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Physiology of Qxytetracycline Resistant and Sensitive

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Physiology of Qxytetracycline Resistant and Sensitive PHYSIOLOGY OF QXYTETRACYCLINE RESISTANT AND SENSITIVE STREPTOCOCCUS LACTIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By EMIL MICHAEL MIKOLAJCIK, B. S., M. S ****** The Ohio State University 1959 Approved by Department of Daily Technology ACKNOWLEDGMENT The achievement of this work was made possible through the technical and economic assistance of many persons and organizations. I am grateful for such assistance and deeply indebted to: Dr. W. J. Harper, who assisted with the technical phases of the project and the preparation of the manuscript; Dr. I. A. Gould and staff of The Department of Dairy Technology for having guided and directed my efforts; Dr. W. J. Frajola and Mr. Elson Craig of The Department of Physiological Chemistry, who assisted with the electron microscopy; Grants from The National Institutes of Health and The Ohio Dairy Products Fund for financial aid; The University of Puerto flico, which granted me sabbatical leave and economic assistance; M y wife, Jane, who assisted with her encouragement and perseverance. ii TABLE OP CONTENTS Page INTRODUCTION......................................... 1 REVIEW OP LITERATURE................................ 3 The Physiology of the Genus Streptococcus ........ 3 The Effect of Resistance to Oxytetracycline on Bacterial Metabolism ............................ 11 Effect of Antibiotics on Bacterial Metabolism . 15 SCOPE OP INVESTIGATION................................. 21 GENERAL PROCEDURES..................................... 22 Bacteriological Procedures ........................ 22 Preparation of Cells and Cell F r a c t i o n s ............ 24 Analysis of Metabolic Products .................... 27 Radioactive Tracers Techniques .................... 29 EXPERIMENTAL RESULTS................................... 31 Taxonomic and Morphological Characteristics .... 31 Site of Action of Oxytetracycline...................53 Metabolic Differences between Resistant and Sensitive Cells ................................. 60 The Influence of Antibiotic Binding by Resistant Cells and Cell Fractions on Acid Production of Susceptible Cultures ............................ 70 DISCUSSION............................................. 84 SUMMARY AND CO N C L U S I O N S ............................... 92 APPENDIX............................................... 97 REFERENCES ............................ 112 AUTOBIOGRAPHY........................................ 120 iii LIST OP TABLES Table Page 1 Influence of Large Surface Area and Mechanical Shaking on Acid Production in Milk by Sensi­ tive and Resistant S. lactis 1 0 D ............ 46 2 Turbidity Production of Sensitive and Resistant S. lactis 10D in Sobol's Broth Containing Various Concentrations of S a l t ...................... 47 3 Turbidity Production of Sensitive and Resistant S. lactis 10D in Sobol's Broth of Varying pH . 48 4 Volatile Acid Production in Milk by Sensitive and Resistant S. lactis 10D Cultured in the Presence afld Absence of Oxytetracycline .... 51 5 Titratable Acidity of Milk Cultured with Sensi­ tive and Resistant S. lactis 10D in the Presence and Absence of Oxytetracycline .... 52 6 Distribution of Radioactive Oxytetracycline Among Various Cell Fractions and Washings of Resistant and Sensitive S. lactis 10D With and Without Incubation.................... 56 7 Effect of Washing on the Radioactive Oxytetracy­ cline Binding by Resistant s. lactis 10D Cells Which Had Been Carried in Oxytetracycline-Free Media Prior to U s e .......................... 58 8 Effect of Centrifugation on the Radioactivity of Washings of Resistant and Sensitive S. lactis 10D Cells Exposed to Radioactive Oxytetra­ cycline ....................................... 59 9 Influence of Oxytetracycline upon the Amino Acid Content of Culture Medium, Intracellular Frac­ tion, and Cell Wall of Sensitive and Resistant S. lactis 1 0 D ............................. 61 10 Influence of Oxytetracycline upon the Bio­ synthesis of Carbonyl Compounds in Milk and Sobol's Broth by Resistant and Sensitive S. lactis 1 0 D ............................. 64 iv V Table Page 11 Rate of Biosynthesis of C1^ Amino Acids and Carbonyl Compounds from Radioactive Acetate by Sensitive and Resistant S. lactis 10D C e l l s ......................................... 68 12 Rate of Acid Production by Resistant and Sensi- tive S, lactis in Milk Containing Various Con­ centrations of Oxytetracycline............... 71 13 Effect of Fresh Packed Resistant S. lactis 10D Cells upon Acid Production of Sensitive S. lactis 10D Starter Cultured in Milk Containing Oxytetracycline.............................. 