Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-07-04T22:33:07Z No. of bitstreams: 1 Monica_Scariotto_2018.pdf: 1771323 bytes, checksum: 1281a118a9ea7e2155e71f785f0e816f (MD5) / Made available in DSpace on 2018-07-04T22:33:07Z (GMT). No. of bitstreams: 1 Monica_Scariotto_2018.pdf: 1771323 bytes, checksum: 1281a118a9ea7e2155e71f785f0e816f (MD5) Previous issue date: 2018-03-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / New techniques have emerged in the field of environmental science with the aim of developing water treatments using biodegradable materials, such as some polymers, and secondary materials such as household, industrial and agricultural waste. Films with good adsorption capacity of pollutants can be produced by the electrospinning technique using biodegradable polymers. The adsorption of contaminants can also be efficient by means of modified carbon from natural sources. The objective of the present work was to produce Ecovio® and PCL electrospun films, with and without incorporation of functionalized carbon from tilapia bone residue (CAF), for the adsorption of tetracycline (TC) in aqueous solution. Initially, adsorptive tests were performed to select the physicochemical modification of the CAF that obtained the highest adsorption. Then, polymer films were produced with and without the incorporation of CAF, and subjected to adsorption tests at different pHs along with the isolated CAF. The materials were analyzed by means of Optical Microscopy, Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD). CAF treated with HCl in 150 mesh granulometry obtained the highest adsorptive efficiency and was used in the subsequent tests. The parameters of electrospinning and preparation of solutions presented discrete differences for the obtained fiber diameters. A small reduction in diameters was observed with incorporation of CAF. The electrospinning parameters selected were 14 kV, 1 mL h-1 and 12 cm. The SEM showed that CAF had heterogeneous sites on its surface and, for electrospun films, there was beads formation and differences in the surface of the fibers, according to the solvent used. The FTIR indicated that CAF consists mainly of HAp and collagen. The influence of the pH was verified through adsorptive tests, in which alkaline pH was more efficient, with adsorption of TC (100 mg L-1) of 20.45 mg g-1 (pH 10) and 19.23 mg g-1 (pH 9) for CAF and 19.93 mg g-1 (pH 9) for the Ecovio®85/PCL15/CAF blend of 15% (w/v) with chloroform/DMF solvents. The mechanisms suggested in the adsorption of TC in the present work are diffusion of charges on the surface of carbon and formation of complexes. / Novas técnicas têm surgido no ramo da ciência ambiental com o objetivo de desenvolver tratamentos de água utilizando materiais biodegradáveis, como alguns polímeros, e materiais de uso secundário, como resíduos de fontes domésticas, industriais e agrícolas. Filmes com boa capacidade de adsorção de poluentes podem ser produzidos pela técnica de eletrofiação utilizando polímeros biodegradáveis. A adsorção de contaminantes também pode ser eficiente por meio de carvão modificado proveniente de fontes naturais. O objetivo do presente trabalho visou produzir filmes eletrofiados de Ecovio® e PCL, com e sem a incorporação de carvão funcionalizado a partir de resíduo de osso de tilápia (CAF), para a adsorção de tetraciclina (TC) em solução aquosa. Inicialmente, ensaios adsortivos foram realizados para selecionar a modificação físico-química do CAF que obteve maior adsorção. Em seguida, filmes poliméricos foram produzidos com e sem a incorporação de CAF, e submetidos a ensaios adsortivos em diferentes pH juntamente com o CAF isolado. Os materiais foram analisados por meio de Microscopia Óptica, Microscopia Eletrônica de Varredura (MEV), Espectroscopia de Infravermelho por Transformada de Fourier (FTIR), Análise Termogravimétrica (TGA), Calorimetria Exploratória Diferencial (DSC) e Difratometria de Raios-X (DRX). O CAF tratado com HCl na granulometria de 150 mesh obteve a maior eficiência adsortiva, sendo utilizado nos ensaios subsequentes. Os parâmetros de eletrofiação e preparo de soluções poliméricas apresentaram diferenças discretas para os diâmetros de fibras obtidos. Notou-se uma redução discreta nos diâmetros com a incorporação de CAF. Os parâmetros de eletrofiação selecionados foram 14 kV, 1 mL h-1 e 12 cm. A MEV mostrou que o CAF apresenta sítios heterogêneos em sua superfície e que para os filmes eletrofiados houve a formação de beads e diferenças na superfície das fibras, de acordo com cada solvente utilizado. O FTIR indicou que o CAF é formado por principalmente por HAp e colágeno. A influência do pH foi constatada por meio de ensaios adsortivos, em que pH alcalino se mostrou mais eficiente, com adsorção de TC (100 mg L-1) de 20,45 mg g-1 (pH 10) e 19,23 mg g-1 (pH 9) para o CAF e 19,93 mg g-1 (pH 9) para a blenda de 15% (m/v) de Ecovio®85/15PCL/CAF com solventes clorofórmio/DMF. Os mecanismos sugeridos na adsorção de TC no presente estudo são a difusão de cargas na superfície do carvão e a formação de complexos.