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Genetic Determinants of Antibiotic Resistance in Staphylococcus Aureus and Mycobacterium Smegmatis

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Genetic Determinants of Antibiotic Resistance in Staphylococcus Aureus and Mycobacterium Smegmatis 71-7518 MILLER, Marcia Ann, 1942- GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS AND MYCOBACTERIUM SMEGMATIS. The Ohio State University, Ph.D., 1970 Microbiology Please Note: School lists name as Marcia Ann Wolfe Miller. University Microfilms, A \ERO\Company , Ann Arbor, Michigan Copyright by Marcia Ann Miller 1971 Copyright by Marcia A n n Miller 19 71 71-7518 MILLER, Marcia Ann, 1942- GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS AND MYCOBACTERIUM SMEGMATIS. The Ohio State University, Ph.D., 1970 Microbiology Please Note: School lists name as Marcia Ann Wolfe Miller. University Microfilms,A \ER0X Company , Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS AND MYCOBACTERIUM SMEGMATI3 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Marcia Ann Miller, B.S., M.A. The Ohio State University 1970 Approved by Adviser Academic Faculty of Microbial and Cellular Biology ACKNOWLEDGMENTS I wish to express my appreciation to my advisor, Dr. Melvin S. Rheins, for his guidance throughout the course of my graduate study. I also wish to express my gratitude to fellow graduate students Helen M. Pollock and Robert Z, Maigetter and to Mr. Ivan Kapotenovic for his technical assistance. Most of all, I would like to thank my husband Larry for his constant encouragement, patience, and halpfulnoss, without which this degree would not have been possible. ii VITA November 23, 1942. Born - Fremont, Ohio 1956-1960. ....... Attended Salem-Oak Harbor High School, Oak Harbor, Ohio 1960-1962. ........... Attended Michigan State University, East Lansing, Michigan December 27, 1962. , Married - Larry Reed Miller 1963-1965................ B.S. Ed., Bowling Green State University, Bowling Green, Ohio 1965-196 6 ................ M.A., Bowling Green State University, Bowling Green, Ohio 1966-1968, ....... Research Biologist, Genetics Division, Department of Biology, Bowling Green State University, Bowling Green, Ohio (Drosophila Genetics and NASA Biosatellite Project) 1968-1970............. Ph.D., The Ohio State University, Columbus, Ohio PUBLICATIONS Marcia A. Miller and Shirley A. Harmon, "Genetic Associ­ ation of Determinants Controlling Resistance to Mercuric Chloride, Production of Penicillinase and Synthesis of Methionine in Staphylococcus aureus." Nature. 215:531-2, 1967. Marcia A. Miller and Irwin I. Oster. "Further Observations on Temperature Sensitive Mutations in Multicellular Forms." Proceedings XII International Congress of Genetics.' 1; l4l~. 1968. iii VITA (Continued) FIELDS OF STUDY Major Field: Microbiology Area of Specialization.Microbial Genetics iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS ......................................... ii VITA ...................................................... iii LIST OF TABLES .......................................... vii LIST OF ILLUSTRATIONS .................................. viii INTRODUCTION ........................................... 1 REVIEW OF THE LITERATURE .............................. 5 Drug Resistance and Microbial Genetics ..... 5 Mutagenesis and Extrachromosomal Inheritance . 11 Genetic Studies: Transduction and Lysogeny . 18 MATERIALS AND METHODS .................................. 22 Source and Description of Cultures . ............ 22 Basic Propagation Media ......... ....... 25 Iodometric Method for Activity of Penicillinase 26 Detection of Penicillinase Activity by a Bio-Assay Method ....... 27 Isolation of D-Cycloserine Resistant Mutants of S. aureus ........... 27 Determination of Spontaneous Mutations ..... 28 Acridine Orange and Temperature Treatment . 29 Induction of Mutants by Ultraviolet Irradiation ......................................... 31 Propagation and Titration of Bacteriophages . 32 Transduction Technique ........................... 34 v TABLE OF CONTENTS (Continued) Page Harvesting of Cells for DNA Extraction .... 34 DNA Extraction and Determination of DNA .... 36 Determination of DNA Base Composition ..... 39 RESULTS ................................................ 41 Culture Characteristics and Techniques for Cultivation of M. smegmatis 607 B and .§• aur'eus U 9 W ............. 41 Bacteriophages: Host Range, Plaque Formation, and Propagation ........... ......... 50 Methods for Detection of Penicillinase Activity ..... .............................. 60 Characterization of D-cycloserine Resistant Cells ................ 66 Lack of Reversal of D-cycloserine Inhibition by Mycobactin, a Growth Factor for Specific Mycobacteria ..... 74 Spontaneous Mutation Rates .......... 