TETRACYCLINE RESISTANCE IN ADULT HUMAN GASTROINTESTINAL MICROFLORA - CAN IT TELL THE STORY OF ANTIBIOTIC RESISTANCE IN HUMANS? THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Hanna H. Cortado, B. S. The Ohio State University 2008 Committee members: Professor Hua Wang, Adviser Approved by Professor Macdonald Wick Professor Jeff Culbertson Adviser Food Science and Nutrition Graduate Program ABSTRACT The acquisition of antibiotic resistance (AR) by clinically important bacteria is a worldwide human health concern. The rapid spread of AR has been attributed mainly to horizontal gene transfer events by resistance-encoding mobile genetic elements. Recently a large number of antibiotic resistant (ART) commensal bacteria were found in many retail foods. Characterization of the AR status of human gut microflora, involving infant subjects who have not been exposed to conventional solid foods as well as healthy adults with regular diet intake, can help reveal the potential impact of the food chain on disseminating AR to humans. In this study, adult human microflora associated with the gastrointestinal (GI) tract of 14 healthy individuals is examined for the presence of tetracycline-resistant bacteria and tetracycline-resistance conferring genes. Culture- dependent methods utilized in this study revealed a resistant count ranging from 106-109 CFU-mL-1, with total bacterial counts ranging from 106-1010 CFU-mL-1. A culture- independent approach was also optimized to assess the level of tetM gene pool in the sample microbiome. Among all subjects, the tetM gene pool range from 107 -109 tetM gene copy number per gram, as determined by TaqMan real-time quantitative polymerase chain reaction. Sixty-two out of 78 isolates tested (79%) were able to grow in media containing 140 μg-mL-1 tetracycline. Of these isolates, 39 carried one or more plasmids ii and 8 distinct plasmid profiles were observed. Plasmid-carrying isolates were screened for the efflux tet genes, tetK and tetL as well as the ribosomal protection genes tetM, tetQ and tetW. The tetM gene was found to be most prevalent- observed in 51% (20/39) of the isolates. Chromosomal and plasmid DNA of representative tetM-positive isolates were probed for the 406 bp tetM gene in a DNA-DNA hybridization procedure. Preliminary results indicate the plasmid carriage of the tetM gene in some of the isolates examined. Overall, results indicate a heavy ART bacterial load and considerable AR gene pool in the GI tract of healthy human adults who have not taken antibiotics in the past 3 months. In some of the isolates, the tetM gene was found in plasmids, indicating that the human digestive tract could be a potential site for genetic transfer of AR determinants. The identified dominant AR gene carriers differ from those from infant subjects, and the AR gene pool is about 1-3 log higher than that found in infant subjects. The regular inoculation of the human digestive ecosystem of ART bacteria through the food chain is hypothesized as a significant contributor towards the dynamic shifting of ART isolates and enrichment of AR genes in the gut, as observed in this study. iii ACKNOWLEDGEMENT I wish to thank: My adviser, Dr. Hua Wang, for her guidance and mentorship throughout this research My committee members, Dr. Mick Wick and Dr. Jeff Culbertson for their time and support Members of our lab, Yingli Li, Dan Kinkelaar, Xinhui Li, Lu Zhang, Monchaya Rattanaprasert, Linlin Xiao, Xiaojing Li and Andrew Wassinger for their technical assistance and encouragement This research was supported by Dr. Wang’s OSU start-up fund. iv VITA August 6, 1982 ............................................. Born – Iloilo, Philippines 2003 .............................................................. B. S. Molecular Biology and Biotechnology, University of the Philippines 2003 – 2005 .................................................. Research Assistant, Southeast Asian Fisheries Development Center, Tigbauan Iloilo, Philippines 2006 – present ............................................... Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. Hanna Cortado, Boris San Luis, Leobert dela Peña, Rosario Monsalud and Cynthia Hedreyda. 2005. Local Vibrio isolates exhibit molecular characteristics distinct from reference V. harveyi and V. campbellii strains. Science Diliman. 17(1): 23-30. 2. Fiona L. Pedroso, Evelyn Grace T. de Jesus-Ayson, Hanna H. Cortado, Susumu Hyodo and Felix G. Ayson. 2005. Changes in mRNA expression of grouper (Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status. General and Comparative Endocrinology. 145(3): 237-246. FIELDS OF STUDY Major Field: Food Science and Nutrition v TABLE OF CONTENTS Page Abstract ............................................................................................................................ ii Acknowledgments ........................................................................................................... iv Vita .................................................................................................................................. v List of Tables ................................................................................................................... vii List of Figures ................................................................................................................. viii Chapters: 1. Introduction ............................................................................................................1 2. Literature Review ...................................................................................................6 3. Objectives .............................................................................................................33 4. Prevalence of Tetracycline - Resistant Bacteria and tet genes in the Human Gut ..............................................................34 4.1 Materials and Methods ........................................................................34 4.2 Results .................................................................................................43 4.3 Discussion ...........................................................................................54 5. Conclusion and Future Development ....................................................................61 Appendix ...........................................................................................................................63 Bibliography .....................................................................................................................65 vi LIST OF TABLES Table Page 2.1 Tetracycline resistance genes and corresponding mechanisms.............................18 2.2 Major components of the human gastrointestinal (GI) microflora along the length of the GI tract .......................................................................................23 4.1 Primer sequences used for tet screening of isolates ..............................................41 4.2 tet gene screening of isolates from digestive microflora of healthy adult human subjects .............................................................................................51 vii LIST OF FIGURES Figure Page 2.1 Different routes of antibiotic resistance dissemination from animals to humans .................................................................................................20 4.1 Total plate count and tetracycline-resistant count of bacteria isolated from fecal samples of 9 healthy human subjects ..................................................44 4.2 Standard curve for determination of tetM gene copy numbers using TaqMan real-time PCR .........................................................................................45 4.3 Validation of extraction efficiency .......................................................................47 4.4 Quantification of tetM gene copy numbers in fecal samples of 14 healthy human adults using real-time qPCR .........................................................48 4.5 Assessment of antibiotic resistance in human digestive microflora by conventional plate counting and real-time PCR ..............................................49 4.6 Representative plasmid profiles from isolates ......................................................50 4.7 DNA-DNA hybridization for tetM detection ........................................................52 viii CHAPTER 1 INTRODUCTION During its era, antibiotics were deemed as miracle drugs, one that would put an end to the problem of morbidity and mortality from diseases, until the unwelcome emergence of antibiotic resistance (AR). In the United States alone, as much as $5 billion is spent annually towards treatment of infections caused by antibiotic resistant (ART) bacteria (5). As antibiotic resistance was primarily a human health concern, initial studies concluded that the misuse and overuse of antibiotics in the clinical setting were the main causes of AR emergence. However, an accumulating body of research has expanded this perspective to include antibiotic use in the absence of disease in agriculture and other environmental settings as potential contributors to the problem (4, 11, 14). It is not surprising that extensive measures are undertaken by various agencies throughout the world to address this serious concern. In 1996 the National Antimicrobial