Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Therapeutic hypothermia after cardiac arrest : Studies on neurological and cardiological outcome and prediction of outcome in hypothermia-treated patients resuscitated from out-of-hospital cardiac arrest

The outcome of the successfully resuscitated patient is mainly determined by the extent of hypoxic-ischemic cerebral injury, and hypothermia has multiple mechanisms of action in mitigating such injury. The present study was undertaken from 1997 to 2001 in Helsinki as a part of the European multicenter study Hypothermia after cardiac arrest (HACA) to test the neuroprotective effect of therapeutic hypothermia in patients resuscitated from out-of-hospital ventricular fibrillation (VF) cardiac arrest (CA). The aim of this substudy was to examine the neurological and cardiological outcome of these patients, and especially to study and develop methods for prediction of outcome in the hypothermia-treated patients. A total of 275 patients were randomized to the HACA trial in Europe. In Helsinki, 70 patients were enrolled in the study according to the inclusion criteria. Those randomized to hypothermia were actively cooled externally to a core temperature 33 ± 1ºC for 24 hours with a cooling device. Serum markers of ischemic neuronal injury, NSE and S-100B, were sampled at 24, 36, and 48 hours after CA. Somatosensory and brain stem auditory evoked potentials (SEPs and BAEPs) were recorded 24 to 28 hours after CA; 24-hour ambulatory electrocardiography recordings were performed three times during the first two weeks and arrhythmias and heart rate variability (HRV) were analyzed from the tapes. The clinical outcome was assessed 3 and 6 months after CA. Neuropsychological examinations were performed on the conscious survivors 3 months after the CA. Quantitative electroencephalography (Q-EEG) and auditory P300 event-related potentials were studied at the same time-point. Therapeutic hypothermia of 33ºC for 24 hours led to an increased chance of good neurological outcome and survival after out-of-hospital VF CA. In the HACA study, 55% of hypothermia-treated patients and 39% of normothermia-treated patients reached a good neurological outcome (p=0.009) at 6 months after CA. Use of therapeutic hypothermia was not associated with any increase in clinically significant arrhythmias. The levels of serum NSE, but not the levels of S-100B, were lower in hypothermia- than in normothermia-treated patients. A decrease in NSE values between 24 and 48 hours was associated with good outcome at 6 months after CA. Decreasing levels of serum NSE but not of S-100B over time may indicate selective attenuation of delayed neuronal death by therapeutic hypothermia, and the time-course of serum NSE between 24 and 48 hours after CA may help in clinical decision-making. In SEP recordings bilaterally absent N20 responses predicted permanent coma with a specificity of 100% in both treatment arms. Recording of BAEPs provided no additional benefit in outcome prediction. Preserved 24- to 48-hour HRV may be a predictor of favorable outcome in CA patients treated with hypothermia. At 3 months after CA, no differences appeared in any cognitive functions between the two groups: 67% of patients in the hypothermia and 44% patients in the normothermia group were cognitively intact or had only very mild impairment. No significant differences emerged in any of the Q-EEG parameters between the two groups. The amplitude of P300 potential was significantly higher in the hypothermia-treated group. These results give further support to the use of therapeutic hypothermia in patients with sudden out-of-hospital CA.

Construing and Defining the Out of Control : Addiction in the Media 1968-2008.

The four scientific articles comprising this doctoral dissertation offer new information on the presentation and construction of addiction in the mass media during the period 1968 - 2008. Diachronic surveys as well as quantitative and qualitative content analyses were undertaken to discern trends during the period in question and to investigate underlying conceptions of the problems in contemporary media presentations. The research material for the first three articles consists of a sample of 200 texts from Finland s biggest daily newspaper, Helsingin Sanomat, from the period 1968 - 2006. The fourth study examines English-language tabloid material published on the Internet in 2005 - 2008. A number of principal trends are identified. In addition to a significant increase in addiction reporting over time, the study shows that an internalisation of addiction problems took place in the media presentations under study. The phenomenon is portrayed and tackled from within the problems themselves, often from the viewpoint of the individuals concerned. The tone becomes more personal, and technical and detailed accounts are more and more frequent. Secondly, the concept of addiction is broadened. This can be dated to the 1990s. The concept undergoes a conventionalisation: it is used more frequently in a manner that is not thought to require explanation. The word riippuvuus (the closest equivalent to addiction in Finnish) was adopted more commonly in the reporting at the same time, in the 1990s. Thirdly, the results highlight individual self-governance as a superordinate principle in contemporary descriptions of addiction. If the principal demarcation in earlier texts was between us and them , it is now focused primarily on the individual s competence and ability to govern the self, to restrain and master one's behaviour. Finally, in the fourth study investigating textual constructions of female celebrities (Amy Winehouse, Britney Spears and Kate Moss) in Internet tabloids, various relations and functions of addiction problems, intoxication, body and gender were observed to function as cultural symbols. Addiction becomes a sign, or a style, that represents different significations in relation to the main characters in the tabloid stories. Tabloids, as a genre, play an important role by introducing other images of the problems than those featured in mainstream media. The study is positioned within the framework of modernity theory and its views on the need for self-reflexivity and biographies as tools for the creation and definition of the self. Traditional institutions such as the church, occupation, family etc. no longer play an important role in self-definition. This circumstance creates a need for a culture conveying stories of success and failure in relation to which the individual can position their own behaviour and life content. I propose that addiction , as a theme in media reporting, resolves the conflict that emanates from the ambivalence between the accessibility and the individualisation of consumer society, on the one hand, and the problematic behavioural patterns (addictions) that they may induce, on the other.

