Ispitivanje ekspresije gena za sintezu stafilokoknog enterotoksina A i toksina TSST-1 kod S. aureus u mleku

Abstract

S. aureus je ubikvitaran mikroorganizam, široko rasprostranjen u životnoj sredini. Glavni je uzročnik mastitisa kod mlečnih krava, pa tako uzrokuje velike zdavstvene i ekonomske probleme. Može biti prisutan u mleku i proizvodima od mleka koji se termički ne obrađuju, ali i u termički obrađenim proizvodima naknadnom kontaminacijom od strane čoveka. Bitna karakteristika ovog mikroorganizma je sposobnost stvaranja ekstracelularnih enzima i toksina koji predstavljaju glavni uzrok trovanja hranom. Da bi se sintetisala dovoljna količina enterotoksina koja može izazvati intoksikacije kod ljudi, potrebno je da u hrani bude prisutno više od 105 CFU S. aureus/mL. S. aureus sintetiše više vrsta toksina koji izazivaju različite efekte na ćelije čoveka i drugih sisara. Sinteza toksina zavisi od aktivnosti gena za regulaciju ekspresije stafilokoknih enterotoksina (SEs) i TSST-1. Postoji više faktora koji sprečavaju ili stimulišu sintezu enterotoksina kao što su: temperatura, pH, aw, kiseonik, redoks potencijal i dr. Enterotoksini se sintetišu pri temperaturama od 10°C do 46°C, pri pH od 5 do 9,6, aktivnosti vode od 0,86 do 0,99 i koncentraciji NaCl do 12%. Nalaz enterotoksogenih stafilokoka u hrani ne znači da će doći do trovanja, jer uslovi matriksa hrane i sredine određuju preživljavanje, rast populacije stafilokoka i stvaranje dovoljne količine enterotoksina koja može izazvati oboljenje kod ljudi. Cilj ovog istraživanja bio je da se ispita uticaj različitih temperatura, vremena čuvanja i matriksa na stepen ekspresije gena za sintezu enterotoksina A i toksina TSST-1, kako bi se došlo do novih saznanja o uticaju paragenetskih faktora na rizik nastanka stafilokokne alimentarne intoksikacije. Materijal za eksperimentalnu inokulaciju predstavljalo je kratkotrajno sterilizovano (UHT) i pasterizovano kravlje mleko sa 3,2% mlečne masti i referentni sojevi S. aureus koji sintetišu stafilokokni enterotoksin A i toksin 1 toksičnog šok sindroma. Eksperimentalni rad odvijao se u dve faze. U prvoj fazi eksperimenta, hrana animalnog porekla (pasterizovano i UHT mleko) koja simulira najčešće izvore toksoinfekcije S. aureus kontaminirana je određenom količinom prekonoćne bujonske suspenzije referentnog soja S. aureus, u kojoj je prethodno utvrđen inicijalni broj. Uzorci su čuvani pri temperaturama od 15°C i 22°C tokom 24, 48 i 72 časa, simulirajući neadekvatne uslove koji nastaju akcidentalnim narušavanjem hladnog lanca. Nakon 24, 48, odnosno 72 časa kontaminirano mleko inokulisano je na Baird-Parker agar (SRPS ISO 6888-1:2009), a nakon inkubacije podloga utvrđen je broj izraslih kolonija S. aureus i upoređen sa inicijalnim brojem. Paralelno sa inokulacijom ove podloge, uzeta je određena količina mleka koja je potom tretirana tečnim azotom da bi se konzervisala iRNK tj. da bi se „zamrznuo“ ekspresioni genetski profil S. aureus. Iz zamrznutih uzoraka ekstrahovana je ukupna RNK, koja je potom metodom reverzne transkripcije - Real Time PCR prevedena u odgovarajuću cDNK svakog od ispitivanih gena. Da bi se ispitala korelacija između ekspresije gena za sintezu stafilokoknog enterotoksina i njegove količine u uzorcima, a na taj način isključila i eventualna posttranslaciona promena u sintezi, stafilokoni enterotoksin A je kvantifikovan pomoću dijalizne koncentracije i ELFA metode. Kao rezultat istraživanja sprovednih u ovoj disertaciji može se uočiti statistički značajna razlika između stepena ekspresije sea gena u pasterizovanom mleku čuvanom pri temperaturi od 22°C tokom 24 sata u odnosu na kalibrator (S. aureus u mleku čuvanom pri 8°C), što se poklapa sa postignutim brojem S. aureus od 105 CFU/mL i imunohromatografski detektovanom količinom enterotoksina A. Stepen ekspresije enterotoksina A u pasterizovanom mleku čuvanom pri temperaturi od 15°C tokom 72 sata statistički se značajno razlikuje u odnosu na kalibrator, što se takođe podudara sa detektovanom količinom enterotoksina A. U UHT mleku čuvanom tokom 24 sata pri temperaturi od 22°C, odnosno tokom 48 sati pri temperaturi od 15°C utvrđena je statistički značajna razlika u stepenu ekspresije u odnosu na kalibrator, što se podudara sa detektovanom količinom enterotoksina A i dinamikom promene broja S. aureus. Na osnovu ovih podataka možemo zaključiti da intezitet ekspresije sea gena odgovara količini fizički „de novo“ sintetisanog enterotoksina A. Stepen ekspresije tst gena pokazao je statistički značajnu razliku u odnosu na kalibrator u pasterizovanom i UHT mleku nakon 24 sata pri temperaturi od 22°C, i 48 sati pri temperaturi od 15°C, dok je broj S. aureus pozitivan na TSST-1 prelazio 105 CFU/mL u pasterizovanom i UHT mleku nakon 24 sata čuvanom pri 15°C, odnosno 22°C. Obzirom na mali broj literaturnih podataka o uticaju sastava hrane, temperature i vremena čuvanja kao i drugih paragenetskih faktora na regulaciju patogenosti S. aureus, dobijeni rezultati mogu predstavljati polaznu osnovu za ispitivanje značaja genetskog profila patogenosti S. aureus u hrani na razvoj infekcije kod potrošača.

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.