Bacillus Cereus Spores and Cereulide in Food-Borne Illness

34/2009 RANAD SHAHEEN RANAD University of Helsinki University Division of Microbiology Division Food-Borne Illness Food-Borne Faculty of Agriculture and Forestry Agriculture of Faculty Department of Applied Chemistry and Microbiology Applied Chemistry Department of Spores and Cereulide in and Cereulide Spores Bacillus cereus Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki Helsingiensis in Dissertationes bioscientiarum molecularium Universitatis

RANAD SHAHEEN Bacillus cereus Spores and Cereulide in Food-Borne Illness 34/2009 e Helsinki 2009 ISSN 1795-7079 ISBN 978-952-10-5846-2 Helsinki 2009 ISSN 1795-7079 ISBN 978-952-10-5846-2 Recent Publications in this Series: in Publications Recent Euro 13/2009 Liliya coli Escherichia I) from (Complex Oxidoreductase in NADH:Ubiquinone Transfer and Proton Electron Greco 14/2009 Dario Genomics to Functional Microarrays From Gene Expression: Gorbikova 15/2009 Elena c Oxidase By Cytochrome Translocation and Proton Oxygen Reduction 16/2009 Mari J. Lehtonen in Finland and Host Respons of Isolates a Potato Pathogen - Variation Rhizoctonia Solani as 17/2009 Kristiina Kaste and Chronic Alcohol Preference Studies in Models of Addiction: and CREB in Drug Factors ∆FosB Transcription Nicotine Exposure 18/2009 Mari K. Korhonen and Their Variants Homologue Mismatch Repair Proteins of MutL Functional Characterization Virtanen 19/2009 Heidi Signalling Studies of GDNF Family Ligand–RET Structure-Function 20/2009 Solveig Sjöblom carotovora subsp. Plant Pathogen Erwinia carotovora Quorum Sensing in the Närvänen Tero 21/2009 Understanding for Enhanced Process Granulation — Tools Particle Size Determination during Fluid Bed 22/2009 Kaisa Nieminen Development Cytokinin Signalling in the Regulation of Cambial 23/2009 Marja Pummila Development in Ectodermal Organ Pathways Role of Eda and Troy Alonen Anna 24/2009 Screening Isomers: Synthesis, Structural Characterization, and UDP-glucuronosyltransferase Glucuronide 25/2009 Päivi Lindholm Activity and Neurotrophic Expression Family: Molecular Structure, Novel CDNF/MANF Protein 26/2009 Jelena Mijatovic System in Mice Dopaminergic Nigrostriatal Role of Ret Signaling in the Regulation of the 27/2009 Mirka Jokela-Määttä in Variation Light Environments: Sensitivity in Different of Rhodopsins for High Visual Molecular Tuning within and between Species Absorbance Spectrum and Opsin Sequence Parkash Vimal 28/2009 Factors and Their Receptors Neurotrophic Tselykh Timofey 29/2009 and of Cell Cycle, Transcription Acting at the Crossroad and Mat1 Vig Analysis of MrpL55, Functional Metabolism Regulation 30/2009 Kirsi Harju 1,3-Dipolar Cycloadditions, Solid- Heterocycles. Five-Membered Synthesis of Nitrogen-Containing and Parallel Methods Phase Techniques, Sipilä Timo 31/2009 Ring Aromatic - for Bioremediation Degradation in Sphingomonas Aromatic Plasmids and Cleavage Genes in Soil and Rhizosphere 32/2009 Jack C. Leo Autotransporters Structural and Functional Studies on Trimeric 33/2009 Päivi Uutela and in Studies of Neurotransmitters Mass Spectrometry Liquid Chromatography-Tandem Their Metabolites in the Brain Bacillus cereus spores and cereulide in food-borne illness

Ranad Shaheen

Department of Applied Chemistry and Microbiology Division of Microbiology Faculty of Agriculture and Forestry University of Helsinki

Academic Dissertation in Microbiology

To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Auditorium 2402, at Viikki Biocenter 3, Viikinkaari 1, on December 18th, 2009 at 12 o’clock noon.

Helsinki 2009 Supervisor: Prof. Dr. Mirja S. Salkinoja-Salonen Division of Microbiology Department of Applied Chemistry and Microbiology University of Helsinki, Finland

Cosupervisor: Dr. Maria A. Andersson Division of Microbiology Department of Applied Chemistry and Microbiology University of Helsinki, Finland

Reviewers: Doc. Dr. Maria Saarela VTT Biotechnology Espoo, Finland.

Prof. Dr. Edward R.B. Moore Department of Clinical Bacteriology University of Göteborg Göteborg, Sweden

Opponent: Dr. Carl E. Cerniglia, Director National Center for Toxicological Research, Food and Drug Administration (FDA) Jefferson Arkansas, USA

ISBN 978-952-10-5846-2 (paperback) ISBN 978-952-10-5857-8 (PDF) ISSN 1795-7079 Yliopistopaino Helsinki, Finland 2009

The Cover image: Field emission scanning electron micrograph (FESEM) taken using the instrument Hitachi S-4800 (Tokyo, Japan) of spores of the Bacillus cereus strain UM 98 isolated from milk of dairy silo tank. For the microscopy, the spores were ﬁ xed with phosphate-buffered glutaraldehyde, dehydrated in an ethanol series and dried in hexamethyldisilazane. In the ﬁ gure, the spores adhered to steel surface. The image was taken by Ranad Shaheen. To my beloved Parents and Family Table of Contents

LIST OF FIGURES LIST OF TABLES LIST OF ORIGINAL PUBLICATIONS THE AUTHOR’S CONTRIBUTION ABBREVIATIONS DEFINITIONS ABSTRACT 1. REVIEW OF THE LITERATURE ...... 1 1.1. Taxonomic view on B. cereus ...... 1 1.1.1. B. cereus group ...... 1 1.1.2. The species B. cereus (sensu stricto) ...... 1 1.2. B. cereus spore ...... 2 1.2.1. Ultrastructure of the Bacillus spore ...... 2 1.2.2. Properties of the Bacillus spores and those of B. cereus spores ...... 3 1.2.3. Sporulation and germination of Bacillus spores ...... 5 1.3. Diversity of B. cereus ...... 6 1.4. Interactions of B. cereus with living and nonliving environment ...... 7 1.4.1. Adhesion of B. cereus to nonliving and living surfaces ...... 7 1.4.2. Antagonistic property of B. cereus towards microorganisms ...... 8 1.4.3. B. cereus as a probiotic ...... 8 1.5. Human virulence ...... 10 1.5.1. B. cereus as a human pathogen ...... 10 1.5.2. Cereulide, the emetic toxin of B. cereus ...... 11 1.5.2.1. Structure and properties of cereulide ...... 11 1.5.2.2. Toxic mechanisms and detection of cereulide ...... 13 1.5.2.3. Toxicity of cereulide in whole animals ...... 14 2. AIMS OF THE STUDY ...... 20 3. MATERIALS AND METHODS ...... 21 4. RESULTS AND DISCUSSION ...... 22 4.1. Strategies for isolating and identifying cereulide producers among strains and isolates of B. cereus ...... 22 4.2. Cereulide productivity of B. cereus isolates of geographically and temporally diverse origins ...... 23 4.3. Occurrence of cereulide producing B. cereus in some environmental niches ..24 4.4. Potential for cereulide production in selected infant food formulas ...... 25 4.5. Strategies of B. cereus spores for persistence in the dairy process environment ...... 33 4.6. Are the cereulide producing B. cereus successful in competition with the non producing B. cereus in food? ...... 34 5. SUMMARY AND CONCLUSIONS ...... 36 6. ACKNOWLEDGEMENTS ...... 38 7. REFERENCES ...... 39 LIST OF FIGURES

Figure 1. Transmission electron micrograph of a sporulating culture of the emetic B. cereus strain F4810/72. The culture was grown for 10 days on tryptic soy agar at 22 °C ...... 2

Figure 2. Structure of cereulide...... 11

Figure 3. Gene cluster for the biosynthesis of cereulide in B. cereus AH187 (F4810/72) carrying the plasmid pCER270...... 19

Figure 4. A ﬂ ow chart for distinguishing the cereulide producing B. cereus isolates from the nonproducers in foods or in environmental samples...... 22

Figure 5. Intraspecies antagonistic activity of B. cereus. For testing the sensitivity, the target strain of B. cereus was plated as a lawn and the potential antagonists were spotted on the lawn. In this ﬁ gure the lawn of B. cereus ML60 was spotted with antagonistic strains F4810/72, B208 and ML30 and the nonantagonistic strain RIVMBC0067. The clear zone around the spotted F4810/72, B208 and ML30 show these were antagonistic towards ML60. .35 LIST OF TABLES

Table 1. Surface characteristics reported for B. cereus spores. The studied strains were the type strain ATCC 14579T, one dairy isolate, 3 isolates from patients with diarrhoeal food poisoning and 2 strains isolated from the environment (Tauveron et al., 2006)...... 3 Table 2. Resistance properties of Bacillus spores ...... 4 Table 3. Factors promoting or inhibiting germination of B. cereus spores ...... 6 Table 4. Reported uses of B. cereus as probiotic ...... 9 Table 5. Cases of B. cereus reported with details of emetic or emetic like food poisoning caused by this species ...... 12 Table 6. Selected biochemical properties of the B. cereus emetic toxin (cereulide) ...14 Table 7. Performance of the published in vitro assays for the emetic toxin of B. cereus (cereulide) ...... 15 Table 8. Essential features of three bioassays used for detecting the toxicity caused by cereulide...... 16 Table 9. Summary of the toxic activities of the emetic toxin, cereulide ...... 17 Table 10. Deduced functions of the proteins encoded by the ces operon of B. cereus strain F4810/72 by sequence homology ...... 18 Table 11. Methods used in this thesis ...... 21 Table 12. Cereulide production by the B. cereus strains and isolates analysed in this thesis. All strains were grown on tryptic soy agar plates for 1 d at 28 ºC. The plate grown biomass was analysed for cereulide by LC-MS and for toxicity by the sperm motility inhibition assay. -, trait is negative; +, trait is positive; w= weak with ≤1 mm zone of hemolysis; nd= not determined ..26 Table 13. The strains and isolates of B. cereus investigated in this thesis and found not to produce cereulide. All strains were analysed with LC-MS and were found to contain less than the detection limit of cereulide (<0.0005 μg mg-1 wet wt) ...... 30 Table 14. Screening for cereulide producers among environmental isolates of B. cereus not connected to illness (one isolate per sampled item). Compilation of strains and isolates described in this thesis and elsewhere. .32 LIST OF ORIGINAL PUBLICATIONS:

I. Svensson, B., Monthan, A., Shaheen, R., Andersson M.A. & Salkinoja-Salonen, M.S. Christiansson A. 2006. Occurrence of emetic toxin producing B. cereus in the dairy production chain. International Dairy Journal 16,740-749. II. Shaheen, R., Andersson, M.A., Apetroaie, C., Schulz, A., Ehling-Schulz, M., Ollilainen V-M., Salkinoja-Salonen M.S. 2006. Potential of selected infant food formulas for production of B. cereus emetic toxin, cereulide. International Journal of Food Microbiology 107, 287-294 III. Shaheen, R., Svensson, B., Andersson M.A., Christiansson, A., Salkinoja-Salonen, M.S. 2009. Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks. Accepted for publication in Food Microbiology. IV. Carlin, F., Fricker, M., Pielaat, A., Heisterkamp, S., Shaheen, R., Salkinoja- Salonen, M.S., Svensson, B., Nguyen-the, C., Ehling-Schulz M. 2006. Emetic toxin producing strains of B. cereus show distinct characteristics within the B. cereus group. International Journal of Food Microbiology 109, 132-138. V. Shaheen, R., Andersson M.A., Pirhonen, T., Jääskeläinen, E.L., Svensson, B., Nguyen-the, C., Salkinoja-Salonen, M.S. 2009. Antagonistic activity and cereulide production of foodborne B. cereus may synergistically contribute to human morbidity. Submitted Aug 17,2009.

