis after 8 days incubation Figure 1.9 Cryostat section of spinal cord which grew 37 Dolosigranulum pigrum Figure 1.10 Western blot of extracellular preparation of 38 Dolosigranulum pigrum Figure 2.1