A colibacilose é uma enfermidade causada pela bactéria Escherichia coli. Algumas cepas podem ser potencialmente patogênicas, favorecendo doenças localizadas e/ou sistêmicas, acarretando consideráveis prejuízos econômicos para avicultura brasileira. Com o objetivo de determinar a resistência frente a antimicrobianos por (E. coli) foram analisadas 115 amostras isoladas de aves de produção, provenientes de três diferentes origens: cama de aviários (34 amostras), lesões de celulite (48 amostras) e lesões de aves com sintomatologia respiratória (33 amostras). Os padrões dos antibiogramas realizados seguiram as normativas disponíveis no National Commitee of Laboratory Standards (NCCLS, 2003). Entre os isolados de aves com sintomatologia respiratória, não foi observado resistência aos antimicrobianos: amoxicilina/ácido clavulâmico; cefuroxima, ceftiofur e amicacina, ou seja, três β- lactâmicos e ao aminoglicosídeo testado. Na análise estatística constatou-se diferença significativa entre as amostras de E.coli. de cama, celulite e aves com sintomatologia respiratórias, para as drogas norfloxacina e ciprofloxacina. As análises não apresentaram diferenças significativas para as demais drogas testadas. Os testes não apresentaram diferenças significativas entre as resistências e cada origem, nos seguintes casos: ampicilina, enrofloxacina, ciprofloxacina, ofloxacina, sulfametoxazole/trimetoprim e tetraciclina. Contudo, houve significância para os antimicrobianos: norfloxacina e ciprofloxacina. Os percentuais de resistência para os antimicrobianos avaliados foram: ampicilina (35,7%); amoxicilina/ácido clavulânico (0,9%); cefalexina (8,7%); cefuroxime (2,6%); ceftiofur (4,3%); clindamicina (98,3%); enrofloxacina (18,3%); ciprofloxacina (22,6%); norfloxacina (20%); ofloxacina (18,3%); sulfametoxazol/trimetoprim (47,8%); amicacina (3,5%); gentamicina (25,2%); tetraciclina (59,1%). A múltipla resistência para dois, ou mais agentes antimicrobianos, em cama, celulite e para amostras de sintomatologia respiratória, foi de (35,2%), (31,2%) e (66,6%), respectivamente. Não houve diferença significativa nas proporções de resistência entre as origens e as estações do ano. / Colibacilosis is a disease caused by the bacterium Escherichia coli. Some strains can be potentially pathogenic, favoring illnesses located and/or systemic, causing considerable economic damages for Brazilian poultry production. With the objective to determine the resistance front the antimicrobials for (E. coli) had been analyzed 115 isolated samples of poultry production, proceeding from three different origins: litter of poultry houses (34 samples), injuries of celulitis (48 samples) and injuries of poultry with respiratory signals (33 samples). The standards of the carried through antibiograms had followed normative available in the National Commitee of Laboratory Standards (NCCLS, 2003). It enters the isolated ones of birds with respiratory signals, was not observed resistance to antimicrobials: amoxicillin/ clavuanic acid; cefuroxime, ceftiofur and amikacyn, that is, three β- lactams and to the tested aminoglycosides. In the analysis statistics significant difference was evidenced enters the respiratory samples of E.coli of litter, celulitis and poultry with signals, for the drugs norfloxacin and ciprofloxacin. The analyses had not represented significant differences for the too much tested drugs. The tests had not presented significant differences between the resistances and each origin, in the following cases: ampicilin, enrofloxacin, ciprofloxacin, ofloxacin, sulfamethoxazole/trimethoprim and tetracyclin. However, it had significance for antimicrobials: norfloxacin and ciprofloxacin. The percentages of resistance for evaluated antimicrobials had been: ampicilin (35.7%); amoxicillin/ clavulanic acid (0.9%); cefalexin (8.7%); cefuroxime (2.6%); ceftiofur (4.3%); clindamycin (98.3%); enrofloxacin (18.3%); ciprofloxacin (22.6%); norfloxacin (20%); ofloxacin (18.3%); sulfamethoxazole/trimethoprim (47.8%); amikacyna (3.5%); gentamycin (25.2%); tetracyclin (59.1%). The multiple resistance for two or more agents antimicrobials, in litter, celulitis and for samples of respiratory signals, was of (35,2%), (31,2%) e (66,6%), respectively. It did not have significant difference in the ratios of resistance between the origins and the stations of the year.