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-07-26T14:34:03Z No. of bitstreams: 1 2012 - Elaine Concei??o Liporage de Mendon?a.pdf: 3515906 bytes, checksum: ebf489151f2cd6961ddeeb292cd910a7 (MD5) / Made available in DSpace on 2017-07-26T14:34:03Z (GMT). No. of bitstreams: 1 2012 - Elaine Concei??o Liporage de Mendon?a.pdf: 3515906 bytes, checksum: ebf489151f2cd6961ddeeb292cd910a7 (MD5) Previous issue date: 2012-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The use of antibiotic in the control of intramammary infections and in the elimination of its possible sources in dairy farms is an important control measure. However, the inappropriate use of antibiotics can result in the appearance of resistant strains and compromise the efficiency of the treatment. Besides Staphylococcus spp. are among the main pathogens of bovine mastitis, they are often resistant to antibiotics, especially beta-lactamics, mainly by two distinct mechanisms: the production of extracellular enzyme beta-lactamase, encoded by the blaZ gene, and production of PBP2a or PBP2 ' a penicillin-binding protein with low affinity, encoded by the mecA gene. The expression of mecA gene is constitutive or induced by beta-lactamic antibiotics, such as oxacillin and cefoxitin. The mecA gene is inserted into the chromosome through a staphylococcal mobile genetic element, called staphylococcal cassette chromosome mec (SCCmec). The present study evaluated the phenogenotypical resistance profile to betalactam antibiotics of 250 Staphylococcus spp. isolates, using oxacillin and cefoxitin as markers in order to produce data to the knowledge of resistance in dairy farms located in the South-Fluminense and the Metropolitan regions of the State of Rio de Janeiro to support the implementation of measures to control this disease. The assessment of resistance was made through 8 different phenotypic tests and yielded 54 profiles. Disk diffusion and agar screen with oxacillin were used as "gold standard" for the calculation of sensitivity, specificity and prediction once they are recommended by the CLSI veterinarian as standardized tests. Disk diffusion with cefoxitin achieved the best performance in the prediction of oxacillin resistance. Genotypic detection of mecA do not provided any positive isolate, otherwise mecI and mecRI genes were also detected in 11.6% (29/250) of the studied Staphylococcus spp. Four cassette mec types were detected (I, II, III and IV), being type I the most disseminated one. Gene blaZ was detected in 5.2% (13/250) isolates. From these 13 blaZ positive isolates, the whole system comprising blaR1-blaI-blaZ was detected in 23.1% (3/13) isolates / MENDON?A, Elaine Concei??o Liporage. Caracteriza??o fenogenot?pica da resist?ncia antimicrobiana em Staphylococcus spp. isolados de mastites cl?nicas e subcl?nicas em unidades leiteiras de munic?pios do Rio de Janeiro como subs?dio para implementa??o de medidas de controle. 89 p. Disserta??o (Mestrado em Ci?ncias Veterin?rias). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2012. A utiliza??o de antibi?ticos no controle das infec??es intramam?rias e na elimina??o de prov?veis fontes de infec??o nas fazendas leiteiras se constitui em importante medida de controle. No entanto, o uso inadequado de antibi?ticos no tratamento da doen?a pode gerar o aparecimento de cepas resistentes e comprometer a efici?ncia do tratamento. Bact?rias do g?nero Staphylococcus spp. est?o entre os principais agentes etiol?gicos da mastite bovina e s?o freq?entemente resistentes aos antimicrobianos, em especial aos beta-lact?micos, principalmente por dois mecanismos distintos: a produ??o da enzima extracelular beta-lactamase, codificada pelo gene blaZ, e a produ??o de PBP2a ou PBP2?, uma prote?na ligante de penicilina de baixa afinidade, codificada pelo gene mecA. A express?o do gene mecA ? constitutiva ou induzida por antibi?ticos betalact?micos, como a oxacilina e cefoxitina. O gene mecA est? inserido no cromossomo estafiloc?cico atrav?s de um elemento gen?tico m?vel, denominado cassete estafiloc?cico cromoss?mico mec (SCCmec). O presente estudo avaliou o perfil fenogenot?pico de resist?ncia aos beta-lact?micos em 250 isolados de Staphylococcus spp, utilizando os marcadores oxacilina e cefoxitina, de modo a produzir dados que