Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157:H7 is a food-borne pathogen that causes hemorrhagic enteritis in humans. Cattle are asymptomatic carriers and their feces are the major source of infection. The objective of the first study was to determine the prevalence of E. coli O157:H7 in the gut of cattle at slaughter. Gut contents (rumen, cecum, colon and rectum) were collected from slaughtered cattle (n=815) at a packing plant and prevalence of E. coli O157:H7 was determined. The overall prevalence of E. coli O157:H7 in cattle was 20.6%. The prevalence (%) in the rumen, cecum, colon, and rectum was 4.9, 9.1, 7.7, and 10.3, respectively. Prevalence in rectal content was positively associated (P < 0.01) with that of the rumen or colon and not of the cecum. Pulsed-field gel electrophoresis typing showed that the majority of isolates obtained within the same animal shared a clonal similarity. There was no significant difference in the acid tolerance of ruminal compared to hindgut isolates. It was concluded that hindgut was the major site of prevalence of E. coli O157:H7 in cattle at slaughter. Neomycin, an aminoglycoside, is approved as a feed additive and for use in water to cattle. The objective of the second study was to determine the efficacy of feeding neomycin on fecal shedding of E. coli O157:H7 in cattle. Cattle were randomly assigned to control (n=14) or neomycin (n=10) supplemented group and orally inoculated with nalidixic acid-resistant (NalR) E. coli O157:H7. Neomycin was fed at 10 mg/0.45 Kg body weight for 15 days. Fecal samples and rectoanal mucosal swab (RAMS) samples were collected day before (d -1), on days 1, 3, 5, 10, 13, 17, 20, 24, 27, 31, 34, 38, 41, 44, and 48, and then approximately weekly through day 111. Fecal shedding of NalR E. coli O157:H7 was quantified and prevalence in RAMS was determined. Neomycin significantly reduced prevalence and concentration of E. coli O157:H7 compared to the control. Following two weeks of neomycin feeding, concentration and prevalence were similar between the two groups. Short term neomycin feeding before slaughter may reduce the E. coli O157:H7 load in cattle.

Aminoglycosides are a group of broad-spectrum antibiotics that, under neutral pH conditions, carry a positive charge. The net cationic charge arises from the high number of amino groups in the core structure of aminoglycosides. Previous studies performed have shown that negatively charged citrate ligand-capped gold nanoparticles (AuNPs) can interact with various biomolecules such as aminoglycosides. AuNPs bound to biomolecules have been used in conjugation with various assaying techniques to detect and study compounds in vitro and in vivo. AuNPs also have strong light scattering properties that can be used with a wide variety of imaging and assaying techniques. Our laboratory has previously performed experiments on the aminoglycoside antibiotic ribostamycin sulfate. During this experiment, the concentration dependent rod-like assembly of ribostamycin sulfate was characterized. This experiment used three analytical techniques in conjunction with AuNPs: (1) dynamic light scattering (DLS), (2) UV-Vis absorption spectroscopy, and (3) dark field optical microscope imaging (DFM). This suite of techniques was used to analyze mixtures of ribostamycin sulfate at different concentration with different sized AuNPs. The primary objective of this research was to determine if the techniques used to characterize the self-assembly of ribostamycin sulfate could be generalized and applied to other aminoglycoside antibiotics. The secondary objective of this research was to determine if other aminoglycoside antibiotics formed rod-like assemblies. This study demonstrated that AuNPs can be used to detect self-assembled oligomers for different aminoglycoside antibiotics. In addition, this study also revealed that not all aminoglycoside antibiotics will self assemble into rod-like oligomers similar to ribostamycin. It was observed that the aminoglycoside antibiotic amikacin self assembled into rod-like aggregates similar to ribostamycin sulfate but the aminoglycoside antibiotics neomycin sulfate and streptomycin sulfate did not.

