The problem in this investigation consists of, first, the procurement of several strains of Micrococcus pyogenes var. aureus; second, the comparison of the degree and rate of development of resistance of these organisms to terramycin; and, third, to study the morphological and physiological changes which occur during the development of resistance.

A atividade de antimicrobianos, entre os quais os usados como Antibióticos Promotores de Crescimento (APC´s), quando liberados no solo podem influenciar diretamente na sua biota. Muitas das bactérias presentes em solos cultiváveis e florestais têm papel fundamental na imobilização de minerais e processos de decomposição. Sendo assim, extinguindo-se ou alterando-se as bactérias presentes neste meio, a degradação de compostos xenobióticos, por exemplo, ficaria comprometida, havendo ainda a preocupação do desenvolvimento de bactérias resistentes. Além disso, também são praticamente inexistentes os estudos que tratam a respeito da interação entre antibióticos tetraciclínicos, com constituintes da matéria orgânica do solo no Brasil. Dentro deste contexto, procedeu-se a caracterização de amostras de latossolos oriundas de mata nativa e pastagem, ambas de texturas argilosa e arenosa, bem como os ácidos húmicos extraídos dos mesmos. Foram conduzidos ensaios de sorção dessas amostras com o antibiótico oxitetraciclina, desenvolvendo metodologia de determinação deste composto por LC-MS. Constatou-se que a concentração hidrogeniônica é o fator que mais tem influência no processo de sorção solo-OTC e AH-OTC; com destaque para a condição de pH 5,0, situação da qual mais se aproximam os valores de pH reais dos solos brasileiros. Tal condição apresentou as alterações mais expressivas no que diz respeito às possíveis interações ocorridas entre o antibiótico e os ácidos húmicos. A variável textura do solo também se mostrou importante, seguida pelo teor de matéria orgânica e manejo. / The antimicrobials activity, among then the ones used as growth promoter, when released in soil, could influence directly its biota. Many organisms present in cultivable soils have an important function in metals retaining and decomposition processes. Modifying or excluding these bacterias, de xenobiotic compunds degradation, for example, would be at risk, with the worring of resistant bactérias development. Besides this, the studies envolving tetracyclinic antibiotics interaction with the organic matter in Brazil are scarce. In this contexto, the soil samples characterization from forest and pasture, both with clay and sandy textures, was carried out, and also done with the extracted humic acids from them. Sorption experiments were carried out with the soil and the humic acids, and the oxytetracycline. It was developed a method for OTC determination using LC-MS. It was also found that the pH was the most important variable which influence in the sorption process soil-OTC and HA-OTC, especially at pH 5,0. The most significant modifications concerning the samples interactions with OTC occurred at this condition. The texture also showed important results, followed by the organic matter concentration and soil manure.

The pharmacokinetics of two antimicrobials were investigated in alpacas. Six healthy alpacas were each administered a single dose of 10 mg/kg of oxytetracycline by IV injection and IM injection. In addition, a single dose of 20 mg/kg of florfenicol by IV administration was given to alpacas in a separate study. The pharmacokinetic parameters of oxytetracycline and florfenicol in alpacas were compared to the results previously obtained in llamas. There were significant differences between llamas and alpacas in several of oxytetracycline pharmacokinetic parameters but there were no significant differences in all of florfenicol pharmacokinetic parameters in these two animals. It can be concluded that llamas and alpacas have different oxytetracycline disposition kinetics while they have similar disposition kinetics of florfenicol. The pharmacokinetics of clorsulon, a narrow-spectrum anthelmintic agent, was investigated in llamas following oral administration at a single dose of 14 mg/kg. The plasma levels of clorsulon produced by this dose was lower than the values reported in the clorsulon pharmacokinetic studies carried out in sheep and goats following oral administration at a single dose of 7 mg/kg This suggests the entire dose of clorsulon is not absorbed in llamas. Since the differential equations describing one-compartment system with first-order input and two-compartment system after IV administration with nonlinear elimination kinetics cannot be solved, there is no mathematical expression for the AUC for drugs following these models. The AUC values calculated from the proposed preliminary AUC equations for drugs following these models were compared to the AUC calculated using the trapezoidal rule method based on computer-generated data using the fourth-order Runge-Kutta method. Except for a few exceptions, the predicted AUC from the proposed equations matched the values calculated from the theoretically generated data. / Graduation date: 2004

