View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M University AUTOINDUCER 2-BASED QUORUM SENSING RESPONSE OF ESCHERICHIA COLI TO SUB-THERAPEUTIC TETRACYCLINE EXPOSURE A Dissertation by LINGENG LU Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2004 Major Subject: Food Science and Technology AUTOINDUCER 2-BASED QUORUM SENSING RESPONSE OF ESCHERICHIA COLI TO SUB-THERAPEUTIC TETRACYCLINE EXPOSURE A Dissertation by LINGENG LU Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved as to style and content by: Suresh D. Pillai Michael E. Hume (Chair of Committee) (Member) James J. Zhu Luc R. Berghman (Member) (Member) Gary R. Acuff Alan R. Sams (Member) (Head of Department) Rhonda K. Miller (Chair of Food Science and Technology Faculty) August 2004 Major Subject: Food Science and Technology iii ABSTRACT Autoinducer 2-based Quorum Sensing Response of Escherichia coli to Sub-therapeutic Tetracycline Exposure. (August 2004) Lingeng Lu, B.M.; M.M., Shanghai Medical University Chair of Advisory Committee: Dr. Suresh D. Pillai Autoinducer 2 (AI-2) is a quorum sensing signal employed by bacteria to coordinate their response to environmental stresses. The objective of this study was to determine the relationship between presence of AI-2 molecules, exposure to sub-therapeutic tetracycline, the expression of genes associated with the conjugal transfer of antibiotic resistance plasmids, and the conjugal transfer of these plasmids in Escherichia coli. The studies showed that AI-2 activity increased in Tets E. coli in the presence of tetracycline (2 µg/mL) under both batch and continuous culture conditions. The presence of AI-2 molecules induced tetracycline tolerance development in Tets E. coli. The studies showed that the survival rates of Tets E. coli exposed to AI-2 molecules were significantly higher compared to the cells not exposed to AI-2 molecules or cells that were exposed to only LB (Lauria-Bertani) broth. Molecular analyses using real-time PCR indicate that the expression of at least one conjugation-associated gene (trbC) is increased 9-fold in cells exposed to AI-2 molecules in the presence of sub-therapeutic tetracycline compared to its negative controls. The transconjugation frequency of the plasmid RP4 carrying the tet(A) gene increased between 10-100 fold in the presence of AI-2 molecules. In companion studies, AI-2-like activity was detected in fish, tomatoes, cantaloupes, carrots and milk samples. Interestingly, ground beef and poultry meat contained substances that appear to inhibit AI-2 activity. Collectively, these iv results highlight the potential importance of bacterial quorum sensing signals such as AI-2 in the response of bacterial cells to environmental stimuli and the possible role of quorum sensing signals in the quality and safety of foods. v DEDICATION My doctor of philosophy and this dissertation are dedicated with my greatest gratitude and deepest love to my wife and brothers and sisters-in-law and mother-in-law and in memory of my grandparents and parents vi ACKNOWLEDGMENTS My sincerest appreciation is expressed to Dr. Suresh D. Pillai, my advisor, for his academic advice. With patience, attention, wisdom and broad academic knowledge, he led me through this project and all of my graduate studies. The benefits I gained from him are long-term, lasting throughout my future professional career. He is becoming my model for my academic life. I appreciate Drs. Michael E. Hume, Luc R. Berghman, James J. Zhu and Gary R. Acuff for serving as my committee members and dissertation advisors with consideration, affection, and profound knowledge. The benefits I gained will last throughout my life. My gratitude also goes to Drs. Bonnie L. Bassler at Princeton University, Venessa Sperandio at Southwestern Medical Center in Dallas, William E. Bentley at University of Maryland, Lai King Ng at National Microbiology Laboratory in Canada, James W. Golden and Duan Liu at Texas A&M University for kindly providing many of the bacterial strains used in the study. I thank Drs. Allen Byrd, Leon F. Kubena, Tony Poole, Haiqi He and Kenneth Bischoff at USDA, Peter Feng at FDA, Ellen W. Collisson, Alan R. Sams, Rhonda K. Miller, Steven C. Ricke, James Keeton, Lee A. Cartwright, Mr. Robert L. Pottberg, Ms. Elizabeth Hirschler, Ms. Jo Ann Pilkey, and Ms. Pattie F. Horsman for their support and care during my studies. I also thank my ever-present lab companions: Everado Vega, Kenneth Widmer, James Totten, Matthew Roe, Dr. Seema Endley, Jennon Smith, Dr. Palmy Rajan Jesudhasan, Reema Singh, Kamlesh Soni, Deepak Srikumar, Isabel Espinosa, Jessica Cardenas, Robert Spence and Leroy Cortinas. I am happy to have worked