Análise da expressão gênica de Bacillus cereus s.s. em leite contaminado experimentalmente e pasteurizado ao longo da shelf-life
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Data
2019-04-10
Autores
Lima, Joyce Aparecida Santos
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Universidade Estadual Paulista (Unesp)
Resumo
Bacillus cereus sensu stricto (s.s.) tem o potencial de produzir toxinas gastrointestinais, resistir a condições adversas, como tratamentos térmicos e pasteurização; produzir enzimas e formar biofilmes, causando diversos surtos ao redor do mundo relacionados com a sua presença em alimentos contaminados. Entretanto, não é bem elucidado o dano que o tratamento térmico causa à esta bactéria e à sua expressão gênica. Assim, o objetivo deste estudo foi verificar se a pasteurização e o armazenamento em refrigeração ao longo da shelf-life seriam capazes de influenciar a expressão dos genes relacionados com a produção de toxinas diarreicas, proteases e à formação de biofilmes em leite experimentalmente contaminado com B. cereus s.s.. Trinta litros de leite cru foram tindalizados e experimentalmente contaminados com aproximadamente 108 UFC.mL-1 de B. cereus s.s. SAMN07414939 (Bio Project PRJNA390851). Uma amostra foi coletada logo após a contaminação (LTC – T0) e então o leite foi pasteurizado e envasado em embalagens de polietileno com 1L de capacidade (LCP - T1, T2 e T3) e mantido em refrigeração até o momento das análises. Posteriormente, a extração de RNA total das amostras foi realizada, seguido pelo cDNA e qPCR utilizando os genes plcr, hblA, hblC, hblD, nheA, nheB, nheC, cytk e npr para avaliação da expressão gênica. A expressão dos genes se manteve ao longo de toda a shelf-life, sem diferença estatística significativa entre os momentos avaliados para os genes plcr, hblA, hblC, hblD, nheA, nheB, nheC, cytk e npr, os quais persistiram com seu potencial de causar síndrome diarreica, formar biofilme e produzir proteases. Tais resultados realçam a importância de uma matéria-prima de qualidade e boas práticas de fabricação e higiene dentro das plantas manipuladoras de alimentos.
Bacillus cereus sensu stricto (s.s.) has the potential to produce gastrointestinal toxins, resists to adverse conditions, such as heat-treatment and pasteurization; the ability to form biofilms and produce enzymes, causing several outbreaks worldwide related to its presence in contaminated food. However, it is not well elucidated what damage the pasteurization causes to this bacterium and its gene expression. Thus, the aim of this study was to verify if pasteurization and storage during shelf-life in refrigeration were able to influence the expression of genes related to the production of diarrheal toxins, proteases and biofilm formation in milk experimentally contaminated with B. cereus s.s. Thirty liters of raw milk were sterilized by tyndallization and experimentally contaminated with 108 vegetative cells of B. cereus s.s. SAMN07414939 (Bio Project PRJNA390851). A sample was collected right after contamination (LTC – 0h); then the milk was pasteurized and then packed in 1 L capacity polyethylene bags and samples LCP - T1, T2 and T3 were collected. Posteriorly, the total RNA were extracted from the samples and the cDNA and qPCR were performed using the primers plcr, hblA, hblC, hblD, nheA, nheB, nheC, cytk and npr for gene expression quantification. The gene expression was maintained along the shelf-life, without significant statistical difference between the moments evaluates for the plcr, hblA, hblC, hblD, nheA, nheB, nheC, cytk and npr gene expression, which persisted with its potential to cause diarrheal syndrome, form biofilm and produce proteases. These results highlight the importance of a quality raw milk and good manufacturing and hygiene practices within food handling plants.
Bacillus cereus sensu stricto (s.s.) has the potential to produce gastrointestinal toxins, resists to adverse conditions, such as heat-treatment and pasteurization; the ability to form biofilms and produce enzymes, causing several outbreaks worldwide related to its presence in contaminated food. However, it is not well elucidated what damage the pasteurization causes to this bacterium and its gene expression. Thus, the aim of this study was to verify if pasteurization and storage during shelf-life in refrigeration were able to influence the expression of genes related to the production of diarrheal toxins, proteases and biofilm formation in milk experimentally contaminated with B. cereus s.s. Thirty liters of raw milk were sterilized by tyndallization and experimentally contaminated with 108 vegetative cells of B. cereus s.s. SAMN07414939 (Bio Project PRJNA390851). A sample was collected right after contamination (LTC – 0h); then the milk was pasteurized and then packed in 1 L capacity polyethylene bags and samples LCP - T1, T2 and T3 were collected. Posteriorly, the total RNA were extracted from the samples and the cDNA and qPCR were performed using the primers plcr, hblA, hblC, hblD, nheA, nheB, nheC, cytk and npr for gene expression quantification. The gene expression was maintained along the shelf-life, without significant statistical difference between the moments evaluates for the plcr, hblA, hblC, hblD, nheA, nheB, nheC, cytk and npr gene expression, which persisted with its potential to cause diarrheal syndrome, form biofilm and produce proteases. These results highlight the importance of a quality raw milk and good manufacturing and hygiene practices within food handling plants.
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Palavras-chave
Biofilmes, Biologia molecular, Expressão gênica, Microbiologia