THE AUTHOR’S CONTRIBUTION:

Paper I Ranad Shaheen identiﬁ ed and measured the emetic toxin produced by the strains and isolates of B. cereus in the dairy production chain and interpreted the results. Paper II Ranad Shaheen wrote the article, planned and carried out the experimental work. Paper III Ranad Shaheen designed the experiments, interpreted the results and wrote the paper. She was responsible for all experimental work except the phenotypic characterization of the B. cereus isolates, RAPD-PCR and the alkaline and acid resistance analysis of the spores. Paper IV This was a joint European project paper. Ranad Shaheen was responsible for the setup of the emetic toxin producing collection strains and for characterising the toxic properties of these strains. She executed all cereulide analyses in this multi-centre project. Paper V Ranad Shaheen designed the experiments, was responsible for the experimental work and executed most of it, interpreted the results and wrote the paper. ABBREVIATIONS aa amino acid ABC transporter ATP binding cassette in a transporter ADP, ATP adenosine 5`-diphosphate and 5`-triphosphate, respectively ATCC American Type Culture Collection aw water activity AU arbitrary unit bp base pair Caco-2 colon adenocarcinoma cell line CDSs coding DNA sequences CFU colony forming unit DNA deoxyribonucleic acid DPA dipicolinic acid EFSA European Food Safety Authority ESI electron spray ionization EU European Union HeLa human cervical cancer cell line HEp-2 human larynx carcinoma cell line HepG2 human hepato carcinoma cell line 5-HT3 5-hydroxy tryptamine (serotonin) receptor 3 LC-MS liquid chromatography – mass spectrometry log Kow logarithm of the octanol / water partition coefﬁ cient LRIL ligated rabbit ileal loop MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NL The Netherlands NRPS non-ribosomal peptide synthesis O-Ala lactic acid O-Leu 2-hydroxy isocaproic acid O-Val 2-hydroxy isovaleric acid Paju human neural cell line PBS phosphate buffered saline PCR polymerase chain reaction Tris tris-(hydroxymethyl) aminomethane TSB tryptic soy broth SASP small acid-soluble proteins UK United Kingdom USA United States of America VPR vascular permeability reaction

ΔΨm mitochondrial inner membrane transmembrane potential DEFINITIONS depsipeptide a peptide in which one or more of the amide (-CONHR-) bonds are replaced by ester (COOR) bonds dodeca twelve, numerical prefi xes derived from Greek genotype the genetic constitution of a cell or an organism. The genotype is distinct from its expressed features or phenotype infection the pathological state resulting from the invasion of the body by pathogenic microorganisms isolate a taxonomic unit, it means a pure culture that was isolated some time somewhere. non-ribosomal peptide is synthesized by peptide synthetases with no involvement of ribosome. It is made from amino acid precursors (AMP activated amino acid). The precursors and the conversion to hydroxy acid occur only after activation into AMP-amino acids, before or after their incorporation into the peptide. phenotype is any observable characteristic or trait of an organism. species a taxonomic unit, each species consists of one or more strains. The strains of any given species have been properly characterised and fulfi l the defi nition of that specifi c species. strain a taxonomic unit defi ned as the descendants of a single isolate in pure culture and usually made up of a succession of cultures ultimately derived from an initial single colony. toxin biologically produced poisonous compound toxic a substance that damages one or more functions of an organism or of a living cell toxinogenic an organism that produces a toxin virulence refers to the degree of pathogenicity of a given microbe

ABSTRACT

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 favourable 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 it was found that the B. cereus isolates from dairies possessed spores with properties that make them resistant towards cleaning: long survival in hot water with 1% sodium hydroxide (pH > 13), effi cient adherence to steel and to many other surfaces, ability to germinate at + 8 ºC and to form biofi lms in milk. Among the dairy silo isolates were cereulide producers, but these were low in frequency (1.1 %). 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 fi rst 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. Review of the Literature

1. REVIEW OF THE LITERATURE

1.1. Taxonomic view on B. cereus (Helgason et al., 2000; Kolstø et al., 2002). These three species differ in virulence, The genus Bacillus is a large and diverse which is encoded by genes located on group of bacteria belonging to the plasmids recognized as mobile genetic family Bacillaceae, Phylum Firmicutes. elements (Van der Auwera et al., 2007). The species in this genus are aerobic These include the cry gene encoding δ- or facultatively anaerobic, endospore endotoxins of B. thuringiensis, pXO1 forming, rod shaped gram positive bacteria plasmid carrying genes for the anthrax widely distributed in nature, especially toxin complex and the pXO2 encoding in the soil (Harwood, 1989). B. cereus the poly--D-glutamic acid capsule of B. group known as the causative agents of anthracis as well as the positive regulator food-borne illness belongs to Group I of of the virulence factor AtxA located on the genus Bacillus (Gibson and Gordon, pXO1 (Stenfors Arnesen et al., 2008). The 1974). Other species in the genus Bacillus B. cereus emetic toxin genes (ces) are also including B. subtilis (Kramer and Gilbert, present on a large plasmid (Hoton et al., 1989; Shinagawa, 1990), B. licheniformis 2005; Ehling-Schulz et al., 2006; Rasko et (Salkinoja-Salonen et al., 1999), B. al., 2007). thuringiensis (Jackson et al., 1995; Beattie B. anthracis causes the serious human and Williams, 1999) and B. pumilus (From disease anthrax (Mock & Fouet, 2001). et al., 2007) are increasingly recognized as The species B. thuringiensis is an insect food poisoning agents. pathogen which produces insecticidal δ-endotoxins during sporulation and is 1.1.1. B. cereus group commercially used for crop protection The B. cereus group comprises six (Aronson & Shai, 2001). recognised species, B. cereus, B. The species B. mycoides and B. anthracis, B. thuringiensis, B. mycoides, pseudomycoides are phenotypically B. pseudomycoides, B. weihenstephanensis distinguishable from the species B. cereus (Gordon et al., 1973; Lechner et al., sensu stricto by their rhizoidal colony 1998; Granum 2002; Jensen et al., 2003; shape and whole cell fatty acid composition Stenfors Arnesen et al., 2008). In general, (Nakamura, 1998). B. weihenstephanensis species of the B. cereus group have a is the psychrotolerant species within the B. low G+C content of DNA (35%) (Ravel cereus group, characterized by the ability and Fraser, 2005), hydrolyze lecithin and to grow aerobically at 7 C or lower in do not ferment mannitol to acid (Parry agitated liquid culture but not at 43 C, et al., 1983; Fritze, 2004). B. cereus, possessing a signature sequences in the B. thuringiensis and B. anthracis are major cold shock gene cspA and in the 16 members of a single species B. cereus rDNA sequence (Lechner et al., 1998). sensu lato. They are genetically closely related based on genome sequence data 1.1.2. The species B. cereus (sensu both in the gene content and in synteny stricto) (Helgason et al., 2000; Rasko et al., The species B. cereus is a gram positive 2004; Didelot et al., 2009) with 99% rod-shaped bacterium generally 0.9 similarity of the 16S rRNA gene sequence to 1.2 m by 2 to 4 m. The spore is

1 Review of the Literature ellipsoidal, central or paracentral, rarely 1.2. B. cereus spore distending the sporangia (Gordon et al., 1973). B. cereus is widely distributed in 1.2.1. Ultrastructure of the Bacillus the habitats like soil (Vilain et al., 2006, spore Jensen et al., 2003), water (Østensvik All Bacillus species can form heat stable et al., 2004; Jensen et al., 2003), foods endospores (Harwood, 1989; Henriques (Stenfors Arnesen et al., 2008) as well as and Moran, 2007). The bacterial the mammalian and insect guts (Jensen et endospore is a resting, dormant, tough, al., 2003). B. cereus species grows from non reproductive structure and it is the 4 to 55 C (Roberts et al., 1996) at pH of most resistant living structure known 4.3 to 9.3 and requires a minimum water (Atrih and Foster. 1999). Endospores activity (aw) of 0.93 (Forsythe, 2000). formed by bacillus and related aerobic Three amino acids L-valine, L-leucine endospore-forming Firmicutes are a and L-threonine, are essential to B. cereus strategy to survive during unfavourable growth (Agata et al., 1999). Its growth is conditions. inhibited by lactoferrin at a concentration The structures of the mature spore of 1000 μg/ml (Sato et al., 1999). B. cereus are: 1. The core which is the analog species have phenotypes differentiating it of the vegetative cell protoplast as it from the other members of the B. cereus contains DNA, ribosomes, tRNA and a groups: Cells of B. cereus (sensu stricto) high concentration of dipicolinic acid are motile, do not have parasporal crystals, (DPA) and of Ca+2 (Setlow, 2006). It are resistant to penicillin and to gamma contains only 25 – 50% of water, i.e. the phage (Gordon, 1973; Granum, 2007; content of free water is extremely low Apetroaie et al., 2005). such that the macromolecular movement is greatly restricted (Cowan et al., 2003). 2. The germ cell wall is composed of

Inner membrane Inner spore coat Core Cortex Outer membrane Outer spore coat

Exosporium Figure 1. Transmission electron micrograph of a sporulating culture of the emetic B. cereus strain F4810/72. The culture was grown for 10 days on tryptic soy agar at 22 °C. Courtesy of Maria A. Andersson. The spore parts are named as described in F. Mayer (1999).

2 Review of the Literature

Properties Range Length of exosporium 2-3.2 μm Number of appendages 1-23 per spore Length of appendages 0.6-2 μm Length of the hair like fi laments (nap) of glycoprotein 27-35 nm peptidoglycan identical to that of the many other bacillus species (Hachisuka vegetative cell (Setlow, 2006). 3. The et al., 1984). The appendages consist of cortex consists of a peptidoglycan which protein as the main part together with a is different from the vegetative cell small amount of carbohydrate and lipid peptidoglycan (Atrih and Foster 1999). (Kozuka and Tochikubo 1985). There was The cortex is essential for the formation a large difference in the protein profi les of of a dormant spore and for the reduction the appendages of different strains of B. of its water content (Andersson, 1998). cereus (Stalheim and Granum, 2001) and a 4. The spore coat complex consisting of variation in the surface characteristics of B. several layers of different proteins, mostly cereus spores between strains (Andersson spore-specifi c and important for resistance and Rönner, 1998; Husmark and Rönner, towards chemicals and lytic enzymes 1990; Hachisuka et al., 1984; Tauveron et (Setlow, 2006). 5. The exosporium al., 2006) (Table 1). which is a loose fitting balloon-like structure (Yan et al., 2007). The B. 1.2.2. Properties of the Bacillus cereus exosporium contains more than 20 spores and those of B. cereus spores proteins (Steichen et al., 2003; Todd at al., Several properties reported for the spores 2003), amino and neutral polysaccharides, of B. cereus make them a problem for the lipids and ash (Matz et al., 1970). Alanine food industry. B. cereus spores are highly racemase protein is a major component of resistant to adverse conditions such as the exosporium of B. cereus spores (Yan et heat, dehydration, desiccation, radiation, al., 2007). It converts reversibly L-alanine disinfectants and cleaning agents to D-alanine (Steichen et al., 2003; Todd (Table 2). The spores of B. cereus are et al., 2003). The structures of the spores hydrophobic and adhere to the processing of the B. cereus emetic strain F4810/72 equipment and subsequently form biofi lm are shown in Figure 1. (Andersson et al., 1995, Peng et al., 2001). The exosporium of the Bacillus The resistance properties reported for the spores is surrounded by a hair like spores of B. cereus are also a problem for external protein layer. It has been a human health. B. cereus spores are highly suggested that these proteins are tightly resistant to acidity in a range of media absorbed on spore surface after the cell simulating the conditions in the human lyses or are included between the coat stomach after food ingestion. The decrease and the exosporium (Charlton et al., 1999; in the spore counts was less than 1.5 log Todd et al., 2003). B. cereus spores are CFU ml -1 after 6 h of incubation at pH 1 covered with appendages not present in and 1.5 (Clavel et al., 2004).

3 Review of the Literature

The conditions prevailing during the spores. Dipicolinic acid (pyridine-2, sporulation: the temperature (Gonzales 6-dicarboxylic acid) is responsible for et al., 1999) and the composition of the the reduction of the spore core water sporulation medium (De Vries et al., 2005) content during sporulation and for the UV affect the properties of the formed spores. photochemistry of the spore DNA. This The heat resistance of the B. cereus spores molecule comprises ~5 to 20% of the dry increases with the increase in sporulation weight of Bacillus spores. It is chelated temperature (Gonzalez et al., 1999). B. with divalent cations, mainly Ca+2 (Setlow cereus spores showed higher survival at 2007). The small acid-soluble proteins 90 C when the spores were produced at (SASP) in the spore core play an important 37 C as compared to 15-20 C (Gounina- role of spore resistance. SASP proteins Allouane et al., 2008). (α, β) represent 5-10 % of the total core Spores of B. cereus have extreme protein which is sufﬁ cient to saturate and metabolic dormancy with respiratory protect the spore DNA (Setlow and Setlow activity of low as 10-4 of the maximum rate 1995, Setlow 2007) especially against UV for vegetative cells metabolizing substrate radiation. SASP also play a role in the (Andersson, 1998). osmoresistance of spores (Ruzal et al., Several components are important 1994; Tovar-Rojo et al., 2003). for the resistance properties of the Table 2. Resistance properties of Bacillus spores Property Due to Resistance to heat wet heat low water content of spores. mineral ions. stability of spore proteins. saturation of the spore DNA with α/β type SASP.

dry heat saturation of the spore DNA with α/β type SASP. Resistance to radiation -radiation low core water content.

