De acordo com a Food and Agriculture Organization of the United Nations (FAO, 2013), o consumo mundial de carne de frango tem aumentado de maneira significativa nas últimas décadas. Por outro lado, a preocupação dos produtores de alimentos, com a inocuidade de seus produtos também tem aumentado na mesma proporção. Durante as etapas da operação de abate de maneira geral, as contaminações cruzadas são as principais causas de disseminação de microrganismos patogênicos nos produtos obtidos e causadores de gastroenterites no consumidor. Espécies da enterobactéria Salmonella se enquadram como um dos maiores riscos desse tipo de doença devido a sua associação com os inúmeros surtos ocorridos a nível mundial, após o consumo desses produtos. Algumas enterobactérias possuem um gene blaKPC, que codifica a enzima carbapenemase, que confere resistência a antibióticos carbapenêmicos, agravando mais a situação. Alguns fatores de virulência encontrados nesse gênero de bactéria podem ainda estar associados a capacidade de adesão e formação de biofilmes em superfícies inertes, dificultando operações de higienização nas linhas de processamento. Assim sendo, a presente pesquisa objetivou a verificação da capacidade de estirpes de Salmonella spp isoladas de aviário e linha de abate de frangos de um frigorífico no estado do Rio Grande do Sul produzirem biofilmes e apresentarem resistência a antibióticos carbapenêmicos. Na avaliação da produção de biofilme, foi empregada a técnica de microplacas de polietileno e produção de cápsula segundo Stepanovic et al. (2004) e Rodrigues et al. (2006), respectivamente.Foram pesquisados os fatores de virulência de salmonelas, representados pelos genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH,e spvC, utilizando o método de Reação de cadeia de Polimerase (PCR), descrita por Borges et al. (2013). As estirpes foram submetidas ao teste de resistência a antibióticos carbapenêmicos pelo método de disco difusão com carbapenêmicos segundo Clinical and Laboratory Standards Institute-CLSI (2010) e pesquisa do gene de resistência a carbapenêmicos blaKCP, pela técnica de PCR, segundo NAAS et al. (2007). Obteve-se quatro perfis genéticos das estirpes de Salmonella spp.: perfil 1: genes ifpA, agfA, invA e avrA; perfil2: genes ifpA, agfA, sefA, invA e avrA; perfil 3: genes invA e avrA; perfil4: genes ifpA,agfA e invA. Observou-se a resistência das estirpes somente ao antibiótico imipenen. Entre as 36 estirpes de Salmonella spp. isoladas, todas foram consideradas produtoras de biofilme in vitro, sendo que 69% destas, apresentaram-se como fortes produtoras, 25% como moderadas, e apenas 6% como fracas produtoras. O método Agar Vermelho Congo não se mostrou eficiente para teste presuntivo de produção de biofilme para estirpes de Salmonella spp. Não foi evidenciado o gene blaKPC nas estirpes de Salmonella spp. isoladas na presente pesquisa. / According to Food and Agriculture Organization of the United Nations (FAO, 2013), the world consumption of meat of chicken has been higher significantly in the last decades. On the other hand, the concern of the food producers with the safety of their products also has increased in the same proportion. During the steps of the slaughter operation in general, cross contamination are the main causes of pathogenic microorganisms dissemination in the obtained products and the causes of gastroenteritis in the consumer. Species of the Enterobacter Salmonella fall as the highest risks of this kind of disease, due to their association with countless outbreaks which occurred worldwide, after the consumption of these products. Some enterobacter have a blaKPC gene, which codes for the carbapenemase enzyme that confers resistance to carbapenems antibiotics, aggravating the situation. Some virulence factors found in this bacteria genes can also be associated to the ability of adherence and the formation of biofilms in inert surfaces, making it difficult the operations of sanitation in the processing lines. Thus, the present research aimed to verify the capacity of Salmonella spp. strains isolated from aviary and slaughter line of chicken in a fridge of Rio Grande do Sul state to produce biofilms and be resistant to carbapenems antibiotics. In the evaluation of biofilm production, it was used the polyethylene microplates and capsule production technique according to Stepanovic et al. (2004) and Rodrigues et al. (2006), respectively. The virulence factors of salmonella were researched, represented by the genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, e spvC, using the Polymerase Chain Reaction (PCR) method described by Borges et al. (2013). The lineages were submitted to the carbapenems antibiotic resistence test by the disk diffusion method with carbapenems according to the Clinical and Laboratory Standards Institute-CLSI (2010), and the research of the resistance to carbapenems gene blaKCP, by the strains Salmonella spp.: profile 1: genes ifpA, agfA, invA and avrA.; profile 2: genes ifpA, agfA, sefA, invA and avrA; profile 3: genes invA and avrA; profile 4: genes ifpA, agfA and invA. It was observed the resistance of the strains only to the imipenen antibiotic. Among the other 36 cultures of Salmonella spp. isolated, all were considered to produce biofilm in vitro, of which 69% were strong producers, 25% moderate, and only 6% were low producers. The method Congo Red Agar was not efficient to the presumptive test of biofilm production for the Salmonella spp. strains. It was not evidenced the gene blaKPC in Salmonella spp. strains isolates in this research.

