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Resistance to the tetracycline antibiotics occurs primarily by efflux and ribosome protection mechanisms, however a tetracycline inactivating enzyme, TetX, was identified 15 years ago, although little is known about this mechanism. The gene encoding this enzyme was identified on a 𝘉𝘢𝘤𝘵𝘦𝘳𝘰𝘪𝘥𝘦𝘴 transposon and results from DNA sequence analysis and studies of bacterial culture media suggested that 𝘵𝘦𝘵𝘟's protein product might be a NADPH-requiring oxidase (𝘚𝘱𝘦𝘦𝘳 𝘉𝘚 𝘢𝘯𝘥 𝘚𝘢𝘭𝘺𝘦𝘳𝘴 𝘈𝘈. 𝘑 𝘉𝘢𝘤𝘵𝘦𝘳𝘪𝘢𝘭. 171(1): 148-153, 1989). We have expressed a copy of 𝘵𝘦𝘵𝘹 gene, 𝘵𝘦𝘵𝘹2, in Escherichia coli, and purified the enzyme to high purity. We showed that TetX2 is a monomeric 44 kDa cytoplasmic protein and UV-Vis and HPLC studies established that TetX contained an FAD cofactor. Continuous and stopped enzyme assays have been developed and established that that the enzyme requires 0₂ and NADPH for tetracycline degradation. Liquid chromatographic mass spectrometry (LC -MS) analysis of TetX reaction products using oxytetracycline (461 Da) as a substrate indicated that the enzyme catalyses the incorporation of one oxygen atom into oxytetracycline, resulting in a compound of 477 Da with no antibiotic activity. Steady state kinetic analysis demonstrated that TetX2 has a broad substrate specificity with the capacity to inactivate several members of the tetracycline family tested. Identification of the inactivated tetracycline product revealed that the tetracycline inactivation process is a TetX2 catalyzes tetracycline oxidation reaction. These studies provide the first biochemical analysis of a tetracycline inactivating enzyme. / Thesis / Master of Science (MSc)

Liposomes are self-assembled lipid vesicles made from phospholipids that are safe and suitable for drug encapsulation and localized drug delivery. Liposomal formulations are characterized by low toxicity and improved therapeutic index (by changing drug biodistribution) and liposomes encapsulating antifungal or anticancer drugs have already been approvedby regulatory agencies.One area of application for liposomes is localized antibiotic delivery. Antibiotics target bacteria, but specific types of infections(namely biofilms or intracellular infections)that required high or prolonged antibiotic administration have long been a challenge for antibiotic treatments. Liposomal delivery of antibiotics can improve their therapeutic index while minimizing their adverse effects. When it comes to methods of antibiotic encapsulation, however,most reports to date follow the methods developed for anticancer drugs for encapsulating antibiotics. This oversight causes discrepancies in the literature, mainly because of the significantly different chemical structures of antibiotics and cancer drugs. Furthermore, most antibiotics are highly sensitive to temperature fluctuations, which is concerning, given most liposomal preparation methods involve extreme temperature fluctuations. The aim of my thesis was to explore these missing links in the literature by answering these questions: (1) will liposome preparation method affect encapsulation efficiency of antibiotics?And (2) does liposomal preparation method adversely affect the efficacy of antibiotics?Investigating these questions led to further insight into the optimal process for achieving high encapsulation efficiencies for different antibiotics and for further avoiding damage due to harsh processing conditions. We found that different preparation methods are better for different types of antibiotics, being the one that promotes a large aqueous space better for hydrophilic drugs and the one that creates oligolamellar and large unilamellar vesicles better for more hydrophobic drugs. The steps in liposome preparation methods such as heating and sonication can affect the stability of the antibiotics. / Thesis / Master of Applied Science (MASc) / When antibiotics are administered, orally or intravenously, they should pass through different tissues to arrive to the site of infection; this can cause dilution and/or intoxication. To overcome these problems, drug delivery vehicles have been used to encapsulate and deliver antibiotics, improving their therapeutic index while minimizing their adverse effects. Liposomes are vesicles composed of at least one lipid bilayer, with an inner aqueous compartment. Liposomes are an attractive vehicle to deliver antibiotics because they can encapsulate both hydrophobic and hydrophilic antibiotics, they have low toxicity, and they can change the bio-distribution of the drug. In mythesis, I addressedtwo main questions regarding liposomal antibiotic encapsulation:(1) will liposome preparation method affect encapsulation efficiency of antibiotics, and(2) does liposome preparation method adversely affect the efficacy of antibiotics. While investigating these questions,I also identified certain outstanding biases in the liposomal characterization methods.

This problem has been concerned with, first, the collection of sixteen species of plants belonging to Cactaceae family; second, the drying of these and the extraction of the oleoresins thereof; third, the determination of the extent to which these substances inhibit the growth of ten gram-positive and ten gram-negative bacterial organisms; and fourth, a determination of the possible utilization of these extracts a prophylactic or chemotherapeutic agents.

