Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2018 Mechanistic characterization of resistome and microbiome dynamics across diverse microbial habitats Andrew John Gasparrini Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Bioinformatics Commons, Genetics Commons, and the Microbiology Commons Recommended Citation Gasparrini, Andrew John, "Mechanistic characterization of resistome and microbiome dynamics across diverse microbial habitats" (2018). Arts & Sciences Electronic Theses and Dissertations. 1697. https://openscholarship.wustl.edu/art_sci_etds/1697 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Genetics and Genomics Dissertation Examination Committee: Gautam Dantas, Chair Daniel E. Goldberg Andrew L. Kau Barbara B. Warner Timothy A. Wencewicz Mechanistic Characterization of Resistome and Microbiome Dynamics Across Diverse Microbial Habitats by Andrew J. Gasparrini A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2018 St. Louis, Missouri © 2018, Andrew J. Gasparrini Table of Contents List of Figures ................................................................................................................................ vi List of Tables ............................................................................................................................... viii Acknowledgments.......................................................................................................................... ix Abstract of the Dissertation ......................................................................................................... xiii Chapter 1: Introduction ................................................................................................................... 1 1.1 Next-generation approaches to understand and combat the antibiotic resistome ................. 1 1.1.1 The promise of antibiotics and the threat of resistance ................................................................ 1 1.1.2 Common mechanisms of antibiotic action and antibiotic resistance ........................................... 5 1.1.3 The golden age of antibiotics ....................................................................................................... 7 1.1.3 High-throughput resistance discovery.......................................................................................... 9 1.1.4 Next-generation approaches to resistance .................................................................................. 19 1.1.5 Conclusions ................................................................................................................................ 25 1.2 Genomic and functional techniques to mine the microbiome for novel antimicrobials and antimicrobial resistance genes: .................................................................................................. 27 1.2.1 Introduction ................................................................................................................................ 27 1.2.2 Rise in Antimicrobial Resistance and Decline in Antimicrobial Discovery .............................. 29 1.2.3 Brief History of Antimicrobials ................................................................................................. 30 1.2.4 Brief History of Antimicrobial Resistance ................................................................................. 32 1.2.5 Culture-based methods underestimate functional diversity of microbiomes ............................. 35 1.2.6 Function based methods for resistance discovery ...................................................................... 37 1.2.7 Sequence-based methods for resistance discovery .................................................................... 38 1.2.8 Function-based methods for antimicrobial discovery from microbiomes ................................. 43 1.2.9 Sequence-based methods for antimicrobial discovery from microbiomes ................................ 46 1.2.10 Future directions in antimicrobial and antimicrobial resistance discovery .............................. 49 1.3: Antibiotics and the preterm infant gut microbiome and resistome .................................... 52 1.3.1 Introduction ................................................................................................................................ 52 1.3.2 Techniques for studying development of the infant gut microbiota and the developing resistome ............................................................................................................................................. 52 1.3.3 A longitudinal, sequencing based interrogation of the preterm infant gut microbiota ............... 54 1.3.4 Antibiotics disrupt the preterm gut microbiota richness and composition ................................. 55 ii 1.3.5 Antibiotic therapy enriches for antibiotic resistance genes and multidrug resistant bacteria .... 56 1.3.6 The predictable response of the preterm infant gut microbiota to antibiotic therapy ................ 58 1.3.7 Implications for future research ................................................................................................. 59 1.4 Scope of the thesis ............................................................................................................... 60 1.5 Acknowledgments ............................................................................................................... 63 Chapter 2: Persistent metagenomic signatures of early life antibiotic treatment in the infant gut microbiota and resistome .............................................................................................................. 64 2.1 Introduction ......................................................................................................................... 64 2.1.1 Abstract ...................................................................................................................................... 64 2.1.2 Introduction ................................................................................................................................ 64 2.2 Results ................................................................................................................................. 66 2.2.1 Metagenomics based analysis of effect of antibiotics on the preterm infant gut microbiota ..... 66 2.2.3 Antibiotic resistome of preterm infant gut microbiota ............................................................... 72 2.2.4 Persistence of multidrug resistant Enterobacteriaceae in the preterm infant gut microbiota ..... 77 2.2.5 Persistent metagenomic signature of antibiotic treatment in premature infants ........................ 80 2.3 Materials and Methods ........................................................................................................ 82 2.3.1 Sample and metadata collection ................................................................................................. 82 2.3.2 Metagenomic DNA extraction ................................................................................................... 82 2.3.3 Metagenomic sequencing library preparation ............................................................................ 83 2.3.4 Rarefaction analysis ................................................................................................................... 83 2.3.6 Construction of metagenomic libraries from infant gut samples for functional selection ......... 84 2.3.7 Functional selections for antibiotic resistance ........................................................................... 87 2.3.8 Amplification and sequencing of functionally selected fragments ............................................ 88 2.3.9 Assembly and annotation of functionally selected fragments .................................................... 90 2.3.10 Bacterial isolation from infant stools ....................................................................................... 92 2.3.12 Isolate sequencing library preparation ..................................................................................... 93 2.3.13 Assembly of isolate genomes ................................................................................................... 93 2.3.14 Isolate genomic analysis .......................................................................................................... 93 2.3.15 Enterococcus biofilm formation assay ..................................................................................... 94 2.4 Discussion ........................................................................................................................... 96 2.5 Acknowledgments ............................................................................................................... 97 2.6 Supplementary Figures .......................................................................................................