Publicación a texto completo no autorizada por el autor / Identifica molecularmente los serotipos de cepas sospechosas de Salmonella spp., aisladas mediante protocolos microbiológicos estandarizados, a partir de 272 muestras pareadas de hisopados rectales y vaginales de reproductoras primerizas clínicamente sanas de un criadero del distrito de Pachacamac. El ADN de los aislados sospechosos, evaluados hasta pruebas bioquímicas, es extraído y analizado mediante la técnica de PCR múltiple, para detectar la presencia de los genes inv A, prot6E y fliC específicos para Salmonella spp., Salmonella Enteritidis y Salmonella Typhimurium, respectivamente; encontrándose en 12 cepas aisladas que amplifican al gen inv A; de éstas, 10 (83.3%) amplifican para el gen fliC y ningún aislado amplifica el gen prot6E. Estos resultados confirman que Salmonella Typhimurium es el patógeno predominante en cobayos reproductoras al primer parto en esta crianza comercial. / Tesis

O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of the external aldimine was shown to be pH dependent with a pK of 8.1 ± 0.3 most likely representing a general base that accepts a proton from the a-amine of cysteine to facilitate a nucleophilic attack on C4r of the PLP imine. Formation of the geminal diamine was also shown to be pH dependent with two pK values having an average value of 8.1. One of the groups most likely represents the general base which accepts a proton from the a-amine of cysteine while the second group likely interacts with the amino acid side chain to orientate the amino acid into the correct configuration.

O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.

