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Physiologic and Molecular Studies on Oral Gnaerobic Spirochetes and Procaryotes Found in Blood

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Physiologic and Molecular Studies on Oral Gnaerobic Spirochetes and Procaryotes Found in Blood PHYSIOLOGIC AND MOLECULAR STUDIES ON ORAL GNAEROBIC SPIROCHETES AND PROCARYOTES FOUND IN BLOOD Richard McLaughlin Faculty of Dentistry McGill University, Montreal March, 1999 A Thesis Submitted to the Faculty of Graduate Shidies and Research in Partial Fulllllment of the Requirements of the Degree of Doctor of Philosophy O Richard McLaughlin, 1999 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AON4 Ottawa ON KI A ON4 Canada Canada Your fik Votre réference Our file Notre rtifdrence The author has granted a non- L'auteur a accordé une licence non exclusive Licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom7 vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwïse de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. TABLE OF CONTENTS Page Abstract ix Resumé xi Acknowledgments xiii Claim of contribution to knowledge xiv List of figures xvi List of tables xiu List of Abbreviations ni CHAPTER 1. Introduction and Literature Review 1. Morphology of Spirochetes 1. Mucoid Layers .. 11. Outer Membrane Sheath -.. 111. Axial Fibrils iv. ProtopIasmic Cylinder v. Peptidoglycan vi. Spirochete Genomes vii. Sphencal Bodies II. Taxonomy of Spirochetes III. Ecology of Spirochetes IV. Periodontal Disease 1. An Overview -. Il. Spirochetes as Etiological Agents S.. Ill. Classification of Novel IsoIates iv. Spirochete Culturability v. Treatment of Periodontal Disease vi. Adherence vii. Cytotoxic Effects S.. Vlll. Iron Sequestration v. Role of the Immune System in Gingivitis and Periodontitis 1. Introduction .* II. interleukin- 1 ... 111. Turnor-Necrosis Factor-a and Lymphotoxin vi . Arachidonic acid v. Meta110 proteinases vi. Stimulation of the Immune System by Treponema denticola vii. s-ary VI. Spirochetes as the Etiological Agent of Alzheimer's Disease 1. Introduction .. LI. Alzheimer's disease *** 111. Pathology iv. Biochemicai Abnonnalities VII. Examination of Blood from Heaithy Humans 1. Introduction .. LI. Phylogenetic Identification of Microbial Cells with Cuitivation .. LU. Nonculture Methods for Identification of Microorganisms iv. Norma flora of the Hurnan Body v. B lood Culture DL. Mycoplasma X. Procaryotes or Eucaryotes XI. Outline of the Thesis Guidelines Regarding Doctoral Theses Containing Quotations From PubIished or Submitted Manuscripts Chapter 2. An inexpensive solid medium for obtaining colony-forming units of oral spirochetes 60 Abstract Paper Acknowledgements Chapter 3. Rapid identification of oral anaerobic spirochetes using restriction fragment lengtb polymorphism of the 16s ribosomal gene Introduction to Chapter 3 Abstract Introduction Materials and methods Bacterial strains Genomic DNA isolation and PCR amplification Restriction fkagment length polymorphisms DNA cloning Results and Discussion Acknowledgements Chapter 4. Factors affecting the formation of spherical bodies in the spirochete Treponema denticola Introduction to Chapter 4 Abstract Paper Acknowledgements Chapter 5. Alzheimer's disease may not be a spirochetosis Introduction to Chapter 5 Abstract Introduction Materials and Methods Subjects B lood work-up Results Discussion Acknowledgements Chapter 6. Naturally-occurring pleomorphic microorganisms in human bIood Introduction to Chapter 6 Abstract Introduction Materials and Methods Blood Electron rnicroscopy Average microscopie field DNA extraction PCR amplification and cloning Sequencing Indirect immunofluorescence FIuorescence in situ hybridization CeIl culture Results and discussion Acknowledgements vii Chapter 7. Summary and Conclusions References ABSTRACT Spirochetes are helical bacteria consisting of an outer sheath, a protoplasmic cylinder and periplasmic flagella. AU oral anaerobic spirochetes (OAS) are species within the genus Treponemu. They are important causative agents of periodontitis. This thesis examines some aspects of the physiology of OAS. As well, a novel symbiotic bacteriun found in the blood of healthy hurnans was studied as a consequence of my work with OAS. Our Iab has been instrumental in rendering routine and reliable pwth of OAS in viîro. An inexpensive medium which rernains molten at 37OC and solidifies at 25OC was found for the enumeration of colony-forming units of OAS. New Oral Spirochete (NOS) medium with the addition of 0.5% gelatin-0.5% Noble agar met the above criteria. Clinical