This paper aimed to describe some lab and histological findings from an experimental infection of Trypanosoma (Trypanozoon) evansi Steel, 1885 (Balbiani, 1888) in rats (Rattus novergicus) belonging to the Wistar strain, testing the effectiveness of three medications. These animals were divided in 4 groups of 10 each and treated to three different chemical treatments after the detection of more than eight tripomastigotes for visual side of the blood smears at the light microscope with 1000 increase. Was tested injectable oxytetracycline 10%, benzonidazol per oss and injectable diminazene 7% while one infected and untreated group remained as control. Evaluation of treatment efficacy was performed through daily blood smears stained by Panotico Rápido® for 60 days. The rats were necropsied aiming histological examination. Treatment with diminazene was the most efficient since no parasites having significant difference (p < 0.05) between males and females of that group, which presented survive after the treatment of 14,4 days more than the males. Histological alterations findings were not specific. / Este trabalho objetivou verificar os achados laboratoriais e histológicos da infecção experimental por Trypanosoma (Trypanozoon) evansi (Steel, 1885) Balbiani, 1888, em ratos (Rattus norvegicus) da linhagem Wistar, testando a eficácia de três medicamentos. Foram utilizados 40 ratos, divididos em quatro grupos de 10 cada, sendo cada grupo composto por 5 machos e cinco fêmeas, os quais foram tratados com três quimioterápicos distintos após a detecção de parasitemia superior a oito tripomastigotas por campo visual do esfregaço sangüíneo ao microscópio de luz (1000 vezes). Testou-se oxitetraciclina 10% injetável, benzonidazol comprimidos de 100mg e aceturato de diminazeno 7% injetável comparados com um grupo controle, experimentalmente infectado, mas não tratado. As avaliações foram feitas por contagens diárias da presença de hemoparasitas em esfregaço sangüíneo, corado por Panótico Rápido®, por 60 dias. Os ratos foram necropsiados e avaliados histologicamente à procura de alterações microscópicas dos órgãos afetados. O tratamento feito com aceturato de diminazeno foi o mais eficaz no controle da parasitemia, havendo diferença significativa (p < 0.05) entre machos e fêmeas desse grupo, as quais apresentaram período de sobrevida após o tratamento de 14,4 dias superior aos machos. Histologicamente as alterações encontradas nos órgãos afetados foram inespecíficas.

O cultivo de camarÃes à uma importante atividade econÃmica e tomou-se a indÃstria que mais cresce no Nordeste brasileiro atualmente. Problemas com enfermidades sÃo comuns na aqÃicultura e pelo menos sete espÃcies de bactÃrias do gÃnero Vibrio tÃm sido registradas infectando camarÃes durante diferentes estÃgios de cultivo. A oxitetraciclina (OTe) tem sido utilizada mundialmente contra esses patÃgenos. Na presente pesquisa, a distribuiÃÃo e depleÃÃo de resÃduos de OTC no tecido de Litopenaeus vannamei foram avaliadas depois de 7 dias de exposiÃÃo à raÃÃo medicada contendo 4g.kg-1. Foram conduzidos dois experimentos: em tanques sob condiÃÃes controladas em laboratÃrio e em gaiolas instaladas em um viveiro de uma unidade de cultivo. Os animais medicados foram amostrados periodicamente, durante o tratamento e pÃs-tratamento. Os nÃveis de OTC foram analisados por cromatografia lÃquida de alta eficiÃncia-CLAE. Embora em ambos os ambientes experimentais o tratamento tenha sido semelhante, os picos de OTC foram diferentes. Depois de 7 e 12 dias de exposiÃÃo, as concentraÃÃes no tecido dos camarÃes foram 17,21 ~g.g-l e 3,78 ~g.g-l nos estudos realizados em laboratÃrio e campo, respectivamente. A concentraÃÃo mÃdia observada nos ambientes em estudo manteve-se dentro dos limites da concentraÃÃo inibitÃria mÃnima-CIM, referida para o tratamento de vibrioses. Durante as primeiras 72 horas apÃs cessado o tratamento, resÃduos da droga reduziram muito rapidamente, sendo eliminados mais lentamente depois deste perÃodo. O nÃvel residual de seguranÃa de 0,10 ~g.g-l, imposta pela Comunidade EuropÃia, foi alcanÃado no 16Â. dia de pÃs-tratamento. Depois do 25Â. dia nenhum resÃduo da droga pÃde ser detectado. Entretanto, devido à influÃncia de fatores ambientes neste processo, recomenda-se um perÃodo de eliminaÃÃo mais longo como medida de seguranÃa para obtenÃÃo da eliminaÃÃo completa da droga. / The cultivation of shrimp is an important economic activity and took up the industry that more grows in the Brazilian Northeast today. Problems with diseases are common in aquaculture and at least seven species of bacteria of the genus Vibrio have been recorded infecting shrimp during different stages of cultivation. The oxytetracycline (OTE) has been widely used against these pathogens. In this research, distribution and residue depletion of OTC in tissues of Litopenaeus vannamei were assessed after 7 days of exposure to medicated feed containing 4g.kg-1. Two experiments were conducted: in tanks under controlled conditions in laboratory cages and installed in a nursery, a unit of culture. The treated animals were sampled periodically during the treatment and post-treatment. The levels of TBT were analyzed by high performance liquid chromatography-HPLC. Although in both environments the experimental treatment was similar, the peaks of OTC were different. After 7 and 12 days of exposure, concentrations in the tissue of the shrimp were 17.21 ~ ~ 3.78 gg gg-le-l in studies conducted in laboratory and field, respectively. The average concentration observed in the environments under study was within the limits of the minimum inhibitory concentration, MIC, referred for the treatment of vibriosis. During the first 72 hours after the treatment ceased, residues of the drug reduced very quickly and is eliminated more slowly after this period. The residual level of safety of 0.10 gg ~-l, imposed by the European Community, was reached at 16 Â. days post-treatment. After the 25 Â. days no residue of the drug could be detected. However, due to the influence of environmental factors in this process, it is recommended a longer period of disposal as a security measure to achieve the complete elimination of drugs

