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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.

Antibiotics form the cornerstone of modern medicine, facilitating advancements in numerous healthcare fields and contributing to unprecedented increases in human life expectancy. However, the efficacy of these life-saving drugs is now jeopardized by the rise of antimicrobial resistance. This growing threat is exacerbated by the slow pace of new antibiotic discoveries. The drug discovery process is both time-consuming and costly, and efforts to identify novel antibiotics often result in the rediscovery of known antibiotics, further hindering progress. To combat antibiotic resistance and facilitate the discovery of novel drugs, several approaches can be employed. First, understanding the mechanisms of resistance found in environmental bacteria is crucial for preparing against the potential mobilization of these resistance mechanisms into pathogenic bacteria. Second, developing tools that make the drug discovery process less costly and time-consuming can accelerate the discovery rate and broaden participation in drug discovery efforts. Finally, understanding how bacteria synthesize natural product antibiotics provides invaluable information that can be leveraged in drug discovery efforts, including synthetic biology approaches. This thesis addresses efforts and challenges in the various aspects of drug discovery. To enhance our understanding of environmental resistance mechanisms, I conducted a screen for ciprofloxacin-inactivating enzymes and characterized the activity of a previously reported ciprofloxacin-inactivating enzyme, CrpP. These findings highlight the difficulties associated with discovering synthetic antibiotic-inactivating enzymes. To contribute to drug discovery, I expanded the Antibiotic Resistance Platform and developed a streamlined version to improve antibiotic dereplication efforts, thereby accelerating the natural product discovery process. Additionally, I investigated the mechanism of β-serine biosynthesis, a nonproteinogenic amino acid found in the antibiotic edeine. By elucidating how β-serine is synthesized, this information can be applied to synthetic biology approaches for drug discovery. / Thesis / Doctor of Philosophy (PhD) / Antibiotics used in medical treatments today often originate from natural sources like environmental bacteria and are known as natural product antibiotics. These natural product antibiotics are essential for treating bacterial infections and play a crucial role in modern medicine, including surgery and cancer treatment. However, the increasing problem of antimicrobial resistance and the lack of new drugs being discovered threatens the effectiveness of these life-saving medicines. To combat antibiotic resistance and protect the use of antibiotics, we need to understand how bacteria resist antibiotics, develop better methods for discovering new antibiotics, and gain insights into how bacteria produce natural product antibiotics. This thesis addresses these challenges by trying to find bacteria that can break down antibiotics, improving a tool for drug discovery, and understanding how bacteria make the antibiotic known as edeine. These efforts advance our understanding of antibiotic resistance and pave the way for developing new and effective antibiotics.

Introduction: Streptococcus pneumoniae is the main bacterial cause of pneumonia, bacteremia and meningitis. Incidence rates have decreased since the initiation of pneumococcal vaccines, but antibiotic resistant strains continue to emerge and place a heavy burden on healthcare systems to treat such serious resistant infections. This study looks at risk factors that increase a patients probability of contracting a drug resistant strain of S. pneumo. Methods: Confirmed cases of S. pneumo were acquired through the Active Bacterial Core Surveillance program from 2009-2012 for the state of Georgia. Cumulative incidence rates, odds ratios and Pearson’s chi square were calculated to test for trends. Multi-logistic regression model was designed to control for covariates. Antibiotic Susceptibility results were analyzed by resistant profiles through WHONET. Results: Cumulative incidence rates have decreased significantly, however antibiotic resistant and multidrug resistant strains have increased. Incidence rates for children less than five and adults over 65 have decreased, however, the burden of disease remains in young to middle adults. Antibiotic resistant strains have shifted from penicillin to erythromycin and cefotaxime. Discussion: Interventions need to be targeted towards young to middle aged adults. Antibiotic stewardship programs should seek uniform guidelines to battle the increasing emergence of multidrug resistant strains.

Twenty-eight Holstein and crossbred calves of both genders were used to evaluate the effect of milk replacer antibiotics on abundance of selected antibiotic resistance genes (ARG) in the feces. Calves were blocked by breed, gender, and birth order, and assigned to one of three treatments at birth. Treatments were control (containing no antibiotics in the milk replacer), subtherapeutic (neomycin sulfate and oxytetracycline hydrochloride each fed at 10 mg/calf/d), and therapeutic (no antibiotics in the milk replacer until d 36, then neomycin sulfate and oxytetracycline hydrochloride each fed at 1000 mg/calf/d for 14 d). Calves were fed milk replacer twice daily at 0600 h and 1800 h. Fecal and respiratory scores and rectal temperatures were recorded daily. Calves were weighed at birth and weaning to calculate average daily gain. Beginning at six weeks of age fecal grab samples were collected from heifers at 0600 h, 1400 h, 2000 h, and 2400 h for 7 d, while bull calves were placed in metabolism crates for collection of all feces and urine. DNA was extracted from feces, and ARG corresponding to the tetracyclines (tetC, tetG, tetO, tetW, and tetX), macrolides (ermB, ermF), and sulfonamides (sul1, sul2) classes of antibiotics along with the class I integron gene, intI1, were measured by quantitative polymerase chain reaction (qPCR). No tetC or intI was detected. There was no significant effect of antibiotic treatment on the absolute abundance (gene copies/ g wet manure) of any of the ARG except ermF, which was lower in the antibiotic-treated calf manure probably because host bacterial cells carrying ermF were not resistant to tetracycline or neomycin. All ARG except tetC and intI were detectable in feces from 6 weeks onwards, and tetW and tetG significantly increased with time (P < 0.10), even in control calves. Overall, the majority of ARG analyzed for were present in the feces of the calves regardless of exposure to dietary antibiotic. Feed antibiotics had little effect on the ARG monitored; other methods for reducing the ARG pool should also be investigated. / Master of Science

Streptococcus pneumoniae (pneumococcus) is a commensal gram-positive colonizer of the human nasopharynx capable of causing diseases including otitis media, pneumonia, bacteremia, and meningitis. Although it is often a harmless colonizer, there is a high rate of mortality and morbidity among the immunocompromised, elderly, and young children. While these infections can often be treated with antibiotics, resistance to numerous antibiotics is increasing. Antibiotic resistance is a well-studied dilemma; however, little information is known of how bacteria take up certain antibiotics. Because most antibiotics cannot diffuse freely across the bacterial cell wall, we hypothesize that metabolite transport proteins participate in the uptake of certain classes of antibiotics.

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.

The coupled transcription-translation system previously developed for Streptomyces lividans was modified such that it functioned using purified ribosomal subunits, a crude initiation factor preparation and a high speed supernatant fraction. This system was used to investigate antibiotic resistance mechanisms in two Streptomyces which synthesise inhibitors of translation. Resistance to either pactamycin in Streptomyces pactum or celesticetin in Streptomyces caelestis was due to ribosome modification. In each case, high level resistance was attributed solely to one ribosomal subunit, the 30S subunit of the S. pactum ribosome and the 50S subunit of the S. caelestis ribosome. Shotgun cloning experiments have enabled a pactamycin resistance determinant from S. pactum to be isolated in S. lividans. However, in the original pactamycin resistant clone the plasmid was unstable and in the absence of pactamycin selection pressure, only a deleted form could be recovered. When ribosomes from resistant subclones were analysed, it appeared that a ribosome modification system from S. pactum had been cloned. Ribosome reconstitution studies indicated that a property of 16S rRNA was responsible for resistance. Since the cloned resistance determinant was not homologous to 16S rRNA (as judged by Southern analysis), pactamycin resistance in S. pactum is probably due to post-transcriptional modification of 16S rRNA.