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Antibiotic Resistance in Poultry Gastrointestinal Microbiota and Targeted Mitigation

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Antibiotic Resistance in Poultry Gastrointestinal Microbiota and Targeted Mitigation Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yang Zhou, B.S. Graduate Program in Food Science and Technology The Ohio State University 2016 Dissertation Committee: Hua Wang, Advisor Monica Giusti Michael Lilburn Zhongtang Yu Copyright by Yang Zhou 2016 Abstract The rapid emergence and spread of antibiotic resistance (AR) is a major public health concern. The poultry industry worldwide represents the largest segment in food animal production. The prevalence and abundance of antibiotic resistant (ART) bacteria in poultry and poultry products have been a recognized food safety challenge. The large amount of ART bacteria-rich feces from industrial poultry production and its release further contaminate water and soil, impacting the environmental AR gene pool. Therefore, revealing contributing factors to AR in poultry production and developing targeted control strategies has critical impacts on AR mitigation in the ecosystem. This study examined 1) the natural occurrence of AR in gastrointestinal (GI) tract of chicken without antibiotic exposure; 2) the impact of antibiotic administration route on AR ecology in chicken GI microbiota, and 3) the efficacy of commensal Lactobacillus crispatus CG-2 inocula in AR mitigation in poultry rearing. The results of this study contributed to an improved understanding of AR ecology in food-producing animals. ii The first chapter is a literature review covering the origin, propagation, and dissemination of AR, as well as AR mitigation strategies. AR status in food-producing animals, particularly poultry, and recent achievements in AR mitigation were also reviewed to lay the foundation for research presented in this dissertation. In the second chapter, the fecal microbiota of chickens from 1st to 4th week, grown in the teaching farm without any antibiotic treatment, were examined to assess the early establishment of AR gene pools and the constitution of natural microbiota in chicken GI r r r r tract. At least 5.5 Log10 (gene copies/g) of Tet , Amp , Erm and Sul encoding gene pools were found in the chicken GI tract within the first week of life without exposure to antibiotics. The sizes of the AR gene pools varied among different sources of chickens. In the 4th week post-hatch, over 96% bacteria in layer chicken’s natural gut microbiota were Firmicutes, and blaCMY-2 gene pool remained stable in the natural gut microbiota during Week 4. These results suggest that the AR gene pools were established and remained stable in chicken gut microbiota without antibiotic intervention during the first 4 weeks of life. iii In the third chapter, several culture-recovered commensal bacterial strains isolated from chicken feces were identified and examined for their potential contribution to AR ecology. Three Ampr E. coli (E. coli CA-1, E. coli CA-4 and E. coli CA-20) strains were found resistant/insusceptible to β-lactam, erythromycin, daptomycin, vancomycin and linezolid, but susceptible to tetracycline and quinolones. A Lactobacillus crispatus strain designated CG-12 was isolated and found susceptible to most antibiotics examined, but resistant/insusceptible to quinolone and daptomycin. The results were consistent with data by whole genome sequence analysis. The identified and characterized E. coli strains have been used in later studies as ART marker strains (Chapter 4), and the Lactobacillus crispatus was later examined for its efficacy in modulating GI microbiota (Chapter 5). In the fourth chapter, the impact of oral resistant bacteria exposure and antibiotic administration methods on microbiota and AR gene pools in poultry GI tract was examined using leghorn chicken with natural gut microbiota. It was found that without r antibiotic exposure, Amp gene (blaCMY-2) pool established in feces of chicks and persisted + in GI microbiota of chickens orally inoculated with the blaCMY-2 E. coli cocktail. Study also found that oral exposure to 300kg/mg of Amp in chickens inoculated with ART iv bacteria led to rapid enrichment of the corresponding AR gene pool and phylum Proteobacteria, and sharp decrease of phylum Firmicutes in feces. However, when administered via intramuscular injection, the same dosage of Amp, led to significantly less increase of Proteobacteria and decrease of Firmicutes. The Ampr marker strains were detected in certain blank control chickens in different cages in the same facility, indicating possible ART bacteria dissemination through environmental exposure. Shift of fecal microbiota and dominant bacterial population were consistent with the dynamics of the targeted AR gene pool. These results confirmed that the impact of certain antibiotics on gut microbiota can be significantly reduced by avoiding the mainstream oral antibiotic administration route in the chicken model. Our study also indicates that additional control strategies for the spread of bacteria in the environment may be also important to reduce ART bacteria dissemination in food animal production. In the fifth chapter, the growth inhibition activity of antibiotic-susceptible Lactobacillus crispatus strain CG-12 has been characterized both in vitro and in vivo. Results from in + vitro studies showed that the strain could inhibit the growth of the blaCMY-2 E. coli strains isolated from natural chicken fecal microbiota. In an in vivo study, Lactobacillus v crispatus CG-12 was inoculated into chicken model to test its colonization resistance + against the blaCMY-2 E. coli strains in gut microbiota. This study found that Lactobacillus crispatus CG-12 reduced the prevalence of blaCMY-2 gene in newly established GI microbiota. However, inoculation of Lactobacillus crispatus CG-12 had limited impact on the targeted ART E. coli strains from their colonization to proliferation. Data from this study suggested that Lactobacillus crispatus was prevalent in neonatal GI microbiota of chicken, and that antibiotic-susceptible Lactobacillus crispatus CG-12 could reduce early established AR gene pool. But its colonization resistant activity requires further evaluation against other ART populations. Finally, future direction of AR mitigation in poultry GI microbiota was discussed. vi Dedication This dissertation work is dedicated to my family and many friends. A special gratitude to my loving parents, who offered encouragement and support on every big decision I made in my life. vii Acknowledgement I would like to express my sincere gratitude to my advisor Dr. Hua Wang for her continuous support throughout my entire Ph. D study and related research, for her patience, inspiration and encouragement. Her guidance helped me throughout the research and her insight will continue to benefit my future work. I also thank my committee members, Dr. Monica Giusti, Dr. Michael Lilburn and Dr. Zhongtang Yu, for their support and encouragement throughout my Ph.D. program. Their insightful comments inspired me to broaden my knowledge from various perspectives. My sincere thanks also go to my colleagues, Lu Zhang, Qianying Yao, Yu Li and Ying Huang, for the stimulating discussions in the lab, for the unconditional help during experiment and for all the fun we have inside and outside the office. Also I thank the supportive staffs in OARDC Chicken Research Center for offering me help and advice for my study. Last but not the least, I would like to thank my family, to my loving parents, aunts and uncles for supporting me throughout writing this dissertation and my life in general. viii Vita April 1980 ………………………… Yang Zhou 2012 ………………………………. B. S. Life Science, Fudan University 2012 – Present ………………………Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Publications Lu Zhang,Ying Huang,Yang Zhou, Timothy Buckley, Hua H. Wang. 2013. Antibiotic Administration Routes Significantly Influence the Levels of Antibiotic Resistance in Gut Microbiota. Antimicrobial agents and Chemotherapy. 57(8):3659-3666 Zhou Y, Zhang L, Huang Y, Wang H. 2014. The Impact of Antibiotic Administration Routes and Environmental Exposure on Antibiotic Resistance Ecology in Poultry Gut ix Microbiota. ASM general meeting. Boston, MA (Peer-reviewed manuscript in preparation). Zhou Y, Zhang L, Huang Y, Wang H. 2015. Antibiotic Administration Routes and Environmental Exposure Influence Antibiotic Resistance Ecology in Poultry Gut Microbiota. 4th Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens. Washington, D.C Field of Study Major Field: Food Science and Technology x Table of Contents Abstract ………………………………………………………………………… ii Dedication ……………………………………………………………………… vii Acknowledgements …………………………………………………………….. viii Vita ……………………………………………………………………………… ix List of Tables ……………………………………………………………………. xiv List of Figures …………………………………………………………………… xv Chapters 1. Literature Review………………………………………………………………. 1 1.1 The big picture……………………………………………………………….... 1 1.2 Antibiotic usage and antibiotic resistance in animal and poultry production … 5 1.3 Antibiotics and resistance …………………………………………….……… 12 1.4 Dissemination of antibiotic resistance……………………….……………….. 30 1.5 Commensal bacteria, gut microbiota and antibiotic resistance ………………. 38 1.6 Mitigation strategies for antibiotic resistance……………………….………... 43 2. Natural microbiota and Antibiotic Resistance Gene Pools in Chicken Gut……. 62 2.1 Abstract………………………………………………………………………... 62 2.2 Introduction……………………………………………………………………
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  • Vaginal Bacteriome of Nigerian Women in Health and Disease: a Study with 16S Rrna Metagenomics