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacterial cause of diarrhea. ETEC bacterial adherence to the small intestinal epithelial cells and delivery of enterotoxins cause diarrhea in children living in developing countries and international travelers. Currently, there are no vaccines licensed for ETEC associated children’s diarrhea and travelers’ diarrhea. Recently, toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] (toxoid fusion), adhesin MEFA (multiepitope fusion antigen) CFA/I/II/IV (CFA MEFA), and toxoid-adhesin MEFA CFA/I/II/IV-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] (CFA-toxoid MEFA) are demonstrated to induce neutralizing antitoxin and/or anti-adhesin antibodies in intraperitoneal (IP) or subcutaneous (SC) immunized mice, suggesting these antigens are potential candidates for ETEC subunit vaccines. However, these antigens have not been examined for immunogenicity using intradermal (ID) or intramuscular (IM) routes, the routes perhaps are more suitable for human vaccine administration. In this study, toxoid fusion 3xST[subscript aN12S]-mnLT[subscript R192G/L211A], CFA/I/II/IV MEFA, alone or combined, or toxoid-adhesin MEFA CFA-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] were ID or IM immunized to mice (8 mice per group) induced antigen-specific antibodies were titrated, and antibody neutralization activities were assessed in vitro. Data showed that mice ID or IM immunized with the toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] antigen developed anti-LT and anti-STa antibodies and mice immunized with the CFA/I/II/IV MEFA developed antibody responses to all seven adhesins (CFA/I, CS1-CS6). In addition, mice co-administered ID or IM with toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] and CFA/I/II/IV MEFA, or with toxoid-adhesin MEFA CFA-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] developed antibodies to both toxins and all seven adhesins. Antibody neutralization studies of the serum samples of the immunized mice showed that the induced antibodies neutralized enterotoxicity of LT and STa and/or inhibited adherence of ETEC or E. coli bacteria producing any of these seven adhesins. These data confirmed immunogenicity of these ETEC subunit vaccine target antigens and provide useful information for vaccine development against ETEC diarrhea.

A colibacilose é uma enfermidade causada pela bactéria Escherichia coli. Algumas cepas podem ser potencialmente patogênicas, favorecendo doenças localizadas e/ou sistêmicas, acarretando consideráveis prejuízos econômicos para avicultura brasileira. Com o objetivo de determinar a resistência frente a antimicrobianos por (E. coli) foram analisadas 115 amostras isoladas de aves de produção, provenientes de três diferentes origens: cama de aviários (34 amostras), lesões de celulite (48 amostras) e lesões de aves com sintomatologia respiratória (33 amostras). Os padrões dos antibiogramas realizados seguiram as normativas disponíveis no National Commitee of Laboratory Standards (NCCLS, 2003). Entre os isolados de aves com sintomatologia respiratória, não foi observado resistência aos antimicrobianos: amoxicilina/ácido clavulâmico; cefuroxima, ceftiofur e amicacina, ou seja, três β- lactâmicos e ao aminoglicosídeo testado. Na análise estatística constatou-se diferença significativa entre as amostras de