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. / 2999-01-01

Development of antibiotic resistance is an increasingly important issue. It has created some problematic bacterial infections that are extremely difficult to treat. Although, antibiotics remain front-line therapy for conquering bacterial infections, their indiscriminate use is no longer viewed as benign. Many facotrs have contributed to the increased use of antimicrobial agents. Practitioners maintain that they cannot refuse patient demands for antibiotics. The purpose of this proejct is to increase public awareness of the appropriate use of antibiotics. Dorothea Orem's Self-Care Theory of Nurisng is the theoretical basis for this education program created to assist nursing students in educating the public about appropriate antibiotic use. The program includes an assessment tool, learning objectives, and educational module for nurses and health professionals, an educational model for public presentation, a resource guide, and an evaluation tool. Nurses are in a unique position to provide prevention and awareness strategies to the public on the consequences of antibiotic use.

Antibiotic use in agricultural ecosystems has the potential to increase resistance to antibiotics in soil microbial communities since 40-95% of an antibiotic dose administered to livestock is excreted intact or as metabolites. Exposure to antibiotics is also known to alter microbial community composition, biomass, and physiology, but the potential influences of antibiotic residues on the essential ecosystem processes that microbes regulate, e.g., carbon and nitrogen cycling are not well understood. I investigated the effects of antibiotic residues associated with dairy cattle operations on soil microbial communities and the ecosystem processes they regulate. I examined the effects of antibiotic exposure on the biogeochemical functioning of soil microbial communities by measuring the activity of extracellular enzymes associated with organic matter processing and nutrient mineralization in soils collected from dairy cattle operations across the United States. At each experimental station paired sites were identified by local managers that represented sites with high and low stocking rates of dairy cows who had been treated prophylactically with antibiotics to prevent mastitis. Responses varied among individual enzymes, but I found an overall significant decrease in total hydrolytic enzyme activity under high cattle stocking rates indicating a change in the functioning of the microbial community in soils exposed to antibiotic laden manure. Principle components analysis suggest that while some of the variation in enzyme activities are associated with the abundance of antibiotic resistance genes, soil organic matter (total organic, mineralizable, and particulate organic carbon) was the most significant variable accounting for differences in enzyme activities. This reflects an inherent challenge in studies of antibiotic exposure in agricultural landscapes: the difficulty of distinguishing direct effects of antibiotic residues from the organic matter and nutrient subsidy associated with manure applications. To address this concern I conducted a series of incubation experiments manipulating soils to isolate the influences of antibiotics, manure resource subsidies, and bovine microbiome inoculants into soils. Specifically, I examined soil respiration and antibiotic resistance gene counts using qPCR following treatment with cephapirin, pirilimycin and a positive and negative control. I found that pre-exposure to antibiotics and manure is important in modulating the response of microbial communities (soil respiration, and gene copy numbers of AmpC and TetO) to further antibiotic exposure. I conclude that antibiotics themselves have a direct effect on soil communities and their functioning that is additive to the effect of manure (i.e., as a resource subsidy). This effect is mediated by the history of previous exposure to antibiotics, i.e., cattle stocking density. These results suggest that antibiotic residues from dairy cattle operation may have significant effects on microbial communities and the biogeochemical cycling they regulate in agricultural ecosystems. / Master of Science / Antibiotic usage has become a widespread health concern. As development of new antibiotics slows down and bacteria resistant to all but last resort antibiotics become more common the scientific and health communities have begun researching antibiotic resistance. Most antibiotics used in the United States are not used on people but are instead used on farm animals to promote growth and increase production. These antibiotics are normally given to animals before they are even sick in order to stop any potential infections. As a result of this many of the antibiotics used go through the animal unused and are therefore released in waste product like urine and manure. That same manure is often spread onto fields as a fertilizer to allow crops to grow. This means that antibiotics are being applied to fields and pastures along with manure. These antibiotics are then killing soil microbial communities (bacteria and fungus that live within soil) while simultaneously creating a store of antibiotic resistance genes. Antibiotic resistance genes are what render bacteria immune to antibiotics themselves. In addition to the immunity the soil microbes may be less efficient at nutrient cycling, the process through which nutrients in the dirt are transformed to a form useable by plants, meaning more fertilizers and manure may be needed to reap the same amount of crop from the same area of land. This research found that antibiotics, when applied with manure, are leading to changes in soil microbial communities as well as a decrease in ecosy+stem functioning. This work is significant because it indicates that antibiotic resistance has implications beyond just public health, it could be affecting food growth and have real economic consequences.