possam contribuir para o conhecimento da resist?ncia antimicrobiana em algumas propriedades leiteiras das regi?es Sul-Fluminense e Metropolitana do Estado do Rio de Janeiro com o objetivo de subsidiar a implementa??o de medidas de controle dessa enfermidade. A avalia??o da resist?ncia foi feita a partir de 8 diferentes testes fenot?picos, sendo obtidos 54 perfis. Os testes de difus?o em disco simples e ?gar screen com oxacilina foram utilizados como ?padr?o ouro? para os c?lculos dos valores de sensibilidade, especificidade e predi??o por serem preconizados pelo CLSI veterin?rio. O teste de difus?o em disco simples com cefoxitina foi o de melhor desempenho na predi??o da resist?ncia a oxacilina. Na avalia??o genot?pica, n?o foi detectado qualquer isolado positivo para o gene mecA, j? os genes mecI e mecRI foram detectados igualmente em 11,6% (29/250) dos Staphylococcus spp avaliados. Foram detectados os quatro tipos de cassete mec analisados (I, II, III e IV), sendo o tipo I o que teve mais ampla distribui??o entre as regi?es estudadas. Gene blaZ foi detectado em 5,2% (13/250) dos isolados, sendo que nestes, todo o sistema blaZ-blaI- blaR1 foi detectado em 23,1% (3/13) dos isolados.

Este estudo teve como objetivo desenvolver e validar micrométodo simples e sensível para quantificação de cefuroxima plasmática utilizando CLAE-UV com a finalidade de aplicação no monitoramento das concentrações de cefuroxima de pacientes submetidos à cirurgia de revascularização do miocárdio (RM) com CEC no esquema de doses administradas em bolus. Os tempos de retenção para o fármaco e padrão interno (guaifenesina) foram 5,3 e 8,7 minutos respectivamente, com um tempo de corrida de 15 minutos, utilizando coluna de fase reversa C18 (25 cmX4,6 mm, 5 micra) e fase móvel binária constituída de tampão acetato de amônio e trietilamina 0,025 M pH 4,2 e acetonitrila (80:20, v/v), fluxo de 1,0 mL/min, detecção no ultravioleta, λ=274nm em sistema isocrático de eluição. A validação deste método analítico investigada através dos limites de confiança apresentou sensibilidade de 0,1 µg/mL (LD) e limite inferior de quantificação (LIQ) de 0,20 µg/mL, linearidade na faixa compreendida 0,2 µg/mL a 200 µg/mL e 4,37% e 2,95% para precisão intra- e inter-dias, respectivamente. Boa exatidão (98,75%) e alta seletividade foram registradas para o método. Através de um protocolo de estudo para antibioticoprofilaxia das infecções cirúrgicas investigaram-se dez pacientes com indicação de cirurgia eletiva de revascularização do miocárdio com circulação extracorpórea. Realizou-se o monitoramento das concentrações plasmáticas após a dose de ataque de 1,5 g, seguido da manutenção realizada através de bolus em tres doses de 0,75 g 6/6 horas. Uma vez que as concentrações plasmáticas de cefuroxima obtidas na sexta hora (vale) foram inferiores à recomendada 16 µg/mL (4x MIC), recomenda-se o aumento de 0,75 g 6/6 horas para 1,5 g mantendo-se o intervalo entre doses de forma a atingir aquela requerida na antibioticoprofilaxia das cirurugias cardíacas. / The objective of the study was to validate na analytical method to determine cefuroxime in plasma by high performance liquid chromatography (HPLC - UV) for clinical purposes in surgical patients submitted to elective cardiac surgery of myocardial revascularization with cardiopulmonary bypass after drug administration as IV boluses. Retention times for the analite and its internal standard (guaifenesin) were 5.3 and 8.7 minutes, respectively; run time was 15 minutes, using a reversed phase colunm C18 (250X4.6 mm, 5 micron) and a binary mobile phase of ammonium acetate/trietilamine 0.025 M pH 4.2 and acetonitrile (80:20, v/v), flow rate 1 mL/min, ultraviolet detector, λ=274nm isocratic elution system. Validation of confidence limits presented 0.1 µg/mL sensitivity (LD) and lower limit of quantification (LLOQ) of 0.20 µg/mL, linearity in the range 0.2 µg/mL to 200 µg/mL and 4.37% e 2.95% for intra- / interday precisions, respectively. Good accuracy (98.75%) and high selectivity were obtained. The study protocol for antibiotic prophylaxis of surgical infections was designed for ten patients with indication of elective cardiac surgery of myocardial revascularization with cardiopulmonary bypass. Loading dose of 1.5 g followed by maintenance dose of 0.75 g every six hours by IV boluses were applied and plasma drug monitoring was done. Based on data obtained cefuroxime plasma concentrations at time dose interval were lower than 16 µg/mL (4x MIC) at the trough, consequently it is recommended to increase the maintainance dose from 0.75 g 6/6 h up to 1.5 g 6/6h, to reach the minimum required for the antibiotic prophylaxis of cardiac surgeries.