E.coli. de cama, celulite e aves com sintomatologia respiratórias, para as drogas norfloxacina e ciprofloxacina. As análises não apresentaram diferenças significativas para as demais drogas testadas. Os testes não apresentaram diferenças significativas entre as resistências e cada origem, nos seguintes casos: ampicilina, enrofloxacina, ciprofloxacina, ofloxacina, sulfametoxazole/trimetoprim e tetraciclina. Contudo, houve significância para os antimicrobianos: norfloxacina e ciprofloxacina. Os percentuais de resistência para os antimicrobianos avaliados foram: ampicilina (35,7%); amoxicilina/ácido clavulânico (0,9%); cefalexina (8,7%); cefuroxime (2,6%); ceftiofur (4,3%); clindamicina (98,3%); enrofloxacina (18,3%); ciprofloxacina (22,6%); norfloxacina (20%); ofloxacina (18,3%); sulfametoxazol/trimetoprim (47,8%); amicacina (3,5%); gentamicina (25,2%); tetraciclina (59,1%). A múltipla resistência para dois, ou mais agentes antimicrobianos, em cama, celulite e para amostras de sintomatologia respiratória, foi de (35,2%), (31,2%) e (66,6%), respectivamente. Não houve diferença significativa nas proporções de resistência entre as origens e as estações do ano. / Colibacilosis is a disease caused by the bacterium Escherichia coli. Some strains can be potentially pathogenic, favoring illnesses located and/or systemic, causing considerable economic damages for Brazilian poultry production. With the objective to determine the resistance front the antimicrobials for (E. coli) had been analyzed 115 isolated samples of poultry production, proceeding from three different origins: litter of poultry houses (34 samples), injuries of celulitis (48 samples) and injuries of poultry with respiratory signals (33 samples). The standards of the carried through antibiograms had followed normative available in the National Commitee of Laboratory Standards (NCCLS, 2003). It enters the isolated ones of birds with respiratory signals, was not observed resistance to antimicrobials: amoxicillin/ clavuanic acid; cefuroxime, ceftiofur and amikacyn, that is, three β- lactams and to the tested aminoglycosides. In the analysis statistics significant difference was evidenced enters the respiratory samples of E.coli of litter, celulitis and poultry with signals, for the drugs norfloxacin and ciprofloxacin. The analyses had not represented significant differences for the too much tested drugs. The tests had not presented significant differences between the resistances and each origin, in the following cases: ampicilin, enrofloxacin, ciprofloxacin, ofloxacin, sulfamethoxazole/trimethoprim and tetracyclin. However, it had significance for antimicrobials: norfloxacin and ciprofloxacin. The percentages of resistance for evaluated antimicrobials had been: ampicilin (35.7%); amoxicillin/ clavulanic acid (0.9%); cefalexin (8.7%); cefuroxime (2.6%); ceftiofur (4.3%); clindamycin (98.3%); enrofloxacin (18.3%); ciprofloxacin (22.6%); norfloxacin (20%); ofloxacin (18.3%); sulfamethoxazole/trimethoprim (47.8%); amikacyna (3.5%); gentamycin (25.2%); tetracyclin (59.1%). The multiple resistance for two or more agents antimicrobials, in litter, celulitis and for samples of respiratory signals, was of (35,2%), (31,2%) e (66,6%), respectively. It did not have significant difference in the ratios of resistance between the origins and the stations of the year.

