Food-Related Gram-Positive Bacteria: Enterotoxin A Expression in Staphylococcus aureus and a New Regulation Mechanism in Lactococcus lactis

Sammanfattning: Popular Abstract in English Foods, microorganisms, and humans have been tightly associated from the start of human history. Foods are not only the source of nutrients for humans, but also good media for microorganisms. Although we humans discovered the existence of microorganisms relatively recently, we have been dealing with them for hundreds and thousands of years. We have used some microorganisms to transfer raw food materials to ready-to-eat products, e.g. beer, wine, soy sauce, cheese, and so on, for preservation purposes and/or good flavour. On the other hand, microorganisms have spoiled foods, even causing food poisoning diseases in humans and cattle. Control of pathogens is an important and on-going task, which continues to influence human health. Many effective measures, such as heat processing (e.g. pasteurization of milk), irradiation (e.g. UV lamps for surface sterilization), high-pressure processing, low-temperature storage, chemical preservation (e.g. organic acids), packaging (e.g. modification of atmosphere), water activity (e.g. dryness) and so on, are studied and performed in order to prevent the growth of pathogens. Most of these physical and chemical treatments affect (to a greater or lesser extent) the nutritional value and flavour. Nowadays, people prefer foods that are fresher, less processed, and nutrient-rich. This leads to opportunities and challenges for food researchers and industry. More information is needed to understand microorganisms and their mechanisms of virulence. With the development of natural science and technology, studies of microbial pathogens are not only limited to the growth behaviour of microbes. Nowadays, we can study the molecular mechanisms of virulence and their genetics by using tools such as PCR and high-throughput DNA sequencing. Staphylococcus aureus is an important pathogen of humans and animals. It is associated with both infection and intoxication Overall, staphylococcal food poisoning (SFP) is said to be the fourth most common causative agent of food-borne illness in the EU, but this depends on the region. Due to the generally unsevere symptoms, people may not report it or go to hospital; this leads to underestimation of the total number of cases. S. aureus is carried in humans and other warm-blooded animals. About 25% of healthy humans carry S. aureus in their nose and skin all the time, and about 50% of people have it intermittently. Due to this, one of the most important sources of S. aureus contamination in food is people. Other sources of contamination can be mainly raw materials and food-processing equipment. The challenges in controlling SFP are not only to prevent bacterial growth in food, but also to prevent the formation of enterotoxins. Staphylococcal enterotoxins are extremely thermo-tolerant. Normal cooking in the kitchen is good enough to kill the bacteria in food, but is not sufficient to deactivate the enterotoxins entirely. S. aureus produces many types of enterotoxins, and enterotoxin A (SEA) is the one most commonly associated with cases of food poisoning. In this thesis, I have studied the expression and formation of SEA in S. aureus. The growth behaviour was also studied. The results show that there is no great difference regarding the growth rate and final optical density among the various strains tested, using either controlled laboratory conditions or food matrices. But a very important phenomenon was confirmed, namely the huge variation in amounts of SEA produced by the S. aureus strains investigated. It was also found that this variation was linked to the genetic backbone of the SEA gene. From a number of genome sequences of S. aureus analysed, there were found to be two versions of the sea gene. One version of the gene (sea1) is carried by strains that produce high amounts of SEA. The strains that produce minor amounts of SEA have the other version of the sea gene (sea2). Both versions of the gene have a common, endogenous promoter, but the expression of sea2 was found to be less pronounced than that of sea1. The sea gene is encoded by a specific family of bacteriophages (bacterial viruses, also called phages). Based on genome analysis of sea-encoding phages, three major branches were described. One branch contained sea2 and the two other branches harboured sea1 with different genetic surroundings. By using a classic phage-inducing reagent, it was found that one of these branches produced high amounts of SEA after prophage induction. These strains were also genetically mapped to verify phage relationships. By monitoring gene expression, it was possible to compare the levels of sea mRNA with the amount of SEA released from the bacteria. Interestingly, a new transcript associated with the phage life cycle was observed in the prophage-induced branch, producing a boost of SEA. Thus, there appears to be a close connection between the biology of the phage and the amount of SEA formed—as also observed in situ in sausages. However, in the food-processing, storage, and consumption environments, many factors such as temperature, preservatives, and starvation can trigger phage induction. For instance, food preservatives are used widely in food products, e.g. organic acids, which have been used for centuries. It was found that expression of the sea gene was 9 times higher at pH 6.0 and 4 times higher at pH 5.5 (adjusted with acetic acid) than at natural pH. SEA formation peaks at pH 6.0, which is a very common pH in food products. Strains with different prophage background behave similarly, but with differences in the effect of acetic acid on the virulence and degree of expression of SEA. One study was performed to test S. aureus strains with different prophage background in a real food matrix, i.e. frankfurter sausage. The results confirmed that some S. aureus strains are more virulent than others, and that counting the number of living S. aureus bacteria in the product is not a reliable way of judging whether or not the food is safe to eat. In the context of this thesis, I have also investigated the metabolic regulation of weak acids in Lactococcus lactis. Weak acids and lactic acid bacteria (LAB) are frequently used to preserve food products and prevent the growth of S. aureus. LAB are widespread in our environments and an important part of our lives. Many food products are produced with the help of LAB, such as cheese, yoghurt, sauerkraut, and wine. They are also added to food as biopreservatives, mainly because they produce lactic acids together with mixed acids, which lower the pH and therefore inhibit other microorganisms. In this work, I studied three important enzymes of the glycolytic pathway, all dehydrogenases. The aim was to understand the metabolic switch between homolactic fermentation and mixed-acid fermentation in LAB. I also studied the inhibitory effects of natural metabolites such as ADP and ATP, the energy currency molecules of the cell. It was found that the pool of ADP and ATP can effectively inactivate some of the dehydrogenases, e.g. ADH, but it has less influence on GAPDH and LDH. This regulatory mechanism contributes to readjustment of the flux of ATP production in L. lactis, and consequently the production of weak acids.