UV radiation photochemistry of the DNA in spores, the spore photoproducts (SP) generated in the spores upon exposure to irradiation are less lethal than other photoproducts and are repaired in the ﬁ rst minute of spore outgrowth. low water content in the spore core. high level of DPA in the spore core. binding of α/β SASP to spore DNA. Resistance to disinfectants (chlorine dioxide, hypochlorite, spore coat proteins detoxifying the disinfecting ozone, peroxynitrite, hydrogen chemicals (e.g CotA laccase protects against peroxide) hydrogen peroxide) impermeability of the spore inner membrane saturation of DNA with α/β type SASP DNA repair mechanism. Resistance to lysozyme not yet known whether the outer coat functions are the main barrier against lysozyme or if the inner coat layer is essential for the resistance to lysozyme. Compiled from Henriques and Moran. 2007; Setlow 2006

4 Review of the Literature

1.2.3. Sporulation and germination of the spores (Atrih et al.,1998). The spore Bacillus spores core rapidly takes up water so that the core Sporulation involves asymmetric cell water content rises to that in the protoplast division with a copy of the genome of growing cells and the macromolecular partitioned into each of the sister cells. motion and enzyme activity in the core The smaller cell develops into the mature are restored. Factors reported to affect endospore and the mother cell contributes the germination of B. cereus spores are to the differentiation process of the compiled in Table 3. endospore and then autolyses releasing the In B. cereus L-alanine and the purine mature spore into the environment (details ribonucleoside inosine are effective of the sporulation process reviewed by germination-promoting compounds Henriques and Moran 2007). It takes (Gounina-Allouance et al., 2008) and approximately 6 h for the process of D- alanine is an effective inhibitor of L- spore formation of B. cereus to complete alanine-induced germination. The most (Henriques and Moran 2007). rapid germination of B. cereus spores was Germination is a non-log-linear event. observed in a mixture of 0.1 mM L-alanine Some spores form vegetative cells within and 0.1 mM inosine. B. cereus spores failed 2 hours, others only after many hours or to germinate in minimum salts medium even days. For 12 B. cereus strains tested, with glucose plus yeast extract in 0.1 mM 2 out of the 10 strains did not germinate inosine (Warren and Gould 1968). Other and the maximum spore germination was germinants have also been identiﬁ ed, like obtained after 100 min with no additional L-phenylalanine, L-glutamine, a mixture germination was observed up to 160-200 of L-asparagine, glucose, fructose and K+. min (Broussolle et al., 2008). Germination Some mammalian cells like Caco-2 cells occurs without need for synthesising any were reported to induce germination of new macromolecules and all the needs enterotoxigenic B. cereus spores whereas are present in the mature dormant spores HEp-2 cells did not trigger germination (Moir 2006). (Wijnands et al., 2007). In the process of germination, A number of germinantion receptors substances acting as germinants permeate are present in the spores of B. cereus. the outer coat and cortex layers of the B. cereus type strain ATCC14579 spore spores and interact with receptors located may contain seven functional receptors in the inner spore membrane (Hudson et (Hornstra et al., 2006). gerP- encoded al., 2001; Paidhungat and Setlow 2001; protein of B. cereus is believed to be Moir 2006). Then compounds such as important in establishing a coat that is monovalent cations (H+, Na+, K+), divalent permeable to germinants (for details see cations (Ca+2, Mg+2, Mn+2) and DPA are Moir, 2006). Variability of response to released from the spore core (Moir, 2006). inosine or to L-alanine was observed The germ cell wall becomes the bacterial between spores of B. cereus. Some cell wall when the spore germinates. The strains can germinate at low germinant release of Ca-DPA triggers the hydrolysis concentration (i.e. 0.05 mmol L-1) of the spore´s peptidoglycan cortex by (Broussolle et al., 2008). activating the cortex lytic enzymes (Moir, Mild preheating activates spores to 2006). Hydrolysis of the peptidoglycan is germinate, in the presence of germination required for germination and outgrowth of permissive environment. Optimal heating

5 Review of the Literature

Table 3. Factors promoting or inhibiting germination of B. cereus spores Factor Effects Reference Heat activation Heat activation at 75 C for 30 min is the optimum Warren and Gould temperature which allows a rapid rise in germination 1968 in L-alanine + inosine or L-alanine +O-carbamyl-D- serine medium. pH of the medium The effect of pH on germination depends on the Broussolle et al., for germination germinants: 2008

In inosine (1mmol L-1) B. cereus spore germination decreased with a decreasing pH from 7.5 to 3.8. Some strains showed no germination at pH 6.8 and very low or no germination at pH 3.8 for all strains.

In L- alanine (100 mmol L-1) B. cereus spore germination decreased with a pH decreasing from 7.5 to 3.8. NaCl In L-alanine, most of the B. cereus spores germinate Broussolle et al., less efﬁ ciently with the increase of NaCl from 0.06 2008 % to 5 % Sporulation tem- The spores of B. cereus produced at 15 C and 20 C Gounina-Allouane perature showed a higher germination capacity in response to et al., 2008 inosine at concentrations between 0.01 and 10 mmol L-1and L-alanine at concentrations between 1 and 100 mmol L-1 than the spores produced at 37 C. Composition of Germination response enhanced by increasing the Hornstra et al., the sporulation expression of the B. cereus ger operon when B. 2006 medium cereus spores were produced in nutrient rich medium (30 mM amino acid and 10 mM glucose) compared to a medium containing low amounts of nutrients (14 mM amino acid and no glucose). Bacteriostatic Bovicin HC5 is a small spore forming peptide pro- De Carvalho et al., agent duced by Streptococcus bovis HC5. In a concentra- 2007 tion of 80 AU ml-1 bovicin HC5 reduced the outgrowth of B. cereus spores. AU: one arbitrary unit was deﬁ ned as the reciprocal of the highest dilution that showed a zone of inhibition against L. lactis ATCC 19435 with at least 5 mm in diameter. temperature depends on the sporulation 1.3. Diversity of B. cereus temperature. B. cereus spores that were formed at 37 ºC require 80-90 ºC heat B. cereus is widespread in the environment shock for activation, whereas those and possesses a wide range of habitats. formed at room temperature, need only The diversity of B. cereus ranges from heat shock of 70-75 º C (Becker et al., strains used as probiotics to strains 2005). Gamma-radiation, reducing agents that produce virulence factors causing such as thioglycolate or mercaptoethanol serious disease. Most B. cereus strains and oxidizing agents also may activate the produce one or several heat labile protein spores (Andersson 1998). enterotoxins but some strains produce the

6 Review of the Literature heat stable toxin, cereulide. Studies of producing B. cereus were weakly the intraspecies diversity of B. cereus by haemolytic while others were nonhemolytic genetic [M13-PCR, random amplifi cation on sheep blood agar, positive or negative of polymorphic DNA (RAPD), multilocus for tyrosine decomposition, positive or sequence typing (MLST), ribotyping] negative for lecithinase, exhibited different and phenetic [Fourier transform infrared, ribopatterns and showed variation in the (FTIR), protein profi ling and biochemical housekeeping gene adk. Furthermore, typing assays] have shown that B. a new phylogenetic cluster was found cereus strains other than the cereulide among cereulide producing strains of B. producing strains, have a high degree of cereus using multilocus sequence typing heterogeneity (Ehling-Schulz et al., 2005a; (Vassileva et al., 2007; Hoton et al., Vassileva et al., 2007; Svensson et al., 2009). 2004; Andersson et al., 1999; Pirttijärvi et al., 1999; Guinebretière et al., 2008). B. 1.4. Interactions of B. cereus with living cereus isolates from the dairy production and nonliving environment chain, food, soil and other environments have a higher degree of heterogeneity as 1.4.1. Adhesion of B. cereus to compared to clinical isolates (Helgason et nonliving and living surfaces al., 2000; Ehling-Schulz et al., 2005a). B. cereus spores are the most adhesive and Cereulide producing B. cereus was hydrophobic among Bacillus spp. spores initially suggested to represent a specifi c (Rönner et al., 1990). Spores of the B. class of B. cereus (Agata et al., 1996) cereus type strain adhere nonspecifi cally because this class deviated in several to several types of industrially used traits from the cereulide non producers: materials (Hornstra et al., 2007). The weak hemolysis on sheep blood agar, adhesion of B. cereus spores to non living negative for hydrolysis of starch and surface is due to three characteristics: for fermentation of salicin, negative for The higher hydrophobicity of the spore tyrosine decomposition but positive for surface; the low negative spore surface lecithinase (Agata et al., 1996, Shinagawa charge and the presence of appendages 1993, Pirttijärvi et al., 1999, Andersson on the spore surface (Rönner et al., 1990; et al., 2004) and possessing specific Tauveron et al., 2006). ribopatterns (Pirttijärvi et al., 1999). B. cereus spores were found to Several genotype properties grouped the adhere to human cells, shown for Caco- cereulide producing isolates (Ehling- 2 and HEp-2 (A. Andersson et al., 1998; Schulz et al., 2005a; Priest et al., 2004, Wijnands et al., 2007) indicating that Ash and Collins, 1992; Vassileva et al., ingested spores may adhere the intestinal 2007). It was proposed that these belong epithelium (Granum, 2007). The spore to a virulent clone of B. cereus recently coat associated proteins are involved in emerged within the Bacillus population the adhesion of B. cereus spores to Caco-2 (Ehling-Schulz et al., 2005a; Priest et al., cells (Sánchez et al., 2009). The adhesion 2004, Ash and Collins, 1992; Vassileva of enterotoxin producing vegetative B. et al., 2007). However, phenotype and cereus cells to Caco-2 cells may contribute genotype diversity exist even within the to the severity and persistence of B. cereus cereulide producer of B. cereus. Apetroaie causing food poisoning (A. Andersson et et al. (2005) found that some cereulide al., 1998).

7 Review of the Literature

1.4.2. Antagonistic property of B. and for human (Duc et al., 2004, Sánchez cereus towards microorganisms et al., 2009) (Table 4). The antagonistic interaction between The interest of using probiotics as bacteria was described early in 1877 animal feed supplements has increased in when Pasteur and Joubert noticed that recent years due to the increased number some E. coli strains interfered with the of antibiotics banned from animal use growth of B. anthracis present in infected as growth promoters in the European animals (cited by Oscáriz and Pisabarro Union in 2006 (Council of the European 2001). B. cereus produces bacteriocins Union, 2003). Spore forming bacteria are with antagonistic action towards other technically easy to use as probiotics for organisms. E.g. zwittermicin (ZmA) animal feed because the spores survive produced by B. cereus strain UW85 has a storage at ambient temperature as well as broad spectrum of activity towards plant heating during the making of animal feeds. pathogenic fungi and bacteria (Stabb B. cereus var. toyoi is licensed by the et al., 1994; Handelsman et al., 1990; European Food Safety Authority (EFSA) Kevany et al., 2009). Cerein 7A is a to be used as animal feed supplement. bacteriocin produced by B. cereus Bc7. It B. cereus Bactinsubtil, B. cereus is 3949 Da in size, sensitive to proteolytic var. vietnami (Subtyl) and B. cereus enzymes and active against gram positive (Biosubtyl DL) are three commercially but not gram negative bacteria (Oscáriz available probiotic strains (Hoa et al., et al., 1999). It is produced at the end of 2000) for human use. These strains were the exponential growth phase but before reported to have several advantages for sporulation and it is a membrane active use as a probiotic: they persist in the compound which is highly hydrophobic gastrointestinal tract and the spores are (Oscáriz and Pisabarro 2000). immunogenic (Duc et al., 2004). However, it was found that Biosubtyl DL and 1.4.3. B. cereus as a probiotic Bactinsubtil produce the HbL enterotoxin Probiotics are live organisms that, when and the nonhaemolytic enterotoxin Nhe administered in adequate amounts, confer are produced by Biosubtyl DL and Subtyl a health benefit on the host (Quoted indicating that they are not necessarily safe from: FAO/WHO 2002). They improve for human use (Duc et al., 2004). B. cereus the efficiency of feed, protect against IP5832 (Bactinsubtil) has been used as a infectious disease (Zani et al., 1998), human probiotic for many years but it is enhance the host immune responses or now banned in Europe. Nevertheless, it inhibit tumor growth in animal models is still sold in other countries all over the (cited from De Los Santos et al., 2005). world (Duc et al., 2004; Sánchez et al., Of the 305 species in the genus 2009). Bacillus (DSMZ web site) B. cereus The uses of B. cereus as probiotics belongs to the few that are used as raise a big question of human safety. probiotics. Probiotic B. cereus are used Reports of infection connected to the as animal feed supplements (Zani, et al., probiotic consumption include members 1998; Alexopoulos et al., 2001; De Los of the genus Bacillus and production Santos et al., 2005; Taras et al., 2005; of enterotoxin has been reported. The Lodemann et al., 2008; Schierack et al., single dose of spores probiotics is up to 2009), for aquaculture (Ravi et al., 2007) 109 spores/g or 109 spores/ml (cited from

8 Review of the Literature

Table 4. Reported uses of B. cereus as probiotic Probiotics Comments Quoted by For human use Bactisubtil from (B. cereus Used for oral bacteriotherapy Hoa et al., 2000; Duc et IP 5832, Marion Merrell S.A. and bacterioprophylaxis of al., 2004 Bourgoin-Jallieu, France). gastrointestinal disorders in Biosubtyl DL (National Institute humans. of Vaccines and Biological Substances, Da Lat, Vietnam). Subtyl (B. cereus var.vietnami) (Pharmaceutical Factory 24, Ho Chi Minh City, Vietnam). For veterinary use (animal feeds) B. cereus DQ915582 This strain was isolated from a Ravi et al., 2007 marine sediment and was found to be effective in inhibiting the larval pathogens of shrimps. B. cereus Cen Biot Reported to reduce the Zani et al., 1998 prevalence of diarrhoea and to improve feed conversion and weight gain in pigs. B. cereus var. toyoi (Toyocerin®) Licensed by the European Food Taras et al., 2005; De Safety Authority (EFSA) to be Los Santos et al., 2005; used as a supplement to animal Lodemann et al., 2008 feed. Does not secrete toxin. Improved feed effi ciency in broilers. Induces an increase of lactobacilli in the duodenum or in the caecum and reduction of E. coli or of enterococci in the intestine or faeces. Decreases the incidence of diarrhoea and the mortality in piglets. Pacifl or (B. cereus CIP 5832) Shown to be benefi cial for the Alexopoulos et al., 2001, survival and growth of piglets. Duc et al., 2004 Withdrawn from production due to the presence of Nhe enterotoxin.

