Ispitivanje ekspresije gena za sintezu stafilokoknog enterotoksina A i toksina TSST-1 kod S. aureus u mleku / Investigation of expression of staphylococcal enterotoxin A and TSST-1 toxin genes of S. aureus in milk

S. aureus is an ubiquitous microorganism, widely distributed in the environment. It is the main cause of mastitis in dairy cows so it causes major health and economic problems. It can be present in milk and milk products that are not thermally treated, but also in thermally processed products by subsequent contamination by humans. An important feature of this microorganism is the ability to produce extracellular enzymes and toxins that are the major cause of food poisoning. In order to synthesize a sufficient amount of enterotoxin that can induce intoxication in humans, more than 105 CFU of S. aureus / mL should be present in the food. S. aureus synthesizes several types of toxins that cause different effects on the human cells and cells of other mammals. The synthesis of toxin depends on the activity of the gene regulating the expression of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1). There are several factors that prevent or stimulate the synthesis of enterotoxins such as temperature, pH, water activity, oxygen, redox potential, etc. Enterotoxins are synthesized at temperatures from 10°C to 46°C, at pH 5 to 9.6, water activities ranging from 0.86 to 0.99 and a concentration of NaCl to 12%. Finding enterotoxogenic staphylococci in food does not mean that there will come to poisoning, because the conditions of the food and environment matrix determine their survival, the growth of the staphylococci population and the creation of a sufficient amount of enterotoxins that can cause disease in humans. The aim of this study was to investigate the influence of different temperatures, storage times and matrix on the degree of gene expression for the synthesis of enterotoxin A and toxin TSST-1 in order to gain new insights about the effect of paragenetic factors on the risk of staphylococcal alimentary intoxication. The material for experimental inoculation was short-time sterilized (UHT) and pasteurized cow milk with 3.2% milk fat and reference strains of S. aureus which synthesizes staphylococcal enterotoxin A and TSST-1. Experimental work took place in two phases. In the first stage of the experiment, pasteurized and UHT milk, which simulates the most common sources of S. aureus toxoinfection, were contaminated by a certain amount of overnight broth suspension of the reference strain S. aureus, in which the initial number was previously determined. Samples were stored at temperatures of 15°C and 22°C for 24, 48 and 72 hours, simulating inadequate conditions resulting from accidental cold chain disturbance. After 24, 48, and 72 hours, contaminated milk was inoculated to Baird-Parker agar (SRPS ISO 6888-1: 2009), and after the incubation of the substrate, the number of grown S. aureus colonies was compared with the initial number that was determined in beginning of the experiment. At the time this substrate was inoculated, certain amount of milk was taken, which was then treated with liquid nitrogen to conserve iRNA, i.e. in order to "freeze" the expression genetic profile of S. aureus. From frozen samples, the total RNA was extracted, which was then converted by the reverse transcription method - Real Time PCR into the corresponding cDNA of each of the examined genes. In order to investigate the correlation between gene expression for the synthesis of staphylococcal enterotoxin and its amount in the samples, and thereby exclude the possible posttranslational change in synthesis, staphylococcal enterotoxin A was quantified by dialysis and ELFA methods. As a result of the investigations carried out in this thesis, a significant difference between the degree of expression of the sea gene in pasteurized milk stored at 22°C during 24 hours in relation to the calibrator (S. aureus in milk stored at 8°C) can be observed, which matches the achieved S. aureus number of 105 CFU / mL and an immunochromatographically detectable amount of enterotoxin A. The degree of expression of enterotoxin A in pasteurized milk stored at a temperature of 15°C for 72 hours significantly differs from the results of the calibrator, which also match the detected amount of enterotoxin A. In UHT milk stored for 24 hours at a temperature of 22°C, and for 48 hours at a temperature of 15°C, a significant difference in the degree of expression of sea gene relative to the calibrator was found, which matches the detected amount of enterotoxin A and the dynamics of the change in the number of S. aureus. Based on these data, it can be concluded that the intensity of expression of the sea gene corresponds to the amount of physically "de novo" synthetized enterotoxin A. The degree of expression of the tst gene showed a significant difference from the calibrator value in pasteurized and UHT milk after 24 hours at a temperature of 22°C and 48 hours at a temperature of 15°C, while the number of S. aureus positive for TSST-1 exceeded 105 CFU / mL in pasteurized and UHT milk after 24 hours stored at 15°C and 22°C. Given the small number of literature data on the effect of food composition, temperature and storage time, as well as other paragenetic factors on the regulation of S. aureus pathogenicity, the obtained results can be the starting point for examining the significance of the genetic profile of S. aureus pathogenicity in the development of the infection in consumers.