Plant promoters are the principle cis-acting regulatory sequences responsible for the temporal and spatial expression of genes. One method for isolating plant promoters is based on the ability of a common soil bacterium, <i> Agrobacterium tumefaciens </i>, to transfer a specific segment of DNA (T-DNA) into plant cells. This specific T-DNA has been shown to integrate stably into the recipient plant genome. If the T-DNA contains a promoterless marker gene, then T-DNA integration events occurring adjacent and downstream to a promoter region can be detected by the activation of the marker gene. These T-DNA-mediated gene fusions, consisting of an unknown plant promoter sequence and the coding sequence of a marker gene, can be isolated using the marker gene as a promoter tag. The key objective of this work was to develop a novel, bifunctional selectable marker gene and assess its use as: a selectable marker gene in bacterial and plant transformation systems, and as a promoter tag for T-DNA promoter-tagging studies in dicots. A bifunctional fusion gene was produced between phosphinothricin acetyltransferase and neomycin phosphotransferase (PAT::NPT II), by fusing an NPT II coding sequence to the 3' terminus of the PAT gene. The PAT gene product confers tolerance to a non-selective herbicide L-phosphinothricin (Ignite, Hoechst AG). The neomycin phosphotransferase ('npt II') gene allows for direct selection of transformed cells with the antibiotic, kanamycin. Using an <i>in vivo Escherichia coli </i> selection system, a translational fusion gene between these two reporter genes was achieved. The resulting protein had activities of both parent enzymes. This was demonstrated both in transformed <i>Escherichia coli</i> and in transformed <i>Nicotiana tabacum</i> and <i>Brassica napus</i> plants. Using this bifunctional selectable marker gene, a T-DNA promoter tagging vector, pBAU2, was constructed and its utility was demonstrated in <i>Nicotiana tabacum</i>. One of the <i>N. tabacum</i> promoter tagged events was selected for subsequent promoter isolation studies. The promoter from this regenerant was isolated by screening a Lambda subgenomic library and also by thermal asymmetric interlaced (TAIL-)PCR. The isolated upstream regulatory sequence was fused to a reporter gene, â-glucuronidase ('gus'), and subjected to a preliminary evaluation in <i> Nicotiana tabacum</i> and in <i>Brassica napus</i>.

RNAs are biomolecules ubiquitous in all living cells. Usually, they fold into complex molecular structures, which often mediate their biological function. In this work, models of RNA folding have been studied in detail. One can distinguish two fundamentally different approaches to RNA folding. The first one is the thermodynamic approach, which yields information about the distribution of structures in the ensemble in its equilibrium. The second approach, which is required to study the dynamics of folding during the course of time, is the kinetic folding analysis. It is much more computationally expensive, but allows to incorporate changing environmental parameters as well as time-dependent effects into the analysis. Building on these methods, the BarMap framework (Hofacker, Flamm, et al., 2010) allows to chain several pre-computed models and thus simulate folding reactions in a dynamically changing environment, e. g., to model co- transcriptional folding. However, there is no obvious way to identify spurious output, let alone assessing the quality of the simulation results. As a remedy, BarMap-QA, a semi-automatic software pipeline for the analysis of cotranscriptional folding, has been developed. For a given input sequence, it automatically generates the models for every step of the RNA elongation, applies BarMap to link them together, and runs the simulation. Post-processing scripts, visualizations, and an integrated viewer are provided to facilitate the evaluation of the unwieldy BarMap output. Three novel, complementary quality measures are computed on-the-fly, allowing the analyst to evaluate the coverage of the computed models, the exactness of the computed mapping between the individual states of each model, and the fraction of correctly mapped population during the simulation run. In case of deficiencies, the output is automatically re-rendered after parameter adjustment. Statistical evidence is presented that, even when coarse graining the ensemble, kinetic simulations quickly become infeasible for longer RNAs. However, within the individual gradient basins, most high-energy structures only have a marginal probability and could safely be excluded from the analysis. To tell relevant and irrelevant structures apart, a precise knowledge of the distribution of probability mass within a basin is necessary. Both a theoretical result concerning the shape of its density, and possible applications like the prediction of a basin’s partition function are given. To demonstrate the applicability of computational folding simulations to a real-world task of the life sciences, we conducted an in silico design process for a synthetic, transcriptional riboswitch responding to the ligand neomycin. The designed constructs were then transfected into the bacterium Escherichia coli by a collaborative partner and could successfully regulate a fluorescent reporter gene depending on the presence of its ligand. Additionally, it was shown that the sequence context of the riboswitch could have detrimental effects on its functionality, but also that RNA folding simulations are often capable to predict these interactions and provide solutions in the form of decoupling spacer elements. Taken together, this thesis offers the reader deep insights into the world of RNA folding and its models, and how these can be applied to design novel biomolecules.