Duc et al., 2004). This is higher than 2000). Furthermore, the species B. cereus the infective dose of the pathogenic B. is closely related to B. anthracis (Didelot cereus for diarrhea (105- 107 spores per et al., 2009). The emetic toxin producing consumption). There is at least one report strains of B. cereus and the B. anthracis on vegetative cells and spores of Bacilli have their pathogenic determinants detected in mesenteric lymph nodes and located on plasmids. A case in which B. spleen of a mouse after giving bacillus cereus acquired toxin genes from one of spores as a probiotic (Spinosa et al., the anthracis plasmids (Hoffmaster et

9 Review of the Literature al., 2004) proves that plasmid exchange (Takabe and Oya, 1976, Mahler et al., between these close species and strains 1997, Pirhonen et al., 2005, Dierick et al., occurs in nature. Thus, using B. cereus 2005, Pósfay-Barbe et al., 2008) (Table 5). as a probiotic can be a problematic and In addition to cereulide, the second toxin needs special attention. Lack of virulence of B. cereus with known connection to traits in any specifi c strains of B. cereus fatal poisoning in human is cytotoxin K should be checked carefully before any (Lund et al., 2000). recommendation for the use as a probiotic. Health authorities have not declared B. cereus food poisoning as a reportable 1.5. Human virulence disease in any country and therefore its incidence is underreported. Food poisoning 1.5.1. B. cereus as a human pathogen due to B. cereus may be misdiagnosed due B. cereus belongs to the Hazard group to the similarity of symptoms with other 2 organisms as defi ned in the European types of food poisoning, for example legislation (European Commission, 1993). Staphylococcus aureus intoxication and In 1950 B. cereus was fi rst recognized to Clostridium perfringens (Shinagawa, cause food borne illness. S. Hauge isolated 1990). Patients with diarrhoeal or emetic in Norway in 1955 B. cereus, inoculated it syndromes usually do not seek medical into a sterile vanilla sauce to 106 cfu per care due to the short duration of the ml and consumed it (Hauge, 1955). Severe symptoms (< 24 h) (Granum, 2007) which abdominal pain, diarrhoea and rectal adds to underestimation of the reported tenesmus followed 13 h after consuming rate of the illness caused by B. cereus. of the contaminated sauce. This allowed Furthermore B. cereus may have been Hauge to describe B. cereus as a causative involved in in the unclarified outbreaks agent of food poisoning (Hauge, 1955). involving heated food from which no B. cereus is the aetiological agent viable bacteria could be isolated. of two major types of foodborne illness, In Finland the causative agent of 19 the emetic syndrome and the diarrhoeal outbreaks out of 55 in year 2005 remained syndrome. The diarrhoeal syndrome unidentified (Niskanen et al., 2007). In involves diarrhea and abdominal pain most countries (for instance UK, NL and initiating 8 h to 16 h after the ingestion USA) > 90% of the registered outbreaks of B. cereus in food, followed by remain unclear, i.e. the causative agent is toxin production in the small intestine not identifi ed (Pirhonen, 2009). In Finland (Kramer and Gilbert, 1989; Granum and this percentage is exceptionally low (40%, Lund 1997). The emetic syndrome is i.e. >50% of the registered outbreaks are characterized by nausea and vomiting 1 h clarifi ed). to 5 h after the ingestion of the heat-stable Several other virulence factors have emetic toxin cereulide, preformed in the been found in B. cereus contributing to the food (Granum and Lund, 1997; Beecher, virulence of this species: phospholipase- 2002). The dose of the emetic toxin, C which gives the capability to hydrolyse cereulide, causing acute serious illness in lecithin, the major component of the human was reported as  8 g of cereulide mammalian cell membrane (Gilmore et per kg of body weight (Jääskeläinen al., 1989), cereolysin AB and haemolysin et al., 2003b). The outcome of emetic BL which contribute to the haemolytic food poisoning may be serious and fatal activity of B. cereus (Beecher, 2002) and

10 Review of the Literature tyrosin monooxygenase responsible for These strains contained a homologue of the formation of melanins protecting the the B. anthracis pXO1-encoded PA gene, cells and spores of B. cereus from various pagA, but not the pXO2-encoded poly-D- environmental factors (Claus and Decker, glutamic acid capsule biosynthetic genes 2006). Beecher et al. 1995 reported that (Hoffmaster, et al., 2004). hemolysin BL produced by B. cereus contributes to the virulence for B. cereus 1.5.2. Cereulide, the emetic toxin of endophthalmitis. B. cereus B. cereus has been implicated in a variety of local and systemic 1.5.2.1. Structure and properties of infections in immunocompetent and cereulide immunocompromised hosts where B. Cereulide is a small cyclic nonribosomally cereus was the only microrganisms synthesized dodecadepsipeptide, 1152 identified: pneumonia, endocarditis, Da in size (Fig 2). It consists of three meningitis, periodontitis, osteomyelitis, repeating units of two amino acid residues wound infection, necrotizing fascilitis and and two hydroxy acids (D-O-Leu, D-Ala, myonecrosis, septicemia and infection of L-O-Val and L-Val)3 (Agata et al. 1994; the central nervous system of term and Isobe et al., 1995). Cereulide has been preterm neonates (Hilliard et al., 2003; reported produced also by isolates of B. Miller, 1997; Kotiranta et al., 2000; Gaur weihenstephanensis (Thorsen et al., 2006; et al., 2001; Schoeni and Wong 2005; Hoton et al., 2009). Hoffmaster, et al., 2004; Sada et al., The chemical structure of cereulide 2009; Nishikawa et al., 2009; Lebessi et resembles that of valinomycin, [-D-O- al., 2009). B. cereus strain G9241 was Val-D-Val-L-O-Ala-L-Val-]3 produced by isolated from the sputum and the blood of Streptomyces fulvissimus, Streptomyces a patient with life-threatening pneumonia. tsusimaensis and Streptomyces griseus

Figure 2. Structure of cereulide. Courtesy of prof. M.S. Salkinoja-Salonen and Raimo Mikkola

11 Review of the Literature Okahisa et Fricker et al., 2007 al., 2008 Pósfay- Barbe et al., 2008 Reference et al., Takabe 1976 Mahler et al., 1997 Hormazábal et al., 2004 Pirhonen et al., 2005; Jääskeläinen et al., 2003b Dierick et al., 2005 Fricker et al., 2007 ciency and prolonged seizures. fi ces ) gene sequence. ces ) gene sequence. . B. cereus was isolated from the food remnants, patient ileum and colon. B. cereus was isolated from remnants of the food. B. cereus c for the ces gene. Toxicity was detected by HEp-2 cytotoxicity assay in the remaining food.  Toxicity 346 persons suffered from food poisoning caused by cereulide.  346 persons suffered 9 years old girl developed fulminant hepatitis, renal and pancreatic insuf  A  No toxicity assay was carried out. An outbreak with 50 affected. An eleven years old boy died with mycocardial degeneration and fatty liver.  An outbreak with 50 affected.  Symptoms started 1-6 h after ingestion.  Acute gastroenteritis. was isolated from the intestine, peritoneal exudate, cooked food and a sheet with patient’s  B. cereus vomit. The son died after developing a fulminant liver failure.  Acute gastroenteritis to father and 17 year old son.  The symptoms started 30 min after ingestion.  Emetic  Emetic toxin was detected in the remaining food, bile, plasma and intestinal content of patient by HEp-2 cell vacuolation, HepG2 and rat liver mitochondrial assays. The clinical symptoms and microbiological examination linked the cases with cases of food borne illness.  Two emetic toxin produced by adults with severe food poisoning involved both emesis and diarrhoea.  Two  Emetic of the remaining food was detected by sperm bioassay.  Toxicity  Cereulide was measured in the remaining food by LC-MS method. fatal for the 7 years old girl who died due to severe metabolic acidosis and liver  Five children of a family, failure.  The symptoms (vomiting) started 6 h after ingestion. was detected in the gut and spleen of patient.  B. cereus  17 children in a day care centre. possessing the ces gene was detected in remaining food using real time PCR assay with  B. cereus primers derived from the cereulide synthetase ( Toxicity was detected by HEp-2 cytotoxicity assay in the remaining food.  Toxicity  Single case. possessing cereulide synthesis genes was detected in the remaining food using real time PCR assay  B. cereus using primers derived from cereulide synthetase (  The symptoms appeared 2 h after ingestion. possessing cereulide synthesis genes was detected in the remaining food using PCR with primers  B. cereus speci fi reported with details of emetic or like food poisoning caused by this species B. cereus gs ower fl paste covered with sticky rice cake with sauce spaghetti and pesto of pasta with minced meat dish Japan sweet red bean Geneva reheated pasta Japan Noodle Switzerland reheated NorwayFinland dried fi reheated dish Belgium Pasta salad Germany reheated rice Country Food involved Description of the case Germany Cauli Table 5. Cases of Table

12 Review of the Literature

(Agata et al., 1994, Andersson et al., factor (Turnbull et al., 1979). Research 1998b; Mikkola et al., 1999, Makarasen on the emetic toxin was hampered by et al, 2009). Cereulide is a potassium ion the fact that rodents are insensitive to selective ionophore, a highly lipophilic the orally given toxin (Yokoyama et al., molecule (log Kow ~6.0, Teplova et al., 1999). Therefore primates were needed 2006) and therefore likely to be efﬁ ciently for each test until Hughes et al. (1988) absorbed from the gut into the blood developed an in vitro assay based on stream (Paananen et al., 2002). Cereulide the vacuolisation of the human larynx does not haemolyse rabbit or sheep carcinoma cells (HEp-2 cells). These erythrocytes and has no phospholipase C authors tested samples connected to food or any other enzymic activity (Shinagawa poisoning and cultured isolates connected at al., 1995). Cereulide is highly heat to food poisoning in rice. They noticed stable (121 C for 90 min), stable upon that the extracts obtained from some exposure to pH from 2 to 11 and to the isolates caused vacuoles in the HEp-2 proteolytic activity of pepsin and trypsin cells. Sakurai et al. 1994 observed that the (Table 6) therefore it may be found in food vacuoles formed in the HEp-2 cells were even when no live B. cereus is found due swollen mitochondria. to heating of food (Pirhonen et al., 2005; Agata et al. (1994) extracted and Jääskeläinen et al., 2003b). purified the factor which causes the Cereulide begins to accumulate in the vacuolation in HEp-2 cells from the B. cereus cultures in the stationary phase culture supernatant of B. cereus strain of the growth independent of sporulation NC7401 connected to a case of emetic (Häggblom et al., 2002). No cereulide was syndrome food poisoning and named the produced by B. cereus in TSB at 10 ± 1 ºC toxin cereulide. Shinagawa et al., 1995 (Häggblom et al., 2002) or by plate grown and Agata et al. (1995) found that the cells at ≤ 8 ºC (Thorsen et al., 2009). factor causing vacuolisation of HEp-2 cells also caused vomiting when fed to 1.5.2.2. Toxic mechanisms and detection rhesus monkey (Macaca mulatta) and the of cereulide house musk shrew (Suncus murinus) and In the 1970’s, B. cereus was recognized as concluded that this was the emetic toxin. the causative agent of a speciﬁ c type of B. Cereulide was chemically synthesized by cereus foodpoisoning which is the emetic Isobe et al., 1995 and shown to possess syndrome. Rhesus monkey was used as the same emetic and pathogenic activities. the experimental animal to demonstrate Andersson et al. (1998a) developed a that the heat stable toxin produced by B. bioassay based on loss of the motility of cereus isolates from emetic outbreaks boar spermatozoa upon 24 h exposure to was associated with the emetic syndrome the toxin. Finlay et al. (1999) developed (Melling et al., 1976). The monkey the metabolic staining assay MTT using feeding test and ligated rabbit ileal loop as an indicator 3-(4,5-dimethylthiazol-2- (LRIL) methods were used with success yl)-2,5-diphenyltetrazolium bromide, a to show that the factors responsible for water soluble yellow tetrazolium salt. This the vomiting and the diarrhoeal illnesses salt was converted to an insoluble purple were distinct. The LRIL and the vascular formazan (MTT) in HEp-2 cells but not permeability reaction (VPR) methods in cells exposed to the emetic toxin. This were found to be of value for studying allowed detecting the cytotoxicity of the diarrhoeal toxin but not the vomiting cereulide towards the HEp-2 cells.

