The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.

In the last decades, there has been an escalating consumption of antibiotics with the number of antibiotic prescriptions increasing worldwide. Overuse or inappropriate use of antibiotics has resulted in a major increase in the development of multi-drug resistant pathogens. Antimicrobial resistance is one of the world’s most serious public health problems with great implication in terms of morbidity, mortality, and costs. To date, there has been no formal antibiotic use study conducted in the West Wollega zone of Ethiopia to assess antibiotic utilization. The objective of this study was to determine the pattern of antibiotic use in two hospitals in the West Wollega zone of Ethiopia, namely Gimbie Adventist Hospital and Nedjo Hospitals, using drug utilization metrics and the costs associated. In addition it was to assess the correlation between diagnosed infectious diseases and antibiotic prescriptions. This study was a cross-sectional, retrospective, descriptive review of antibiotic usage in the two hospitals in the year 2007. Prescriptions dispensed in the first month of each quarter of 2007 were reviewed. The number of prescriptions screened, antibiotic courses started, antibiotic days by specific agent and overall, the cost of individual and all antibiotics, the number and type of infectious diseases diagnosed were collected from which core drug use indicators were calculated. The correlation between infectious disease diagnosed and the antibiotic days prescribed were analyzed. A total of 18568 antibiotic and non-antibiotic prescriptions were reviewed retrospectively in the four months of the study period, 47 percent of which contained at least one antibiotic. The average number of antibiotics per prescription was 1.33 and 1.09 whilst the percentage of injectable antibiotics prescribed was 83.2 percent and 3.76 percent to outpatients and inpatients respectively. Antibiotics prescribed from the Essential Drug List (EDL) and List of Drugs for District Hospital (LDDH) were 63.0 percent, 74.8 percent, and 90.8 percent and 76.1 percent for outpatients and inpatients respectively. 98.6 percent of outpatient and 97.0 percent inpatient prescribed antibiotics were actually dispensed. Penicillins and quinolones were the most prescribed antibiotics in both inpatient and outpatient departments constituting 43.46 percent and 24.08 percent respectively. The antibiotic utilization ratio, incidence of outpatient antibiotic use, incidence of inpatient antibiotic use, the number of Defined Daily Doses (DDD)/1000inhabitants/year and DDD/100 Occupied Bed Days (OBD) for the zone was 0.16, 17.25, 23.56, 158.61, and 70 respectively. Antibiotic cost constituted 33.7 percent of all expenditure on drug, cost of antibiotic per patient care day and cost per antibiotic day was 3.84 Ethiopian Birr (ETB) ($0.40) and 6.29 ETB ($0.66) respectively. The correlation between infectious diseases diagnosed and antibiotic prescription shows significant variation. At outpatient departments alone an average number of antibiotic courses started was 2.7 at Gimbie Adventist Hospital and 7.6 for Nedjo Hospital. When overall antibiotic days prescribed and required was compared in both hospitals, there were 2.4 and 5 times more antibiotic days prescribed than were required for Gimbie and Nedjo Hospitals respectively. This suggests that the overuse of antibiotic is worse in the government hospital (Nedjo Hospital) than in the mission hospital (Gimbie Adventist Hospital). This study suggested that there was overuse of antibiotics in the West Wollega hospitals although further investigation is needed to identify its underlying causes and nature. It is recommended that the health personnel, the hospital management, the zonal and regional Health Bureau, the regulatory bodies and Non-Governmental Organizations (NGOs) work hand-in-hand to promote the rational use of antibiotics in this region so that the consequences and financial cost of antimicrobial resistance can be prevented.

A study has been made on the growth stimulatory-effect of antibiotics when fed to chicks as supplements to different rations. A total of 9 biological tests employing 1280 chicks were carried out. Chicks fed the Connecticut ration and modifications thereof showed a significant growth response to aureomycin supplementation of the rations. The efficiency of feed utilization was improved by the addition of aureomycin to the rations in this experiment. The effect of aureomyoin has been tested in rations in which the protein level has been adjusted to 16%, 17.9%, 19.5% and 22% respectively. The basal rations used consisted of ground corn and wheat as the cereal grain and soybean oil meal and fishmeal as the protein supplements along with additional vitamins and minerals to balance the ration. The results showed that aureomycin did not lower the dietary requirement of the chick for protein. A test was made on the effect of antibiotics on growth of chicks when fed in rations which were deficient in specific amino acids. A wheat ration deficient in lysine and a corn ration deficient in tryptophan have been used. The results showed that antibiotics did not lower the dietary requirement of the chick for either lysine or tryptophan. Antibiotics were fed in chick rations containing different amounts of available carbohydrates. It was found that antibiotics did not lower the dietary requirement of the chick for carbohydrate. A comparison of aureomycin and penicillin showed no difference in growth stimulating properties between the two antibiotics under the conditions of the different experiments. Aureomyoin supplement (aurofac) promoted the same growth response at 0.05 and 1.0% levels. The greatest growth response to antibiotics was obtained when the chicks were fed a diet adequate in available carbohydrates and protein and balanced in amino acids. / Land and Food Systems, Faculty of / Graduate

