Antibiotic Resistance in Aquaculture Production DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ying Huang M.S. Graduate Program in Food Science and Technology The Ohio State University 2014 Dissertation Committee: Dr. Hua H. Wang, Advisor Dr. John H. Litchfield Dr. Gireesh Rajashekara Dr. Zhongtang Yu Copyrighted by Ying Huang 2014 Abstract The rapid emergence of antibiotic resistance (AR) has become a major public health concern. Recent findings provided solid evidences suggesting that multiple risk factors contributed to AR development, enrichment, dissemination and persistence, and a comprehensive understanding of AR ecology is essential for targeted mitigation. Following the investigation of AR in aquaculture products from China, a study on AR in fish and aquaculture production-related samples from a U.S. fish farm with controlled practices and no history of antibiotic applications was further conducted to better understand the potential impact of aquaculture production practice on the prevalence of antibiotic resistant (ART) bacteria in the aquaculture ecosystem. Phenotypic resistant populations against sulfamethoxazole with trimethoprim (Sul/Tri), tetracycline (Tet), erythromycin (Erm) or cefotaxime (Ctx) were screened by conventional plating with the corresponding antibiotics, followed by population assessments with denaturing gradient gel electrophoresis (DGGE) and 16S rDNA next generation sequencing (NGS). Despite the absence of antibiotic application in the farm, our results showed that antibiotic resistant (ART) bacteria were abundant in all samples examined, including fish intestine, surface rinsing water, feed, pond water and mud samples. By NGS, a total of 569 genera were identified in Tetr and Ctxr bacteria from five types of samples. Certain correlations of ART bacteria between different types of ii samples were observed. Various AR genes measured by quantitative PCR (qPCR) were significantly more abundant in fish intestine and feed than farm environmental samples. Certain dominant ART bacteria subpopulations in feed and pond water were also identified in fish intestine. About 79% of 4747 ART isolates from aquaculture samples showed resistance to more than one antibiotic. Some ART isolates showed MIC of Sul/Tri, Tet, Erm or Ctx no less than 512 μg/ml. Various AR genes were detected in ART isolates including sul1, sul2, tetS, tetL, tetM, ermB or ermC. Identified AR gene carriers belong to 18 genera. In addition, the AR traits in many isolates were quite stable, even in the absence of selective pressure. A fosmid genomic library generated from a pooled DNA of 6 ART isolates was used to screen for new AR genes against tetracycline. Tetr clones were further subjected to subcloning using pBluescript vector, followed by DNA sequence analysis. A 4.7-kb fragment from a Tetr subclone T61 contained two divergently transcribed open reading frames (ORFs). The larger ORF encoded a 398-amino-acid protein with 88% identity to a major facilitator superfamily (MFS) transporter and 87% identity to class D tetracycline/H+ antiporter. The smaller ORF encoded a 199-amino-acid protein with 86% identity to TetR family transcriptional regulator. Deletion mutagenesis confirmed the involvement of the two ORFs in Tetr. The original host of the new tetD variant, designated tetD(Y), was identified as Providencia sp. iii Our results suggested that additional risk factor(s), rather than direct exposure to antibiotics, are responsible for AR in aquaculture production. Results would contribute to an improved understanding of AR ecology in the aquaculture production system. iv Acknowledgments I would like to thank Dr. Hua Wang, for offering me precious encouragement, guidance, and support throughout my entire Ph.D. study. Also thank her for providing valuable advice on many aspects of my life. I would also like to thank my committee members: Dr. John Hyland Litchfield, Dr. Gireesh Rajashekara and Dr. Zhongtang Yu, for their kind help and patience throughout my Ph.D. study. I would also like to thank Dr. Laura Tiu, who is the aquaculture extension specialist and has helped me get access to the samples. I am very grateful to all of my lab mates in these four years: Lu Zhang, Xinhui Li, Linlin Xiao, Andrew Wassinger, Wenfei Wang, Yi Shao, Lei Ye, Qianying Yao, Yang Zhou, Yu Li and He Yan for their help and encouragement during my research. We had a wonderful time together. I want to thank all my friends and my family for their unconditional support. v Vita 2007................................................................B.S. Bioengineering, China Agricultural University 2010................................................................M.S. Food Science, China Agricultural University 2010 to present ..............................................Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Publications Peer-reviewed articles: Zhang L, Huang Y, Zhou Y, Buckley T & Wang HH. (2013) Antibiotic administration routes significantly influence the levels of antibiotic resistance in gut microbiota. Antimicrobial agents and chemotherapy. 57 (8): 3659-3666. Ye L, Zhang L, Li X, Shi L, Huang Y & Wang HH. (2013) Antibiotic-Resistant Bacteria Associated with Retail Aquaculture Products from Guangzhou, China. Journal of Food Protection. 76 (2): 295-301. Zhang L, Kinkelaar D, Huang Y, Li Y, Li X & Wang HH. (2011) Acquired Antibiotic Resistance: Are We Born With It? Applied and Environmental Microbiology. 77(20): 7134-7141. vi Xie Y, An H, Hao Y, Qin Q, Huang Y, Luo Y & Zhang L. (2011) Characterization of an anti-Listeria bacteriocin produced by Lactobacillus plantarum LB-B1 isolated from koumiss, a traditionally fermented dairy product from China. Food Control. 22(7): 1027- 1031. Hao Y, Zhao L, Zhang H, Zhai Z, Huang Y, &Zhang L. (2010) Identification of the bacterial biodiversity in koumiss by denaturing gradient gel electrophoresis and species- specific PCR. Journal of Dairy Science. 93(5): 1926-1933. Huang Y, Luo Y, Zhai Z, Zhang H, Yang C, Tian H, Li Z, Feng J, Liu H & Hao Y. (2009) Characterization and application of an anti-Listeria bacteriocin produced by Pediococcus pentosaceus 05-10 isolated from Sichuan Pickle, a traditionally fermented vegetable product from China. Food Control. 20(11): 1030-1035. Li R, Zhai Z, Yin S, Huang Y, Wang Q, Luo Y, & Hao Y. (2009) Characterization of a rolling-circle replication plasmid pLR1 from Lactobacillus plantarum LR1. Current Microbiology. 58 (2): 106-110. Yin S, Hao Y, Zhai Z, Li R, Huang Y, Tian H, & Luo Y. (2008) Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4. FEMS Microbiology Letters. 285(2): 183-187. Hao Y, Huang X, Mei X, Li R, Zhai Z, Yin S, Huang Y, & Luo Y. (2008) Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli. Protein Expression and Purification: 60(2), 221-224. Presentations at conferences: Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. 2014. Antibiotic resistant microbiome and its potential evolution in aquaculture production. 2014 ASM General Meeting. Boston, MA. 5/20/2014 Zhou Y, Zhang L, Huang Y, Wang HH. The impact of antibiotic administration routes and environmental exposure on antibiotic resistance ecology in poultry gut microbiota. 2014 ASM General Meeting. Boston, MA. Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. 2014. Identification of a new tetD subgroup from domestic aquaculture products by functional metagenomics. 2014 OVIFT Annual Meeting. Columbus, OH. 3/17/2014 Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. 2013.. Antibiotic resistance in aquaculture products from domestic production. 2013 OARDC Annual research meeting. Columbus, OH. vii Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. 2013. Antibiotic resistance in aquaculture products from domestic production. 2013 Ohio Branch ASM General Meeting. Ashland, OH. Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. .2012. Antibiotic resistance in aquaculture products from domestic production. 2012 IFT Annual Meeting. Las Vegas, NV. Huang Y, Zhang L, Ye L, Wang WF, Tiu L, Wang HH. .Antibiotic resistance in aquaculture products from domestic production. 2012 PHPID Annual Meeting. Columbus, OH. Zhang L, Kinkelaar D, Huang Y, Wang HH. 2011. Acquired antibiotic resistance: drug, food Exposure or are we born with it? 2011 ASM General Meeting. New Orleans, LA. Fields of Study Major Field: Food Science and Technology viii Table of contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Table of contents ................................................................................................................ ix List of Tables ..................................................................................................................... xi List of Figures .................................................................................................................. xiii Chapter 1 : Literature Background and Rationale of the Study .......................................... 1 1.1 Antibiotics and their modes of