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde. / Objective: Nystatin is an effective topical antifungal agent widely used in the treatment of otomycosis. Though it is safe for external ear use, current recommendations are to avoid its use in cases of tympanic membrane perforation. The objective of our study was to test the security of Nystatin when applied directly to the middle ear of a guinea pig model. Methods: We performed an experimental study with 18 Hartley guinea pigs that were divided into two groups. Exposing middle ears from one group to Nystatin (group I) and from the other to the ototoxic neomycin (group II), we compared results of auditory brainstem response (ABR) testing at three intervals during the study. Each animal’s contralateral ear was injected with a physiological solution (NaCl). At the end of the study, we performed a histological analysis of the animals’ cochleae using a scanning electron microscope. Results: Average hearing loss in the Nystatin group was 13.0 dB which was similar to the results obtained in the NaCl-exposed ears (4.0 dB in group I and 15.1 dB in group II). Average hearing loss in the neomycin group was 39.3 dB, which represents a clinically significant difference (p<0.001). Scanning electron microscope evaluation revealed intact cochlear hair cell architecture in the Nystatin and normal saline groups, compared to important destruction in the neomycin group. Conclusion: Nystatin does not cause hearing impairment or cochlear hair cell damage when exposed directly to the middle ear of a guinea pig model.

Para efetivamente tratar uma infecção, é necessário que o antibiótico possua atividade antimicrobiana adequada e seja capaz de inibir o crescimento do microrganismo patogênico. O doseamento microbiológico é uma metodologia indicada para a análise do antimicrobiano de forma simples, quando comparado com outras metodologias. A Food and Drug Administration (FDA) tem encorajado uma abordagem proativa para introduzir inovações e benefícios associados ao processo de produção farmacêutica. A Qualidade por Design Analítico (AQbD) ajuda no desenvolvimento de métodos analíticos robustos e de baixo custo, que são aplicáveis durante todo ciclo de vida do produto. Os métodos microbiológicos tradicionais, de forma geral, apresentam baixa reprodutibilidade e alta incerteza. Desta forma, justifica-se o desenvolvimento de métodos microbiológicos alternativos para a análise de antimicrobianos empregando-se os conceitos de Qualidade por Design Analítico, com a finalidade de melhorar a reprodutibilidade e reduzir a incerteza final. O objetivo deste trabalho foi aplicar o conceito de Qualidade por Design Analítico (AQbD) no desenvolvimento de método colorimétrico para análise de sulfato de neomicina. O sulfato de neomicina é um antimicrobiano aminoglicosídeo amplamente empregado no tratamento de infecções cutâneas ou mucosas, tais como queimaduras, úlceras, e dermatites infecciosas. Métodos cromatográficos como a cromatografia líquida de alta eficiência em fase reversa, de pareamento iônico ou cromatografia iônica com derivatização (pré ou pós-coluna) são utilizados para a análise de aminoglicosídeos, inclusive sulfato de neomicina. Contudo, de acordo com as farmacopeias, o método microbiológico é o método analítico de escolha para a análise de sulfato de neomicina e outros aminoglicosídeos. A análise colorimétrica é um método amplamente utilizado para a detecção e quantificação de diferentes substâncias, incluindo o crescimento microbiano em estudos de eficácia terapêutica. Neste trabalho, propomos o uso de resazurina como marcador colorimétrico. O indicador sofre uma reação de oxido-redução na qual altera a coloração em resposta à redução química resultante do crescimento celular. O uso de microplacas para a análise colorimétrica é uma alternativa ao método realizado em tubos de ensaio. Uma alternativa ao uso de espectrofotômetros para a análise colorimétrica é o uso de aparelhos smartphones, pois são equipados com CPUs rápidas, câmeras de alta resolução e sensores de imagem. O processamento da imagem captada pela câmera do dispositivo é utilizado como um analisador colorimétrico. Portanto, a aplicação dos conceitos de Qualidade por Design Analítico (AQbD) possibilitou o desenvolvimento racional de método microbiológico colorimétrico para análise de sulfato de neomicina. / To effectively treat an infection, the antibiotic must have adequate antimicrobial activity and be capable of inhibiting the growth of the pathogenic microorganism. The microbiological assay is an indicated methodology for