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Optimization of Peptide Nucleic Acid Fluorescence in Situ Hybridization (PNA-FISH) for the Identification of Microorganisms in Food Matrices

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Optimization of Peptide Nucleic Acid Fluorescence in Situ Hybridization (PNA-FISH) for the Identification of Microorganisms in Food Matrices Optimization of Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) for the identification of microorganisms in food matrices. A Dissertation presented to the University of Porto for the degree of Doctor in Chemical and Biological Engineering by Rui Jorge Alves Rocha Supervisor: Doctor Nuno F. Azevedo Co-supervisor: Doctor Carina Almeida Co-supervisor: Professor Maria J. Vieira Porto, March 2018 This work was funded by the following projects and grants: POCI-01-0145-FEDER-006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy - UID/EQU/00511/2013) and POCI-01-0145- FEDER-006684 (Center of Biological Engineering - UID/EQU/00511/2013) funded by the European Regional Development Fund (ERDF), through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds, through FCT - Fundação para a Ciência e a Tecnologia. NORTE‐01‐0145‐FEDER‐000005 - LEPABE-2-ECO-INNOVATION and NORTE-01-0145-FEDER-000004 - BioTecNorte operation, supported by North Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). PIC/IC/82815/2007 - DNA mimics, ENMed/0003/2014 - NanoDiaBac and PTDC/DTP-PIC/4562/2014 - Coded-FISH, funded by national funds through FCT - Fundação para a Ciência e a Tecnologia. PhD Fellowship SFRH/BDE/51910/2012 supported by national funds through FCT - Fundação para a Ciência e a Tecnologia. Agradecimentos Em primeiro lugar quero salientar que tudo o que possa escrever nesta secção relativamente a todos as pessoas aqui mencionados pecará invariavelmente pela sua falta de justiça quanto à sua verdadeira importância para a conclusão de mais uma etapa pessoal, profissional e académica na minha vida. Em primeiro lugar, gostaria de agradecer ao Professor Nuno Azevedo pela oportunidade concedida para integrar o projecto da Biomode S. A. e a abertura dada para fazer o Doutoramento também ligado à Biomode. Agradecer ainda a sua disponibilidade, preocupação, conselhos, sugestões e contribuições científicas que muito valorizaram o trabalho que foi sendo desenvolvido aos longo destes últimos anos. Seguidamente quero agradecer à Dra. Carina Almeida igualmente pela sua preocupação, conselhos, sugestões e contribuições científicas, ao longo dos últimos anos. Gostaria, no entanto, ainda de salientar a sua disponibilidade para o esclarecimento de todas as dúvidas (que foram muitas), todo o conhecimento científico transmitido e a capacidade de reinterpretação da minha escrita. Quero ainda agradecer a possibilidade dada pela Professora Maria J. Vieira para poder desenvolver o meu plano de trabalhos no LIBRO. Agradecer ainda o apoio financeiro recebido, indispensável para a realização do Doutoramento por parte da Fundação para a Ciência e Tecnologia e da Biomode S. A. Não poderia deixar de salientar a simpatia, camaradagem, apoio e paciência demonstrada por todos os elementos de laboratório com os quais me cruzei, na Biomode, no BEL e no LIBRO. Esta lista não ficaria completa sem um agradecimento muito especial aos meus Amigos da Cooperativa e da Irmandade do Chamade, a boa disposição, alegria, os bons momentos, bem como todo o companheirismo e acima de tudo a verdadeira amizade, que desde sempre tivemos. v Page Não poderia nesta secção esquecer a importância dos meus Pais desde o meu nascimento, já que tudo aquilo que consegui atingir, foi graças ao seu esforço, dedicação e exigência. E também à minha irmã pelo seu apoio, preocupação e disponibilidade em todos os momentos. Por fim um agradecimento ainda mais especial à minha Pipoquinha pela sua alegria contagiante, pelo apoio, carinho e motivação dados incondicionalmente ao longo destes últimos anos. A todas as pessoas que contribuíram para aquilo que sei e que sou um sincero OBRIGADO! vi Page Abstract Foodborne illnesses remain until today as a major health problem. They result from the consumption of contaminated food with bacteria, viruses, fungi, parasites, toxins or chemicals, causing an array of more than 250 different known diseases. Overall, the World Health Organization estimates the occurrence of more than 600 million food- related illnesses in a single year and over 420,000 deaths. One in every 10 people fall ill as a result of consumption of contaminated food. The high impact of foodborne diseases has raised awareness in consumers, companies and national/international organizations for the production and distribution of safe food products. As such, over the years an improvement in the monitoring and cleaning methods has been observed for controlling agents responsible