13 Review of the Literature

Table 6. Selected biochemical properties of the B. cereus emetic toxin (cereulide) Properties Condition Reference pH stability pH 2 for 2 h at room temperature Mikami et al., 1994; Melling and Capel 1978 pH 11 for 2 h at room temperature Shinagawa et al., 1995 pH 12 for 2 h at room temperature Mikami et al., 1994; Melling and pH 12 for 4 h at room temperature Capel 1978 Shinagawa et al., 1995 Sakurai et al., 1994 Heat stability stable at 121 ºC for 20 min Mikami et al., 1994; Shinagawa et stable at 126 ºC for 90 min al., 1995 stable at 4 ºC for 2 months Melling and Capel 1978 stable at -80 C for 24 h Melling and Capel 1978 Sakurai et al., 1994 Protease stability resistant to trypsin (2 mg / ml) for 2 h Mikami et al., 1994, Melling and at 37 C Capel 1978 resistant to pepsin at 500 μg / ml Shinagawa et al., 1995 resistant to 2% pronase (w/v), pH 7.4 in Sakurai et al., 1994 PBS at 37 C for 24 h Sakurai et al., 1994 resistant to 2% trypsin (w/v), pH 8 in 0.2 M Tris-HCl buffer at 37 C for 24 h Andersson et al., 1998a resistant to proteinase K (100 μg ml-1, pH 7 at 37 C for 2 h Inactivated at pH 80 min at 121 C or 60 min at 150 C Rajkovic et al., 2008 9.5 by

Hydrophobicity highly hydrophobic (log Kow ~6.0) Teplova et al., 2006 Solubility in Water No Agata et al., 1994 Methanol Yes Andersson et al., 1998a Ethanol Yes This thesis (paper II) Pentane Yes Häggblom et al., 2002

A quantitative chemical assay was 1.5.2.3. Toxicity of cereulide in whole introduced by Häggblom et al. (2002) animals based on liquid chromatography followed In order to determine the dose of cereulide by ion trap mass spectrometry (HPLC- which may cause illness, Rhesus monkey MS). This method allows measuring the and house musk shrew have been exact contents of the molecule cereulide in used as the animal models. Cereulide the B. cereus biomass as well as in food caused emesis in the house musk shrew or samples from environmental origins following an oral dose of 12.9 g per (Table 7). kg body weight and 9.8 g per kg by A rapid sperm bioassay was developed intraperitoneal injection. In the rhesus in 2004. It allows detecting the toxicity of monkey the dose causing vomiting was B. cereus bacterial extract in a short period  70 g per animal (approximately 10 of time (Andersson et al., 2004). Table 8 g per kg body weight) (Agata et al., shows the advantages of using the rapid 1995; Shinagawa et al., 1995). Using the sperm bioassay over bioassay methods house musk shrews for animal feeding previously developed for detecting the tests, it was found that cereulide causes toxicity of cereulide. emesis through the serotonin receptor 5- HT 3 which stimulates the vagus afferent nerve that enervates the stomach (Agata

14 Review of the Literature Jääskeläinen et al., 2003b Andersson et al., 2007 Mikami et al., 1994 Finlay et al., 1999 Andersson et al., 2004 Kawamura-Sato et al., 2005 Jääskeläinen et al., 2003a Reference B. cereus B. cereus (cereulide) B. cereus cally detects cereulide in bacteria Detects mitochondrial toxin. Screen for cereulide producing isolates. Generates the toxicity titre. Detects mitochondrial toxicity. Generates the toxicity titre of a given extract. Detects a functional electron transfer chain (cytochrome c-a/a3). Detect mitochondrial toxicity. cells. Growth of the target Detects toxins uncoupling the mitochondrial respiration. Speci fi or in food. Generates quantitative result. Toxicity target and the outcome of Toxicity assay uorescene fl cation of the mass ions c for cereulide. Formation of vacuoles in the mitochondria. Loss of the mitochondrial ability to convert the yellow tetrazolium salt into an insoluble purple formazan. Loss of the mitochondrial ability to emit yellow orange protein content Rapid oxygen reduction in the mitochondria. Quanti fi using JC-1 staining indicating loss of transmembrane potential. speci fi Toxicity end point Toxicity Assay Human cell lines and primary cells as the toxicity targets HEp-2 cells HEp-2 cells HeLa, Paju, Calu-3, Caco-2 Animal primary cells, cell lines and organelles as the toxicity targets Boar spermatozoa Loss of motility. Mouse Hepa-1 cellsRat liver mitochondria Measurement of the increase cell (RLM) Chemical assay LC-MS Table 7. Performance of the published in vitro assays for emetic toxin Table

15 Review of the Literature

Table 8. Essential features of three bioassays used for detecting the toxicity caused by cereulide. HEp-2 cell vacuolation Boar sperm assay Rapid sperm assay (Hughes et al., (Andersson et al., microassay (Andersson 1988) 1998a) et al., 2004) Cultivation time for 1 d 10 d 1 d B. cereus (20-30 ºC) Quantity of B. 50 ml of broth culture 500 mg wet wt of 5 to 10 mg wet wt of cereus biomass plate grown cells plate grown biomass needed Time to prepare one > 2 h 12 h 15 to 30 min extract Solvent used to Water methanol methanol extract the B. cereus biomass Extraction protocol Autoclaving the culture Blend the biomass 200 l of methanol and centrifugation. The into 100 ml of added, heated in water cell free supernatant methanol, soak bath for 15 min at 100 obtained by ﬁ ltration is overnight, evaporate C used for the assay with vacuum and redissolve the residue with a small volume of methanol Exposure time of 1 d 1 to 4 d 5 to 15 min target cells Time needed to 1 h 10 min 10 min prepare the exposed cells for microscopy Threshold for 5-10 ng per ml of the 1 ng per exposure 0.3 ng per exposure detection of liquid culturea cereulide a Quoted from Constantin. 2008

et al., 1995). Repeated feeding with an 1.5.2.3. Biosynthesis of cereulide emetic toxin containing culture of the B. Many microbial peptides are non- cereus strain F4810/72 at 16 occasions in ribosomally synthesized by peptide rhesus monkeys showed no development synthetases. In the chemical structure of of tolerance to this toxin indicating that cereulide peptide bonds and ester bonds the emetic toxin is poorly antigenic alternate. The presence of D- amino in contrast to the staphylocococcal acids and a cyclic structure are often enterotoxins known to produce resistance found in the products of nonribosomal in the animals (Melling & Capel 1978). peptide synthetases (NRPS). Cereulide is Recently Yabutani et al. (2009) tried to non-ribosomally produced by a peptide raise antibodies against cereulide but failed synthetase (Toh et al., 2004; Horwood because of the non immunogenic chemical et al., 2004; Ehling-Schulz et al., 2005b; character of the cereulide molecule. The Yabutani et al., 2009). effects of cereulide on several biological The genes (ces) responsible for the targets are described in Table 9. synthesis of cereulide were identified (Toh et al., 2004; Horwood et al., 2004) and found located on a large plasmid,

16 Review of the Literature

Table 9. Summary of the toxic activities of the emetic toxin, cereulide Target organisms Observed toxic effect Reference Whole organism Emesis in primates Emesis. Turnbull et al., 1979; Shinagawa (rhesus monkey and et al., 1995; Agata et al., 1995, human), Suncus Jääskeläinen et al., 2003b murinus (house musk shrew)

Primary cells Human natural killer Inhibition of the cytotoxicity and Paananen et al., 2002 cells (NK) production of cytokines by NK cells. Swelling of mitochondria and apoptosis. Boar sperm cells Inhibition of motility, dissipation Andersson et al., 1998a, 2007 of the mitochondrial membrane potential. Human cell lines HepG2 cells Inhibition of RNA synthesis and cell Andersson et al., 2007 proliferation. HeLa, Caco-2, Calu Dissipation of the mitochondrial Jääskeläinen et al., 2003b

3 and Paju cells m. HEp2 cells Inhibition of the growth and Mikami el al., 1994 induction of acid production. Organelles Human hepatocytes Swelling of the mitochondria. Mahler et al., 1997 Mouse liver Acceleration of oxygen consumption, Sakurai et al., 1994, Yokoyama mitochondria swelling of the mitochondria. et al., 1999 Fetal porcine Necrotic cell death, inhibition of Virtanen et al., 2008 Langerhans islets insulin secretion. HEp-2: Human laryngeal carcinoma cells; HepG2: human hepatocellular carcinoma cells; HeLa: human cervical cancer cells; Paju: human neural cells; Calu-3: human lung carcinoma cells; Caco-2: human colon carcinoma cells pCER270 (Hoton et al., 2005; Ehling- cluster of approximately 24 kb i.e. 10% Schulz et al., 2006; Rasko et al., 2007). of the pCER270 (Ehling-Schulz et al., The plasmid pCER270 of the strain B. 2006). Sequence analysis showed that the cereus F4810/72 (Rasko et al., 2007) and ces gene cluster contains 7 coding DNA that from the strain NC7401 by Yabutani sequences (CDSs): cesH, cesP, cesT, cesA, et al. (2009) have been fully sequenced. cesB, cesC and cesD. Details of the ces The plasmid pCER270 contains 270,082 genes are explained in Table 10. The gene nt with a GC content of 34% and with cluster for cereulide biosynthesis is shown 71% of coding region. It has 250 genes, in Figure 3. CesH is located at the 5’ end 235 genes code for proteins and 15 are of cesP. CesT is located downstream from pseudo genes. (http://www.ncbi.nlm. cesP while cesA overlaps with cesT. CesC nih.gov/sites/entrez?Db=genome&C and cesD are located in the 3’ part of the md=ShowDetailView&TermToSear ces gene cluster. To date the ces genes ch=22569; access date 07/08/2009). The are only found in strains producing the enzymatic machinery for the biosynthesis emetic toxin and a plasmid lacking the of cereulide consists of the ces gene ces gene has been found in strains which

17 Review of the Literature . B. B. licheniformis . B. brevis brevis and B. subtilis B. brevis Comments 58 % identities to putative hydrolases /acyltransferase from group members. cereus 32–38% identity and approx. 60% similarity to the 4’- phosphopantetheinyl transferase from involved in the nonribosomal synthesis of gramicidin S and surfactin, respectively. 35% identity, 53% similarity to GrsT from 53% similarity to GrsT 35% identity, 33% identity and 56% similarity to BacT from 33% identity and 56% similarity to BacT incorporation of L-Val. May be involved in the transport of cereulide or confer self resistance towards cereulide May be involved in the transport of cereulide or confer self resistance towards cereulide strain F4810/72 by sequence homology B. cereus Function Acyltransferase transferase ABC transporter ATP- binding protein (putative ABC transporter; permease Mass (kDa) Length aa Total Bp End (nt) Start (nt ) 37886 38668 782 26036140 36895 755 31 251 28.9 Hydrolase/ Phosphopantheteinyl 35158 35871 713 237 27.6 II thioesterase Type 25017 3514116958 10124 25003 3374 8045 2681 385.713A Cereulide synthesase 304.305 Cereulide synthesase B Activate the precursors for D-O-Leu and D-Ala activation and Activation of a precursor L-O-Val, 15917 16813 896 298 15094 15900 806 268 29.859 Putative permease Product Product Name CesH CesP CesT CesA CesB Ces C Ces D Table 10. Deduced functions of the proteins encoded by ces operon Table http://www. Quoted from http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=22569, access date June/25, 2009, Rasko et al., 2007, Ehling-Schulz 2006 uniprot.org/uniprot/?query=cereulide&sort=score,

18 Review of the Literature do not produce the emetic toxin (Ehling- weihenstephanensis isolates producing Schulz et al., 2006). So far no study has cereulide identified using LC-MS. This shown transfer of the plasmid carrying made other authors to suggest that the the ces gene between species. Thorsen plasmid could be subject to lateral transfer et al. (2006) reported the presence of B. among the species of B. cereus group (Vassileva et al., 2007).

Figure 3. Gene cluster for the biosynthesis of cereulide in B. cereus AH187 (F4810/72) carrying the plasmid pCER270. The ﬁ gure obtained from http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDe tailView&TermToSearch=22569, access date 25/06/2009

19 Aims of the Study

2. AIMS OF THE STUDY

The aim of this doctoral thesis was to understand the potential of cereulide producing B. cereus in causing food poisoning.

The speciﬁ c aims were:

1. Assess the prevalence of cereulide producing B. cereus in different ecological niches (soil, water, foods, dairy farm, dairy industry)

2. Identify the conditions promoting cereulide production by B. cereus in food, using infant food formulas as an example.

3. Reveal the biological strategies of B. cereus spores enabling these to persist and propagate in food industry, using dairy silo tanks as an example.

4. Identify properties of B. cereus cereulide producing strains relevant for the risk of food poisoning.

20 Materials and Methods

3. MATERIALS AND METHODS

Table 11. Methods used in this thesis Method Described in Reference Extraction of cereulide from different materials Extraction of cereulide with methanol from Paper I, II, III, IV, V Andersson et al., 2004 plate grown bacteria for rapid detection Extraction of cereulide with pentane from Paper II food Extraction of cereulide with ethanol from Paper II food Biological assays for toxicity Inhibition of boar sperm motility Paper I, II, III, IV, V Andersson et al., 1998a, 2004 Chemical assay HPLC-MS for cereulide Paper I, II, III, IV, V Jääskeläinen et al., 2003a Isolation method Isolation and enumeration of B. cereus in Paper II, V ISO standard 7932, 2004 food Methods for characterization of B. cereus Haemolysis B. cereus on blood agar Paper V Andersson et al., 2004 Starch hydrolysis Paper V Pirttijärvi et al., 1999 Ribopattern analysis Paper II, III, V Pirttijärvi et al., 1999 RAPD-PCR Paper III Nilsson et al., 1998 PCR detection of the cold shock gene in B. Paper III Francis et al., 1998 cereus Assay of antagonism Paper V Methods for preparation and characterization of B. cereus spores Preparation of spores Paper III Magnusson et al., 2006 Adherence onto micro plates Paper III Adherence onto stainless steel Paper III Resistance of spores in hot alkaline or acid Paper III solutions Scanning electron microscopy and methods for fl uorescence detection Scanning fl uorometry Paper III Epifl uorescence microscopy Paper III Field emission scanning electron Paper III microscopy

21 Results and Discussion

4. RESULTS AND DISCUSSION

4.1. Strategies for isolating and the occurrence of cereulide producing B. identifying cereulide producers cereus in several ecological niches, the among strains and isolates of B. dairy production chain and in milk based cereus infant food formulas (papers I, II, IV, V). I combined two independent methods to In this study, we identiﬁ ed the cereulide score cereulide production by the B. cereus producers among the B. cereus strains isolates: the rapid sperm bioassay to detect and isolates of many origins and explored the toxicity and to show that the toxin

Sample (food, milk environment) homogenization

Plate culture on blood agar, tryptic soy agar (TSA), mannitol egg yolk-polymyxin (MYP) agar

Rapid sperm bioassay of individual colonies

Colonies that inhibit sperm motility Colonies that do not inhibit sperm motility

Liquid chromatography –ion Cereulide non producing B. trap-mass spectrometry cereus

Detect the molecular ions at m/z of No peak at m/z of 1175 (Na + adduct), + + + + 1175 (Na adduct), 1192 (K adduct), 1192 (K adduct), 1171 (NH4 adduct) + + + 1171 (NH4 adduct) or 1154 (H ) or 1154 (H )

