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ORCID

http://orcid.org/0000-0002-4551-4633

Date of Award

Winter 12-15-2018

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Increasing antibiotic resistance in pathogens is a serious public health challenge, with over two million antibiotic resistant infections in the United States leading to at least 23,000 deaths and an estimated $55 billion in excess healthcare and societal costs. Antibiotic resistance has risen steadily in both pathogenic and benign bacteria since antibiotics’ introduction to agriculture and medicine 70 years ago. A dramatic reduction in the number of antibiotics approved for human use has accompanied this rise in antibiotic resistance, leading to the alarming prospect of a post-antibiotic era. Understanding the evolutionary origins, genetic contexts, and molecular mechanisms of antibiotic resistance in diverse environments is crucial to anticipating and mitigating the spread of antibiotic resistance. It is similarly critical to understand the effect of antibiotic exposure on the microbial communities, such as the human gut microbiota, which serve as reservoirs for antibiotic resistance genes available for horizontal transfer to pathogenic organisms. In this work, I take a multipronged biochemical, microbiologic, and genomic approach to surveil existing and emerging antibiotic resistance threats in environmental and human-associated microbial habitats, and understand the effects of antibiotic perturbation on microbial community and resistome dynamics.

To understand the effect of antibiotic perturbation on microbial community and resistome dynamics, I examined a population of very and extremely preterm infants who received extensive antibiotic exposure during their hospitalization in the neonatal intensive care unit. I use complementary sequencing and culture-based methods to analyze 448 stools collected longitudinally over the first 20 months of life from 41 preterm infants, as well as 17 antibiotic-naïve near term infants that served as developmental controls. Using random forests, I define a program of healthy microbiota assembly in antibiotic naïve infants, and demonstrate acute but transient deviations from this program in antibiotic treated preterm infants. With a combination of sequencing isolates cultured from infant stool and functional metagenomics, I show that a consequence of early life antibiotic treatment in preterm infants is prolonged carriage of multidrug resistant Enterobacteriaceae and an enriched gut resident resistome.

I address the emergence of antibiotic resistance from environmental and commensal reservoirs to pathogens through the lens the tetracycline destructases, a family of flavoenzymes capable of inactivating diverse tetracycline substrates. I conduct a bioinformatic survey of functionally selected metagenomes to show that these enzymes are widespread in diverse microbial communities, and characterize their spectrum of activity and mechanism of action to confirm that these enzymes are functionally and biochemically novel and illuminate sequence-function relationships underlying the family. I identify an inhibitor of these enzymes that prevents tetracycline degradation in vitro and rescues tetracycline activity against resistant bacteria, and show that this inhibitor works by both competing for substrate binding and sterically restricting critical FAD cofactor dynamics. Lastly, I identify a Pseudomonas aeruginosa clinical isolate from a cystic fibrosis patient that encodes a functional tetracycline inactivator with 100% nucleotide identity to one previously identified in our bioinformatic survey, signaling the clinical arrival of this family of enzymes.

Language

English (en)

Chair and Committee

Gautam Dantas

Committee Members

Daniel E. Goldberg, Barbara B. Warner, Timothy A. Wencewicz, Andrew L. Kau,

Comments

Permanent URL: https://doi.org/10.7936/dx5f-hr58

Available for download on Saturday, October 31, 2020

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