and shared the fun with this group. vii My appreciation is extended to my friends at Texas A&M including Michael Sawilowsky, Chen Shuen-ei, Wang Limn, Liu Xingchu, Cao Guangtong, Li Xin, Charles A. Hernandez, Damon Drinnon, Zhang Ping, Shen Shixue, Cai Jianfeng, Zhang Cheng, Li Yuqian, Gu Xinsheng, Xiao Yumin, Zhang Xiaodong and Lai Runzhi. It has been an unforgettable time at Texas A&M University in College Station. My sincere gratitude also goes to my conversation partners Ms. Hayden Hefflefinger and Ms. Abigail Grass for their inspiration. I was honored to be a Tom Slick Fellow at Texas A&M University. I wish to thank The Tom Slick Fellowship committee for their valuation. The honor encouraged me to work my best. The completion of this dissertation is not an end but just commencement. In my life, my dear family is my harbor for sailing, and is my backbone to stand up and bravely face any challenge. Without their selfless support and love, I would not have the chance for climbing the steep mountain of knowledge. Thanks to God for giving me the wisdom to explore the whole new world, giving me the key to open the treasure of knowledge, and offering me opportunities to experience the spirit of sophisticated culture and science. viii TABLE OF CONTENTS Page ABSTRACT……………………………………………………………………….. iii DEDICATION…………………………………………………………………….. v ACKNOWLEDGMENTS…………………………………………………………. vi TABLE OF CONTENTS………………………………………………………….. viii LIST OF TABLES ………………………………………………………………… xi LIST OF FIGURES………………………………………………………………... xiii CHAPTER I INTRODUCTION………………………………………………….. 1 Rationale ……………………………………………………………. 1 Overall objective of the study……………………………………….. 3 Specific objectives ………………………………………………….. 3 II LITERATURE REVIEW…………………………………………… 4 Sub-therapeutic antibiotic administration in animals ………………. 4 Tetracycline resistance development in enteric bacteria …………..... 6 Bacterial quorum sensing …………………………………………… 9 III AUTOINDUCER 2-BASED RESPONSE INDUCES TETRACYCLINE TOLERANCE IN ESCHERICHIA COLI UNDER SUB-THERAPEUTIC EXPOSURE……………………..... 32 Overview……………………………………………………….......... 32 Introduction…………………………………………………….......... 33 Materials and Methods……………………………………………..... 35 Results……………………………………………………………….. 37 Discussion…………………………………………………………… 46 ix CHAPTER Page IV AUTOINDUCER 2 (AI-2) PRODUCTION IN ENTERIC BACTERIA IN RESPONSE TO SUB-THERAPEUTIC ANTIBIOTIC EXPOSURE ………………………………………....... 50 Overview………………………………………………………………. 50 Introduction……………………………………………………………. 51 Materials and Methods………………………………………………… 53 Results…………………………………………………………………. 56 Discussion……………………………………………………………... 71 V AUTOINDUCER 2 (AI-2)-BASED QUORUM SENSING PROMOTES trbC GENE EXPRESSION AND CONJUGAL TRANSFER OF tet(A) GENE IN RESPONSE TO SUB- THERAPEUTIC TETRACYCLINE EXPOSURE IN ESCHERICHIA COLI …………………………………………............ 74 Overview………………………………………………………………. 74 Introduction……………………………………………………………. 75 Materials and Methods………………………………………………… 77 Results…………………………………………………………………. 85 Discussion……………………………………………………………... 92 VI AUTOINDUCER-2 (AI-2)-LIKE ACTIVITY ASSOCIATED WITH FOODS AND ITS INTERACTION WITH ORGANIC ACIDS USED AS FOOD ADDITIVES………………….. 96 Overview………………………………………………………………. 96 Introduction……………………………………………………………. 97 Materials and Methods………………………………………………… 99 Results…………………………………………………………………. 103 Discussion……………………………………………………………... 109 VII A NON-INDOLE-BASED AIRBORNE QUORUM SENSING MOLECULE CAN CONFER TETRACYCLINE TOLERANCE IN PHYSICALLY SEPARATED ESCHERICHIA COLI AND SALMONELLA NEWPORT STRAINS……………………………………………………………… 114 Overview………………………………………………………………. 114 Introduction…………………………………………………………..... 115 Materials and Methods…………………………………………........... 117 Results…………………………………………………………………. 119 Discussion……………………………………………………………... 124 x CHAPTER Page VIII SUMMARY……………………………………………………….. 128 Autoinducer 2-based response induces tetracycline tolerance in Escherichia coli under sub-therapeutic exposure………………………………………………………….... 128 Autoinducer AI-2 activity in response to sub-therapeutic antibiotic exposure in enteric bacteria…………………………….. 129 AI-2-based quorum sensing promotes trbC gene expression and conjugal transfer of tet(A) gene in response to sub-therapeutic tetracycline exposure in Escherichia coli………………………………………………….... 130 Autoinducer 2(AI-2)-like activity associated with foods and its interaction with organic acids used as food additives……………………………………………………… 130 A non-indole-based airborne quorum sensing molecule can confer tetracycline tolerance in physically separated Escherichia coli and Salmonella Newport………………………… 131 IX CONCLUSIONS ………………………………………………….. 132 REFERENCES………………………………………………..................................... 133