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Information to Users INFORMATION TO USERS The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 00S1079 The survival ofStaphylococcus aureus in abscesses from streptozotocin-induced diabetic mice Harvey, Kevin Michael, Ph.D. The Ohio State University, 1990 Copyright ©1990 by Harvey, Kevin Michael. All rights reserved. UMI 300N.ZeebRd. Ann Arbor, MI 48106 THE SURVIVAL OF STAPHYLOCOCCUS AUREUS IN ABSCESSES FROM STREPTOZOTOCIN-INDUCED DIABETIC MICE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kevin Michael Harvey, B.A. ******* The Ohio State University 1990 Dissertation Committee: Approved by: Frank A. Kapral, Ph.D. Bernard U. Bowman, Jr., Ph.D. Thomas F. DeMaria, Ph.D. Abramo C. Ottolenghi, Ph.D. , Adviser Jepartment of Medical Norman L. Somerson, Ph.D. Microbiology and Immunology Copyright by Kevin Michael Harvey 1990 DEDICATION To my wife, Betsy, whose love and faith provided the encouragement to reach this point. To my sons, Christopher and Brendan, for making the work worth the effort, and to my family especially my parents, for their love and support. ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Frank A. Kapral, for his patience and guidance. My sincerest thanks to my committee members, Dr. Norman L. Somerson, Dr. Abramo C. Ottolenghi, Dr.Thomas F. DeMaria, and Dr. Bernard U. Bowman, for their suggestions and comments. I would also like to thank Judy Hart and Shelley Smith for their technical assistance and friendship. My gratitude is extended to the support staff in the "kitchen" and office for all their help. I also wish to thank all my colleagues who started their research with me especially, EAF, ALL, JLP, XZ, and GL, for their friendship. VITA May 17, 1958.............. Born - Pittsburgh, Pennsylvania 1980...................... B. A. cum laude. University of North Texas, Denton, Texas 1980-198 1................ Junior Chemist, Kolmar Laboratories, Inc., Denton, Texas 1981-198 4................ Chemist, Sasco Cosmetics, Inc., Carrollton, Texas 1984-Present.............. Graduate Research Associate, Department of Medical Microbiology and Immunology, The Ohio State University, Columbus, Ohio PUBLICATIONS Harvey, K. M., J. Hart, and F. A. Kapral. Survival of Staphylococcus aureus in intraperitoneal abscesses from diabetic mice. In: Jelijaszewicz, J. (ed) The Staphylococci. Zbl. Bakt. Suppl. 15. Gustav Fischer Verlag, Stuttgart (In Press). FIELDS OF STUDY Major Field: Medical Microbiology and Immunology Major Area of Study: Host-Parasite Interactions iv TABLE OF CONTENTS DEDICATION ........................................... ii ACKNOWLEDGEMENTS....................................... iii VITA ................................................. iv TABLE OF CONTENTS.................................... V LIST OF T A B L E S ......................................... vii LIST OF FIGURES.......................................... ix LIST OF P L A T E S ............................ xi CHAPTER I. INTRODUCTION .............................. 1 II. MATERIALS & METHODS........................ 15 M i c e ....................................... 15 Staphylococcal strains and animal inoculation.......................... 15 Determination of blood glucose ............ 16 Generation of diabetic m i c e ................ 17 Evaluation of staphylococcal survival within a b s c e s s e s ............................ 18 Bactericidal assay ........................ 18 Histological sections ...................... 19 Lipid e x t r a c t i o n ...................... 20 Fractionation of lipids .................... 21 Collection of individual lipids ............ 22 Esterification of free fatty acids and gas chromatographic analysis .............. 23 Trimethylsilyl ether derivatives of monoglycerides and gas chromatographic analysis.............................. 24 Determination of glucose concentration in intraperitoneal abscesses ............ 26 Chemicals and reagents .................... 27 III. RESULTS.................................... 28 Generation of diabetic m i c e ................ 28 Survival of staphylococci in abscesses within diabetic mice . .................... 29 vi Histology of intraperitoneal staphylococcal a b s c e s s e s ............................ 32 Analysis of lipids in intraperitoneal abscesses.............................. 33 Glucose concentration within abscesses . 35 IV. DISCUSSION ................................... 38 BIBLIOGRAPHY ........................................ 88 LIST OF TABLES Table Page 1. Average blood glucose (mg/dl ± SD) before and after inoculation with streptozotocin (STZ) or citrate (CIT) ........................... 54 2. Percent positive mice made diabetic with streptozotocin and controls receiving citrate.................................... 55 3. Average blood glucose (mg/dl ± SD) before and after infection with S. aureus 18Z ........ 57 4. Total lipids extracted from S. aureus 18Z intraperitoneal abscesses at 5 d a y s .... 73 5. Total lipids extracted from 10 day S. aureus 18Z intraperitoneal abscesses .............. 74 6. Total triglycerides extracted from 5 day S,. aureus 18Z intraperitoneal abscesses .... 75 7. Total triglycerides extracted from 10 day S. aureus 18Z intraperitoneal abscesses . 76 8. Composition of free fatty acid fraction extracted from 5 day S. aureus 18Z intraperitoneal abscesses ..... ........ 77 9. Composition of free fatty acids extracted from 10 day S. aureus 18Z intraperitoneal abscesses.............................. 78 10. Free fatty acids extracted from 5 day S. aureus 18Z intraperitoneal abscesses . 79 11. Free fatty acids extracted from 10 day S. aureus 18Z intraperitoneal abscesses . 80 12. Total amount of monoglycerides extracted from 5 day S. aureus 18Z intraperitoneal abscesses.................................. 81 vii 13. Total monoglycerides extracted from 10 day S. aureus 18Z intraperitoneal abscesses . 82 14. Total unsaturated and saturated monoglycerides extracted from S. aureus 18Z intraperitoneal abscesses at 5 and 10 days . 83 15. Sensitivity of Staphylococcus aureus strains TG and 303 grown in different media to oleic acid and 1- and 2-monoolein................ 84 16. Evaluation of lipemic effect of abscess homogenate on Glucostix (Ames) ............ 87 * viii LIST OF FIGURES Figure Page 1. Fasting blood glucose concentrations of mice before and after treatment with streptozotocin or citrate ................... 56 2. The survival of Staphylococcus aureus strain 18Z in the abscesses from control and diabetic m i c e .............................. 64 3. Survival curves of s. aureus strain PG114 in abscesses of control and diabetic mice . 65 4. The survival of S. aureus strain P78 in intraperitoneal abscesses from control and diabetic animals .......................... 66 5. The survival of S. aureus strain P78-22 in the abscesses from control and diabetic animals.................................... 67 6. Bactericidal activity of the total lipid pool extracted from the abscesses of control and diabetic mice 5 days post infection with S. aureus 1 8 Z ...................... 68 7. Bactericidal activity of the total lipid pool extracted from 5 day old abscesses of control and diabetic mice infected with S. aureus 1 8 Z ........................................ 69 8. Bactericidal activity of the total lipid pool extracted from the abscesses of control and diabetic animals 10 days post infection with S. aureus 1 8 Z .............................. 70 9. The bactericidal activity of total lipids extracted from abscesses of control or diabetic mice at 10 days post infection with S. aureus strain 1 8 Z ...................... 71 10. Fasting blood glucose concentrations of mice infected with S. aureus strain P78-22 .... 85 ix 11. The glucose concentration of S. aureus strain P78-22 abscesses in control and diabetic m i c e .................. 86 x LIST OF PLATES Plate Page I. Ixnmunocytochemical staining of pancreas islets from a normal mouse using the indirect immunoperoxidase technique to detect insulin
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