A atividade de antimicrobianos, entre os quais os usados como Antibióticos Promotores de Crescimento (APC´s), quando liberados no solo podem influenciar diretamente na sua biota. Muitas das bactérias presentes em solos cultiváveis e florestais têm papel fundamental na imobilização de minerais e processos de decomposição. Sendo assim, extinguindo-se ou alterando-se as bactérias presentes neste meio, a degradação de compostos xenobióticos, por exemplo, ficaria comprometida, havendo ainda a preocupação do desenvolvimento de bactérias resistentes. Além disso, também são praticamente inexistentes os estudos que tratam a respeito da interação entre antibióticos tetraciclínicos, com constituintes da matéria orgânica do solo no Brasil. Dentro deste contexto, procedeu-se a caracterização de amostras de latossolos oriundas de mata nativa e pastagem, ambas de texturas argilosa e arenosa, bem como os ácidos húmicos extraídos dos mesmos. Foram conduzidos ensaios de sorção dessas amostras com o antibiótico oxitetraciclina, desenvolvendo metodologia de determinação deste composto por LC-MS. Constatou-se que a concentração hidrogeniônica é o fator que mais tem influência no processo de sorção solo-OTC e AH-OTC; com destaque para a condição de pH 5,0, situação da qual mais se aproximam os valores de pH reais dos solos brasileiros. Tal condição apresentou as alterações mais expressivas no que diz respeito às possíveis interações ocorridas entre o antibiótico e os ácidos húmicos. A variável textura do solo também se mostrou importante, seguida pelo teor de matéria orgânica e manejo. / The antimicrobials activity, among then the ones used as growth promoter, when released in soil, could influence directly its biota. Many organisms present in cultivable soils have an important function in metals retaining and decomposition processes. Modifying or excluding these bacterias, de xenobiotic compunds degradation, for example, would be at risk, with the worring of resistant bactérias development. Besides this, the studies envolving tetracyclinic antibiotics interaction with the organic matter in Brazil are scarce. In this contexto, the soil samples characterization from forest and pasture, both with clay and sandy textures, was carried out, and also done with the extracted humic acids from them. Sorption experiments were carried out with the soil and the humic acids, and the oxytetracycline. It was developed a method for OTC determination using LC-MS. It was also found that the pH was the most important variable which influence in the sorption process soil-OTC and HA-OTC, especially at pH 5,0. The most significant modifications concerning the samples interactions with OTC occurred at this condition. The texture also showed important results, followed by the organic matter concentration and soil manure.

