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Faculty of Graduate Studies and Research M.Sc. Comparative t Faculty of Graduate Studies and Research M.Sc. Comparative Growth and Locomotion of Anaerobie Oral Spirochetes by Antonia Klitorinos A thesis submitted to the Faculty of Graduate Studies and Research, McGi:' University, in partial fulfillment of the requirements for the degree of Master of Science. Department of Microbiology and Immunology McGill University Montreal, Quebec, Canada © March 1991 1 1 / - 1 ABSTRACT A positive relationship has been established between the number of oral spirochetes in diseased periodontal pockets and the severit~ of periodontal disease. Elucidation of the role that spirochetes play in periodontal disease has been hampered by the difficulty of growing these organisms. Growth studies were carried out in or,jer to obtain a better und~rstanding of the nutritional requirements of three specÎes of oral spirochetes. Growth was followed by cell counts and turbidity measurements. Long-chain fatty acids were shown to support the growth of ail treponemes studied with the exception of Treponema ~anskjj. Short-chain fatty acids and rabbit serum were found to be essential for growth. Glucuronic acid was shown to stimulate growth. Locomotion of oral spirochetes has been suggested to ba an invasive factor for tissue penetration. Video time-Iapse microscopy using darkfield optics was employed to study the locomotory behaviour of spirochetes in media of different densities. Optimal migration of spirochetes was found to be viscosity-dependent. Treponema denticola ATCC 35404 and T. dentjcola ATCC 35405 exhibited the greatest mean speeds at 0.2 - 0.3 % (wt.lvol.) Noble agar. Optimal motility for T. vjncentjj ATCC 35580 and 1. socranskjj ss. socranskii ATCC 35536 was achieved at 0.35 - 0.40 % (wt./vol.) Noble agar. 1 i '" .., 1 RESUME Il existe une association positive entre les nombres de spirochètes buccaux trouvés dans les poches des gencives et la sévérite de la périodontite. Le rôle joué par ces microbes dans le développement de la périodcntite n'a pas été élucidé, surtout à cause de la grande difficulté dans la culture de ces organismes. En effet, des études entreprises au sujet des besoins nutritifs nécessaires à la culture des spirochètes ont établi une meilleure connaissance de trois espèces de spirochètes buccaux. La croissance fut suivie en comptant le nombre de bacteries viables en fonction de la transmltance à l'aide de spectrophotomètre. Les acides gras à longue chaîne aidèient la croissance tous les spirochètes, à l'exception de T. socranskii. tandis que les acides gras à courte chal'ne et le sérum de lapin se sont avérés absolument nécessaires. L'acide glucuronique stimula la croissance. Il fut suggéré que la virulence dépend directement de la mobilité des spirochètes buccaux. La microscopie avec illumination sur fond noir, combiné avec le vidéo-accéléré a été utilisé dans If étude du comportement migratoire des spirochètes dans des milieux de culture de différentes consistances. La migration optimale des spirochètes dépend directement de la densité des milieux. Les I. Win.1jcola ATCC 35404 et ATCC 35405 affichaient la plus grande vitesse moyenne avec 0.2 - 0.3 % (w/v) d'agar Nob!e. La mobilité optimale pour T. vincentii et T. ~ocranskil fut établie à des concentrations de 0.35 - 0.40 % (w/v) d'agar Noble. t 11 ACKNOWLEDGEMENTS These investigations were carried out in the Lyman Duff Medical Sciences Laboratory and S+rathcona Anatomy and Dentistry Laboratory, McGili University, Montreal. First and foremost, 1 would like to express my heartfelt gratitude to my parents and my brother for their support and encouragement. 1 would like ta thank Dr. E.C.S Chan for his supervision and guidance throughout the course of my study. Finally, 1 would like to thank Dr. P. Noble for his technical assistance in the mati lity study and Dr. J. Dealy for the use of the Wells-Brookfield Micro-Viscometer and his expert advice in rheology. 1 iii 1 TABLE OF CONTENTS Page ABSTRACT .... ......................................................................................................... .i RESUME .................................................................................................................. .ii ACKNOWLEDGEMENTS .................................................................................... .iii LIST OF TABLES ..................................................................................................vi LIST OF FIGURES ..............................................................................................vii .NTRODUCTION .....................................................................................................1 LlTERATURE REVIEW: 1. HISTORY .............................................................................................................3 II. GENERAL CHARACTERiSTICS OF SPIROCHETES ................................. 