The experiments reported in the present dissertation were undertaken to obtain further evidence for the possible roles of protein, RNA and DNA macromolecules in radiation-mutagenesis in Drosophila melanogaster. Several antibiotics were tested for their modifying effects on the frequency of radiation-induced sex-linked recessive lethals. Pre-radiation treatment with actinomycin D significantly reduces the frequency of induced mutations in germ cell stages assumed to include spermatids and spermatocytes. These results are consistent with the hypothesis of a role of proteins in the stabilization (repair) of radiation-induced premutational lesions. Puromycin, a specific inhibitor of protein synthesis is ineffective in the modification of induced mutation frequencies in Drosophila melanogaster. Mitomycin C, is itself a potent mutagen in all germ cell stages, peak mutagenicity occurring in spermatid stages. In combination with ɣ-rays, mitomycin C shows an overall additivity of effect. Mutation frequencies due to mitomycin C are not altered by pre- or concurrent treatment with actinomycin D. This may indicate a different mechanism for mutagenesis by mitomycin C and radiation. / Science, Faculty of / Zoology, Department of / Graduate

A number of 14-membered lactones, derived from 42 or 43 were used to investigate a series of alkylation, hydride reduction and hydrogenation reactions. These reactions yielded diastereoselective product distributions which were rationalized as proceeding through a [3434] conformation. A simple model for determining these low energy conformations was developed using MM2 calculations, energy trends of acyclic molecules, X-ray crystallography and NMR spectroscopy. In order to unambiguously identify ring conformations, a method to generate polar maps of the large ring lactones was developed. These polar maps quickly differentiated complex ring conformations as well as identifying the symmetry elements of a conformation. With the aid of polar maps, a new solid state conformation for 14-membered rings was discovered for two macrolides synthesized in this project. Furthermore, three additional unidentified examples of this conformation were recognized from the literature. Energy calculations showed this twisted conformation to be of very low energy. The discovery of the twist conformation led to an improved nomenclature for large rings which considers the number of bonds separating both corner and pseudo corner atoms. Under this proposed nomenclature, the twist conformation was designated the [34'3'4'] conformation. During this investigation, a useful technique for assigning the stereochemistry of E and Z-trimethylsilyl (TMS) enol ethers was developed using ¹H NMR nuclear Overhauser effect difference spectroscopy (NOEDS) experiments. One advantage of this technique over the widely used ¹³C NMR method is that only one isomer is required to accurately assign the Eor Z. stereochemistry. [Formula Omitted] / Science, Faculty of / Chemistry, Department of / Graduate

