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.

Ancient Philosophers on Mental Illness

This dissertation is about ancient philosophers notions of mental illness, from Plato onwards. Mental illness here means disorders that, in ancient medical thought, were believed to originate in the body but to manifest themselves predominantly through mental symptoms. These illnesses were treated by physical means, which were believed to address the bodily cause of the illness, conceived of as an elemental imbalance or a state of cephalic stricture , for example. Sometimes the mental symptoms were addressed directly by psychotherapeutic means. The first and most important question explored concerns how the ancient philosophers responded to the medical notion of mental illness, and how they explained such illnesses in their theories of physiology and psychology. Although the illnesses are seldom discussed extensively, the philosophers were well aware of their existence and regarded their occurrence an indication of the soul s close dependence on the body. This called for a philosophical account. The second question addressed has to do with the ancient philosophers role as experts in mental problems of a non-medical kind, such as unwanted emotions. These problems were dubbed diseases of the soul , and the philosophers thus claimed to be doctors of the soul. Although the distinction between mental illnesses and diseases of the soul was often presented as rather obvious, there was some vagueness and overlap. There is still a third question that is explored, concerning the status of both mental illnesses and diseases of the soul as unnatural conditions, the role of the human body in the philosophical aetiologies of evil, and the medico-philosophical theories of psycho-physiological temperaments. This work consists of an introduction and five main chapters, focusing on Plato, Aristotle, the Stoics and Galen, and the Sceptics, the Epicureans and later Platonists. The sources drawn on are the original Greek and Latin philosophical and medical texts. It appears that the philosophers accepted the medical notion of mental illness, but interpreted it in various ways. The differences in interpretation were mostly attributable to differences in their theories of the soul. Although the distinction between mental illness and diseases of the soul was important, marking the boundary between the fields of expertise of medicine and philosophy, and of the individual s moral responsibilities, the problematic aspects of establishing it are discussed rather little in ancient philosophy. There may have been various reasons for this. The medical descriptions of mental illness are often extreme, symptoms of the psychotic type excluding the possibility of the condition being of the non-medical kind. In addition, the rigid normativeness of ancient philosophical anthropologies and their rigorous notion of human happiness decreased the need to assess the acceptability of individual variation in their emotional and intellectual lives and external behaviour.

Role of heat treatment in the processing and quality of oat flakes

This thesis reports on investigations into the influence of heat treatment on the manufacturing of oat flakes. Sources of variation in the oat flake quality are reviewed, including the whole chain from the farm to the consumer. The most important quality parameters of oat flakes are the absence of lipid hydrolysing enzymes, specific weight, thickness, breakage (fines), water absorption. Flavour, colour and pasting properties are also important, but were not included in the experimental part of this study. Of particular interest was the role of heat processing. The first possible heat treatment may occur already during grain drying, which in Finland generally happens at the farm. At the mill, oats are often kilned to stabilise the product by inactivating lipid hydrolysing enzymes. Almost invariably steaming is used during flaking, to soften the groats and reduce flake breakage. This thesis presents the use of a material science approach to investigating a complex system, typical of food processes. A combination of fundamental and empirical rheological measurements was used together with a laboratory scale process to simulate industrial processing. The results were verified by means of industrial trials. Industrially produced flakes at three thickness levels (nominally 0.75, 0.85 and 0.90 mm) were produced from kilned and unkilned oat groats, and the flake strength was measured at different moisture contents. Kilning was not found to significantly affect the force required to puncture a flake with a 2mm cylindrical probe, which was taken as a measure of flake strength. To further investigate how heat processing contributes to flake quality, dynamic mechanical analysis was used to characterise the effect of heat on the mechanical properties of oats. A marked stiffening of the groat, of up to about 50% increase in storage modulus, was observed during first heating at around 36 to 57°C. This was also observed in tablets prepared from ground groats and extracted oat starch. This stiffening was thus attributed to increased adhesion between starch granules. Groats were steamed in a laboratory steamer and were tempered in an oven at 80 110°C for 30 90 min. The maximum force required to compress the steamed groats to 50% strain increased from 50.7 N to 57.5 N as the tempering temperature was increased from 80 to 110°C. Tempering conditions also affected water absorption. A significantly higher moisture content was observed for kilned (18.9%) compared to unkilned (17.1%) groats, but otherwise had no effect on groat height, maximum force or final force after a 5 s relaxation time. Flakes were produced from the tempered groats using a laboratory flaking machine, using a roll gap of 0.4 mm. Apart from specific weight, flake properties were not influenced by kilning. Tempering conditions however had significant effects on the specific weight, thickness and water absorption of the flakes, as well as on the amount of fine material (<2 mm) produced during flaking. Flake strength correlated significantly with groat strength and flake thickness. Trial flaking at a commercial mill confirmed that groat temperature after tempering influenced water absorption. Variation in flake strength was observed , but at the groat temperatures required to inactivate lipase, it was rather small. Cold flaking of groats resulted in soft, floury flakes. The results presented in this thesis suggest that heating increased the adhesion between starch granules. This resulted in an increase in the stiffness and brittleness of the groat. Brittle fracture, rather than plastic flow, during flaking could result in flaws and cracks in the flake. These would be expected to increase water absorption. This was indeed observed as tempering temperature increased. Industrial trials, conducted with different groat temperatures, confirmed the main findings of the laboratory experiments. The approach used in the present study allowed the systematic study of the effect of interacting process parameters on product quality. There have been few scientific studies of oat processing, and these results can be used to understand the complex effects of process variables on flake quality. They also offer an insight into what happens as the oat groat is deformed into a flake.