72 14 Effect of Fresh Resistant S. lactis 10D Starter upon Acid Production of Sensitive S. lactis 10D Organisms Cultured in Milk Containing Oxytetracycline.............................. 75 15 Effect of Fresh Resistant S. lactis 10D Starter upon Acid Production of H-15 Commercial Starter Cultured in Milk Containing Oxytetracycline.............................. 76 16 Effect of Simultaneous Addition of Resistant and Sensitive Starters upon Acid Production in Milk Containing Oxytetracycline .............. 78 17 Effect of Resistant Starter on Measurement of Oxytetracycline by the Disc" Assay Method . 79 18 Effect of Method of Preparing Resistant Cells on Acid Production of Sensitive S. lactis 10D Starter Cultured in Milk Containing Oxytetracycline . .......................... 81 19 Effect of Resistant Inactive Starter upon the Acid Production of Sensitive S. lactis 10D in Milk Containing Oxytetracycline . '. 82 20 Influence of Resistant S. lactis 10D Starter upon the Acid Production of Sensitive S. lactis 10D Cells Cultured in Milk Containing Chlortetra- cycline and Penicillin ...................... 83 21 Influence of Large Surface Area and Mechanical Shaking.................... 97 vi Table Page 22 Comparative Amino Acid C o n tent.................... 98 23 Rate of Biosynthesis of Carbonyl Compounds.... 10J 24 Rate of Acid P r o d u c t i o n ......................... 102 25 Effect of Fresh Packed Resistant Cells on Acid Production...................................... 103 26 Effect of Resistant Cultures on Sensitive S t a r t e r ........................................ 104 27 Effect of Resistant Cultures on H-3 Starter . 105 28 Effect of Resistant Cultures on H-9 Starter . 106 29 Effect of Resistant Cultures on H-15 Starter . 107 30 Effect of Simultaneous Addition of Resistant and Sensitive Starters ........................ 108 31 Effect of Method of Preparing Resistant Cells . 109 32 Effect of Inactive Resistant Starter ............ 110 33 SHlortetracycline and Penicillin Binding Properties of Resistant Starter .............. Ill LIST OP FIGURES Figure Page 1 Cross Section of Sensitive S. lactis 10D .... 35 2 Cross Section of Resistant S. lactis 10D .... 37 3 Fixed Cell of Sensitive S. lactis 1 0 D .... 40 4 Fixed Cell of Resistant S. lactis 1 0 D .... 42 5 Growth Curve of S. lactis 1 0 D ................ 54 6 Influence of 00 mg. Resistant Cells on the Acid Production of Sensitive Starter.......... 74 7 Influence of Resistant Starter on Acid Produc­ tion of H-15 Commercial S t a r t e r ............ 77 vii INTRODUCTION The successful manufacture of cheese and cultured dairy products is dependent upon satisfactory growth and acid production by bacterial starters in fluid milk. This milk-starter relationship has entailed many technical problems, one of which has been starter failures due to foreign inhibitory substances in the milk. Antibiotics, which have been utilized as the major means of mastitis control for the past 15 years, contribute materially to many starter failures, and thus are of con­ cern to the processors of cultured dairy products. Effec­ tive means of detecting and eliminating antibiotics in milk, or means of overcoming their deleterious effect on starter organisms, have been a major objective of the dairy industry and many Investigators. The development of antibiotic resistant starter cultures would appear to offer a solution to the problem. However, lactic acid producing bacteria which have been made resistant to antibiotics do not produce acid at a rate and quantity satisfactory for manufacturing purposes. Elucida­ tion of the fundamental cause of decreased acid production by normal numbers of resistant bacteria and understanding of 1 the physiological differences between these bacteria and their sensitive counterparts opens an avenue which could conceivably lead to a solution of the antibiotic problem in the manufacture of products dependent upon starter cultures. REVIEW OF LITERATURE The physiology of Streptococcus lactis and alterations in its metabolic processes due to oxytetracycline* are the major areas of interest related in this investigation. These considerations as they apply to the genus Streptococcus are presented in this review as separate sections. The Physiology of the Genus Streptococcus Compounds Derived from Carbohydrate Metabolism Many of the bacteria comprising the genera Lactobacillus, Streptococcus, and Bacillus are homofermenta- tive. Major characteristics of these homofeimentative bacteria are: (a) production of lactic acid as the principal product of carbohydrate catabolism; (b) respiration by the flavo-protein system; and (c) lack of the enzyme, catalase (41, 87). In contrast, the heterofermentative organisms are reported to produce, in addition to lactic acid, acetic acid and carbon dioxide (4l). Even though the genus Streptococcus is considered
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