the analysis of the antimicrobial in a simple way, when compared with other methodologies. The Food and Drug Administration (FDA) has encouraged a proactive approach to introducing innovations and benefits associated with the pharmaceutical production process. Analytical Design Quality (AQbD) assists in the development of robust, low cost analytical methods that are applicable throughout the product life cycle. Traditional microbiological methods, in general, have low reproducibility and high uncertainty. Thus, it is justified the development of alternative microbiological methods for the analysis of antimicrobials using the concepts of Quality by Analytical Design, in order to improve reproducibility and reduce final uncertainty. The objective of this work was to apply the concept of Quality by Analytical Design (AQbD) in the development of a colorimetric method for the analysis of neomycin sulfate. Neomycin Sulfate is an aminoglycoside antimicrobial widely used in the treatment of cutaneous or mucosal infections, such as burns, ulcers, and infectious dermatitis. Chromatographic methods such as reverse phase high performance liquid chromatography, ion-pairing or ion chromatography with derivatization (pre or post-column) are used for the analysis of aminoglycosides, including neomycin sulfate. However, according to pharmacopoeias, the microbiological method is the analytical method of choice for the analysis of neomycin sulphate and other aminoglycosides. Colorimetric analysis is a widely used method for the detection and quantification of different substances, including microbial growth in studies of therapeutic efficacy. In this work, we propose the use of resazurin as a colorimetric marker. The indicator undergoes an oxidation-reduction reaction in which it alters the coloration in response to the chemical reduction resulting from cell growth. The use of microplates for colorimetric analysis is an alternative to the method carried out in test tubes. An alternative to the use of spectrophotometers for colorimetric analysis is the use of smartphones because they are equipped with fast CPUs, high resolution cameras and image sensors. The image processing captured by the device\'s camera is used as a colorimetric analyzer. Therefore, the application of the concepts of Quality by Analytical Design (AQbD) allowed the rational development of a microbiological colorimetric method for analysis of neomycin sulfate.

Os ensaios microbiológicos são utilizados para avaliar a atividade antimicrobiana desde a descoberta do uso dos antibióticos. Apesar dos ensaios microbiológicos serem amplamente empregados para determinação da potência de antibióticos em formas farmacêuticas, uma vez que fornecem a medida da atividade biológica, apresentam limitações quanto a baixa reprodutibilidade e tempo de análise. O objetivo deste trabalho foi desenvolver, otimizar e validar ensaio colorimétrico rápido em microplaca para determinar a potência da neomicina e bacitracina em produtos farmacêuticos. Metodologias de análise fatorial e análise de superfície de resposta foram utilizadas no desenvolvimento e otimização da escolha do microrganismo, da composição do meio de cultura, da proporção de inóculo, e das concentrações de cloreto de trifeniltetrazólio e dos antibióticos. Os métodos otimizados foram validados pela avaliação da linearidade, precisão, exatidão e robustez. Análise estatística mostrou equivalência entre o método de difusão em ágar e o método colorimétrico rápido em microplaca para ambos antibióticos. Além disso, o ensaio em microplaca apresentou vantagens em relação à reprodutibilidade, sensibilidade, tempo de incubação e quantidades necessárias de meio de cultura e soluções para ambos os antibióticos. / Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite of microbiological assays have been widely employed to determine antibiotic potency of pharmaceutical dosage forms, once they provide a measure of biological activity, they have limitations regarding the low reproducibility and increased time of analysis. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin and bacitracin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and antibiotic concentration. The optimized bioassays methods were validated by the assessment of linearity, precision, accuracy, and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assays and rapid colorimetric microplate bioassays. In addition, microplate bioassay had advantages concerning the reproducibility, sensitivity of response, time of incubation, and amount of culture medium and solutions required.

