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  1. 1. BACILLUS CEREUS Hin-chung Wong Department of Microbiology Soochow University _________________________________________________________ 1. INTRODUCTION 2. OCCURRENCE IN FOOD AND ENVIRONMENT 3. CHARACTERISTICS AND TAXONOMY 4. SPORE AND GERMINATION 5. ISOLATION AND ENUMERATION 6. TYPING 7. CONTROL 8. VIRULENCE FACTORS 8.1. Syndromes of B. cereus Food Poisoning 8.2. Phospholipase and Sphingomyelinase 8.3. Cereolysin 8.4. Haemolysin BL 8.5. Nonhaemolytic enterotoxin 8.6. Enterotoxin T 8.7. Cytotoxin K 8.8. Detection of enterotoxins 8.9. Emetic Toxin 8.10. Bioassays of Enterotoxins and Emetic Toxin 9. CONCLUSIONS 10. REFERENCES _________________________________________________________ 1
  2. 2. 1. INTRODUCTION The Genus Bacillus was established in 1872 with B. subtilis as type species. B. cereus was added fifteen years later. Several accounts of food poisoning attributed to members of the genus Bacillus appeared in the European literature before 1950. During this period, a number of papers also described the isolation of Bacillus species other than B. anthracis from a variety of non-gastrointestinal infestions. Since the early 1950's, particularly during recent years, there have been an increasing number of well-documented reports substantiating the role of B. cereus as a food poisoning organism. An accumulating number of reports implicate both B. subtilis and B. licheniformis as potential food poisoning agents. The pattern of their repeated occurrence in association with episodes of food poisoning suggests a significant involvement. However, application of the standard toxin-testing methods used for B. cereus to isolates of B. subtilis and B. licheniformis associated with gastrointestinal illness have so far failed to indicate what mode of pathogenic action these organisms might have. B. brevis was isolated in large numbers wither alone, or with B. cereus, from the suspected foods in food poisoning outbreaks (Kramer et al., 1982). Here is a review of B. cereus as a foodborne pathogen. 2. OCCURRENCE IN FOOD AND ENVIRONMENT B. cereus has a wide distribution in nature, frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals (Stenfors Arnesen et al., 2008). It has been isolated from foods that were not involved in foodborne illness outbreaks. It is also present in the stools of 14 to 15% of healthy humans. It is frequently isolated from milk and dairy products (Ahmed et al., 1983). In milk, B. cereus causes a defect known as 'bitty' cream or sweet curdling. It is found in rice, rice products, oriental dishes and ingredients (Blakey and Priest, 1980; Chung and Sun, 1986). A variety of foods have been implicated in food-poisoning (Table 1). Emetic syndrome caused by B. cereus is highly associated with rice and rice products (Johnson, 1984). 2
  3. 3. Table 1. In a report by Blakey and Priest, 56% of the peas, beans and cereals samples contained B. cereus, 1x102 to 6x104 organisms/g, and large number of samples with lecithinase positive organisms. During normal cooking procedure, B. cereus in the sample could increase to 107 cells/g (Blakey and Priest, 1980). B. cereus was isolated from 9, 35, 14 and 48% of raw milk, pasteurized milk, Cheddar cheese and ice cream samples, respectively (Ahmed et al., 1983). In a local study, B. cereus occurred in 17% of fermented milks, 52% of ice creams, 35% of soft ice creams, 2% of pasteurized milks and pasteurized fruit- or nut-flavored reconstituted milks, and 29% of milk powders, mostly in fruit- or nut-flavored milk mixes (Wong et al., 1988a). B. cereus was found in 3
  4. 4. 71.4% and 33.3% in spring and in autumn samples of full-fat milk in mainland China, respectively, and the average count among the positive samples was 11.7 MPN/ml (Zhou et al., 2008). Dried milk products and infant food are known to be frequently contaminated with B. cereus, 261 samples of infant food distributed in 17 countries were collected and 54% were contaminated with B. cereus reaching levels from 0.3 to 600/g (Becker et al., 1994). Chinese 'take-out' foods appear to be particularly vulnerable to B. cereus infection and it has been shown that suspensions (2%) of seed flours and meals from diverse botanical origins were found to be excellent sources of nutrients for growth (Beuchat and Ma-Lin, 1980). Of 433 honey samples collected in Argentina, 27% yielded B. cereus isolates and 14% yielded other species of Bacillus. The Argentinian B. cereus isolates were compared with