Desde su descubrimiento, los antibióticos han marcado un hito importante en el tratamiento de enfermedades y han permitido la realización de intervenciones quirúrgicas antes consideradas como letales. Sin embargo, el uso excesivo de antibióticos puede alterar la microbiota, causando disbiosis y aumentando la predisposición al desarrollo de diversas patologías a largo plazo. Antibióticos de amplio espectro como la Oxitetraciclina (OTC) se utilizan de manera desmesurada en diversas industrias agroalimentarias. La relación entre el mecanismo de acción de OTC y alteraciones de la microbiota es aún desconocida. Actualmente, se conoce que OTC funciona como inhibidor de la síntesis de proteínas durante la fase de elongación. Dicho fase de la síntesis de proteínas es altamente conservada entre las bacterias y no explicaría el efecto diferenciado entre géneros bacterianos asociados a disbiosis. Al unirse a la subunidad menor 30S del ribosoma, existe la posibilidad de que OTC también actúe en otras fases de la síntesis proteica. Entre ellas destaca la iniciación de la traducción del ARNm al presentar al menos dos mecanismos alternativos, utilizados diversamente entre las bacterias. El primero utiliza los factores de iniciación IF1, IF2 e IF3, mientras que el segundo utiliza principalmente IF1 e IF2. En el presente estudio se evalúa a OTC como posible inhibidor de la iniciación de la síntesis de proteínas dependiente de los tres factores mediante métodos bioquímicos y análisis informático de modelamiento estructural. Los resultados indican que IF1 es susceptible a OTC, probablemente por su posicionamiento cercano al antibiótico en el ribosoma. Como consecuencia, OTC induce una mayor estabilización de IF1 en el ribosoma, que va en aumento a través de los complejos intermediarios de iniciación, alcanzando un incremento del 40%. La estabilización de IF1, reducción de su capacidad de disociación del ribosoma, conllevaría a una inhibición de la formación del complejo de iniciación 70S. Los resultados aquí expuestos sugieren un nuevo mecanismo de acción de OTC durante la iniciación de la síntesis de proteínas. El entendimiento del nuevo mecanismo de acción de OTC contribuye con una explicación novedosa y base para el entendimiento de disbiosis mediada por el fármaco. Además, los resultados proporcionan las bases de futuras investigaciones para el desarrollo de nuevos antibióticos que actúen en el mismo blanco molecular. / Antibiotics have stablished an important milestone since their discovery, allowing the treatment of infectious diseases and surgical procedures otherwise considered lethal. However, recent studies show that the use of antibiotics can alter the microbiota, causing dysbiosis and leading to the development of diverse pathologies. In particular, broad- spectrum antibiotics such as Oxytetracycline (OTC) are widely used in agricultural and food industries. The relationship between the molecular mechanism of OTC and microbiota modifications is still unknown. The current model suggests that OTC inhibits the elongation phase of protein synthesis inhibitor. This phase of protein synthesis is highly conserved among bacteria and cannot explain the differentiated effect of OTC among bacterial genus. OTC could act in other phases of protein synthesis since the drug binds to 30S small ribosomal subunit. Among these, mRNA translation initiation stands out since it is represented by at least two alternative mechanisms in bacteria. The first mechanism uses initiation factors IF1, IF2 and IF3, while the second uses mainly IF1 and IF2. In the present study, OTC has been evaluated as a potential protein synthesis inhibitor acting at initiation of translation. Particularly, the study focuses in the mechanism that uses all three initiation factors employing biochemical methods and computer analysis of structural modelling. The results indicate that IF1 is susceptible to OTC, probably due to the near positioning with respect to the antibiotic in the ribosome. Consequently, OTC increases the stabilization of IF1 in the ribosome up to 40% along all intermediate initiation complexes. IF1 stabilization would inhibit of 70S initiation complex formation, suggesting a new mechanism of action for OTC during translation initiation. This finding would explain how OTC causes dysbiosis and provides further basis for future research of new antibiotics with a similar molecular mechanism. / Tesis

Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / The imminent threat of antibiotic resistant pathogens that have emerged in clinical settings over the past several decades demands novel solutions in the form of both species- and/or strain-specific diagnostic technologies and treatments. Such new developments would aid in the improved management of bacterial infections by accurate diagnosis and targeted bacterial killing, which would mitigate the continued spread of antibiotic resistance as a result of broad-spectrum antibiotic application The cell surface of bacteria presents a unique opportunity towards development of these modalities, as bacterial cell walls possess both universal and unique features that can be targeted by chemical functionalities without the requirement of cell penetration. This work has sought to take advantage of naturally existing and non-natively installed bacterial cell wall chemical functionalities for which we can develop novel chemoselective chemistries and unique peptides that incorporate those chemical functionalities to enable targeted, biocompatible methods of bacterial labeling and targeting. We initially began these endeavors with the goal of improving upon existing readily reversible iminoboronate chemistry with acetylphenyl boronic acid (APBA), which selectively labels bacteria that contain amine-presenting cell wall lipids (e.g. PE and Lys-PG). In our efforts to improve upon the binding potency of this chemical motif, we synthesized a panel of APBA analogues with varying substituents to modulate amine-binding affinity. We additionally characterized these analogues capacity to form thiazolidinoboronates with free and N-terminal cysteine. Furthermore, we applied an APBA dimer presenting phage library towards identification of potent and selective APBA-presenting peptide binders of 1) a cationic antimicrobial peptide (CAMP) implicated in cancer, human beta defensin 3 (hBD3), and 2) colistin-resistant strains of bacteria that attain their resistance through a variety of different mechanisms. This high-throughput technology afforded identification of peptides that are indeed protein or species/strain selective binders, thus enabling targeted labeling of these important biomolecules. In our continued efforts to identify highly potent and selective bacterial targeting chemistries, we also developed an irreversible chemistry that enables the incorporation of chemical motifs, APBA and semicarbazide, into the cell walls of bacteria through cell wall remodeling mechanisms, which then undergo rapid conjugation with fluorescent and turn-on fluorescent reactive partners. While this alternative approach to bacterial detection requires a primary cell-wall incorporation step, the incorporation and subsequent labeling of these chemical motifs are both highly efficient, which enhances the potency of this bacterial labeling approach The chemical approaches to targeted bacterial labeling herein highlight our ability to develop several species- and strain-selective bioorthogonal chemical probes towards the goal of discovering targeted bacteria binding modalities. Beyond identification of such targeted bacterial binding molecules, we hope to translate these findings into effective, narrow-spectrum antibiotics, which is an endeavor currently being pursued in our laboratory. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Indiana University-Purdue University Indianapolis (IUPUI) / The pulps of 112 permanent teeth of monkeys were surgically exposed and left open to the oral environment for 25 to 27 hours. Then one-third of the exposed, infected pulps were capped with a paste of calcium hydroxide and tap water; one-third were treated with an antibiotic paste compased of erythromycin estolate 10 per cent, streptomycin sulfate 10 per cent, and starch q.s. as the vehicle; and one-third were treated with a paste of zinc oxide powder, one drop of eugenol and one drop of formocresol. The teeth were extracted after one and two years post-operative intervals and were microscopically evaluated. The calcium hydroxide treated teeth had the highest percentage of satisfactory pulpal responses (91.6 per cent). The antibiotic treated teeth were considered to have had satisfactory pulpal responses in 87.5 per cent and the zinc oxide, eugenol and formocresol treated teeth showed satisfactory pulpal response in only 58.0 per cent of the teeth treated with this paste. After two years, several of the antibiotic teeth were considered to have had a satisfactory pulp response even though the calcific bridges were incomplete. The histologic findings were encouraging with calcium hydroxide and the antibiotic paste and warrant clinical investigations.

