Since their discovery over 40 years ago, considerable knowledge has been obtained on the diversity, and structure-function relationships of aminoglycoside acetyltransferases (AACs), responsible for antibiotic resistance among priority clinical pathogens. In recent years, investigations have expanded to biochemical characterizations of AACs found in environmental reservoirs. The successful design of next-generation aminoglycosides (AGs) depends on an up-to-date understanding of the broader AG resistome. Towards this goal, I present the first structural analysis for the unique apramycin modifying enzyme, ApmA. Apramycin is a veterinary antibiotic that is in development for clinical use. The atypical chemical scaffold provides inherent protection from many clinically relevant resistance mechanisms. Prior to the work presented herein, apmA was an uncharacterized apramycin resistance element among environmental species. I heterologously expressed and subsequently purified ApmA to characterize the nature of resistance towards this unique aminoglycoside. The results report the first acetyltransferase of the left-handed β-helix (LβH) superfamily involved in AG detoxification. Secondly, I completed a comprehensive characterization of ApmA utilizing a structurally diverse panel of AGs for susceptibility testing, protein engineering, steady-state kinetics, and x-ray crystallography. Through these approaches, I establish the structural and functional features that define ApmA’s place within the LβH superfamily and set it apart from other known AACs. The biochemical data presented describes a chemical mechanism dependent on the substrate specificity. Furthermore, I describe the molecular determinants behind AG-modification of clinically relevant AGs. Lastly, I describe the first comprehensive structural and functional study of clinical and environmental Antibiotic_NAT (A_NAT) inactivating enzymes. A pan-family antibiogram was obtained and mapped to the reconstructed phylogeny for the A_NAT family. Crystallographic analysis of representatives from each clade was completed with our collaborators from the University of Toronto. Through the analysis of several ligand-bound A_NAT complexes, I contributed to the elucidation of structural features responsible for substrate specificity. The collective findings from these chapters have extended the protein landscape involved in AG-acetylation from one commonly used fold to three distinct architectures, each unique in underlying chemical mechanism and dissemination. / Dissertation / Doctor of Philosophy (PhD) / Pathogens continue to learn new ways to protect themselves from antibiotics. With the discovery of new antibiotics becoming more challenging, global antibiotic resistance has the potential to become the next global pandemic. One solution is to redesign traditional antibiotics to escape resistance. A reliable, effective class of antibiotics currently under development are aminoglycosides. There is considerable knowledge into the sequence-structure-function relationships of proteins traditionally regarded as the sole contributors to a form of aminoglycoside resistance. My work describes the use of computational and biochemical techniques to investigate resistant elements beyond what we know is prevalent in clinical pathogens. Through these efforts I uncover structurally, and mechanistically distinct proteins capable of broad-spectrum, high-level aminoglycoside resistance produced by bacteria in various environments. These results are invaluable for the informed design of less-resistance prone aminoglycosides and antibiotic stewardship programs to limit these forms of resistance from becoming clinically prevalent.

Proteus mirabilis PM13 is a well characterized chloramphenicol-sensitive isolate which spontaneously gives rise to resistant colonies on solid media containing chloramphenicol (50ug/ml) at a plating efficiency of between 10-4 and 10-5 per cell per generation. When a chloramphenicol resistant colony is grown in liquid medium in the absence of the antibiotic for I50 generations a population of predominantly sensitive cells arises. The cat gene responsible for the phenomenon is chromosomal, and has been cloned from P.mirabilis PMI3 with DNA prepared from cells grown in the absence or the presence of chloramphenicol. Recombinant plasmids which confer resistance to chloramphenicol carry an 8.5-kb PstI fragment irrespective of the source of host DNA. The location of The cat gene within the PstI fragment was determined by Southern blotting with a cat consensus 'active - site' oligonucleotide (5'-CCATCACAGACGGCATGATG-3') corresponding to the expected amino acid sequence of the active site region of chloramphenicol acetyltransferase. DNA sequence analysis has revealed a high degree of homology between the P. mirabllls cat -gene and the type I ca-t variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. The mechanism for the appearance and disappearance of chloramphenicol resistance in P. mirabilis appears to be associated with a host-specific trans-acting element which controls cat gene expression. A precedent for such a control network is given by phase variation in Salmonella typhimurium, where an invertible DNA segment controls the transcription of a trans-acting regulatory element. A comparison of the 5' regions of the S.typhimurium flagellin genes in and H2, which are alternately expressed by a flip-flop control mechanism with the 5' region of P.mirabilis cat show blocks of homology. Whether or not this homology is significant in the regulation of cat gene expression has not been determined.

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Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (𝑃 < 0.05). There was a tendency (𝑃 = 0.06) for higher proportion of persistent diarrhea (≥14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (𝑃 > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Antibiotic-resistant bacterial strains continually arise and their increasing prevalence poses significant clinical and societal challenges. Functional analyses of resistant mutants and the study of general stress responses perturbed by antibiotic treatment have yielded valuable insights into how resistance arises through mutations. However, less is known about the population dynamics and communal interactions that underlie the development of resistance through mutations. In this work, we utilize systems approaches to study the functional dynamics of bacterial populations evolving antibiotic resistance. We follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the populations less resistant constituents, in part, by producing indole, a signaling molecule generated by actively growing and unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and wholegenome sequencing reveals that this bacterial altruism is enabled by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other more vulnerable cells, enhancing the survival capacity of the overall population in stressful environments. Deeper studies into cooperative strategies bacteria use to evade antibiotics may prove critical for the rational design of more effective antimicrobial interventions. / 2031-01-01