isolates recovered from honey from other countries using rep-PCR fingerprinting with primers BOX, REP and ERIC, restriction fragment length polymorphism analysis of a 16S rRNA gene fragment (16S rRNA PCR/RFLP), and morphological and biochemical tests. Results showed a high degree of diversity, both phenotypic and genotypic among the isolates of B. cereus (Lopez and Alippi, 2007). The B. cereus isolates from food are highly toxigenic. All the isolates from local dairy products lysed rabbit erythrocytes; 98% showed verotoxicity, 68% showed cytotonic toxicity for CHO cells (Wong et al., 1988a). In another study of 136 strains of B. cereus isolated from milk and cream, 43% and 22% showed toxicity to human embryonic lung cell when the isolates were cultured in brain heart infusion and milk, respectively (Christiansson et al., 1989). In milks, B. cereus growed rapidly and produced cytotonic and cytotoxic toxins (Wong et al., 1988b). Toxin production of B. cereus in milk at low temperature was also evaluated (Christiansson et al., 1989). For the B. cereus isolated from seafood, 48% isolates produced both the hemolysin BL (HBL) and nonhemolytic (NHE) enterotoxins, and 94% and 50% produced NHE or HBL toxins, respectively. The presence of at least one of the three genes of the NHE complex was detected in 99% of the isolates; 69% of the isolates possessed all three genes. Only one B. cereus isolate possessed the cereulide synthetase gene, ces (Rahmati and Labbe, 2008). 4
  5. 5. Spores of Bacillus species were found in 52.8% of the rice samples with an average concentration of 32.6 CFU/g. Eighty three of the 94 isolates were identified as B. cereus and 11 were identified as B. thuringiensis. B. mycoides was the predominant isolate in one rice sample. The ces gene was not identified in any of the isolates. By contrast 56.6% B. cereus isolates possessed the hblA and hblD genes and 89.1% isolates possessed the nheA and nheB genes. As determined by commercial assay kits, 53.0% of the 83 B. cereus isolates produced both NHE and HBL enterotoxins whereas 93.9% were positive for either one or the other (Ankolekar et al., 2009). The enterotoxin genes hblA, hblC, hblD, nheA, nheB and nheC occurred in B. cereus isolates from full-fat milk products with frequencies of 37.0%, 66.3%, 71.7%, 71.7%, 62.0% and 71.7% respectively. Nine B. thuringiensis isolates were also identified from six pasteurized milk samples, and most of them harbored six enterotoxic genes and the insecticidal toxin cry1A gene. The single B. mycoides isolate harbored nheA and nheC genes (Zhou et al., 2008). In Nethelands, presence of genes encoding three enterotoxins (hemolysin BL [HBL], nonhemolytic enterotoxin [NHE], and cytotoxin K) and the ability to produce cereulide were analyzed and the genes for NHE are found in more than 97% of the isolates, those for HBL in approximately 66% of the isolates, and the gene for cytotoxin K in nearly 50% of the isolates (Wijnands et al., 2006). In Korea, a multiplex PCR assay was used to evaluate the distribution of 10 different toxigenicity-related genes among 1,082 B. cereus strains isolated from dried red peppers, rice and Sunsik. The examined foods were free of the emetic toxin but not free of enterotoxins and the distribution of enterotoxigenic genes was significantly different among the B. cereus isolates from various sources (Kim et al., 2009). Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of B. cereus-like bacteria above 104 cfu/g. The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain (Rosenquist et al., 2005). 5
  6. 6. 3. CHARACTERISTICS AND TAXONOMY B. cereus is a Gram-positive, motile, facultative, aerobic sporeformer. Dimensions of vegetative cells are typically 1.0- 1.2 μm by 3.0-5.0 μm. The ellipsoidal spores are formed in a central or paracentral position without swelling the sporangium. The organism does not ferment mannitol and has a very active phospholipase (lecithinase) system. B. cereus is keyed as citrate (+), arabinose (-), Gram (+), aerobic sporeformer. Characteristics and other biochemical characteristics are listed in Table 2 and 3 (Gilbert et al., 1981). 6
  7. 7. The bacilli tend to occur in chains; the stability of the chains determines the form of the colony, which varies greatly in different strains. The G+C content of the DNA is reported to be 32-33 moles % (determined by Tm) and 33-37 moles (analysis). Subspecies that have been designated include: a. Bacillus cereus var. fluorescens Laubach: Produces yellow-green fluorescent pigment. b. Bacillus cereus var. albolactis de Soriano: Acid formed from lactose. c. Bacillus cereus var. mycoides Smith, Gordon and Clark: Colony rhizoid; non-motile; acetoin formation variable; few strains grow at 37C. B. cereus is differentiated from other common related Bacillus species in 7