Cereulide producing B. cereus and amylosin producing B. subtilis and B. mojavensis : characterization of strains and toxigenicities

B. cereus is one of the most frequent occurring bacteria in foods . It produces several heat-labile enterotoxins and one stable non-protein toxin, cereulide (emetic), which may be pre-formed in food. Cereulide is a heat stable peptide whose structure and mechanism of action were in the past decade elucidated. Until this work, the detection of cereulide was done by biological assays. With my mentors, I developed the first quantitative chemical assay for cereulide. The assay is based on liquid chromatography (HPLC) combined with ion trap mass spectrometry and the calibration is done with valinomycin and purified cereulide. To detect and quantitate valinomycin and cereulide, their [NH4+] adducts, m/z 1128.9 and m/z 1171 respectively, were used. This was a breakthrough in the cereulide research and became a very powerful tool of investigation. This tool made it possible to prove for the first time that the toxin produced by B. cereus in heat-treated food caused human illness. Until this thesis work (Paper II), cereulide producing B. cereus strains were believed to represent a homogenous group of clonal strains. The cereulide producing strains investigated in those studies originated mostly from food poisoning incidents. We used strains of many origins and analyzed them using a polyphasic approach. We found that the cereulide producing B. cereus strains are genetically and biologically more diverse than assumed in earlier studies. The strains diverge in the adenylate kinase (adk) gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three patterns), tyrosin decomposition, haemolysis and lecithine hydrolysis (two phenotypes). Our study was the first demonstration of diversity within the cereulide producing strains of B. cereus. To manage the risk for cereulide production in food, understanding is needed on factors that may upregulate cereulide production in a given food matrix and the environmental factors affecting it. As a contribution towards this direction, we adjusted the growth environment and measured the cereulide production by strains selected for diversity. The temperature range where cereulide is produced was narrower than that for growth for most of the producer strains. Most cereulide was by most strains produced at room temperature (20 - 23ºC). Exceptions to this were two faecal isolates which produced the same amount of cereulide from 23 ºC up until 39ºC. We also found that at 37º C the choice of growth media for cereulide production differed from that at the room temperature. The food composition and temperature may thus be a key for understanding cereulide production in foods as well as in the gut. We investigated the contents of [K+], [Na+] and amino acids of six growth media. Statistical evaluation indicated a significant positive correlation between the ratio [K+]:[Na+] and the production of cereulide, but only when the concentrations of glycine and [Na+] were constant. Of the amino acids only glycine correlated positively with high cereulide production. Glycine is used worldwide as food additive (E 640), flavor modifier, humectant, acidity regulator, and is permitted in the European Union countries, with no regulatory quantitative limitation, in most types of foods. B. subtilis group members are endospore-forming bacteria ubiquitous in the environment, similar to B. cereus in this respect. Bacillus species other than B. cereus have only sporadically been identified as causative agents of food-borne illnesses. We found (Paper IV) that food-borne isolates of B. subtilis and B. mojavensis produced amylosin. It is possible that amylosin was the agent responsible for the food-borne illness, since no other toxic substance was found in the strains. This is the first report on amylosin production by strains isolated from food. We found that the temperature requirement for amylosin production was higher for the B. subtilis strain F 2564/96, a mesophilic producer, than for B. mojavensis strains eela 2293 and B 31, psychrotolerant producers. We also found that an atmosphere with low oxygen did not prevent the production of amylosin. Ready-to-eat foods packaged in micro-aerophilic atmosphere and/or stored at temperatures above 10 °C, may thus pose a risk when toxigenic strains of B. subtilis or B. mojavensis are present.

Rigorous scaling law for the heat current in disordered harmonic chain

We study the energy current in a model of heat conduction, first considered in detail by Casher and Lebowitz. The model consists of a one-dimensional disordered harmonic chain of n i.i.d. random masses, connected to their nearest neighbors via identical springs, and coupled at the boundaries to Langevin heat baths, with respective temperatures T_1 and T_n. Let EJ_n be the steady-state energy current across the chain, averaged over the masses. We prove that EJ_n \sim (T_1 - T_n)n^{-3/2} in the limit n \to \infty, as has been conjectured by various authors over the time. The proof relies on a new explicit representation for the elements of the product of associated transfer matrices.

Reynolds stress and heat flux in spherical shell convection

Context. Turbulent fluxes of angular momentum and heat due to rotationally affected convection play a key role in determining differential rotation of stars. Aims. We compute turbulent angular momentum and heat transport as functions of the rotation rate from stratified convection. We compare results from spherical and Cartesian models in the same parameter regime in order to study whether restricted geometry introduces artefacts into the results. Methods. We employ direct numerical simulations of turbulent convection in spherical and Cartesian geometries. In order to alleviate the computational cost in the spherical runs and to reach as high spatial resolution as possible, we model only parts of the latitude and longitude. The rotational influence, measured by the Coriolis number or inverse Rossby number, is varied from zero to roughly seven, which is the regime that is likely to be realised in the solar convection zone. Cartesian simulations are performed in overlapping parameter regimes. Results. For slow rotation we find that the radial and latitudinal turbulent angular momentum fluxes are directed inward and equatorward, respectively. In the rapid rotation regime the radial flux changes sign in accordance with earlier numerical results, but in contradiction with theory. The latitudinal flux remains mostly equatorward and develops a maximum close to the equator. In Cartesian simulations this peak can be explained by the strong 'banana cells'. Their effect in the spherical case does not appear to be as large. The latitudinal heat flux is mostly equatorward for slow rotation but changes sign for rapid rotation. Longitudinal heat flux is always in the retrograde direction. The rotation profiles vary from anti-solar (slow equator) for slow and intermediate rotation to solar-like (fast equator) for rapid rotation. The solar-like profiles are dominated by the Taylor-Proudman balance.