Cereulide producing B. cereus

Figure 4. A fl ow chart for distinguishing the cereulide producing B. cereus isolates from the nonproducers in foods or in environmental samples. Plate grown biomass of B. cereus is suspended in methanol. Viable bacteria and spores are inactivated by heating this methanol suspension in a boiling water bath. This treatment also inactivates any protein toxins in the same extracts. Boar sperm cells are obtained from commercially sources. The sperm can be handled at ambient temperature with no need for special cabinets. The sperm cells are exposed to ≤ 5 vol % of the heated methanol extract or its methanol dilution (10x, 100x….) for 5-15 min at 21 C. Cereulide inhibits the spermatozoa motility. Methanol, in the absence of cereulide, is tolerated by the sperm cells up to 5 vol % for 30 min. Outcome of the bioassay is a toxicity titre of the tested extract. The detection threshold is 0.9± 3 ng of cereulide mg-1 wet wt of the extracted material. Extracts that inhibit the motility of the boar sperm cells are fractionated by liquid chromatography and screened by mass spectrometry for the cereulide specifi c mass ions. 22 Results and Discussion is in the active form, and the chemical cereulide was found with isolates from assay (LC-MS) to specifi cally detect the food connected and not connected to an cereulide molecules and to accurately emetic food poisoning. High amounts determine the concentration of cereulide of cereulide (>1 g mg -1) were found in the bacterial extracts (papers I, II, IV, in two isolates originating from dairy V). Figure 4 describes the strategy used to milk (A116, A16). Similarly by high isolate and identify cereulide producing B. amounts of cereulide has been reported cereus. The rationales behind the steps of of endophytes (NS 115, NS 117, NS 58, the rapid sperm assay are explained in the NS 88) of live spruce trees, one isolate legend. (HS-10b ) from brown paper and of one isolate (B308) from a risotto connected 4.2. Cereulide productivity of B. cereus to food poisoning in Finland (Apetroaie- isolates of geographically and Constantin et al., 2008; Jääskeläinen temporally diverse origins 2008). Inspection of the origin of these strains (Table 12) revealed no obvious B. cereus isolates from food, environment connection of cereulide productivity to the and food borne illness representing 191 geographic, temporal or material origins independent samples were collected from of the strains. The differences were strain eight countries to serve as a European dependent. reference collection (EU project QLK1- B. cereus strains identifi ed by me as CT-2001-00854). In my thesis I analysed cereulide producers were subsequently the toxicities and cereulide contents of used in several other studies by other these isolates. The results were published authors: Ehling-Schulz et al., 2005b; in (Papers IV, V and Ehling-Schulz et al., Apetroaie et al., 2005; Apetroaie- 2005 a & b) and are compiled in Tables 12 Constantin et al., 2008, Fricker el al., & 13 in this thesis. 2007; Dommel, 2008. These strains were In Table 12 and 13, I divided the used to identify the cereulide biosynthesis B. cereus strains and isolates into four genes (Ehling-Schulz et al., 2005b). The categories based on the content of mutant strains prepared by disruption cereulide: high (>1 g mg-1 of wet wt of the peptide synthetase genes were cells), medium (0.1-1 g mg-1 of wet confirmed by me using the LC-MS to wt cells), low (0.0005-0.1 g mg-1 of be no longer cereulide producer. The wet wt cells) producers and (Table 13) thirty eight strains identifi ed as cereulide non producers (<0.0005 g mg-1 of wet producers by the sperm assay and the wt cells). These numbers refer to the LC-MS assay all possessed the ces cereulide content of B. cereus biomass gene, detected by the PCR method or by grown for 24 h at 28 C on tryptic soy plasmid profiling followed by southern agar. The results revealed differences of hybridization blot (paper IV, Apetroaie 100 fold and more between the content et al., 2005, Apetroaie-Constantin et of cereulide in cells of different strains al., 2008, Ehling-Schulz et al., 2005a, grown in the same laboratory, extracted Dommel, 2008). and measured under identical conditions The strain IH 41385 identified (Table 12). as low producer (0.5 -1 ng cereulide Medium (0.1-1 g mg-1) and low mg-1 bacterial biomass) and the emetic (0.0005-0.1 g mg-1) production of reference strain F4810/72 identified as

23 Results and Discussion medium producer (240-600 ng mg -1 (Christiansson et al., 1999, Svensson et al., bacterial biomass) (Paper IV) were also 2004; Vissers et al., 2007; Bartoszewicz studied by Dommel, 2008. The author of et al., 2008; Banyko & Vyletova, 2009) that study found a lower level of mRNA but only 1-2% of all B. cereus isolates transcripts cesP, cesA and cesB in the low retrieved from farms and dairies were producer strain IH 41385 as compared emetic toxin producer. Our data thus show to the emetic reference strain F4810/72. that emetic toxin producing B. cereus were The reasons for the vast differences in rare in these environments. the expression of the toxin genes are None of the B. cereus isolates from yet unknown but unlikely to be related farms identifi ed as emetic toxin producers to promoter strength of the ces operon came from soil (0/374) (Table 4 Paper I). (Dommel, 2008). I also investigated additional soil isolates In the very large number of B. cereus (strains WSBC 10310, WSBC10441, strains that I investigated (191), I found INRA SZ) from Germany and France two B. cereus strains GR285 and NVH and found no cereulide producer (Table 506 that were toxic in the sperm bioassay 14). These results may indicate that soil but did not contain cereulide (Table 13). is not the main reservoir of emetic toxin We conclude that both the sperm bioassay producing B. cereus. Similar fi nding was and the LC-MS should be used in order reported by Altayar and Sutherland 2005 to confi rm the identity of cereulide in the where none of the 101 isolates from soil toxic B. cereus. and animal faeces were emetic toxin producer. Also a recent study by Hoton 4.3. Occurrence of cereulide producing et al., 2009 reported only one emetic B. cereus in some environmental toxin producer out of 543 isolates from niches soil. However, a study in Japan which investigated the occurrence of emetic B. This chapter deals with the occurrence cereus in soil from rice field, 10 out of and significance of cereulide producing 20 isolates were emetic toxin producer B. cereus in certain ecological niches: the (Ueda and Kuwabara, 1993). May be this dairy production chain and in milk based explain why in Japan most cases of emetic infant food formulas. B. cereus food poisonings are due to the In paper I we identified 34 (1.9 %) ingestion of rice dishes. emetic toxin producers among the 1757 B. We found cereulide producing B. cereus isolates from the dairy farm: milk, cereus in dairies and milk (Paper I). This soil, grass, dung, rinsing water, bedding of raises the question whether cereulide the cows and air (Table 4, paper I). Forty producers are also present in dairy milk four emetic toxin producers (1.1 %) were derived foods like milk based infant food found among the 3911 B. cereus isolates formulas. I investigated 100 isolates from from milk sampled from eight Swedish infant formulas and found 11 cereulide dairy plants (Table 5, paper I). The results producers. The B. cereus isolates from the in Paper I show that dairy production infant foods produced similar amounts of chains are a potential but not a major cereulide as those isolated from emetic source of emetic B. cereus. food poisonings. I found cereulide The presence of B. cereus spores producing B. cereus among the strains in farm environment is well known isolated from commercial baby foods

24 Results and Discussion in Germany (Table 2, paper II). These poisoning, we found that 25 (29%) were findings indicate potential for cereulide cereulide producer. This percentage production in commercial infant foods. indicates that emetic foodpoisoning is Spores of cereulide producing B. relatively common among the cases of cereus are more resistant to heat (paper foodpoisoning connected to B. cereus. IV) than those of B. cereus not producing According to the international standard cereulide. Evaporation exposes milk for identifying the causative agents of to heat. This may promote the survival foodpoisoning outbreaks, identifi cation of of spores of emetic toxin producing B. B. cereus is based on one or two isolates cereus in dried milk products compared per case. When the primary plate is blood to those of non producers, explaining the agar, colonies exhibiting remarkable zone high frequency of cereulide producing B. of haemolysis are recommended to be cereus in infant food formulas based on selected by the currently valid standard dried milk (paper II). Further studies are protocols of ISO (Standard 7932, 2004), necessary along this line. NMKL (2003) and FDA (1998). All Another finding was that cereulide cereulide producing B. cereus isolates producers are more common in food than identifi ed in our laboratory were weakly in other environments (Table 1, paper V). haemolytic (≤ 1mm) or non hemolytic Thirty fi ve (24%) out of the 144 isolates (Table 12). Therefore the representation from food connected or not connected to of cereulide producing strains among the food borne illness were cereulide producer B. cereus isolates from food poisoning (Table 1, paper V). In contrast to this, 1- outbreaks is likely underestimated in 2% of the environment isolates (Table laboratories following the most widely 14) were cereulide toxin producer. This used international standards protocols. result shows that cereulide producing B. cereus is relatively common among food 4.4. Potential for cereulide production isolates. in selected infant food formulas Cereulide producing B. cereus share several defi ciencies distinguishing them I investigated 8 infant food formulas to from the cereulide nonproducers: Weak or determine their amenability to support no haemolysis on blood agar, inability to cereulide production. I used the sperm hydrolyse starch or to decompose tyrosine assay to determine the toxicity and (Table 12; Agata et al., 1996; Andersson et the LC-MS analysis for identifying al., 2004; Apetroaie et al., 2005; Pirttijärvi and quantifying cereulide in the food. et al., 1999; Shinagawa, 1993), inability to I found that 50 to 200 g of cereulide grow < 10 ºC (Table 5, Paper IV) and the accumulated within 24 h at 21-23 ºC very slow germination of spores (Table 2, in 100ml of the infant food formulas Paper IV). These negative physiological composed of both milk and cereals, when traits may explain why cereulide the food had been inoculated with > 105 producers usually are poor competitors in cfu ml-1 of vegetative cells of B. cereus the environment. F4810/72. The amount of cereulide When investigating a collection of accumulated in the infant food would 86 B. cereus isolates (one isolate per have caused serious illness if consumed. case) originating from the remnants Jääskeläinen et al. (2003) reported that the of food connected to cases of food food caused serious emetic food poisoning

25 Results and Discussion Reference for the description of the strain Starch hydrolysis lysis HBL Haemo- nd+ ndnd+ nd w nd w - nd - w - nd - a c a, b Paper IV + - w - This thesis, d + nd -+ nd+ - - w b w - - Paper II Papers II, IV Ces gene +nd+ - -+ - w nd - w+ w - nd - - -+ w This thesis, d This thesis, d nd This thesis, d - w This thesis, d - This thesis, d Paper IV

-1  g mg ≤ 1 mm zone of hemolysis; nd= not determined wet wt of bacteria Cereulide measured The measured traits sperm assay ++ 0.0005-0.1 0.0005-0.1 + + - - - w - - This thesis, d This thesis, d ++++ 0.0005-0.1+ 0.1-1 0.1-1 0.1-1 + 0.1-1 + -+ w 0.0005-0.1 0.0005-0.1 - + + - - This thesis, d w w - - This thesis, d This thesis, d strains and isolates analysed in this thesis. All strains were grown on tryptic soy strains and isolates analysed in this thesis. B. cereus FinlandFinlandFinland CakeUKUK Risotto Cake + + Rice + 0.0005-0.1 > 0.1 + 0.1-1 + 0.1-1 nd - - Paper IV Country Source Toxicity UK UKUKUKUK Rice Rice Rice Rice + + + + 0.0005-0.1 0.0005-0.1 0.0005-0.1 0.1-1 + + + - - - - w w - - - This thesis, d This thesis, d This thesis, d UK Pancake + 0.1-1 UK UK UK UK UKUKUK Fried rice Vomit Vomit + + + 0.1-1 0.0005-0.1 0.1-1 + - - - This thesis, d UK Germany Rice + 0.1-1 Origin and source Food borne isolates connected to illness B315 B412 FinlandB308 B208 F3080B/87 CakeF3350/87 + 0.1-1 Strain F4108/89 F3605/73 F3942/87 F3752A/86 F6921/94 LMG17604 F3942/87 F3976 F3876/87 F4426 F5881/94 F4552/75 F4810/72 RIVM BC 00051RIVM BC 00052 NL NL Rice + 0.1-1 F47/94 RIVM BC 00061RIVM BC 00062MHI 1305 NL NL Vomit + 0.0005-0.1 + - w - This thesis, d Table 12. Cereulide production by the Table agar plates for 1 d at 28 ºC. The plate grown biomass was analysed for cereulide by LC-MS and for toxicity by the sperm motility The plate grown biomass was analysed for cereulide by LC-MS and toxicity the sperm motility agar plates for 1 d at 28 ºC. -, trait is negative; +, positive; w= weak with inhibition assay.