3 i. SlimeLayer ................................................................................... 5 ii. OuterSheath .................................................................................5 iii.Axial Fibril. .................................................................................6 iv.ProtoplasmicCylinder .............................................................. 7 III. TAXONOMY OF SPIROCHETES .................................................................. .7 i. Borrelja ..........................................................................................8 ii. Leptospjra .....................................................................................9 iiLSpjrochaeta ............................................................................... 10 iv.Crjstjspjra ..................................................................................1 0 v. Trepooema ................................................................................... 11 IV. CLASSIFICATION OF ORAL SPIROCHETES ......................................... 11 V. THE ISOLATION AND MAINTENANCE OF ORAL SPIROCHETES ...... 12 VI. LOCOMOTION OF SPIROCHETES ............................................................ 14 ! -----------------------~~ --~~ ~ 1 VII. PERIODONTAL DISEASE. .......................................................................... 16 i. Clinical Characteristics of Periodontal Disease ........ 16 iL Positive Numerical Correlation between Oral Spirochetes and Penodontal Disease .............................. 18 iii. Evidence of Spirochetal Invasion of the GingivaITissue ..................................................... 20 VIII. PUTATIVE PERIODONTOPATHIC MECHANISMS ............................. 22 1. Toxic Metabolic End Products and Enzymes Released by Oral Spirochetes may Contribute to Periodontal Disease ............... ,....................................... 22 iL Inhibition of Cell Proliferation ......................................... 24 IX. CELLULAR AND HUMORAL IMMUNITY ................................................... 25 X. A METHOD FOR THE DETECTION OF ORAL BACTERIA ...................... 26 MATE RIALS AND METHODS.......... ............... ............... .. ................................ 27 RESULTS: 1. GROWTH STUDIES .................................................................... 50 Il. EFFECT OF AGAR DENSITY ON GROWTH .......................... 64 III. COLLAGEN BREAKDOWN TEST ............................................ 68 IV. ISOLATION OF COLONIES ...................................................... 68 V. CHEMOT AXIS .............................................................................69 i. Hard agar plug technique ................................................. 69 ii. Weil plate technique ........................................................72 iii. Test-tube method ............................................................73 iv. Bacteria in plug method .................................................. 73 v. Chemical in pond method ............................................... 74 VI. MOTILITY EXPERIMENTS ........................................................................... n DiSCUSSiON .........................................................................................................84 , BiBLIOGRAPHY ....................................................................................................95 ./ r1. 1 LIST OF TABLES Table Page 1. Morphological characteristics of four oral spirochetes.. ..... 28 2. Comparative growth of oral anaerobic spirochetes ln growth media (expressed in % values as calculated from cell counts) ....................................................................................63 3. Comparative growth of oral anaerobic spirochetes in growth media (expressed in % values as calculated from 0.0 readings) ................................................................................ 65 4. Chemotacti(; behavior of I.dentjcola ATCe 35404 and A TCe 35405 to a variety of test-chemicals by the hard agar plug ter.:hn,:que ....................................................................... 70 5. Average locomotory speeds of spirochetes in NOS medium of different Noble agar concentrations ........................... 78 6. Range of persistence of spirochetes in NOS medium of different Noble agar concentrations ................................................ 81 1 LIST OF FIGURES Figure "age 1 . Schematic diagram of a spiror.ht:tte cell and its cross-section ......................................................................4 2. Schematic representation depicting how a spirochete cell swims (Bergls Model) ............... ' ............................................
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