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Foodborne Staphylococcus Aureus: Identification and Enterotoxin Production in Milk and Cheese.
Hrušková, Vendula ; Španová, Alena (oponent) ; Kmet,, Vladimír (oponent) ; Kaclíková, Eva (vedoucí práce)
Foodborne diseases caused by bacteria are an actual issue worldwide. To produce food, which is safe for human consumption, data about food-borne pathogen virulence is required to complement the already existing knowledge about the bacterial growth and survival in food. There is also a growing need for rapid and sensitive methods to detect these pathogens. In this dissertation, the real-time PCR-based method for the detection of S. aureus in food using selective enrichment and the impact of environmental factors on S. aureus growth and enterotoxin production in milk and cheese are described. We developed a rapid and sensitive method for the detection of S. aureus in food using selective enrichment and a new species-specific real-time PCR. The method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to the microbiological method. We investigated the effect of three different temperatures, 8°C, 12°C and 20°C on S. aureus growth and SED production in pasteurized milk and on growth, sed gene expression and SED production in Brain heart infusion. The experiments were performed in small-scale fermentors for six days and gene expression was followed by qRT-PCR. SED production was measured using Enzyme-Linked ImmunoSorbent Assay (ELISA). In BHI the growth pattern was the same at 20°C and 12°C but delayed in the latter. At 8°C there was no growth. In milk, growth was lower compared to BHI. sed mRNA was detected at 20°C and 12°C after 4 and 7 hours respectively in BHI and the production occurred during the exponential phase of growth. In milk the SED production at 20°C and 12°C occurred earlier in growth but a lower total amount was produced compared to BHI. At 8°C, there was no SED production like in BHI. The combined effect of low temperature, 12°C, and the presence of competing background microflora derived from raw milk on the growth of S. aureus and SED production in pasteurized milk was further investigated. An inhibitory effect on S. aureus growth and enterotoxin production was observed and the impact of competition was greater than the impact of low temperature. The enterotoxin production was low and correlated with the growth. By lowering the amount of competing microflora and increasing the inoculation level of S. aureus, only a slight increase in enterotoxin production occurred. In the next stage, two different cheese matrices were inoculated with S. aureus to simulate a post-contamination scenario in cheese manufacture. Samples were collected over period of 4 weeks. Critical food factors, like competing microflora and pH, which are responsible for down- and up-regulation of the virulence of S. aureus, were monitored. We tried to indentify if there are situations in which: (i) no growth but enterotoxin formation is observed, and (ii) growth and no enterotoxin formation occurs.
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Foodborne Staphylococcus Aureus: Identification and Enterotoxin Production in Milk and Cheese.
Hrušková, Vendula ; Španová, Alena (oponent) ; Kmet,, Vladimír (oponent) ; Kaclíková, Eva (vedoucí práce)
Foodborne diseases caused by bacteria are an actual issue worldwide. To produce food, which is safe for human consumption, data about food-borne pathogen virulence is required to complement the already existing knowledge about the bacterial growth and survival in food. There is also a growing need for rapid and sensitive methods to detect these pathogens. In this dissertation, the real-time PCR-based method for the detection of S. aureus in food using selective enrichment and the impact of environmental factors on S. aureus growth and enterotoxin production in milk and cheese are described. We developed a rapid and sensitive method for the detection of S. aureus in food using selective enrichment and a new species-specific real-time PCR. The method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to the microbiological method. We investigated the effect of three different temperatures, 8°C, 12°C and 20°C on S. aureus growth and SED production in pasteurized milk and on growth, sed gene expression and SED production in Brain heart infusion. The experiments were performed in small-scale fermentors for six days and gene expression was followed by qRT-PCR. SED production was measured using Enzyme-Linked ImmunoSorbent Assay (ELISA). In BHI the growth pattern was the same at 20°C and 12°C but delayed in the latter. At 8°C there was no growth. In milk, growth was lower compared to BHI. sed mRNA was detected at 20°C and 12°C after 4 and 7 hours respectively in BHI and the production occurred during the exponential phase of growth. In milk the SED production at 20°C and 12°C occurred earlier in growth but a lower total amount was produced compared to BHI. At 8°C, there was no SED production like in BHI. The combined effect of low temperature, 12°C, and the presence of competing background microflora derived from raw milk on the growth of S. aureus and SED production in pasteurized milk was further investigated. An inhibitory effect on S. aureus growth and enterotoxin production was observed and the impact of competition was greater than the impact of low temperature. The enterotoxin production was low and correlated with the growth. By lowering the amount of competing microflora and increasing the inoculation level of S. aureus, only a slight increase in enterotoxin production occurred. In the next stage, two different cheese matrices were inoculated with S. aureus to simulate a post-contamination scenario in cheese manufacture. Samples were collected over period of 4 weeks. Critical food factors, like competing microflora and pH, which are responsible for down- and up-regulation of the virulence of S. aureus, were monitored. We tried to indentify if there are situations in which: (i) no growth but enterotoxin formation is observed, and (ii) growth and no enterotoxin formation occurs.
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