Orientador: Lucia Helena Innocentini Mei / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T20:49:29Z (GMT). No. of bitstreams: 1 Kleinubing_SirleneAdriana_M.pdf: 3412137 bytes, checksum: 4c9018ce01be800e79a8dbd49b20fe27 (MD5) Previous issue date: 2013 / Resumo: O presente trabalho fundamenta-se no desenvolvimento de micropartículas de polímeros naturais para utilização em sistemas de liberação gastrorresistentes, onde o fármaco passa pelo meio gástrico intacto, permitindo sua absorção apenas no meio entérico. O fármaco selecionado neste estudo foi a oxitetraciclina (OTC), um antibiótico que ao ser ingerido pode causar irritação na mucosa gástrica, limitando seu uso na forma de liberação convencional. O método escolhido para preparação das micropartículas foi por coacervação complexa em um único estágio. Os polímeros naturais escolhidos foram o alginato de natureza aniônica e a quitosana de natureza catiônica, por serem biocompatíveis, não tóxicos e de fácil degradação. Caracterizações morfológicas das micropartículas, com e sem fármaco, para análise da superfície e do seu interior foram realizadas por microscopia eletrônica de varredura (MEV). A distribuição da OTC nas micropartículas foi determinada por Microscopia confocal multifóton baseada na fluorescência inerente deste fármaco, enquanto que a identidade química e a interação entre os biopolímeros de alginato e quitosana foram investigadas por Espectrômetro de Infravermelho com Transformada de Fourier (FTIR). A análise térmica do material foi realizada por Calorimetria Exploratória Diferencial (DSC). A modulação da liberação da OTC das micropartículas foi feita por recobrimento das mesmas em leito fluidizado, com uma dispersão polimérica à base de Acril-EZE® MP, um polímero sintético gastrorresistente de natureza aniônica. O estudo da cinética de liberação in vitro do fármaco foi realizada por método espectrofotométrico, em meio de dissolução ácido e meio tamponado pH6,8, simulando o perfil de dissolução nos fluidos gástricos e intestinais. Pela microscopia confocal a laser verificou-se que a encapsulação da OTC foi bastante eficiente. A espectroscopia FTIR foi útil para se observar deslocamentos das bandas de absorção do alginato e da quitosana, comprovando interação iônica entre os grupos carboxílicos do alginato e os grupos amina da quitosana. A solução de Acril-EZE® MP se mostrou adequada para o estudo da modulação da liberação controlada da OTC microencapsulada na matriz escolhida. Nos estudos do perfil de liberação deste fármaco, verificou-se uma liberação inicial rápida em meio ácido nas micropartículas não recobertas. Após o recobrimento com Acril-EZE® MP, com 50% de ganho de massa, a liberação da OTC neste meio foi reduzida, obtendo-se o efeito de gastrorresistência desejado. Em meio básico a liberação foi gradual, sendo que o fármaco foi totalmente liberado em cinco horas, para as micropartículas com e sem recobrimento. Deste modo, o sistema estudado mostrou um grande potencial para aplicação em sistemas de liberação controlada de OTC in vivo, desde que sejam realizados os testes necessários, podendo ser produzido em curto prazo em escala comercial / Abstract: The present work focused on the development of microparticles based on natural polymers for use in gastro-release systems, where drug-sensitive gastric medium may follow intact until its enteric controlled delivery. The oxytetracycline (OTC) was selected in this study, since it is an antibiotic that, when ingested, may cause irritation of the gastric mucosa, limiting its use in conventional treatment. The method chosen to prepare microparticles was the complex coacervation in a single stage. Natural polymers chosen were the polianionic alginate and the policationic chitosan because they are biocompatible, nontoxic and easy degradation. Morphological characterizations of the surface and interior of the microparticles, with and without drug, were made by scanning electron microscopy (SEM). The distribution of OTC in the microparticles was determined by multiphoton confocal microscopy, taking the advantage of the inherent fluorescence of OTC. By infrared spectroscopy with Fourier Transform - FTIR it was possible to follow chemical identities of each component and the interaction between the biopolymers chitosan and alginate by the displacement of the absorption bands of each biopolymer. The modulation of OTC release from microparticles was made by coating these microparticles in fluidized bed with a polymer dispersion based on acryl-EZE ® MP, a synthetic anionic polymer gastro-resistant. The kinetics of in vitro release of OTC was performed by spectrophotometric method, in acid dissolution medium buffered medium pH6,8 to study the both environment of interest, ie gastric and intestinal. By confocal laser microscopy it was verified that the encapsulation of OTC was quite efficient. A solution of acryl-EZE ® MP was adequate to study the modulation of microencapsulated controlled release of OTC in the matrix chosen. Kinetic studies showed rapid release of OTC at the begining of the experiments in acidic medium, for uncoated microparticles. After coating with acryl-EZE ® PM, 50% mass gain, the suppression of OTC "burst effect", in the range of two hours, was significantly reduced, meaning the material has a great potential to avoid the gastric resistance desired. In basic medium the release was gradual, and was completed in five hours, for both coated and uncoated microparticles. Thus, the system studied showed a great potential for application to short-term delivery systems for OTC in vivo, subject to further studies, also aiming its commercial viability / Mestrado / Ciencia e Tecnologia de Materiais / Mestra em Engenharia Química