A colibacilose é uma enfermidade causada pela bactéria Escherichia coli. Algumas cepas podem ser potencialmente patogênicas, favorecendo doenças localizadas e/ou sistêmicas, acarretando consideráveis prejuízos econômicos para avicultura brasileira. Com o objetivo de determinar a resistência frente a antimicrobianos por (E. coli) foram analisadas 115 amostras isoladas de aves de produção, provenientes de três diferentes origens: cama de aviários (34 amostras), lesões de celulite (48 amostras) e lesões de aves com sintomatologia respiratória (33 amostras). Os padrões dos antibiogramas realizados seguiram as normativas disponíveis no National Commitee of Laboratory Standards (NCCLS, 2003). Entre os isolados de aves com sintomatologia respiratória, não foi observado resistência aos antimicrobianos: amoxicilina/ácido clavulâmico; cefuroxima, ceftiofur e amicacina, ou seja, três β- lactâmicos e ao aminoglicosídeo testado. Na análise estatística constatou-se diferença significativa entre as amostras de E.coli. de cama, celulite e aves com sintomatologia respiratórias, para as drogas norfloxacina e ciprofloxacina. As análises não apresentaram diferenças significativas para as demais drogas testadas. Os testes não apresentaram diferenças significativas entre as resistências e cada origem, nos seguintes casos: ampicilina, enrofloxacina, ciprofloxacina, ofloxacina, sulfametoxazole/trimetoprim e tetraciclina. Contudo, houve significância para os antimicrobianos: norfloxacina e ciprofloxacina. Os percentuais de resistência para os antimicrobianos avaliados foram: ampicilina (35,7%); amoxicilina/ácido clavulânico (0,9%); cefalexina (8,7%); cefuroxime (2,6%); ceftiofur (4,3%); clindamicina (98,3%); enrofloxacina (18,3%); ciprofloxacina (22,6%); norfloxacina (20%); ofloxacina (18,3%); sulfametoxazol/trimetoprim (47,8%); amicacina (3,5%); gentamicina (25,2%); tetraciclina (59,1%). A múltipla resistência para dois, ou mais agentes antimicrobianos, em cama, celulite e para amostras de sintomatologia respiratória, foi de (35,2%), (31,2%) e (66,6%), respectivamente. Não houve diferença significativa nas proporções de resistência entre as origens e as estações do ano. / Colibacilosis is a disease caused by the bacterium Escherichia coli. Some strains can be potentially pathogenic, favoring illnesses located and/or systemic, causing considerable economic damages for Brazilian poultry production. With the objective to determine the resistance front the antimicrobials for (E. coli) had been analyzed 115 isolated samples of poultry production, proceeding from three different origins: litter of poultry houses (34 samples), injuries of celulitis (48 samples) and injuries of poultry with respiratory signals (33 samples). The standards of the carried through antibiograms had followed normative available in the National Commitee of Laboratory Standards (NCCLS, 2003). It enters the isolated ones of birds with respiratory signals, was not observed resistance to antimicrobials: amoxicillin/ clavuanic acid; cefuroxime, ceftiofur and amikacyn, that is, three β- lactams and to the tested aminoglycosides. In the analysis statistics significant difference was evidenced enters the respiratory samples of E.coli of litter, celulitis and poultry with signals, for the drugs norfloxacin and ciprofloxacin. The analyses had not represented significant differences for the too much tested drugs. The tests had not presented significant differences between the resistances and each origin, in the following cases: ampicilin, enrofloxacin, ciprofloxacin, ofloxacin, sulfamethoxazole/trimethoprim and tetracyclin. However, it had significance for antimicrobials: norfloxacin and ciprofloxacin. The percentages of resistance for evaluated antimicrobials had been: ampicilin (35.7%); amoxicillin/ clavulanic acid (0.9%); cefalexin (8.7%); cefuroxime (2.6%); ceftiofur (4.3%); clindamycin (98.3%); enrofloxacin (18.3%); ciprofloxacin (22.6%); norfloxacin (20%); ofloxacin (18.3%); sulfamethoxazole/trimethoprim (47.8%); amikacyna (3.5%); gentamycin (25.2%); tetracyclin (59.1%). The multiple resistance for two or more agents antimicrobials, in litter, celulitis and for samples of respiratory signals, was of (35,2%), (31,2%) e (66,6%), respectively. It did not have significant difference in the ratios of resistance between the origins and the stations of the year.