  8. 8. Table 4 (Kramer et al., 1982). Common characteristics: Gram-positive, catalase positive, cells grown on glucose agar contain intracellular globules unstainable by fuchsin, grow in 7% NaCl and in Sabouraud dextrose broth and/or agar, hydrolyze starch and casein. Phylogenetic analysis shows that the B. cereus group of bacteria are closely related group (Fig. 1) (Stenfors Arnesen et al., 2008). Conjugative behavior shows that these Bacillus species are closely related. Conjugation and mobilisation of pXO16 and pAW63 conjugative systems and mobilisable plasmid pC194, in LB medium, milk and rice pudding, occurs in B. thuringiensis strains and emetic B. cereus, with the highest transfer frequencies in milk (Van der Auwera et al., 2007). 8
  9. 9. 9
  10. 10. B. cereus grows in the temperature range 10-48C, with optimum growth occurring between 28 and 35C. Minimum pH for growth in meat is 4.35. In other report, pH range for cell growth is 4.9-9.3. The generation time in laboratory media at 30C is 18-27 min and 26-31 min in rice (Gilbert and 10
  11. 11. Kramer, 1986). The heat resistance of B. cereus spores has been studied. Linerar and non-linear survivor curves were observed, and nonlinear curves have been described (Johnson, 1984). Calculated D95C values ranged from 1.2 to 20.2 min for eight B. cereus, with an average z value of 9.2C. Similar D95C values have been reported, e.g. 2.5-36.2 min, 5.0-36.0 min, 1.5-36 min. Plasmids have been identified in B. cereus. Plasmids of molecular weight ranged from 1.6 to 105 MDa. Bacteriocin production could be attributed to a 45 MDa plasmid (pBC7), and tetracycline resistance to a 2.8 MDa plasmid (pBC16) (Bernhard et al., 1978). The 11 isolates from an outbreak revealed an identical plasmid profile, with all isolates having four plasmids ranging in molecular mass from 34 to 5.1 Mda, one had an additional band at 26 Mda, while no similar plasmid profile was obtained from other reference strains (DeBuono et al., 1988). 4. SPORE AND GERMINATION B. cereus produces elliptical shaped endospore with dominant central position, no distended sporangium. The spore when liberated from the sporangium is encased in a loose fitting exosporium. On germination the spore coat undergoes rapid lysis while the vegetative cell is emerging. Since spores of B. cereus may survive heat processing, spore germination is important in B. cereus study. Germination of bacterial spores is generally occurs through a series of sequential steps. Once the initial 'trigger reaction' has been activated, germination continues in the absence of the inducer. After the 'trigger' steps, the various spore properties are changed sequentially in the following order: loss of heat resistance, release of dipicolinic acid (DPA) and Ca2+ into the medium, increase in spore stainability, beginning of phase darkening and decrease of the optical density of spore suspension as cortex peptidoglycan is hydrolyzed and the products released to the medium, and finally, the onset of metabolic activity as measured by oxygen uptake. Activity of trypsin-like enzyme may be involved in the mechanism of the 11
  12. 12. breaking of dormancy in spores of B. cereus. The germination of B. cereus spore is partially prevented by several inhibitors of trypsin-like enzymes (leupeptin, antipain, and tosyl-lysine-chloromethyl ketone). A synthetic substrate of trypsin also inhibited germination. A crude extract of germinated B. cereus spores contained a trypsin-like enzyme whose activity is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10-30% of their dipicolinic acid content (Boschwitz et al., 1983). Fragments of lactoferrin, an iron-binding glycoprotein, are formed by heating tryptone in the presence of sodium thioglycolate and these fragments inhibited outgrowth of B. cereus spores. Lactoferrin is an iron-binding glycoprotein, and transferrin, an analogous iron-binding protein also shows these same properties. Also, bacteriostatic activity of nitrite may be due to the production of new bacteriostatic agents when nitrite reacts with protein in the presence of heat (Custer and Hansen, 1983). Inactivation of B. cereus spores during cooling from 90C occurs in two