78 The Effect of Acridine Orange and Elevated Temperature on Penicillin and D-cycloserine Resistant Cells ......... .............. 78 The Mutagenic Effect of Ultraviolet Radiation on Penicillin and D-cycloserine Resistant Cells 82 Transduction of Penicillin and D-cycloserine Resistance . ................ 85 Extraction of DNA . 87 Determination of DNA Base Composition ......... 87 DISCUSSION ................................................ 103 SUMMARY ................................................ 119 BIBLIOGRAPHY .............................................. 121 vi LIST OF TABLES Table Page • 1. Composition of Synthetic Medium ....... 23 2. Reagents used in Deoxyribonucleic Acid Extraction Procedures ........................... 35 3. Amino Acid Requirements for M. smegmatis 607 B and S. aureus U9W in a Synthetic Medium . ........... ..... 51 4. Titer of Various Mycobacteriophage After Six Hours of Incubation for Propagation at 37 C with M. smegmatis 607 B in Varying Concentrations of Tween 80 .... 55 5. Phage Typing Patterns Exhibited by Various Mycobacteriophages on Several Strains of Mycobacteria Employing the Spot-Plate M e t h o d ......................................... 56 6. Degree of Resistance to D-cycloserine Demonstrated by Various Organisms on Several Types of Semi-Solid Laboratory Media 70 7. Spontaneous Mutation Rate to Drug Sensitivity of D-cycloserine Resistant Cloned Cells of S. aureus U9W and M. smegmatis 607 B ........................... 79 8. The Effect of Mutagenic Agents Upon Loss of Penicillin and D-cycloserine Resistance in Strains S. aureus U9W and smegmatis 607 B ........... 81 9, Transduction of the Penicillinase and D-cyloserine Determinants to Penicillin and D-cycloserine Sensitive Cells of Strain U9V/ of S. aureus ........... 86 vii LIST OF ILLUSTRATIONS Figures Figure Page 1. Effect of Tween 80 in varying concentrations on the propagation of mycobacteriophage D29 with host M. smegmatis 607 B as indicated by a drop in phage titer with increase in per cent of Tween 80 incorporated into the culture medium. ....................... 54 2, The effect of various broth media on the degree of resistance to D-cycloserine demonstrated by S. aureus (penicillin resistant), S. aureus (penicillin sensitive) and M. smegmatis 60T~B ........... 73 3, Antagonism of D-cycloserine inhibition of aureus growth by heated preparations containing L-alanine . ..................... 76 4. Survival curve of M, smegmatis 607 B after irradiation with ultraviolet rays .............. 84 Plates Plate Page I M. smegmatis 607 B. Typical rough colony formation on TSA after 72 hours incubation at 37 C in the dark ....................... 43 II M. smegmatis 607 B on Lowenstein-Jensen m e d i u m ........................................... 46 III S. aureus U9W on TSA, demonstrating differences in pigmentation . ................ 49 IV Plaques of various mycobacteriophage plated on M. smegmatis 607 B.... .................. 59 V Phage susceptibility of S. aureus U9V/ (penicillin resistant variant) to staphylococcal and mycobacteriophages .... 62 viii LIST OF ILLUSTRATIONS (Continued) Plate Page •VI Agar-plate modification of the iodometric assay for production of penicillinase in the penicillin resistant variant of S. aureus U9W 65 VII Detection of penicillinase activity determined by a bio-assay method, using a penicillin sensitive variant of S. aureus for noting inhibition by residual penicillin , 68 VIII Densitometer trace and corresponding ultraviolet photograph of DNA from M. smegmatis 607 B .............................. 91 IX Ultraviolet photographs of mycobacterial DNA coming to equilibrium during a 24 hour run . 93 X Ultraviolet photographs of mycobacterial DNA coming to equilibrium during a 24 hour run . , 95 XI Densitometer trace and corresponding ultraviolet photograph of DNA from S. aureu3 U9W, Pase+ D-CSr . ............. .. 98 XII Ultraviolet photographs of staphylococcal DNA coming to equilibrium during a 24 hour run ........... 100 XIII Ultraviolet photographs of staphylococcal DNA coming to equilibrium during a 24 hour run ............. 101 ix INTRODUCTION The discovery of numerous antimicrobial agents during the past 30-40 years has greatly altered both the treatment and relative importance of many diseases. Following the spectacular discoveries of many antimicrobial agents, however, was the disturbing recognition of the emergence of drug resistant pathogenic microorganisms. These strains have become important not only in clinical medicine but to the molecular biologist as well where these organisms have served as tools for genetic research. Our knowledge of the function and regulation of nucleic acids has paralled g advancements in antimicrobial therapy. Studies on the mechanism of drug resistant strains have contributed to the clarification of
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