26 Results and Discussion Reference for the description of the strain Starch hydrolysis lysis HBL Haemo- ++ - - w - - - Paper IV a, b + - w - This thesis, d nd nd w - Papers I, V + nd w - a, b ndnd nd nd nd wnd nd nd - w e - a, e Papers I, V Ces gene nd nd nd nd This thesis, d

-1  g mg wet wt of bacteria Cereulide measured + 0.1-1 + 0.1-1 + 0.0005-0.1 + - w - Paper IV + 0.0005-0.1 nd nd nd nd a + >1 The measured traits + 0.1-1 + >1 sperm assay + ND cial ﬁ lling illness illness liquid from an arti kidney a building dairy farm a building ﬁ dairy farm UKJapan Faeces Person with + 0.0005-0.1 + nd w - Paper IV UK Person with Finland Indoor wall of Sweden Milk from Country Source Toxicity Finland Indoor wall of FinlandFinlandFinland Infant food Infant food + + Meat pastry + 0.0005-0.1 0.0005-0.1 0.1-1 + nd - nd w w - -V Paper IV Papers II, IV, Finland Rice mushSweden + Milk from 0.1-1 Finland Infant food + 0.0005-0.1 nd N nd nd Paper II Finland Meat pastry RIVM BC 00075 RIVM BC 00067RIVM BC 00068 NLRIVM BC 124 NL NL NL Faeces Faeces Faeces + Faeces + + 0.1-1 + 0.0005-0.1 0.0005-0.1 0.0005-0.1 + + - + nd - - - w - - - Paper IV a, b Paper IV F3351/87 NC7401 F3876/87 IH41385 Finland Dialysis 7/PK4 A16 Strain Origin and source Specimens implicated with human illness 1/1 LKT Foods not connected to illness CIF1 CIF3 MIF1 B116 B203 JP31 RIVM BC 00379 A116 NL Chicken + 0.0005-0.1 + - w - Paper IV Table 12 cont. Table

27 Results and Discussion Jääskeläinen et al., Jääskeläinen et al., e Reference for the description of the strain Starch hydrolysis lysis HBL Haemo- Ehling-Schulz et al., 2005, d + nd ndnd nd nd w - Paper IV Papers I, V ndnd nd nd w w - - Papers I, V Papers I, V nd nd w - Papers I, V nd nd wnd - nd nd nd Papers I, V Paper III nd nd nd nd d nd nd nd nd c nd nd w - Papers I, V nd nd nd nd c + nd w - b + nd w - Paper I nd nd w -V Papers I, III, + nd - - Paper IV Ces gene

-1  g mg Jääskeläinen 2008, wet wt of bacteria Cereulide measured c + 0.1-1 + 0.1-1 + 0.1-1 + >1 + 0.1-1 + >1 + 0.1-1 + >1 + 0.1-1 + 0.1-1 + >1 The measured traits sperm assay from farm bedding from a live spruce tree dairy plant from a live spruce tree dairy plant from a live spruce tree dairy plant dairy farm from a live spruce tree Apetroaie-Constantin et al., 2008, b Sweden Rinsing water Finland Sweden Dairy plant + 0.1-1 SwedenSwedenSweden Dairy plant Milk + Milk + 0.1-1 + 0.0005-0.1 ND + - w - Papers III, IV Sweden Sawdust Finland Brown paper + >1 Sweden Milk from Germany Baby food + 0.0005-0.1 nd nd w - Papers II, IV Finland Endophyte Finland Endophyte Sweden Milk from Sweden Milk from Finland Endophyte Sweden Milk from Finland Endophyte Country Source Toxicity V1 3/pk1 HS-10b LU1 MHI 87 LU37 GR177 GR651 Strö 10 NS 88 GR516 NS 117 NS 117 GR314 Jo 331 NS 58 Origin and source Foods not connected to illness mjA1 not connected to illness Environments NS 115 Strain Apetroaie et al., 2005, Table 12 cont. Table a 2003.

28 Results and Discussion of two adult persons contained 100-200 by reconstituting dry powder with water, μg of cereulide per100 g. inoculated, treated and analysed similarly I found that the infant food formula (Figure 2, paper II). The inoculated strain composed of dried milk as well as B. cereus F4810/72 grew in both foods cereals accumulated 60 times more to similar densities during 24 h (Figure 2 cereulide when the reconstituted formula paper II). The result shows that these two was incubated in stationary position as infant food formulas differed in supporting compared to shaking (60 rpm) (Figure cereulide production and environmental 2, paper II). I concluded from these data factor other than the temperature, time, that the non shaking condition promoted inoculum and incubation conditions the production of cereulide at least affected the production of cereulide. in these foods. It is possible that non I also investigated cereulide shaking acts as a stressor on the emetic production in three dairy based formulas B. cereus causing it to produce more amended with cereal or rice, three dairy cereulide. Similar finding was reported only formulas and one rice formula by Rajkovic et al (2006) who showed that with no dairy component. The highest more cereulide accumulated in slurries of concentration of cereulide (200 to 300 g penne, potato, rice and as well as in whole /100 ml) was found in the formulas with consumer milk when incubated in static dairy plus cereal and those with dairy position as compared to shaking. plus rice (Figure 4, paper II). The lowest Although cereulide production is concentration of cereulide was measured growth phase dependent when tested from the formula based on dairy only. in laboratory media (Häggblom et al., Therefore, cereulide accumulated to 2002), I nevertheless found that growth highest density in the infant food formulas of cereulide producing B. cereus to 8 containing farinaceous ingredients like log cfu /ml (Figure 1, Paper II) did not grain and rice when inoculated with necessarily lead to accumulation of the strain F4810/72. Emetic outbreaks cereulide. Cereulide was detected in the have frequently been associated with cereal plus milk based formula 24 h after farinaceous rather than proteinaceous the food was inoculated with 105 cfu foods. It is interesting to note that most per ml whereas in the milk based infant emetic producing strains of B. cereus formula accumulation of cereulide was including the F4810/72 do not hydrolyse detected after 24 h only when inoculated starch (Table 12; Agata et al., 1996, with 100 times more of B. cereus, 107 Apetroaie et al., 2005; Shinagawa 1993) cfu per ml (Figure 2, paper II). Both infant indicating that a nutritional stress factor foods supported the growth of B. cereus may have upregulated the cereulide F4810/72 similarly, i.e. to > 108 cfu per production. However, the substrate ml. These ﬁ ndings show that the factors preferences of different cereulide producer affecting the accumulation of cereulide strains of B. cereus may differ from one in foods differ from those affecting the another, as was shown by Apetroaie- growth of the producer strain in the food. Constantin et al. (2008). I also found that 1000 times cereulide I studied the influence of diluting accumulated in the dairy and cereal based formulas with water on the accumulation formula than in the formula based on of cereulide by B. cereus in cereal and dairy only. Both formulas were prepared milk based infant formulas. I found that

29 Results and Discussion This thesis , a Paper IV Paper IV Paper IV Description of the strain Paper I Paper IV Paper IV This thesis, a Paper IV Paper IV This thesis, a Paper IV Paper IV Paper IV This thesis ,a Paper IV toxicity assay - - - - wet wt) -1 μ g mg France - CountryFinland Sperm - FinlandFinland - - Sweden - FranceNorway - - UK UK GermanyUK NL - FinlandFrance - - SwedenSweden - + sh soup), NVH 1105- investigated in this thesis and found not to produce cereulide. All strains were analysed investigated in this thesis and found not to produce cereulide. B. cereus gs), NVH 0165-99 (dear steak), 0226-00 (turkey), 0230-00 our), B154 (cake) KA96 (raw milk), MA57 milk) Strains and isolates their sources Food connected to illness B106 (potato fl 98HMPL63 (cooked salsify) NVH 200 (meat dish with rice ), 141/1-01 (vegetarian pasta), 0075-95, NVH 0154-01 ( fi (oriental stew), NVH 0309-98, 0391-98 (vegetable puree), 0500-00 (potatoes in cream sauce), NVH 0597-99 (mixed spices), 0674-98 (scrambled eggs), ( fi 0784-00 (ground beef), NVH 0861-00 (ice cream), 1104-98 98 (topping on steak), NVH 1230-88, 1230-98 (oriental stew), 1518-99 (soft ice cream), NVH 1519-00 (stew with deer meat), 1651-00 (caramel pudding) F352/90(chow mein), F528/94 (chow mein and rice), F2081A/98 (cooked chicken), F2085/98 (cooked rice), F3371/93( chop suey), F3453/94 (rice), F4430/73 (pea soup), F4433/73 (meat loaf), FH3502/72 (DSM 2301) F352/90, F284/78, F0210/76, F1942/85, F2081B/98 (cooked chicken), F2404B/79, F2769/77 (lobster plate), F284/78 (pork pie), F3003/73, F4094/73, F4096/73, F4346/75, F4370/75, F4431/73, F4432/73, F4429/71, F4430/73 (pea soup), F4433/73, F4815/94, DSM4282, LMG17605, LMG17615 (pork pie) MHI 203 (pasta) Specimens implicated with human illness F837/76 (DSM 4222) (postoperative infection), DSM 8438 RIVM BC0063 (faeces), RIVMBC 0070, BC0090 BC 91 (faeces from a control person), RIVM BC 122 (faeces) IH41064 (faeces) , B217(faeces) Food not connected to illness CIF13 (infant food) (dry formula) B102 (meat pie) I21 (cooked I20 (cooked leek), INRA A3 (starch), INRA 1(zucchini puree), INRA INRA I16 C24 (pasteurized vegetables), INR C3 INRA carrot), INRA (milk protein) PA C57 (potato puree), INRA (pasteurized vegetables), INRA A32 (zucchini purée) C1 (pasteurized zucchini), INR INRA VI273 (milk), UM37 MA60 JO164 Go95 (milk), JO 160 (milk, JO164 (milk), GR 285 (milk) with LC-MS and were found to contain less than the detection limit of cereulide (<0.0005 Table 13.The strains and isolates of Table

30 Results and Discussion This thesis b, c, d Description of the strain This thesis, Paper IV Paper IV Paper IV Paper IV Paper IV Paper IV Paper IV This thesis , a Paper IV Paper IV This thesis toxicity assay - CountryGermany Sperm - Finland - NL Germany - GermanyGermany - Norway - - FranceNorwaySwedenFinland - - - - Norway + Hoornstra et al., 2006 d Pirttijärvi et al., 1999; c Hallaksela et al., 1991, b Ehling-Schulz et al., 2005, Strains and isolates their sources Food not connected to illness WSBC 2454 (pasteurized milk) WSBC10210, WSBC 10206, RIVM BC 485 (chicken ragout), 934 (lettuce), 938 BC 964 (kebab) not connected to illness Environments WSBC 10441 (soil) WSBC10310 (soil), WSBC WSBC 1020, WSBC 10030, WSBC 10208, WSBC 100035, WSBC 10211, WSBC 10204 (pasteurized milk), 100042, WSBC 10466 WSBC 10395 (raw milk), WSBC 10377 (raw milk), WSBC 10286 (cream), WSBC 10483 (tobacco) (red rice), MHI 124 (baby food), 13 32 food) (dry formulas)NVH 445 (meat), 449 (spices), NVH 506 (spices) Germany SZ (soil) INRA NVH 460 (equipment), 512 655 (river water) - IR177 (manure), IR183 IR72(soil), NFFE640 (air), NFFE 647 (water), NFFE664 (water hose of a dairy farm). TSP9 (paper board) NS 61 (endophytes from live spruce trees) NS 113, Table 13 cont. Table a

31 Results and Discussion Paper I Tables 12 & 13 Tables 12 & 13 Tables 12 & 13 Tables Paper I Paper I Paper I Paper I Paper I Paper I Altayar and Sutherland 2005 et al., 2006; Tables 12 &13 Tables et al., 2006; Origins of the strains described in not connected to illness (one isolate per B. cereus Method of emetic toxin detection Emetic 101 0109 assay MTT 18 LC-MS Boar sperm assay, Hoton et al., 2009 21 0 0 Boar sperm assay 6 Boar sperm assay 454358 LC-MS Boar sperm assay, 157 Andersson et al., 1998a; Hoornstra 0 LC-MS Boar sperm assay, 0 LC-MS Boar sperm assay, Hoton et al., 2009 Hoton et al., 2009 LC-MS Boar sperm assay, Hoton et al., 2009 Number of B. cereus isolates 14 11 HEp2 vacuole cell assay et al. 2007 Vassileva Tested Tested isolates eld) 20 10 HEp2 vacuole cell assay Ueda and Kuwabara, 1993 Norway Norway Belgium Belgium Germany, FranceGermany, 3Sweden (dairy farm)Sweden (dairy farm) 374Sweden (dairy farm) 43Sweden (dairy farm) 19 0 44 0Sweden (dairy farm)Sweden (dairy farm) 204 0Finland 12 0 Boar sperm assay 0 Boar sperm assay 8 Boar sperm assay Japan Boar sperm assay 0 Boar sperm assay Belgium Boar sperm assay Belgium Boar sperm assay Japan (rice ﬁ Japan (rice mills) 29 12 HEp2 vacuole cell assay Ueda and Kuwabara, 1993 Country Soil and animal faeces UK Commercial wastewater powders Origin of isolates This thesis Soil Equipment River water Soil Feed Grass Dung Rinsing water in the farmUsed bedding Sweden (dairy farm)Air 339Endophytes from live spruce trees studies Other Environment (stream water, 4soil, lake water) Soil Arthropods (insects and Boar sperm assay isopods) Mammals Soil Air Item Table 14. Screening for cereulide producers among environmental isolates of Table sampled item). Compilation of strains and isolates described in this thesis elsewhere.