A possível indução de resistência bacteriana devido a inclusão de antibióticos, e a pressão dos consumidores por produtos de qualidade, levaram a proibição do uso dos mesmos na alimentação animal. Diante destes acontecimentos, as buscas por alternativas em substituição aos antibióticos vêm sendo bastante enfatizadas na alimentação animal. Assim, os aditivos fitogênicos, extratos vegetais ou extratos herbais são a mais nova opção de produtos naturais que podem substituir os agentes antimicrobianos convencionais. Dessa forma, o presente estudo teve como objetivo avaliar as características zootécnicas de desempenho (consumo, conversão alimentar e ganho de peso) em frangos de corte suplementados com óleo essencial de orégano. Foram utilizados 600 pintos de corte machos, da linhagem Cobb criados até 42 dias de idade sob cama nova de casca de arroz. O delineamento foi inteiramente casualizado com 6 tratamentos, em arranjo fatorial 2x3, sendo: T1 - controle positivo (antibiótico e 0,05% de coccidiostático); T2 - controle negativo (sem aditivos); T3 0,05% de coccidiostático e 0,03% de óleo essencial de orégano) T4 0,03% de óleo essencial de orégano; T5 0,05% de coccidiostático e 0,05% de óleo essencial de orégano; T6 0,05% de óleo essencial de orégano, sendo 10 repetições/tratamento. Considerando-se o período total de criação, houve efeito tanto da adição do óleo essencial de orégano quanto da adição do coccidiostático no consumo de ração. Também houve efeito signifcativo da inclusão do óleo essencial de orégano nas rações para conversão alimentar no período de 1 a 42 dias de idade das aves. Em relação ao ganho médio de peso e ganho de peso total, houve interação entre o óleo essencial de orégano e o coccidiostático, sendo que a não associação do óleo essencial de orégano ou a inclusão de 0,03% do óleo essencial de orégano ao coccidiostático resultam em efeito significativo. A utilização do óleo essencial de orégano na alimentação de frangos de corte resulta em efeito significativo nas características de desempenho. / The induction of bacterial resistance due to the inclusion of antibiotics, and pressure from consumers for quality products, led to prohibition of their use in animal feed. Before these events, the search for alternatives to replace antibiotics have been widely emphasized in animal feed. So phytogenic additives, plant extracts or herbal extracts are the newest option of natural products that can replace the conventional antimicrobial agents. Thus, the present study aimed to evaluate the performance of husbandry characteristics (consumption, feed conversion and weight gain) in broilers supplemented with oregano essential oil. We used 600 male broiler chicks of Cobb created until 42 days under new bed of rice husk. The completely randomized design with 6 treatments in a factorial 2x3: T1 - positive control (antibiotic and 0.05% of coccidiostats), T2 - negative control (no additives), T3 - 0.05% coccidiostat, and 0 , 03% of essential oil of oregano) T4 - 0.03% essential oil of oregano; T5 - 0.05% 0.05% coccidiostat, and essential oil of oregano; T6 - 0.05% essential oil oregano, with 10 replicates / treatment. Considering the total period of creation, there was no effect of either adding essential oil of oregano as the addition of coccidiostat in feed intake. There was also significant effect of inclusion of the essential oil of oregano in feed rations during the period 1 to 42 days old birds. In relation to weight gain and total weight gain, there was an interaction between the essential oil of oregano and coccidiostat, is not that the association of the essential oil of oregano or the inclusion of 0.03% essential oil of oregano result of the coccidiostat in a significant effect. The use of oregano essential oil in the diet of broilers results in significant effect on the performance characteristics.