This diploma thesis focuses on optimization and validation of the method for a mixture of tetracycline antibiotics (oxytetracycline, tetracycline) determination using high performance liquid chromatography with a diode array detector (HPLC–DAD). The aim of this thesis was to determine suitability of the system for the determination of these antibiotics in milk. The theoretical part is focused on individual groups of antibiotics and their mechanism of action, resistances and possibilities of use. Next point of this thesis is legislation, which describes permitted amounts of antibiotics in milk. In this study, the HPLC method and validation parameter is described in detail. The experimental part deals with creation and optimization of the method and its validation. Following validation parameters were verified: system suitability, linearity, method accuracy, limit of detection and quantification, robustness. The results obtained indicate mostly suitable ratio of mobile phase of 0,01 M trifluoracetic acid:methanol:acetonitrile = 80:10:10. The wavelength 355 nm was found as the most feasible for this method after evaluating the result. Determination of robustness showed that the method is robust, and the only significant effect on the robustness was observed in column changes.

Oxytetracycline (OTC) is one of two antibiotics currently available and approved by the U.S. Food and Drug Administration for use as a chemotherapeutic agent in food fish and is widely used in the aquaculture industry. Previous pharmacokinetic studies of OTC have been conducted in cold water and warm water species of fish. However, no pharmacokinetic studies have been conducted on a cool water species such as yellow perch (Perca flavescens). The yellow perch is a cool water game and commercial species with high aquaculture potential. The pharmacokinetic profiles of oxytetracycline (OTC) was determined by measuring plasma concentrations in yellow perch following intraperitoneal (i.p.), intramuscular (i.m.), per os (p.o.), and intracardiac (i.c.) administration at a single dose of 50 mg/kg body weight. Using a modification of a high-performance-liquid-chromatographic (HPLC) technique, the plasma OTC concentrations were determined for each of the four routes of administration. Plasma concentrations were also evaluated in yellow perch exposed to a static 48-hour OTC water bath (100 mg/l). The terminal half-lives (t1/2) of OTC in yellow perch for i.p., i.m., p.o., and i.c. administrations were 112, 124, 50, and 28 h, respectively. The t1/2 for the i.m. route of administration was significantly longer than in any of the published i.m. OTC fish studies to date. However, the times of maximum OTC concentration (tmax) for the i.p., i.m. and p.o. administrations (2, 4, and 15 h, respectively) occurred relatively early in the plasma concentration-time curves. This suggests, that in yellow perch, OTC is initially absorbed very rapidly. The area under the plasma concentration-time curves (AUC) for the i.p., i.m., p.o., and i.c. routes of administration were 1718, 2659, 383, and 134 mcg·h/ml, respectively. No OTC was detected in the plasma of yellow perch following the water bath route of exposure. Finally, in yellow perch, effective therapy (plasma OTC concentrations above MIC values for most bacteria pathogenic to fish — 4 mcg/ml) would be achieved for up to 168 hours following a single i.p. or i.m. injection of 50 mg/kg and for up to 15 hours following a single p.o dose of 50 mg/kg. / Master of Science

Bone discolouration of pig carcasses is a quality concern that has been observed in Ontario slaughter plants. The objectives of this study were to establish the prevalence of pig carcasses showing bone discolouration, its relationship with residues of tetracyclines in bones, and to investigate the use of tetracyclines in feeding programs for grower-finisher pigs as the main risk factor for discolouration. Abattoir data were examined to determine the extent of the problem and the prevalence of bone discolouration during 2006, 2008, 2009, and 2010 was found to be 0.13%, 0.22%, 0.26%, and 0.28%, respectively, indicating that the issue of bone discolouration was present at low levels over the entire period of the study. A controlled trial using feed, water, and injectable tetracycline products to investigate the effect of tetracyclines on residue and bone colour was conducted. Bones were assessed visually for signs of discolouration, and high performance liquid chromatography (HPLC) was used to measure the levels of tetracycline residues in the bones. Results from this trial demonstrated that discolouration could be produced with 660ppm of chlortetracycline (CTC) in feed for 12 weeks even when 33 days of withdrawal time was observed. It was also found that residues of tetracyclines can be present in bones in the absence of discolouration. A retrospective study was conducted to investigate tetracycline use in herds identified as having discoloured bones at slaughter. Positive shipments were associated with dosage and duration of CTC use as well as with length of withdrawal. In conclusion, discoloured bones of pig carcasses were identified at low levels in one large Ontario abattoir; however, further investigation is needed in order to determine the impact it may have on the swine industry. / Ontario Pork and the University of Guelph - Ontario Ministry of Agriculture (OMAFRA) Sustainable Production System Program