isoIates of spirochetes f?om the periodontal pocket need to be readily identified. Restriction fragment Iength polymorphism (RFLP) analysis was used on reference strains of Treponema denticola, T. vincentii, T. phagedenis, and T. socranskii as well as a nurnber of clinical isolates in our laboratory collection. The banding patterns observed allowed discrimination between the different spirochete species. Morphological variations such as spherical-shaped cells of T. denticola, tenned "spherical bodies" are occasionally observed. The omission of the several individual components fiom NOS medium (brain heart infusion, yeast extract, rabbit serum, volatile fatty acids, or thiamine pyrophosphate), the age of the culture and the addition of lactic acid, enhanced the formation of these bodies. J. Miklossy (NeuroReport, 1993) reported that spirochetes were found in blood, cerebrai cortex and cerebrai spinal fluid in autopsied Aizheimer's Disease (AD) subjects. It was suggested by her that spirochetes could be a causative factor in AD. Our laboratory atternpted to duplicate these results and found spirochetes in the blood of only one late stage AD patient suggesting that spirochetes are not one of the causes of AD. During the examination of blood by darkfïeld microscopy, we observed pleomorphic microorganisms. Blood of a healthy human is a sterile environment. Evidence for the existence of bacteria in blood includes light and electron rnicrographs of their morphology, and molecular analysis of their 16s nbosomal RNA and their gyrB gene. RESUMÉ Les spirochètes sont des bactéries hélicoidales formèes d'une membrane externe, d'un cylindre protoplasmique et d'un flagelle périplasmique. Tous les spirochètes anaérobes oraux (OAS) sont des espèces du "genus" Treponerna. Ils sont une cause importante de la périodontite. Cette thèse explore certains aspects de la physiologie des OAS. De plus, cet analyse des OAS a conduit à l'identification et 1 étude d'une nouvelle bactérie symbiotique retrouvée dans le sang de sujets sains. Les membres de notre laboratoire se sont révélés capables de promouvoir la croissance des OAS de façon routinière et hautement reproductible. Un milieu de culture non-dispendieux, qui se maintient sous forme liquide à 37°C mais se solidifie à 2S°C, servant à l'énumération d'unités formatrices de colonies, a été élaboré par nous. Le milieu NOS (New Oral Spirochete) additionné de 0.5% gélatine-0.5% agar Noble recontre les caractéristiques énumérées ci-dessus. Des isolats cliniques de spirochètes provenant de la poche périodontale doivent être identifiés. L'analyse par RFLP (Restriction fragment length polyrnorphism) de souches de référence de Treponema denricola, T. vincentii, T. phagedenis et de T. socranskii ainsi que de nombreux isolats cliniques provenant de notre propre collection a été faite. Les différents patrons de bandes ont permis de discriminer parmi Les différentes espèces de spirochètes. Des différences morphologiques, telles la formation de cellules sphériques de T. denticola, appeleés "spherical bodies" peuvent parfois être observées. L'omission de différents composants du milieu NOS ("brain heart infusionyy,extrait de levure, serurn de lapin, acides gras volatils, ou de la pyrophosphate de thiamine), l'âge de la culture ainsi que l'addition d'acide lactique, favorisent cette morphologie. J. Miklossy (NeuroReport, 1993) a rapporté que des spirochètes out été retrouvés dans le sang, le cortex cérébral ainsi que dans le fluide cérébro spinal lors d'authopsie de patients atteints de la maladie d'Alzeimer. Elle a aussi suggéré qu' il pourrait y avoir une relation causale des spriochètes pour la maladie. Nous avons tenté de répéter ses resultats, mais n'avons trouvé de spirochètes que dans un cas très avancé de la maladie, suggérant ainsi que l'organisme n'est pas une cause de la maladie. Lors de 1' observation d'échantillons de sang à l'aide de la microscopie à champs obscur nous avons relevé la présence de microorganismes pléomorphiques malgré le fait que. Le sang provenant d'humains sains est un environnement stérile. L'evidence de la présence de bactéries dans le sang humain est présenté l'aide de micrographies lumineuses et électroniques de leur morphologie ainsi que par l'analyses moléculaire de 1'ARN 16s ribosomal et de leur gène @- ACKNO'WLEDGMENTS I wodd like to acknowledge my mother, Eleanor McLaughlin, for her love and support and dedicate this thesis to her. 1also acknowledge the late H.R. McLaughlin and Elizabeth Ralph. They are both sorely rnissed. I acknowledge
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