ORCID
https://orcid.org/0000-0003-4247-1209
Date of Award
11-28-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Antibiotic resistance (AR) can be studied at multiple scales. The smallest is at the level of the DNA or amino acid sequence of AR genes, characterizing how AR genes are encoded, regulated, and expressed. Next is the level of protein structure, deciphering the 3D shape of AR proteins and how that shape affects function. The next higher scale is that of biochemistry, studying the kinetics of these proteins’ mechanism of action and interactions with drug molecules. The level of populations studies individual species of bacteria that are resistant to specific antibiotics. The next higher scale, of microbiomes, examines many species co-existing in the same environment with different resistance profiles. Next higher, we can compare different microbiomes in the same regional area (e.g., surfaces in the same room, or hospitals in the same state), and at the highest level study the global spread of AR across international borders. Studying each of these individual aspects necessarily requires the specific expertise of geneticists, structural biologists, biochemists, microbiologists, bioinformaticians, and epidemiologists. But this sub-division does not reflect the reality of the problem where, for example, global spread can be tracked by analyzing specific mutations in AR genes. In recognition of the limitations of focusing on just one aspect of AR research today increasingly is taking an interdisciplinary approach. For my doctoral research I have co-authored research reports which individually focus on one of each of these seven scales of AR. Together, this body of work represents a comprehensive study of AR. In Chapter 2, I describe how we evaluated sequence determinants of tetracycline-inactivating function in the tetracycline destructases family of enzymes (Blake et al. 2023. Under review). Tetracycline destructases are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. Using the amino acid sequences of reported tetracycline destructases, we evaluated the sequence determinants of these enzymes’ function through two complementary approaches: 1) constructing hidden Markov models (HMM) to predict new sequences, and 2) using multiple sequence alignments (MSA) to identify residues important to protein function. Using the HMM-based approach we computationally searched >300 million protein sequences and screened 50 high-scoring candidate sequences in an Escherichia coli host, leading to the discovery of 13 new TDases. The X-ray crystal structures of two of these enzymes from Legionella species were determined, and the ability of anhydrotetracycline to inhibit their tetracycline-inactivating activity was confirmed. Using the MSA-based approach we identified 31 amino acid positions that were 100% conserved across all 114 known TDase sequences. The roles of these positions was analyzed by generating alanine-scanning mutants at these conserved sites in two TDases and evaluating the impact on cell and in vitro activity, structure, and stability. These results expanded the diversity of TDase sequences, and provided valuable insights into the roles of important residues in TDases, and flavin monooxygenases more broadly. In Chapter 3, I describe how different tetracycline resistance genes differentially affect the fitness of strains E. coli within populations expressing different tetracycline resistance genes (Blake et al. 2023. In progress). In addition to enzymatic resistance by tetracycline destructases, bacteria can also become resistant to tetracyclines using ribosomal protection proteins and enzymatic degradation. Here, we compared the fitness profiles of tetracycline resistance genes from these three mechanisms in mono-culture fitness assays and mixed-culture competition assays. We find that while strains expressing tetracycline-inactivating enzymes encode resistance to the greatest number of tetracycline drugs this comes with the cost of significant reductions in growth rate. Conversely, while efflux pumps encode resistance to fewer tetracyclines they do not result in decreased growth rate. We developed a barcode sequencing approach that can quantify the change in abundance of different strains before and after antibiotic selection to determine the relative fitness of strains expressing these genes when competing in mixed cultures. Again, we found that inactivating enzymes have a selective advantage in third-generation drugs indicated by an increase in abundance relative to the other two mechanisms following competition. But this is inverted during selection in earlier-generation drugs where it decreases in relative abundance. Our results highlight the differences between antibiotic resistance genes belonging to the same drug class or mechanism. In Chapter 4, I describe how travel to different countries across the globe and different antibiotic regimens for the treatment and prevention of diarrhea affects AR (Blake et al. 2023. Under review). International travelers are frequently afflicted by acute infectious diarrhea, commonly referred to as travelers’ diarrhea (TD). Antibiotics are often prescribed as treatment or prophylaxis for TD; however, little is known about the impacts of these regimens on travelers’ gut microbiomes and carriage of antibiotic resistance genes (ARG). Here, we analyzed two cohorts totaling 153 US and UK servicemembers deployed to Honduras or Kenya. These subjects either experienced TD during deployment and received a single dose of one of three antibiotics (TrEAT TD cohort), or took once-daily rifaximin, twice-daily rifaximin, or placebo as prophylaxis to prevent TD (PREVENT TD cohort). We applied metagenomic sequencing on 340 longitudinally-collected stool samples and whole genome sequencing on 54 Escherichia coli isolates. We found that gut microbiome taxonomic diversity remained stable across the length of study for most treatment groups, but twice-daily rifaximin prophylaxis significantly decreased microbiome richness post-travel. Similarly, ARG diversity and abundance were generally stable, with the exception of a significant increase for the twice-daily rifaximin prophylaxis group. We also did not identify significant differences between the ARG abundance of E. coli isolates from the TrEAT TD cohort collected from different treatment groups nor timepoints. Overall, we find no significant worsening of gut microbiome diversity or increase in ARG abundance following single-dose treatment for TD, underscoring that these can be effective with low risk of impact to the microbiome and resistome, and identify the relative microbiome risks and benefits associated with the three regimens for preventing TD. In Appendix 1, I describe the structure of the tetracycline destructases, Tet(X6), and how it binds with substrates (Kumar et al. 2023. Communications Biology). In Appendix 2, I describe the design and synthesis of tetracycline destructases inhibitors that block enzyme biochemistry to rescue antibiotic efficacy (Williford et al. 2023. Journal of Medicinal Chemistry). In Appendix 3, I describe how infectious diarrhea disrupts the microbiome and resistome (Boolchandani et al. 2022. Nature Communications). In Appendix 4, I describe the AR gene burden in wastewater effluent from hospitals across the same regional area in northern India (Talat et al. 2023. Microbiology Spectrum). Lastly, in Appendix 5, I describe the technologies and methodologies to characterize hospital-associated multidrug-resistant bacteria (Blake et al. 2021. Cellular and Molecular Life Sciences).
Language
English (en)
Chair and Committee
Gautam Dantas
Recommended Citation
Blake, Kevin, "Antibiotic Resistance at Sequence, Population, and Global Scales" (2023). Arts & Sciences Electronic Theses and Dissertations. 3193.
https://openscholarship.wustl.edu/art_sci_etds/3193