O DNA de plasmídeos naturais de uma cepa de B. stearothermophilus foi clivado com a enzima de restrição Hind III e os fragmentos resultantes foram ligados com T 4 DNA ligase ao vetor pBR 322 (Bolívar et. al., 1977) que já havia sido previamente tratado com Hind III e fosfatase alcalina. A terça parte desta mistura de ligação foi usada para transformar células de E. coli HB 101. Foram obtidos cerca de 3.500 transformantes, dos quais 46% eram recombinantes. Duas cepas que mostraram caracter amilolítico consideravelmente maior que a doadora do gene continham o plasmídeo pBR 322 com uma inserção de 5.4 Kb. O mapa de restrição, tratamento com a enzima BAL 31 e, sucessivas subclonagens (Silva, E.F. et. al.,1986)mostraram que o gene que codifica e expressa a enzima amilolítica está contido em um fragmento de 2 Kb. A enzima produzida pelas células transformadas tem peso molecular de 60.000, é estabilizada por Ca+2, tem um ótimo de temperatura de 72ºC e retém 90% da atividade original após aquecimento a 85°C por 1 hora. Estes resultados, em conjunto com a análise dos produtos de hidrólise desta enzima em cromatografia de papel, sugerem que foi clonada a alfa - amilase de B. stearothermophilus em células de E. coli. Células de duas cêpas de B. subtilis, IA 289 e BD 241 foram transformadas respectivamente com os plasmídeos p USP 33.2 (Silva, E.F. et al., 1986;1987) e p BU 217 ami 2 (Silva, E.F. & Pueyo, M.T.,1988) para produzir em ambos os casos colônias fortemente amiloliticas. Os mecanismos pelos quais as 2 cêpas passaram a apresentar o fenótipo AMY + , são provavelmente diferentes. / The DNA of natural plasmids. from a B. stearothermophilus strain was cut with Hind III endonuclease and the resulting fragments were joined with T4 DNA ligase to the vector pBR 322 (Bolivar et al. , 1977), which had previously been treated with Hind III and alkaline phosphatase. One-third of the ligation mixture was used to transform E. coli HB 101 cells. It was obtained about 3.500 transformants, which included 46% recombinans. Two strains displayng amylolytic act ivity remarkably higher than the donor gene strain, harbored the plasmid pBR 322 with an insertion of 5.4 kb. The restriction map, Bal 31 treatment and successive subcloning (Silva, E.F, et al.,1986) showed that the entire gene which codifies and allows the expression of the amylolytic enzyme is contained in a 2 Kb fragment. The enzyme has a molecular weight of 60.000, is stabilized by Cata, has a temperature optimum at 72°C and retains 90% of the original activity after heating for 1h at 85°C. These features, together with the analysis of hydrolysis produts carried on paper chromatography , suggests that we succeded in cloning the amylase from B. stearothermophilus in E. coli cells. Cells from two B. subtilis strains, IQ 289 and BD 241 were transformed with the plasmid sp USP 33.2 (Silva E. F. et al., 1986 1987) and pBU 271 ami 2 (Silva, E. F. & Pueyo, M.T., 1988) , and produce in both strains, amyiolytic colonies. The methods in which the two strains have got the AMY + fenotype, may be very different.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T14:29:17Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese_Gazzaneo (Biblioteca Central).pdf: 2832068 bytes, checksum: bb29fc38317d9f60a0d6129655b9b7a1 (MD5) / Made available in DSpace on 2017-07-12T14:29:17Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese_Gazzaneo (Biblioteca Central).pdf: 2832068 bytes, checksum: bb29fc38317d9f60a0d6129655b9b7a1 (MD5) Previous issue date: 2016-03-14 / Defensinas são peptídeos antimicrobianos (AMPs) que apresentam atividade contra diversos microrganismos patogênicos, em especial fungos. Embora não totalmente elucidados, há diversos mecanismos de ação propostos para as defensinas, que incluem permeabilização seletiva ou ruptura da membrana plasmática de microorganismos, ação direta em alvos intracelulares, ativação de cascatas de sinalização e aumento da produção de espécies reativas de oxigênio. Desde a sua descoberta e, tendo em vista sua ampla atividade biológica, o uso de defensinas no melhoramento de plantas cultivadas, bem como na produção de novos medicamentos tem sido proposto. Estudos de atividade biológica e possível aplicação biotecnológica das defensinas demandam uma grande quantidade dessas proteínas. Entretanto, o processo de extração da mesma é laborioso, dispendioso e, de acordo com a população ou disponibilidades da espécie vegetal escolhida, não sustentável ecologicamente. Portanto, a utilização de sistemas heterólogos de expressão é uma importante ferramenta para obtenção de defensinas recombinantes em escala industrial. Nesse estudo, um gene de defensina “DeHys”, isolado da Euphorbia hyssopifolia, foi inserido no plasmídeo pET102/D-TOPO e células da linhagem BL21(DE3) de Escherichia coli foram transformada com essa construção. Foi produzida a defensina recombinante Dehys com tamanho aproximado de 24 kDa. Sua identidade foi confirmada por western blot e pela análise do padrão de digestão com proteases. / Defensins are antimicrobial peptides (AMPs) , which present activity against a variety of pathogenic microorganism, especially funghi. Although not completely elucidated, there are a variety of proposed mechanisms of action for defensins, which includes selective microorganisms plasmatic membrane permeabilization or rupture, straight action agains intracellular targets, activation of signaling cascades and the burst of reactive oxygen species. Since it’s discovery and due to it’s wide biological activities, it’s use in crop enhancing, as well as its use in the development of new drugs have been proposed. Defensin’s biological activity and biotecnological application studies demand a reasonable amount of purified protein. However, the extraction processes is laborious, expensive, time consuming and depending on the population or the chosen plant species supply, not ecologically sustainable. So, the use of heterologous expression sytems is an importante tool to obtain purified proteins in industrial scale. In this study, a defensing gene (Dehys) isolated from Euphorbia hyssopifolia was inserted in a p pET102/D-TOPO vector and trasnformed into BL21(DE3) Escherichia coli strains. A recombinat Dehys defensin of approximately 24 kDa was obtained. It’s identity was double-checked using by Western blot and protease digestion pattern analyses.