Foram utilizados 1140 pintos de corte, machos, da linhagem comercial Cobb criados de 1 a 42 dias de idade e alojados em um galpão experimental. Foram testados seis tratamentos, com seis repetições de 40 aves, adotando-se um delineamento experimental inteiramente ao acaso. Os tratamentos foram: T1 Controle; T2 - Antibiótico (Bacitracina de Zinco); T3 - MOS; T4- Ácidos Orgânicos; T5 - MOS + Ácidos Orgânicos, T6- Extratos de Plantas. Os parâmetros de desempenho avaliados foram (consumo de ração, ganho de peso, conversão alimentar e mortalidade), e morfometria intestinal.Os dados foram submetidos à análise de variância e, para a comparação das médias foi utilizado contrastes ortogonais , com um nível de 5% de probabilidade. Houve efeito significativo (p<0,05) tanto para as varáveis de desempenho em todos os períodos analisados, como para as variáveis de morfometria intestinal. Os aditivos alternativos substituíram o antibiótico de forma eficiente em todas as fases de criação, tanto para índices zootécnicos quanto para morfometria intestinal, sendo uma eficiente alternativa de uso. / Were used in 1140 chicks, male, commercial line Cobb raised from 1 to 42 days old and housed in an experimental barn. We tested six treatments with six replicates of 40 birds, adopting a completely randomized experimental design. The treatments were: T1 - control, T2 - Antibiotic (Bacitracin Zinc), T3 - MOS-Organic Acids T4, T5 - MOS + Organic Acids, Plant Extract-T6. The performance parameters evaluated were (feed intake, weight gain, feed conversion and mortality), and morphometry intestinal.Os data were subjected to analysis of variance and, for comparison of means was used orthogonal contrasts, with a level of 5 % Probability. Significant effects (p <0.05) for both variables of performance in all study periods, as for the variables of intestinal morphology. The additives have replaced the alternative antibiotic efficiently at all stages of creation, both in biological indices as in intestinal morphology, with an efficient alternative use.

Orientador: Ana Valéria Colnaghi Simionato Cantú / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T23:35:40Z (GMT). No. of bitstreams: 1 Orlando_EduardoAdilson_M.pdf: 785459 bytes, checksum: 50e5f02a98813d34ba94c694183e9030 (MD5) Previous issue date: 2013 / Resumo: A classe de antibióticos tetraciclinas é amplamente empregada na aquicultura e possui especial dificuldade analítica devido a uma estrutura complexa e grande interação com os componentes da matriz cárnea. Neste trabalho foram avaliadas diferentes técnicas de extração de resíduos de tetraciclinas em filés de tilápias. Analisou-se oxitetraciclina, doxiciclina, tetraciclina e clortetraciclina por HPLC-fluorescência nas condições: gradiente de fase móvel, com fase orgânica composta por MeOH:ACN (1:1, v/v) e fase aquosa pH 7,00 contendo Na2COOH (37,5 mmol L), CaCl2 (17,5 mmol L) e EDTA (12,5 mmol L) e detecção em 385/528 nm (lexc/lem). Foram avaliados os métodos de extração: partição líquido-líquido; extração em fase sólida utilizando as fases estacionárias fenil, C18 e polimérica Oasis-HLB; concentração em resina XAD 16; e QuEChERS; que foram otimizados utilizando planejamento experimental fatorial fracionário e comparados quanto à eficiência de extração. A partição líquido-líquido e o protocolo QuEChERS apresentaram baixas eficiências de extração dos analitos (14-30%). O emprego da resina polimérica Amberlite XAD 16 apresentou boa eficiência (40-60%), mas com baixa precisão e grande consumo de tempo. O emprego de SPE apresentou os melhores resultados, com destaque para a fase fenil (58-76%), sendo então este método validado com relação as figuras de mérito seletividade, linearidade, exatidão, precisão, limite de detecção e quantificação, mostrando-se adequado para a investigação dos analitos em níveis de resíduos em filés de tilápia. Um total de 26 amostras comerciais foram analisadas, sendo que apenas uma delas apresentou resíduo de 42,0 ± 8,4 ng g de Oxitetraciclina, estando abaixo do limite máximo de resíduo no estabelecido no Brasil de 200 ng g / Abstract: The indiscriminate use of antibiotics in aquaculture can generate residues in meat intended for human consumption and cause the arising of bacterial resistance. The tetracycline antibiotics class is widely used and has ultimate analytical difficulties due to a complex structure and large interaction with components of meat matrix. In this work different techniques were evaluated for extraction of tetracycline residues in tilapia fillets. Oxytetracycline, doxycycline, tetracycline and chlortetracycline were analyzed by HPLCfluorescence under the following conditions: mobile phase gradient; organic phase composed by MeOH:ACN (1:1, v/v) and aqueous phase containing Na2COOH (37,5 mmol L), CaCl2 (17,5 mmol L) e EDTA (12,5 mmol L) at pH 7.00 and detection at 385/528 nm (lexc/lem). The