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde. / Objective: Nystatin is an effective topical antifungal agent widely used in the treatment of otomycosis. Though it is safe for external ear use, current recommendations are to avoid its use in cases of tympanic membrane perforation. The objective of our study was to test the security of Nystatin when applied directly to the middle ear of a guinea pig model. Methods: We performed an experimental study with 18 Hartley guinea pigs that were divided into two groups. Exposing middle ears from one group to Nystatin (group I) and from the other to the ototoxic neomycin (group II), we compared results of auditory brainstem response (ABR) testing at three intervals during the study. Each animal’s contralateral ear was injected with a physiological solution (NaCl). At the end of the study, we performed a histological analysis of the animals’ cochleae using a scanning electron microscope. Results: Average hearing loss in the Nystatin group was 13.0 dB which was similar to the results obtained in the NaCl-exposed ears (4.0 dB in group I and 15.1 dB in group II). Average hearing loss in the neomycin group was 39.3 dB, which represents a clinically significant difference (p<0.001). Scanning electron microscope evaluation revealed intact cochlear hair cell architecture in the Nystatin and normal saline groups, compared to important destruction in the neomycin group. Conclusion: Nystatin does not cause hearing impairment or cochlear hair cell damage when exposed directly to the middle ear of a guinea pig model.

Os ensaios microbiológicos são utilizados para avaliar a atividade antimicrobiana desde a descoberta do uso dos antibióticos. Apesar dos ensaios microbiológicos serem amplamente empregados para determinação da potência de antibióticos em formas farmacêuticas, uma vez que fornecem a medida da atividade biológica, apresentam limitações quanto a baixa reprodutibilidade e tempo de análise. O objetivo deste trabalho foi desenvolver, otimizar e validar ensaio colorimétrico rápido em microplaca para determinar a potência da neomicina e bacitracina em produtos farmacêuticos. Metodologias de análise fatorial e análise de superfície de resposta foram utilizadas no desenvolvimento e otimização da escolha do microrganismo, da composição do meio de cultura, da proporção de inóculo, e das concentrações de cloreto de trifeniltetrazólio e dos antibióticos. Os métodos otimizados foram validados pela avaliação da linearidade, precisão, exatidão e robustez. Análise estatística mostrou equivalência entre o método de difusão em ágar e o método colorimétrico rápido em microplaca para ambos antibióticos. Além disso, o ensaio em microplaca apresentou vantagens em relação à reprodutibilidade, sensibilidade, tempo de incubação e quantidades necessárias de meio de cultura e soluções para ambos os antibióticos. / Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite of microbiological assays have been widely employed to determine antibiotic potency of pharmaceutical dosage forms, once they provide a measure of biological activity, they have limitations regarding the low reproducibility and increased time of analysis. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin and bacitracin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and antibiotic concentration. The optimized bioassays methods were validated by the assessment of linearity, precision, accuracy, and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assays and rapid colorimetric microplate bioassays. In addition, microplate bioassay had advantages concerning the reproducibility, sensitivity of response, time of incubation, and amount of culture medium and solutions required.

No description available.