phases, one phase occurs during cooling from 90 to 80C; the second occurs during cooling from 46 to 38C. No inactivation occurs when spores are cooled from a maximum temperature of 80C. Inactivation of spores at temperatures between 46-38C is stimulated by the higher heat treatment (80-90C). Germination of spores is required for 45C inactivation to occur after the spores are heated to 90C for 10 min. Outgrowth of spore halts at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stops outgrowth at swelling; thus protein synthesis may play a role in the 45C inactivation mechanism (Johnson et al., 1984; Johnson and Busta, 1984). Germination of B. cereus spores is more extensive in rice than in trypticase soy broth at <15C and is generally more extensive for diarrheal strains in either medium than emetic strains (Johnson et al., 1983). Germination of B. cereus spores was also inhibited by the growth of lactic acid bacteria or the organic acids produced (Wong and Chen, 1988). B. cereus spores germinate in inosine or in L-alanine as sole germinants. They 12
  13. 13. require both GerI and GerQ germinant receptors for germination in inosine as the sole germinant, whereas the GerL receptor is responsible for most of the response to L-alanine as the sole germinant, with a smaller contribution from the GerI receptor. The GerT protein of B. cereus shares 74% amino acid identity with its homolog GerN which is a Na+ /H+ -K+ antiporter that is required for normal spore germination in inosine. The GerT protein does not have a significant role in germination, while it has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na+ stress (Senior and Moir, 2008) Germination of most of the enterotoxigenic strains of B. cereus adhered to Caco-2 cells is stimulated while another celline, HEp-2, does not trigger germination (Wijnands et al., 2007). The germination stimulated by Caco-2 cells is related to the normal function of germination receptors (Hornstra et al., 2009). Beta-lactamase type I occurs in the sporulated form in penicillin-resistant B. cereus (Fenselau et al., 2008). 5. ISOLATION AND ENUMERATION The guidelines generally used for the confirmation of an outbreak are listed as follows: (a) B. cereus strains of the same serotype should be present in the epidemiologically food, feces and/or vomitus of the affected persons. Or (b) Significant numbers (>105 CFU/g) of B. cereus of an established food poisoning serotype should be isolated from the incriminated food, or feces, or vomitus of the affected persons. or (c) Significant numbers (>105 CFU/g) of B. cereus should be isolated from the incriminated food, together with detection of the organism in the feces and/or vomitus of the affected persons. 13
  14. 14. B. cereus is easily isolated and identified from samples. Clinical specimens and food samples are collected, stored temporary at refrigerating temperature if necessary, diluted if necessary, and plated on selective agar media. Mannitol egg yolk polymyxin agar (MYP) is usually recommended. Polymyxin is the selective agent, and egg yolk and mannitol are differential agents. Typical colonies are rough with a violet-red background, surrounded by white precipitated egg yolk. KG medium is another widely used selective medium. Low level of peptone and the absence of carbohydrate in KG medium facilitated formation of spore (Johnson, 1984). A Columbia base 5% blood agar surface-spread with polymyxin B is recommended by Kramer et al. (Kramer et al., 1982) for better colonly characteristics and colony types are easily differentiated for serological testing. In enumeration by most probable number method, trypticase soy polymyxin broth is recommended. Polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA) is a modified selective agar, and also contains polymyxin and egg yolk. Pyruvate is added to reduce the size of colonies. The authors state that this medium is superior in detecting lecithinase-negative strains of B. cereus, weak and negative egg yolk reacting strains also developed typical colored colonies, grey to turquoise blue, and the color turns to a peacock blue color after 48 h (Holbrook and Anderson, 1980). The Bromthymol blue is replaced with bromcresol purple to give a new medium designated PEMPA (Szabo et al., 1984). The advantage of this modification is decreased incubation time (from 48 h to 22 h) (Szabo et al., 1984). In a comparison of selective media, MYP is still a better medium for easy colony differentiation (Harmon et al., 1984). Two new chromogenic plating media (CBC and BCM) were compared with two standard selective plating media (PEMBA and MYP) recommended by food authorities for isolation, identification and enumeration of B. cereus and the authors addressed that the new chromogenic media represent a good alternative to the conventional standard media (Fricker et al., 2008). B. cereus is differentiated from other nonmotile species by forming diffuse growth in semisolid B. cereus motility medium. It does not form rhizoid growth on predried nutrient agar as B. cereus var. mycoides. B. cereus is usually strong β-hemolytic on trypticase soy sheep blood agar plate, whereas, 14