evaluated extraction methods were: liquid-liquid partition; solid phase extraction (SPE) using stationary phases phenyl, C18 and Oasis HLB; concentration on adsorbent resin XAD 16; and QuEChERS. The methods were optimized using fractional factorial experimental design and compared in terms of extraction efficiency. The liquidliquid partition and QuEChERS protocol showed low extraction efficiencies (14-30%). The use of polymeric resin Amberlite XAD 16 showed a good efficiency (40-60%) but with low precision and large time consumption. The use of SPE showed the best results and was validated according to the merit figures selectivity, linearity, accuracy, precision, limit of detection and quantification, making it adequate for the investigation of analytes at levels of residues in tilapia fillets. A total of 26 real samples were analyzed and only one sample exhibited Oxytetracycline residue of 42,0 ± 8,4 ng g, being below the maximum residue limit established in Brazil of 200 ng g / Mestrado / Quimica Analitica / Mestre em Química

Orientador: Caio Cezar Randi Ferraz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-02T20:01:54Z (GMT). No. of bitstreams: 1 Sousa_EzilmaraLeonorRolimde_D.pdf: 6699561 bytes, checksum: a7e05e747b868e98926396a2a1fc5d99 (MD5) Previous issue date: 2003 / Resumo: O objetivo deste estudo foi investigar a composição da microbiota de canais radiculares infectados associados a abscessos periapicais; analisar a correlação entre espécies bacterianas específicas com a localização clínica dos abscessos periapicais, sinais e sintomas e testar a suscetibilidade de bactérias anaeróbias estritas prevalentes. As amostras microbiológicas foram coletadas de 60 canais radiculares usando pontas de papel estéreis, transportadas em RTF, diluídas, plaqueadas e incubadas em câmara de anaerobiose. Colônias microbianas foram isoladas, caracterizadas e identificadas por métodos padronizados. Das 287 diferentes espécies bacterianas, 201 eram anaeróbias estritas. Uma ou mais (máximo de 14) espécies bacterianas foram recuperadas de 59 (98,3%) canais radiculares, confirmando a característica polimicrobiana das infecções endodônticas. As bactérias anaeróbias estritas mais freqüentemente isoladas foram: Peptostreptococcus prevotii (22/60), Peptostreptococcus micros (19/60), Fusobacterium necrophorum (19/60). Embora menos freqüentes, bactérias facultativas como Gemella morbillorum (19/60), Streptococcus mitis (13/60), e Streptococcus sanguis (11/60)também foram encontradas. O teste de Pearson ou teste exato de Fisher mostrou que houve relação positiva de algumas espécies bacterianas com a localização do abscesso periapical, bem como, com alguns sinais e sintomas de origem endodôntica (p<0,05). Os resultados indicaram predominância de bactérias anaeróbias Gram-positivas e a presença de microbiota mista nos canais radiculares infectados associados a abscessos periapicais. O método do E-test revelou sensibilidade bacteriana das espécies testadas aos antibióticos benzilpenicilina, amoxicilina, amoxicilinalclavulanato de potássio, metronidazol, clindamicina e cefaclor, contudo, certos microrganismos foram resistentes a azitromicina e eritromicina. Enquanto que, nenhum dos microrganismos testados produziu 'beta¿-lactamase / Abstract: The aim of this study was to investigate the composition of the microbiota of infected root canaIs associated with periapical abscesses, to investigate the correlation of specific species of bacteria with the clinical localization of the periapical abscesses, signs, and symptoms, and to test the susceptibility of prevalent strict anaerobic bacteria isolated. Microbiological samples were collected from 60 root canals using sterile paper points, transported in RTF and diluted, plated and incubated in an anaerobic chamber. Microbial colonies were then purified, characterized and identified by established methods. Of the 287 different bacterial species recovered, 201 were strict anaerobes or microphilic species. One or more (maximum of 14) bacterial species were recovered from 59 (98.3%) root canals, showing the polymicrobial characteristic of endodontic infections. The most frequently strict anaerobes isolated were: Peptostreptococcus prevotii (22/60), Peptostreptococcus micros (19/60), Fusobacterium necrophorum (19/60). Although less frequent, facultative bacteria such as Gemella morbillorum (19/60), Streptococcus mitis (13/60), and Streptococcus sanguis (11/60) were also found. The Pearson X 'POT. 2¿ test or Fisher's exat test showed positive relationship among some bacterial species and the localization of periapical abscesses as well as with some endodontic signs and symptoms (p<0.05). Results indicated predominance of Gram-positive anaerobic bacteria in the mixed microbiota of dental root canals associated with periapical abscesses. The E-test revealed bacterial susceptibility to benzylpenicillin, amoxicillin, amoxicillin/potassium clavulanate, clindamycin and cefaclor. All microorganisms tested did not produce 'beta¿-lactamase / Doutorado / Endodontia / Doutor em Clínica Odontológica