The synthesis of 7-N-acetyldemethyllavendamycin butyl amide (47), 7-Nacetyldemethyllavendamycin isopropyl amide (48), 7-N-acetyldemethyllavendamycin amide of piperidine (49), 7-N-acetyldemethyllavendamycin amide of pyrrolidine (50), 7N-acetyldemethyllavendamycin amide of morpholine (51), demethyllavendamycin butyl amide (52), demethyllavendamycin amide of pyrrolidine (53), and demethyllavendamycin amide of morpholine (54) are described. Pictet Spengler condesation of 7-acetamido-2formylquinoline-5,8-dione (28) with tryptophan butyl amide (66), tryptophan isopropyl amide (67), tryptophan amide of piperidine (68), tryptophan amide of pyrrolidine (69), and tryptophan amide of morpholine (70) in an anisole - pyridine solution directly afforded the five lavendamycin amides 47-51. Compounds 52, 53, and 54 were obtained by hydrolysis of 47, 50, and 51 with 70% H2SO4-H20 solution.Aldehyde 28 was prepared according to the following general procedure.Nitration of 8-hydroxy-2-methylquinoline (30) yielded 8-hydroxy-2-methyl-5,7 dinitroquinoline (31). Compound 31 was then hydrogenated and acylated with acetic anhydride to yield 5,7-diacetamido-2-methyl-8-acetoxyquinoline (33). Compound 33 was oxidized by potassium dichromate to give 7-acetamido-2-methylquinoline-5,8-dione (27). Treatment of 27 with selenium dioxide in refluxing 1,4-dioxane afforded compound 28.Compounds 66, 67, 68, 69, and 70 were synthesized from compounds 61,62, 63, 64, and 65. These compounds were deprotected with ammonium formate in the presence of 10% Palladium on charcoal in methanol under an argon balloon at atmospheric pressure.Compounds 61, 62, 63, 64, and 65 were obtained from 58 with butylamine, isopropylamine, piperidine, pyrrolidine, and morpholine respectively in the presence of triethylamine under an argon balloon at atmospheric pressure.Compound 58 was synthesized by the reaction of N-carbobenzyloxytryptophan, with N-hydroxy succinimide, in the presence of N-dicyclohexylcarbodimide in dried and distilled dioxane under an argon balloon at atmospheric pressure.The structures of the novel compounds 58, 47, 48, 49, 50, 51, 52, 53, and 54 were confirmed by 1H NMR, IR, EIMS, and HRMS.The structures of protected and deprotected amides 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 were also confirmed by 1 H NMR and IR spectroscopy. / Department of Chemistry

A thesis submitted to the Faculty of Science, University of the Witwatersrand Johannesburg. In fulfilment of the requirements for the Degree of Doctor of Philosophy. March 2017 / In this PhD thesis, we report for the first time, new methodology for the synthesis of angucycline antibiotic natural products. In particular, for the synthesis of 1,8-dihydroxy-3methyltetraphene-7,12-dione, commonly known as tetrangulol. We also report on the synthesis of 1,10,12-trimethoxy-8-methylbenzo[c]phenanthridine in our quest to synthesise phenanthroviridone from an intermediate product in the synthesis of tetrangulol. The Suzuki-Miyaura coupling reaction between 1,4,5-(trimethoxynaphthalen-2-yl)boronic acid and 2-iodo-3-methoxy-5-methylbenzaldehyde afforded intermediate, 3-methoxy-5methyl-2-(1,4,5-trimethoxynaphthalen-2-yl)benzaldehyde. Conversion of this benzaldehyde into the alkyne, 2-(2-ethynyl-6-methoxy-4-methylphenyl)-1,4,5-trimethoxynaphthalene was accomplished utilizing the Corey-Fuchs reaction. Exposure of the derived acetylene to a catalytic platinum(II)-mediated ring closure yielded the required tetracyclic aromatic product, 1,7,8,12-tetramethoxy-3-methyltetraphene which was converted into tetrangulol. Exposure of the related 3-methoxy-5-methyl-2-(1,4,5-trimethoxynaphthalen-2-yl)benzaldehyde O-phenyl oxime to microwave irradiation in an ionic liquid yielded 1,10,12-trimethoxy-8methylbenzo[c]phenanthridine, instead of the desired natural product phenanthroviridone. We also report on the unexpected synthesis of the benzonaphthopyranone core found in other classes of angucycline antibiotics from oxygen analogs of 2-naphthylbenzyl alcohols when exposed to N-bromosuccinimide. Treatment of (2-(1,4-dimethoxynaphthalen-2yl)phenyl)methanol and related analogues with N-bromosuccinimide under an oxygen atmosphere afforded 12-methoxy-6H-dibenzo[c,h]chromen-6-one, 2-Methoxy-6Hbenzo[c]chromen-6-one and of 6H-benzo[c]chromen-6-one. An investigation into possible mechanisms for this transformation was also conducted. / LG2017

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