La infección por Salmonella spp., es una de las causas más importantes de gastroenteritis en seres humanos a nivel mundial, constituyendo un problema de salud pública. El proceso de beneficio favorece la diseminación de microorganismos y pueden aumentar la frecuencia de Salmonella en el producto final, teniendo como posibles fuentes de contaminación el agua de lavado de las canales o carcasas, ruptura de vísceras durante el eviscerado o condiciones higiénicas deficientes del establecimiento. En el presente estudio se busco determinar la presencia de Salmonella para ello se analizaron 170 muestras de superficie corporal entre carcasas y canales y 170 muestras de hisopado cloacal. Estas muestras fueron adquiridas de 17 centros de beneficio clandestino de Lima Metropolitana. Para la superficie corporal se utilizó el método de enjuague descrito por el USDA/FSIS (2008). Se utilizó la metodología tradicional para el aislamiento de Salmonella. En los centros de beneficio donde no se realizaba eviscerado el porcentaje de muestras de superficie corporal de carcasas que resultaron positivas a Salmonella spp. fue de 21.3% y de 28.8% para hisopado cloacal; mientras que en los centros de beneficio donde se realizaba eviscerado el 25.6% de muestras de superficie corporal de canales estaban contaminadas y se obtuvo un 35.6% de muestras de hisopado cloacal positivas. El grado de concordancia de los resultados para ambos métodos de toma de muestra fue insignificante (k=0.074, k=0.146). En conclusión los resultados de este estudio muestran que Salmonella spp. está presente en los centros de beneficio clandestino de Lima Metropolitana. -- Palabras clave: Salmonella, canales, beneficio, clandestino, hisopado cloacal, superficie corporal / -- Salmonella spp., is one of the most important causes of gastroenteritis in humans worldwide, constituting a public health problem. The slaughtering process favors the spread of microorganisms and may increase the frequency of Salmonella in the final product. The possible sources of contamination are the wash water of the carcasses, the rupture of the viscera during evisceration and the poor hygiene of the establishment. In this study we sought to determine the presence of Salmonella. For this purpose 170 samples of the body surface of carcasses (among eviscerated and non eviscerated) and 170 cloacal swabs were analyzed. These samples were acquired from 17 slaughterhouses in Metropolitan Lima. For the body surface, we used the method of rinsing described by USDA / FSIS (2008). We used the traditional method for the isolation of Salmonella. In slaughterhouses where the evisceration was not carried out the percentage of samples of body surface carcasses tested positive for Salmonella spp., was 21.3%; and 28.8% for cloacal swabs. While in slaughterhouses where the evisceration was carried out, 25.6% of body surface samples were contaminated and 35.6% of the cloacal swabs were positive. The degree of concordance of the results for both sampling methods was insignificant (k = 0.074, k = 0.146). In conclusion the results of this study show that Salmonella spp. is present in the slaughterhouses of Metropolitan Lima. -- Keywords: Salmonella, chicken carcasses, cloacal swabs, body surface, slaughterhouse / Tesis

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A chemically defined medium for Salmonella growth was developed and optimised using supplements of amino acids, nucleosides, vitamins and different carbon sources. The medium developed was compared to commercially available pre-enrichment media BPW and Salmosyst. Growth of Salmonella was significantly higher in BPW and Salmosyst than the medium developed. The amino acid and nucleoside supplement was directly compared with peptone. The results show peptone to be nutritionally superior and promoting better growth of Salmonella. Three different ELISA assays were used to detect Salmonella growing in four different media. The ELISA assay sensitivity was determined and a degree of media interference with the immunoassays was established. Salmonella culture viability was investigated using three different procedures: differential culturing on selective and non-selective media; fluorescence microscopy with BACLIGHT stained cells and flow cytometry analysis of BACLIGHT and BEP stained cells. Flow cytometry was found to be the most consistent, sensitive and rapid procedure for cell viability measurement. Clusters of viable cells unable to grow on solid media and therefore remaining undetectable by cultural methods were identified using flow cytometry. Severely heat injured Salmonella was used to determine media recoverability. The results indicate that media which contain peptone recover injured Salmonella better than chemically defined or other media. Detection of Salmonella was performed using PCR assay after sample pre-enrichment. The amplification of Salmonella DNA extracted using a crude method resulted in an assay sensitivity of 20 Salmonella cells in pure cultures. The specificity of the oligonucleotide primers employed in the PCR assay was confirmed. Non-salmonella organisms present in high numbers interfered with PCR detection of Salmonella. Food components also interfered with PCR amplification and reduced the assay sensitivity. Interference by food components and non-salmonella DNA was eliminated by the use of a 24 hour pre-enrichment followed by a 3 hour secondary enrichment, a rapid DNA extraction and template preparation. Using this system it was possible to detect 3 Salmonella cells per gram of food in the presence of 106 non-salmonella cells within 28 hours.

No description available.

No description available.

Call number: LD2668 .T4 1950 L8 / Master of Science