Bacterial antibiotic resistance is an increasing global health problem, leaving few therapeutic options available for the treatment of pathogenic infections. The development of new antibiotics has been slow since their discovery more than 8 decades, therefore, monitoring the extent and distribution of antibiotic resistance is of great importance. The aim of this study was to determine the presence of antibiotic resistance genes in sludge samples obtained from three wastewater treatment plants (WWTPs) in Sweden. Samples were collected and analyzed for the presence of nalidixic acid (NA), chloramphenicol (CHL), and tetracycline (TC) resistance genes using polymerase chain reaction (PCR). The DNA extracted from Eskilstuna and MälarEnergi sludge showed the presence of NA and TC resistance genes, whereas Örebro sludge was found to have resistance for TC antibiotic genes. To validate the results, PCR detection for resistance genes was performed on Escherichia coli isolates from the sludge samples. Antibiotic susceptibility testing was used to confirm the genetic analysis for antibiotic resistance genes detection in these E. coli. The PCR results for TC resistance genes correlated between sludge PCR analysis and bacterial isolates for all 3 WWTPs. Based on the results obtained from the genotypic analysis of sludge and E coli, incomplete compatibility in regards to NA, and CHL were observed. However on the basis of antibiotic susceptibility testing, E coli isolates from MälarEnergi sludge samples unveiled the majority presence for antibiotic resistance genes. The results suggest that extra monitoring for the wastewater treatment facilities are vital to minimize the rising incidence of antibiotic resistant bacteria.

Bacteria are one of the largest causes of human disease, with millions of deaths every year attributed to bacterial infections, and they have become more difficult to tackle with the widespread emergence of antibiotic resistance. In this thesis, I describe my studies that pursued two approaches: one focus was on using antimicrobial histones as an alternative to treatment for antibiotic resistant bacteria; in another approach the recombinant version of an eggshell cuticle protein was expressed and purified for testing against food-safety pathogens. One major pathogen that is contributing to this challenge of antibiotic resistance is Staphylococcus aureus. The methicillin-resistant strain of S. aureus leads to increased hospital stays and increased mortality in patients. The impact of such pathogens is worsened when bacteria form surface-attached aggregates known as biofilms. Development of new approaches to eradicate antibiotic- resistant biofilms will benefit human health. This study looked at an alternative method to eradicate bacteria compared to traditional antibiotics. Histones with antimicrobial activity were extracted from chicken blood and tested against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus biofilm (MSSA and MRSA). The histone mixture completely eradicated both strains in biofilm form at relatively low concentrations. In addition, the histone mixture also displayed fast kill kinetics against planktonic forms of the two strains. Finally, the interaction of the histone mixture with the bacterial membrane in MRSA biofilms was observed by scanning electron microscopy (SEM). Bacteria treated with the histone mixture showed clear morphological changes, including pore formation and cell collapse. Therefore, the histone mixture purified from chicken red blood cells could prove to be a good alternative to traditional antibiotics for protection against antibiotic-resistant strains of bacteria in their planktonic and biofilm forms. Reduction of food-borne illness is another important aspect in the promotion of human health. A significant contributor to food-borne illness is contaminated table eggs. The unfertilized egg can be contaminated by a variety of pathogens including Salmonella spp. and Bacillus spp. The egg is protected by the eggshell which is traversed by respiratory pores that are normally covered by a cuticle plug to restrict pathogen entry. This cuticle consists of several proteins including ovocaxlyin-32 (OCX-32). OCX-32 has a large number of naturally occurring haplotypes due non-synonymous single nucleotide polymorphisms (SNPs). In this study, the goal was to express five of the most common haplotypes of OCX-32 in Escherichia coli and purify the recombinant protein for assay of its antimicrobial activity. Five constructs that contain the cDNA of common OCX-32 haplotypes (A, B, C, D, and O) with a histidine tag at the C-terminus were generated. The constructs were subcloned into