Relationship Drivers in Provider - Consumer Relationships. Empirical Studies of Customer Loyalty Programs (första upplagan slutsåld / first edition sold out, 'print on demand' 60 €)

The study addressed a phenomenon that has become common marketing practice, customer loyalty programs. Although a common type of consumer relationship, there is limited knowledge of its nature. The purpose of the study was to create structured understanding of the nature of customer relationships from both the provider’s and the consumer’s viewpoints by studying relationship drivers and proposing the concept of relational motivation as a provider of a common framework for the analysis of these views. The theoretical exploration focused on reasons for engaging in customer relationships for both the consumer and the provider. The themes of buying behaviour, industrial and network marketing and relationship marketing, as well as the concepts of a customer relationship, customer loyalty, relationship conditions, relational benefits, bonds and commitment were explored and combined in a new way. Concepts from the study of business-to-business relationships were brought over and their power in explaining the nature of consumer relationships examined. The study provided a comprehensive picture of loyalty programs, which is an important contribution to the academic as well as the managerial discussions. The consumer study provided deep insights into the nature of customer relationships. The study provides a new frame of reference to support the existing concepts of loyalty and commitment with the introduction of the relationship driver and relational motivation concepts. The result is a novel view of the nature of customer relationships that creates new understanding of the forces leading to loyal behaviour and commitment. The study concludes with managerial implications.

Understanding Customer Loyalty and Disloyalty The Effect of Loyalty-Supporting and -Repressing Factors (first edition sold out, 'print on demand' 60 €)

Customer loyalty has been a central topic of both marketing theory and practice for several decades. Customer disloyalty, or relationship ending, has received much less attention. Despite the close relation between customer loyalty and disloyalty, they have rarely been addressed in the same study. The thesis bridges this gap by focusing on both loyal and disloyal customers and the factors characterising them. Based on a qualitative study of loyal and disloyal bank customers in the Finnish retail banking market, both factors that are common to the groups and factors that differentiate between them are identified. A conceptual framework of factors that affect customer loyalty or disloyalty is developed and used to analyse the empirical data. According to the framework, customers’ loyalty status (behavioural and attitudinal loyalty) is influenced by positive, loyalty-supporting, and negative, loyalty-repressing factors. Loyalty-supporting factors either promote customer dedication, making the customer want to remain loyal, or act as constraints, hindering the customer from switching. Among the loyalty-repressing factors it is especially important to identify those that act as triggers of disloyal behaviour, making customers switch service providers. The framework further suggests that by identifying the sources of loyalty-supporting and -repressing factors (the environment, the provider, the customer, the provider-customer interaction, or the core service) one can determine which factors are within the control of the service provider. Attitudinal loyalty is approached through a customer’s “feeling of loyalty”, as described by customers both orally and graphically. By combining the graphs with behavioural loyalty, seven customer groups are identified: Stable Loyals, Rescued Loyals, Loyals at Risk, Positive Disloyals, Healing Disloyals, Fading Disloyals, and Abrupt Disloyals. The framework and models of the thesis can be used to analyse factors that affect customer loyalty and disloyalty in different service contexts. Since the empirical study was carried out in a retail bank setting, the thesis has managerial relevance especially for banks. Christina Nordman is associated with CERS, Center for Relationship Marketing and Service Management at the Swedish School of Economics and Business Administration. The doctoral thesis is part of the Göran Collert Research Project in Customer Relationships and Retail Banking and has been funded by The Göran Collert Foundation.