In the year 2000, 170 countries decided to stand together and solve some primary and common global problems like poor health, water, and sanitation. Obtaining access to safe drinking water is every individual’s right. The UN defines safe drinking water as “the water required for each personal or domestic use must be safe, therefore free from micro-organisms, chemical substances and radiological hazards that constitute a threat to a person's health” (UNDESA, 2014). This thesis focuses on identifying the number of people who potentially receive spring water from a piped source in rural mountainous areas. There are three significant steps and data requirements which are necessary to meet this study’s objective. These are to: 1) obtain data classifying the various sources of drinking water in each country by specifying the percentage of population served by a particular water in rural and urban areas, 2) determine the number of countries which are undeveloped or developing, and 3) identify specific countries which are defined as mountainous (and thus likely to have spring fed piped water systems) based on elevation and slope in order to estimate the number of people living in mountainous areas. Results show that 183.54 (million) people were estimated to receive piped water from the springs in mountainous areas. Approximately, 34% of the population is from the Eastern Asia and South-Eastern Asia regions, and 33% reside in the Latin America and Caribbean Islands. These were followed by Western Asia and North Africa Regions with 15% and Central Asia and Southern Asia Regions with 14% of their population estimated to receive piped spring water.

The general idea of this study was to develop candidate specific IgY antibodies for an oral therapy targeting the ESBL-producing gram negative bacteria. As the family of ESBLs constantly grows and there is lack of their clear classification in the literature, the specific aim was to build a proof of concept study based on the parental enzyme ß-lactamase TEM-1 to investigate different specific IgYs strategies to inhibit the growth of TEM-1 producing E.coli. This research included a bioinformatic analysis of the TEM-1 structure in the context of TEM-derivative ESBLs. Then, two IgY strategies were designed to target the ß-lactamase TEM-1-producing E.coli (BW25113 ΔbamBΔtolC) with IgYs: as a complement to antibiotics (IgYs against the enzyme TEM-1 used in combination with ampicillin) and as an alternative to antibiotics (IgYs against the bacteria TEM-1-producing E.coli without the addition of ampicillin). A good inhibitory effect of (a)TIgY, (a)p2IgY (in the presence of ampicillin) and eIgY, hIgY (without the ampicillin) on TEM-1-producing E.coli was observed in vitro. Moreover, they had the typical configuration of avian antibodies and were highly specific to their antigens. This study presents a model system to develop specific IgYs against a therapeutic target of interest. The activity of these IgYs complementary or alternatively to antibiotics should be further investigated in vivo in an animal infectious model. IgYs developed in this study might also be good candidates for further investigation as a broad-spectrum treatment against a variety of ESBL-producing E.coli. The aTIgY which was developed against the whole TEM-1 might also target its derivatives, as they have similar 3D structure with single amino acids mutations in the sequence. The ap2IgY was generated against catalytic and conservative residues, characteristic for the whole class A of ß-lactamases, thus it might target also the active site of ESBL-s from this class. The strategy used to generate eIgY and hIgY was efficient and IgYs could be generated directly against ESBL-producing bacteria.

No description available.

No description available.