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Please use this identifier to cite or link to this item: http://hdl.handle.net/10400.5/2041

Title: Tipagem molecular de Listeria monocytogenes
Authors: Coutinho, Ana Rute Antunes
Advisor: Brito, Maria Luísa de Castro e
Keywords: Listeria monocytogenes
multiplex-PCR
PFGE
cheese
queijo
prevalência do serogrupo
Issue Date: 2009
Abstract: In this work, a set of 59 isolates and six reference strains of Listeria monocytogenes collected, in the period of March to October of 1992, from nine cheese dairies of the Ribatejo and West region and Algarve were characterized. The isolates were from pasteurized cow's milk, ewe’s milk, mixed (ewe, cow and goat) milk, fresh cheeses and the environments of the cheese dairies where the milk was processed. In three out of the nine cheese dairies, L. monocytogenes was re-isolated after an intensive cleaning and disinfection program. In order to determine the prevalence of the serogroups and the phylogenetic relatedness among the isolates, the techniques of serotyping by multiplex-PCR (MPCR) and the analysis of profiles by pulsed-field gel electrophoresis (PFGE) were used. Multiplex-PCR and PFGE are currently used by several public and private laboratories for the characterization of L. monocytogenes. Multiplex-PCR constitutes a practical alternative to slide agglutination serotyping and separates L. monocytogenes isolates belonging to the twelve serovars into five distinct serogroups. PFGE is considered the gold standard method for subtyping foodborne pathogens, because of its high discriminatory power and reproducibility. The percentage of isolates belonging to serogroup (4b-4d-4e), usually associated with human cases of listeriosis, was 86% (51/59), confirming recent findings by other authors and by our team, with isolates from cheese and related environments. From the 65 isolates analysed, 48 pulsotypes (combined ApaI and AscI profiles) were identified. The relationships among L. monocytogenes strains based on their combined ApaI and AscI PFGE profiles are supported by a cophenetic correlation coefficient (ρ) equal to 0.97. This value explained the goodness of fit of the clustering to the original data and values higher than 0.80 are considered reasonable. A higher SID value was achieved with ApaI (0.80) compared to AscI (0.64). The combination of patterns produced by both enzymes increased the discriminatory power up to 0.99. In accordance with the visual analysis of the dendrogram, 45 dairy isolates with indistinguishable ApaI and AscI profiles were identified, suggesting that these might be isolates of a persistent strain. The comparison of these isolates with 24 isolates of dairy products in the database of CBISA (Colecção de Bactérias do Instituto Superior de Agronomia) showed, at a similarity of 0.71, a subgroup that gathered 10 from these isolates with the 45 mentioned above. The low genetic variability of L. monocytogenes isolates from milk,cheeses and related environments, suggests that some strains of L. monocytogenes can be better adapted to this specific ecological niche. Relatively to the 24 isolates of dairy products in the database of CBISA, the higher prevalence of serogroup (4b-4d-4e) (58%) was also verified. The contamination of cheese with L. monocytogenes can be explained by a decrease in the effectiveness of sanitization procedures in use, or by poor hygienic practices in milking and in cheesemaking. The application of molecular typing techniques to determine the origin of the contamination of the cheeses can lead to a better understanding of the routes of propagation of L. monocytogenes, allowing the planning of more effective prevention measures. Computer-assisted data analysis of PFGE data simplifies the processing of a large number of samples while allowing data sharing if a stringent compliance to standard protocols is achieved. This standardization includes not only a rigorous accomplishment of the Pulse-Net standardized protocol but also standardization of procedures in computer assisted data analysis for pattern normalization and transfers of information intra and inter laboratories. PulseNet Europe recommends settings of both optimization and position tolerance at 1.5% for band comparison. Although complying with these settings, strains visually indistinguishable may still be considered different according to the clustering analysis. Nevertheless, to improve correct interpretation of subtyping data, the availability of a large and diverse PFGE type database is needed. The PFGE analysis of the data presented here suggests the existence of a persistent strain in one or more cheese dairies. The high prevalence of serogroup (4b-4d-4e) among isolates from milk, cheese and dairy environment reported here is particularly concerning, since this group includes isolates usually associated with both sporadic and epidemic cases of human listeriosis. These results stress the risk of listeriosis associated with the consumption of cheese contaminated with L. monocytogenes. The implementation and maintenance of good manufacturing practices (GMPs) together with sanitation standard operating procedures (SSOPs) all along the food chain will contribute to the effective erradication of persistent strains of this pathogenic bacterium.----------------------------------------Neste trabalho, as técnicas de serotipagem por multiplex-PCR (MPCR) e a análise de perfis de DNA por electroforese em campo pulsado (PFGE) foram aplicadas a um conjunto de 65 isolados de Listeria monocytogenes, 59 dos quais com origem em leites pasteurizados de vaca, de ovelha e de mistura, queijos frescos e ambientes de nove fábricas onde estes leites foram processados, de modo a determinar proximidades filogenéticas entre os isolados e a prevalência dos serogrupos. A percentagem de isolados pertencentes ao serogrupo (4b-4d-4e), habitualmente associado a casos de listeriose humana, foi de 86% (51/59), confirmando resultados recentes de outros autores em isolados de queijos e ambientes relacionados. A partir dos 65 isolados produziram-se 48 pulsotipos (perfis ApaI e AscI combinados). No entanto, a análise visual identificou 45 isolados com perfis ApaI e AscI indistinguíveis, sugerindo tratar-se de uma estirpe persistente. A comparação destes isolados com 24 isolados de produtos lácteos existentes na base de dados da CBISA mostrou, a 0,71 de similaridade, um subgrupo que reunia 10 destes isolados com os 45 isolados referidos anteriormente, sugerindo uma adaptação a este tipo de nicho ecológico. Os resultados apresentados enfatizam o risco associado ao consumo de queijo contaminado com L. monocytogenes.
Description: Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
URI: http://hdl.handle.net/10400.5/2041
Appears in Collections:BISA - Dissertações de Mestrado / Master Thesis

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