32 Results and Discussion more cereulide accumulated per gram of the spores from water or from milk to of food (dry wt) when the food was various material surfaces at 4 C, and 3. more diluted: Diluting the formula with spore germination and biofi lm formation water from 15 g dry formula /100 ml in milk environment. which is the recommended density, to I identifi ed four strategies explaining 6, 3 or 1 g/100 ml increased cereulide how the spores may have survived in the accumulation by factors 10 to 50 (Figure dairy silo environment. One strategy was 3, Paper II). Lücking et al. (2009) recently the extreme resistance of spore suspension showed that cereulide production is to hot 1% NaOH shown as an extremely regulated by SpoOA-AbrB regulon but low log 15 min kill of  1.5. This strategy independent from the later steps of the was strengthened by an ability to adhere sporulation process and that AbrB may to stainless steel at 4 C and to germinate act as a repressor of cereulide production. and grow to biofilm in full milk at 21 Diluting the food causes a nutritional C. The second strategy was shown by stress and this may upregulate the SpoOA isolates that adhered as spores to steel gene. The SpoOA product may repress the from cold water. These spores may have transcription factor AbrB, as speculated survived by adhering to steel as they by Lücking et al. (2009), releasing the did not germinate or grow to biofi lm in repressor of the cereulide synthetase full milk and also were not particularly operon, thus explaining why more resistant (log 15 min kill >3) towards hot cereulide was produced. alkaline washing. The third strategy was exhibited by isolates with spores that 4.5. Strategies of B. cereus spores for germinated extremely slowly even though persistence in the dairy process the conditions were permissive for growth environment (+30 ºC). These spores also expressed an extended viability when exposed to This part of my study was initiated by the heating at +90 ºC. The fourth group of observation of Svensson et al. (2004) that survivors consisted of psychrophiles that certain recurrent genotypes (RAPD-PCR) possessed the cold shock gene and were of B. cereus were found in the silo milk of able to grow at 8 ºC. more than one dairy plant in different parts I found that the spores of B. cereus of Sweden. I collaborated with the group RAPD-PCR group 1 (Svensson et al., of Svensson et al. (2004, 2006) to reveal 2004), representing the group most the survival strategies of B. cereus spores prevalent in the dairy silo tanks, possessed to explain how these B. cereus genotypes the survival strategy number 1. Three managed to colonise the dairy silo tanks. isolates from this group had spores that

I took a detailed look on the spores survived in hot alkali with a high D initial ( of 23 isolates of B. cereus selected to > 40 min) and a low 15 min kill value (log represent those RAPD-PCR patterns that of < 0.3). Such extreme tolerance to alkali were frequent in the dairy silo tanks. This may be the highest reported so far for B. study included: 1. assessment of survival cereus. The previous published record was of the spores in liquids used at the dairy by Langsrud et al. (2000) who described industry for cleaning-in-place, 1% sodium a strain of B. cereus of which the spores hydroxide (pH 13.1, +75 ºC) and 0.9% suspended in 1% NaOH at 80 ºC for 20 nitric acid (pH 0.8, +65 C); 2. adhesivity min lost 3 logs of viability.

33 Results and Discussion

The spores of cereulide producing 4342 and C477 and also of B. mycoides strains (n=17) of B. cereus exhibited ATCC 6462T. Cereulide thus was not higher D90C values (P < 0.001) and required for such inhibition. survival rates after 120 min of heating at All strains studied were members of 90 C (P < 0.001) compared to those of the species of B. cereus sensu lato Among cereulide non producing strains (n=83) the genus Bacillus, the species B. cereus is (Table 14, Figure 2, Paper IV). The 17 deﬁ ned as a species that does not produce emetic B. cereus strains also possessed any acid or gas from sugars (e.g. glucose a strategy of germinating extremely or starch) (Slepecky and Hemphill 2006). slowly ( 1 log in 7 d) in rich medium This means that the possibility that the (at +7 C or at + 30 C (Figure 1, Paper antagonism substance would be an organic IV). Postponed germination is likely to acid, is very unlikely. I tested the heat promote chances for survival in dairy stability of the antagonism substance and industry where most milk is heat treated found that the effective agent was heat resulting to inactivation of the spores that sensitive. This means that it is not possible germinated. An example of this strategy to rule out that the antagonistic agent was is one silo tank in a Swedish dairy plant an enzyme or some other protein (e.g. from which 307 B. cereus isolates were bacteriocin), but it rules out (once more) collected during two weeks in the autumn that the agent was cereulide, which is and two weeks in the winter. Among these heat stable. The nature of the antagonistic isolates, we found 40 (13%) emetic toxin substance was not investigated further producers (Table 5, Paper I). The high within this thesis. heat resistance combined with a delayed Antagonism was twice more common germination of the spores may explain (83 %) among cereulide producers why these cereulide producing B. cereus isolated from foods than among cereulide had been successful in surviving in the nonproducers isolated from foods silo tank environments. (36%). Of the 44 cereulide producing isolates or strains originating from foods 4.6. Are the cereulide producing B. implicated with food poisoning, 93 % cereus successful in competition were antagonistic and only 37 % of the with the non producing B. cereus nonproducers (Table 1, paper V). This in food? indicates that the antagonistic property may contribute to the morbidity of the I investigated if antagonism exists cereulide producing B. cereus more than between isolates of cereulide producing to that of the nonproducing representative. and non producing B. cereus (sensu lato). I also found that cereulide producing I found that cereulide producing B. cereus isolates from a food connected to an antagonized the cereulide non producing emetic food poisoning inhibited the B. cereus strains and isolates when growth of cereulide non producers isolated inoculated on the same plate (Figure 5). In from the same food. The capability to paper V, I showed that this novel property antagonise nonproducers may explain why was common but not obligatory connected cereulide producers B. cereus succeeded to cereulide producing B. cereus: 35 (83%) to conquer the food in the case of the of the 42 cereulide producing strains food poisoning (Pirhonen et al., 2005). inhibited the growth of B. cereus ATCC A similar change of B. cereus population

34 Results and Discussion

was observed at the end of shelf life of Reheating kills vegetative B. cereus but bakery products (Table 7, paper V). I may activate the spores to germinate (See found that cereulide producing B. cereus 1.2.3) The emetic B. cereus spores are is relatively common (29%) among food more heat resistant compared with the isolates connected to food borne illness non emetic B. cereus spores (paper IV). (one isolates per item of food per case, Each time the food reheated, there will Table 1, paper V). The antagonistic be selection in favour of the emetic B. property of cereulide producing B. cereus cereus to grow and produce cereulide. The may contribute to the success of emetic B. germinated cereulide producer cells may cereus in some foods and when time and die during subsequent reheating, but the ambient conditions are permissive, leading cereulide is stable and remains. The slow to food borne illness. germination strategy (paper IV) of emetic It important to note that the emetic B. cereus ensures that sufficient spores food poisoning cases described in the remain to survive beyond all heating literature were most of the time associated steps. This sequence of events increase the with reheated cooked food (Mahler et potential of that food for emetic B. cereus al., 1997; Pirhonen et al., 2005; Fricker food poisoning as the time passes on. et al., 2007; Pósfay-Barbe et al., 2008).

35 Summary and Conclusions

5. Summary and Conclusions The work presented in my thesis started item was analysed. We conclude that the at a time when little was known about the dairy production chain is a potential but prevalence of the cereulide producing B. a minor source of cereulide producing B. cereus in food and non-food environments. cereus. The assays for toxicity testing available at that time were not rapid and did not 3. We found that cereulide producing B. allow screening large numbers of isolates. cereus is more common in food than other The contribution of factors other than the environments. Among the 144 food borne toxins to the morbidity of emetic B. cereus B. cereus isolates, we found 35 (24%) was not known. The major outcomes of cereulide producers, in foods connected my work are the following: (29%) or not connected (17%) to illness. Only 12 (1.8%) out of 1041 B. cereus 1. We screened a large number isolates collected from environments other (191) of B. cereus isolates from than food were identified as cereulide food, outbreaks of food borne producers. illness and environments sampled from several countries within an EU project. 4. We searched for cereulide producers The isolates identiﬁ ed as B. cereus were among B. cereus isolates randomly investigated for cereulide production. collected from commercial formulas of I used the rapid sperm bioassay to test dry infant foods. Out of 100 isolates from the strains for toxicity and used the LC- the cereal and dairy based infant foods, MS technique to identify the toxin as 11 were cereulide producers (11%). This cereulide and to quantify its amount in is more than the frequency of cereulide the bacteria. I found more than 100 fold producers among dairy process isolates differences in the amount of cereulide (1.1%) indicating that cereulide producing produced by the strains. The difference B. cereus may be more common in dried in cereulide production was shown to be milk based products than in liquid milk. strain dependent. We conclude that for the purpose of assessing food safety it is not 5. I studied the susceptibility of different sufﬁ cient to draw conclusions based on the infant food formulas to accumulate cfu numbers of B. cereus or the presence cereulide when cereulide producers were of the ces gene but it is necessary to know present. I found that up to 0.1 to 0.4 the toxicity and to measure cereulide in milligrams of cereulide accumulated in the food. 200 ml of presterilised food within 24 h after the food was contaminated with 2. We searched for cereulide producing 105 cfu of cereulide producing B. cereus B. cereus in the dairy production chain and ml-1 and left at room temperature (21- 23 found that cereulide producers do occur C). This amount of cereulide would cause in the farm environment and the dairy serious food poisoning even for an adult production chain but at low frequency, consumer. The cereal and dairy based 1.9% of the total numbers of B. cereus infant food formulas accumulated 1000 isolates from the farms and 1.1% from the times more cereulide than in the formulas dairy plants when one isolate per sample based on dairy only although both types of

36 Summary and Conclusions reconstituted foods supported the growth spores highly resistant to hot 1% sodium of the inoculant B. cereus similarly. We hydroxide may be effectively inactivated conclude that the composition of the by hot 0.9% nitric acid. Eight out of the food had a major impact on cereulide 14 dairy silo tank isolates possessing hot production when a producer strain was alkali resistant spores were capable of present. germinating and forming biofilm in Interestingly, diluting the infant food whole milk, not previously reported for B. formula with water from the recommended cereus. density of 15 g dry weight /100 ml to 1g / 100 ml increased the accumulation 7. We detected that there are B. of cereulide ca. 50 fold. We suggest that cereus strains that kill other B. cereus the cereulide production in B. cereus is strains. This phenomenon, intraspecies enhanced by nutrient stress. Improper antagonism or cannibalism, is for the fi rst cleaning of infant food bottles may thus time described in this thesis. We measured contribute to the risk of emetic food the antagonistic action of strains of many poisoning. origins and found that antagonism was almost universal (93%) among cereulide 6. We investigated the spore survival of producing B. cereus isolates from food B. cereus in dairy industry environment. connected to human illness. We propose We identified 8 RAPD-PCR groups of that the antagonistic potency may increase B. cereus that colonised more than one the morbidity of cereulide producing B. dairy. When 23 representatives of these cereus towards the human consumer. groups were investigated, we identified four strategies to explain their survival 8. We identifi ed the antagonistic activity of their spores in dairy silos. First, high as a trait that may give advantage for survival (log 15 min kill ≤ 1.5) in the cereulide producing B. cereus to gain hot alkaline (pH >13) wash liquid, used prevalence in a food that undergoes at the dairies for cleaning-in-place. several heatings and the logistic chain Second, effi cient adherence of the spores from manufacturer to consumer becomes to stainless steel from cold water. Third, long. The cereulide producers may a cereulide producing group with spores outcompete other biotypes of B. cereus characterized by slow germination in from a given food commodity by the rich medium and well preserved viability antagonistic activity and the superior heat when exposed to heating at 90 ºC. survival of spores of the cereulide Fourth, spores capable of germinating at producers over that of non producer 8 ºC and possessing the psychrotolerance spores. gene, cspA. There were indications that

37 Acknowledgements

6. ACKNOWLEDGEMENTS

The work described in this thesis was carried out at the Department of Applied Chemistry and Microbiology, University of Helsinki. My work was supported by the European Commission, Quality of Life Programme, Key action 1 (Health Food and Nutrition, contract QLK1-CT-2001-00854, project “B. cereus” 2003-2005; the Academy of Finland CoE grants “Microbial Resources”(2002-2007) and “Photobiomics” (2008-2013), and the Finnish Graduate School on Applied Biosciences (ABS). My deep gratitude goes to my supervisor, professor Mirja Salkinoja-Salonen for her inspiration and enthusiasm. She always stood by me whenever I needed her help and support. I will never forget the countless hours of discussion during my time in Finland which opened my horizon to new ideas. I wouldn’t be who I am today without you! Thanks Mirja for every thing you have done for me! I am grateful to my previewers, Doc Dr. Maria Saarela and Prof Dr. Edward R.B. Moore for reviewing my thesis. I want to thank my colleagues: Maria Andersson for all the help and advice and for being a good mentor for me, Camelia Constantin, Douwe Hoornstra, Elina Jääskeläinen, Hanna Sinkko, Irina Tsitko, Jaakko Ekman, Marko Kolari, Mari Raulio, Minnna Peltola, Mari Koskinen, Mirva Kosonen, Raimo Mikkola, Stiina Rasimus, Teemu Kuosmanen and all the others that I had during my studies. You’ve been great colleagues and wonderful friends. I thank my coauthors: Mirja Salkinoja-Salonen, Maria A. Andersson, Anders Christiansson, Birgitta Svensson, Amanda Monthan, Camelia Apetroaie, Anja Schulz, Monika Ehling-Schulz, Veli-Matti Ollilainen, Frédéric Carlin, Martina Fricker, Annemarie Pielaat, Simon Heisterkamp, Christophe Nguyen-the, Elina L. Jääskeläinen, Tuula Pirhonen for sharing their data and expertise. I am grateful to Tuula Suortti for her kind help in many sorts of administration matters especially during the time of writing my thesis. My thanks to Leena Steininger and Hannele Tukiainen for all their help. I thank Pauliina Lankinen and Timo M. Takala and everyone in the division of Microbiology and the technical staff who helped me directly or indirectly to get along with this work. I want to thank my beloved family: my father, my mother my three lovely sisters Manal, Safa and Razan, and my two brothers Qasem and Ala whom I miss a lot. My deepest gratitude to my beloved husband Ahmad who always supported and encouraged me and tried to remove all the obstacles to be able to pursue my science dreams. My thanks goes to my lovely children Motaz, Diana for understanding and encouraging and for Omar who missed me during all the times needed to travel and during the ﬁ nal stages of my PhD work.

Helsinki, October 2009

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