    Vaginal Bacteriome of Nigerian Women in Health and Disease: a Study with 16S Rrna Metagenomics

    Original Article Vaginal bacteriome of Nigerian women in health and disease: A study with 16S rRNA metagenomics Anukam KC1,2,3, Agbakoba NR3, Okoli AC3, Oguejiofor CB4 1Uzobiogene Genomics, 2Department of Endocrinology and Metabolism, St. Joseph’s Health Care, London, ON, Canada, 3Department of Medical Lab Sciences, Faculty of Health Sciences and Technology, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, 4Department of Obstetrics and Gynaecology, Nnamdi Azikiwe University Teaching Hospital, Anambra State, Nigeria ABSTRACT Introduction: The argument on what bacteria make up healthy vagina and bacterial vaginosis (BV) remain unresolved. Black women most often are placed in grade IV vaginal communities as lacking Lactobacillus‑dominated microbes. We sought to determine the vaginal microbiota compositions of healthy and those with BV using 16S rRNA metagenomics methods. Materials and Methods: Twenty‑eight women provided vaginal swabs for Nugent scoring. Fifteen had BV (Nugent score 7–10), whereas 13 were normal (Nugent score 0–3). DNA was extracted and 16S rRNA V4 region amplified using custom bar‑coded primers prior to sequencing with MiSeq platform. Sequence reads were imported into Illumina BaseSpace Metagenomics pipeline for 16S rRNA recognition. Distribution of taxonomic categories at different levels of resolution was done using Greengenes databases. Manhattan principal component analysis was used for similarity clustering. Results: Non‑BV subjects were colonized by 12 taxonomic phyla that represent 182 genera and 357 species. Overall, 23 phyla representing 388 genera and 805 species were identified in BV subjects.Firmicutes represented 95% of the sequence reads in non‑BV subjects with Lactobacillus‑dominated genera and Lactobacillus crispatus–dominated species, followed by Proteobacteria (3.78%), Actinobacteria (0.74%), and Bacteriodetes (0.05%).