Indiana University-Purdue University Indianapolis (IUPUI) / The Centers for Disease Control (CDC) and World Health Organizations (WHO) have highlighted six species of highly drug-resistant bacteria, commonly termed the ESKAPE pathogens, that new antibacterials are urgently needed to treat). The ESKAPE pathogens account for over two-million infections and have healthcare costs upwards of $20 billion dollars annually. Over the past several decades, pharmaceutical companies have drastically reduced their research programs for developing new antibacterial agents. As well, bacteria are predisposed to rapidly generate resistance against these “me too” drugs, making this strategy a temporary stop-gap in our ability to fight these pathogens. This has left the burden to identify new antibiotics that function through fundamentally unique mechanisms of action to academia. Towards this goal, we are developing a unique antibacterial strategy that functions through targeting the bacterial GroEL chaperonin systems. GroEL is a molecular chaperone that helps fold proteins into their functional states. Being an essential protein, inhibiting GroEL activity leads to global aggregation and bacterial cell death. We previously reported a high-throughput screening effort that identified 235 GroEL inhibitors. A subsequent study with a subset of these inhibitors identified several that kill bacteria. To follow-up, we have synthesized 43 analogs of a hit-to-lead molecule, compound 1, containing systematic deletions of substituents and substructures to determine the essential parts of the scaffold for inhibiting GroEL and killing bacteria. Along with inhibiting GroEL, several compound 1 analogs exhibit >50-fold therapeutic windows between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. Evaluation of two lead candidates (1 and 11) in a gain-of-resistance assay indicated that MRSA bacteria were not able to easily generate resistance to this compound class. Compound 1 also exhibited the ability to permeate through already established S. aureus biofilms and maintain its bactericidal effects, whereas vancomycin could not. Having established initial structure-activity relationships for the compound 1 substituents and substructures in this study, future efforts will focus on optimizing the antibacterial effects of lead candidates and reducing their off-target toxicity to human cells.

Two decades ago archaeologists in northern Africa discovered evidence that an antibiotic was somehow included in diet of ancient peoples, possibly affecting the health of the population. It has been proposed that the causative organisms are Streptomyces aureofaciens - ubiquitous, mold-like, tetracycline-producing bacteria that could have contaminated grain products. Upon consumption, tetracyclines are incorporated into developing or remodeling bone, remaining observable under ultraviolet light for thousands of years. The current project focuses on an analysis of Roman-Egyptian human and animal bone from the Dakhleh Oasis in southwestern Egypt (100 BC to AD 360). Confocal Laser Scanning Microscopy (CLSM) is used to determine whether or not the population had been exposed to antibiotics, taking advantage of tetracycline's natural fluorescent properties. Results show that, though nearly every sample shows tetracycline fluorescence described in previous literature, bone from the Kellis I and Kellis 2 cemeteries display distinct differences in florescent patterning. CLSM allows three ­dimensional viewing and high-resolution imaging, lending new perspective and increased accuracy to the analysis. Previously published theories regarding the means of exposure and resulting health affects are reconsidered. Further investigation could have implications that overflow their archaeological context due to the multiple uses modem science has for tetracycline therapy.