pGEX4T-1 vector which encodes Glutathione-S-transferase (GST) upstream of the multiple cloning site. My study developed methods to optimize the expression conditions, and to increase the solubility of the recombinant protein. Various expression strains of E. coli and solubility buffers were tested. In addition, the construct was subcloned into a plasmid containing the small ubiquitin-like modifier (SUMO) fusion tag; the solubility of the new SUMO-OCX-32 haplotype A recombinant fusion protein was evaluated. The best results were obtained by slow dialysis refolding of denatured SUMO-OCX-32 fusion protein. This recombinant protein showed almost complete solubility with minimal precipitation and was tested against the egg-related pathogen, Bacillus cereus. Unfortunately, the SUMO-OCX-32 recombinant protein did not inhibit growth of B. cereus. In my studies reported in this thesis, two very different approaches were taken. A histone mixture was isolated from an abundant starting material, which proved to be highly effective and promising in the eradication of S. aureus biofilms at relatively low concentrations. Alternatively, expression of a soluble recombinant protein for functional activity assay was very challenging and required the optimization of a number of methods to prepare soluble protein for testing. One of the methods tested proved effective in obtaining large amounts of soluble protein. However, further developmental work will be essential to determine if this approach is a viable strategy in acquiring functional protein.

Au cours des dernières années, l’automatisation en bactériologie clinique est devenue très importante du fait de l’augmentation constante du nombre d’échantillons mais nécessite tout de même des améliorations. Une revue a été rédigée pour présenter les nouveaux challenges et opportunités dans la surveillance et la détection des bactéries multi-résistantes. Premièrement nous avons testé de nouveaux outils technologiques innovants permettant la lecture plus précoce des antibiogrammes grâce à des scanners de haute résolution d’images: scanner Advencis Bio-System Incubator et le Scan® 1200 et de réaliser l’ensemencement des antibiogrammes en automatique sur un automate déjà existant le PREVI® Isola (bioMérieux, Marcy l’Etoile, France). Dans un second temps, une analyse rétrospective de la résistance aux antibiotiques observés sur l’hôpital la Timone à Marseille a été réalisé entre 2014 et 2016. Nous avons développer un logiciel d’interprétation automatique des phénotypes basé sur la reconnaissance d’images pour lequel un brevet a été rédigé : le logiciel « ANTI-LOGIC ». Le but de notre troisième travail a été de participer au développement d’une PCR en temps réel permettant de détecter le gène mcr-1. Puis, nous avons travaillé sur des souches multi-résistantes afin de tester un panel constitué de 29 à 32 antibiotiques. Pour la détection des souches résistantes à la colistine, un milieu de culture sélectif a été mis au point et nous avons essayé de trouver une alternative thérapeutique par l’étude de combinaisons d’anciens antibiotiques (colistine-sulfadiazine) pour le traitement et la décolonisation avant greffe fécale de patients infectés ou colonisés par des BMR. / In the last few years, automation in clinical microbiology has become very important due to the constant increase of samples but they need to be improved. A review of the literature was performed to expose the new challenges and the new opportunities in the surveillance and the detection of multi-drug resistance bacteria.For that purpose, we conduct a thorough search to test new innovative technological tools for reading earlier AST with high-resolution image scanner: Advencis Bio-System Incubator and the Scan® 1200 and to carry out the automatic seeding of AST with the PREVI® Isola system (bioMérieux, Marcy l’Etoile, France). In a second step, the retrospective analysis of antibiotic resistance and phenotypes observed at La Timone hospital in Marseille was carried out between 2014 and 2016. We have developped a new software for the automatic interpretation of phenotypes based on image recognition protected by a patent: the "ANTI-LOGIC" software. The aim of our third work was to participate on the development of a real-time PCR to detect the mcr-1 gene. We then worked on multi-resistant strains with Gram-negative bacteria, we have tested a large panel of 29-32 antibiotics to see if we are in a therapeutic stalemate. For the detection of colistin resistant strains, a selective culture medium has been developed. In order to finish this part, with the appearance of colistin-resistant strains, we tried to find a therapeutic alternative by studying combinations of old antibiotics (colistin-sulfadiazine) for the treatment and decolonization before faecal transplantation of patients infected or colonized by multi-resistant bacteria