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.

Operatiivisen tilaus- ja toimitusprosessin kehittäminen sesonkityyppisessä liiketoiminnassa

Tämän tutkielman tutkimusongelmana on operatiivisen tilaus-toimitusprosessin ominaispiirteiden nimeäminen sekä kyseisen prosessin kehittäminen sesonkityyppisessä liiketoiminnassa. Tutkimuksen apuna käytetään prosessin uudelleensuunnittelun keskeisiä teorioita. Tutkielman tavoitteena on luoda viitekehys, jota voidaan soveltaa kehitettäessä tai uudelleen suunniteltaessa tilaus-toimitusprosessia erityisesti sesonkityyppisen liiketoiminnan parissa. Empiirisessä osuudessa testattiin viitekehyksen soveltuvuutta käytäntöön todellisen kohdetapauksen avulla. Tässä tutkimuksessa kehitettävänä oli toimeksiantajayrityksen Oyj Stockmann Abp:n ja sen yhteistyökumppanin Itella Logistics Oy:n välinen tilaus-toimitusprosessi. Useat organisaatiot kehittävät toimintojaan vain omista näkökulmistaan. Tämä johtaa siihen, että toimitusketjun jokainen osa optimoi vain omia toimintojaan ja lopulta asiakkaan tarpeet alkavat unohtua eikä ketjun toimintaa enää kehitetä asiakaslähtöisesti. Tästä syystä tilaus- ja toimitusprosessin kehittäminen tulee perustaa asiakaslähtöiseen prosessin analysointiin. Tässä tutkimuksessa painopistealueita ovat asiakkaalle arvoa tuottavat ja tuottamattomat toiminnot sekä informaation välityksen varmentaminen kaikilla prosessin tasoilla. Seikkaperäinen analysointi vaatii poikkeuksetta prosessin mallintamista ja tämän tueksi laadittavaa prosessia selittävää käsikirjaa. Teoreettisena lopputuloksena on tässä tutkimuksessa syntynyt viitekehys, joka perustuu näihin lähtökohtiin. Empiirisessä osassa todettiin, että tilaus-toimitusprosessia voidaan tehostaa minimoimalla useiden tietojärjestelmien aiheuttamaa ylimääräistä työntekoa. Käytännössä ongelmat ilmenivät järjestelmien rajapinnoissa, jonka takia useat tilaus-toimitusprosessiin liittyvät transaktiot oli syötettävä järjestelmään erikseen. Manuaalinen ja ylimääräinen työsuoritus lisäävät virheiden syntymisen mahdollisuutta, kuluttavat resursseja sekä lisäävät ihmisten työmäärää. Tutkimuksen kirjoittajan näkemyksen mukaan ihanne olisi, jos kaikki transaktiot tehtäisiin ainoastaan kerran yhdessä järjestelmässä. Tämä vaatii kuitenkin informaation vaihdannan lisäämistä, operatiivisten toimintojen uudelleen suunnittelua ja yhteistyön lisäämistä prosessin eri tasoilla.

Genetic polymorphisms and laboratory variables as predictors of blood pressure response to antihypertensive drugs

Hypertension is one of the major risk factors for cardiovascular morbidity. The advantages of antihypertensive therapy have been clearly demonstrated, but only about 30% of hypertensive patients have their blood pressure (BP) controlled by such treatment. One of the reasons for this poor BP control may lie in the difficulty in predicting BP response to antihypertensive treatment. The average BP reduction achieved is similar for each drug in the main classes of antihypertensive agents, but there is a marked individual variation in BP responses to any given drug. The purpose of the present study was to examine BP response to four different antihypertensive monotherapies with regard to demographic characteristics, laboratory test results and common genetic polymorphisms. The subjects of the present study are participants in the pharmacogenetic GENRES Study. A total of 208 subjects completed the whole study protocol including four drug treatment periods of four weeks, separated by four-week placebo periods. The study drugs were amlodipine, bisoprolol, hydrochlorothiazide and losartan. Both office (OBP) and 24-hour ambulatory blood pressure (ABP) measurements were carried out. BP response to study drugs were related to basic clinical characteristics, pretreatment laboratory test results and common polymorphisms in genes coding for components of the renin-angiotensin system, alpha-adducin (ADD1), beta1-adrenergic receptor (ADRB1) and beta2-adrenergic receptor (ADRB2). Age was positively correlated with BP responses to amlodipine and with OBP and systolic ABP responses to hydrochlorothiazide, while body mass index was negatively correlated with ABP responses to amlodipine. Of the laboratory test results, plasma renin activity (PRA) correlated positively with BP responses to losartan, with ABP responses to bisoprolol, and negatively with ABP responses to hydrochlorothiazide. Uniquely to this study, it was found that serum total calcium level was negatively correlated with BP responses to amlodipine, whilst serum total cholesterol level was negatively correlated with ABP responses to amlodipine. There were no significant associations of angiotensin II type I receptor 1166A/C, angiotensin converting enzyme I/D, angiotensinogen Met235Thr, ADD1 Gly460Trp, ADRB1 Ser49Gly and Gly389Arg and ADRB2 Arg16Gly and Gln27Glu polymorphisms with BP responses to the study drugs. In conclusion, this study confirmed the relationship between pretreatment PRA levels and response to three classes of antihypertensive drugs. This study is the first to note a significant inverse relation between serum calcium level and responsiveness to a calcium channel blocker. However, this study could not replicate the observations that common polymorphisms in angiotensin II type I receptor, angiotensin converting enzyme, angiotensinogen, ADD1, ADRB1, or ADRB2 genes can predict BP response to antihypertensive drugs.