for causing illnesses. However, the monitoring of illness-causing agents in food products can be challenging. The detection of microbial pathogens can be achieved with several methodologies, including traditional culture plating techniques, immunological methods, methods based on nucleic acid amplification (PCR and variants) or hybridization (e.g. microarrays), biosensors, among others. Of those, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) has, over the last few years, gained popularity as a reliable tool for pathogen detection, due to their superior performance characteristics when compared to standard FISH (using traditional DNA or RNA as probes) and other commonly employed methods. Listeria monocytogenes is one of the most important foodborne pathogens. Despite the lower incidence levels in comparison to other agents, the illnesses caused by L. monocytogenes have one of the highest hospitalization and mortality rates among foodborne pathogens. In Chapter 2, a PNA-FISH method for the detection of L. monocytogenes is described and validated in a variety of food matrices, namely ground beef, ground pork, milk, lettuce and cooked shrimp. The here described method presents a detection performance similar to the traditional culture method commonly employed for the detection of the pathogen, ISO 11290 and a detection limit of 0.5 CFU/25 g or mL of food sample. Moreover, the results are obtained in two days, which represents a time reduction of more than 50% in comparison to the ISO 11290. The origin of the PNA molecule dates to the yearly 90’s and was rapidly employed for the detection of bacteria in FISH. Despite its wide application in FISH, there is still a lack of information regarding the influence of the several parameters and their interplay vii on the fluorescence outcome of the technique. As such, in Chapter 3, was performed a Page systematic evaluation of the parameters pH, probe and dextran sulfate concentration on the hybridization solution using response surface methodology for protocol optimization in Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The results showed that a probe concentration higher than 300 nM is favorable for both groups of bacteria. Regarding pH and dextran sulfate however, a clear distinction between the two groups was found. While Gram-negative had the best outcome under a high pH (approx. 10), combined with low dextran sulfate concentration (approx. 2% [wt/vol]), Gram- positive species, on the other hand hybridized better with a near-neutral pH (approx. 8) and higher dextran sulfate concentrations (approx. 10% [wt/vol]). This behavior seems to result from the ability of pH and dextran sulfate to influence probe diffusion towards the rRNA target. Similarly to the hybridization step, the fixation/permeabilization step is also crucial for a positive PNA-FISH outcome. In order to assess its influence, an evaluation of different fixation/permeabilization strategies, that consisted on the use of an organic solvent (ethanol), detergent (triton X-100) and an enzyme-based protocol with lysozyme in conjugation with paraformaldehyde, were performed in the same set of Gram-negative and Gram-positive species of Chapter 3. In general, Gram-positive species required harsher fixation/permeabilization conditions in comparison to Gram-negative species, with the exception of B. cereus in triton X-100 and lysozyme. Ultimately, the fixation/permeabilization step recurring to paraformaldehyde and ethanol proved to have a significantly superior performance for all tested species, especially for Gram-positive species (p<0.05). (Chapter 4). In summary, a complete understanding and optimization of a PNA-FISH procedure, as illustrated with the development of a detection method for L. monocytogenes (Chapter 2), is required in order to have a reliable and robust method. The here disclosed optimal parameters (Chapters 3 and 4) found differences between Gram- positive and Gram-negative bacteria that arise from their inherent differences in terms of cell envelope structures, namely their content in peptidoglycan. These protocol variances means that a unified optimal protocol for all microorganisms is unlikely to be obtained. Nonetheless, this optimization effort will greatly assist in the development of new detection methods based on PNA-FISH for other microorganisms. Furthermore, the viii extension of the optimization scope to the remaining steps and components of PNA-FISH Page procedure, is crucial to increase the knowledge necessary in order to have a complete understanding of the technique. Also, the expansion to other bacteria, especially with different cell envelope structures, and also other nucleic acid mimics is also of great relevance. ix Page Resumo As doenças com origem alimentar constituem até aos
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