  15. 15. B. thuringiensis and B. cereus var. mycoides are often weakly hemolytic and B. anthracis is nonmotile and nonhemolytic. Endotoxin crystals are found in B. thuringiensis (Harmon, 1978; Harmon, 1982). Other biochemical tests used in confirming B. cereus are: acid is produced from anaerobic glucose fermentation, nitrate reduction, VP Positive (acetylmethylcarbinol is produced from glucose), tyrosine decomposition, etc. After selective growth on blood agar and mannitol egg yolk polymyxin agar, Kramer et al. (Kramer et al., 1982) recommended the following biochemical tests for confirmation of B. cereus: Glucose +, mannitol -, xylose -, and arabinose -. Serotyping is recommended by a number of investigators (Table 4). B. anthracis and other Bacillus species including B. cereus can also be identified by the rapid API tests with the aid of computer programs (Logan et al., 1985). 6. TYPING A serological typing scheme has been developed at the Food Hygiene Laboratory, England (Gilbert and Kramer, 1986; Gilbert and Parry, 1977; Taylor and Gilbert, 1975). Based on the established type-specificity of the flagellar (H) antigen, the scheme currently comprises a 'routine set' of 28 agglutinating antisera raised against prototype strains from outbreak and non-outbreak foods and clinical specimens supplemented by an additional 'experimental set' of test sera. In approximately 90% of outbreaks the causative serotypes can be established. However, the B. cereus H-specific antisera are not available commercially. Most of the outbreaks associated with a vomiting-type syndrome, foods, clinical specimens or both yielded H-serotype 1 only. But only a few of diarrheal-type outbreaks yielded serotype 1 only (Gilbert and Kramer, 1986; Gilbert and Parry, 1977). Among those strains of B. cereus isolated from different foods and environmental samples, predominance of small cluster of serotypes characteristic of the sources is apparent, notably types H.1, 3, 4, 5, 8 and 12 (all of which are commonly associated with food poisoning), and H.17, 18, 19, 21, 22 (which are implicated with rarely or not at all) (Gilbert and Kramer, 15
  16. 16. 1986). Phage typing scheme has been developed for B. cereus. By using 12 bacteriophages, 10 Myoviridae and 2 Siphoviridae phages isolated from sewage, were employed (Ahmed et al., 1995). Biotypes, fatty acid profiles, and restriction fragment length polymorphisms of a PCR product (PCR-RFLPof the cereolysin AB gene) were compared for 62 isolates of the B. cereus group originated from various foods. The isolates were clustered into 6 biotypes, 10 fatty acid groups, or 7 PCR-RFLP clusters and these schemes may be used in tracking the origination of B. cereus strains (Schraft et al., 1996). A simple amplified fragment length polymorphism (AFLP) method was developed for the epidemiological typing of B. cereus and compared with those obtained by conventional serotyping using flagellar antigens and assessed in relation to epidemiological data. 16 different profiles (each unique to the 15 incidents) were recognized (Fig. 2). In this method, the chromosome DNA is digested by HindIII, ligated to adapters (ACG GTATGC GAC AG and GAGTGC CATACGCTGTCTCGA), and amplified by PCR using primers (GGTATGCGACAGAGCTTA, GGTATGCGACAGAGCTTC, G GTATGCGACAGAGCTTG and GGTATGCGACAGAGCTTT) (Ripabelli et al., 2000). 16
  17. 17. Multilocus sequence typing (MLST) scheme for bacteria of the B. cereus group has been developed. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed (Table 5) for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk (encoding adenylate kinase), ccpA (catabolite control protein A), ftsA (cell division protein), glpT (glycerol-3-phosphate permease), pyrE (orotate phosphoribosyltransferase), recF (DNA replication and repair protein), and sucC (succinyl coenzyme A 17
  18. 18. synthetase, beta subunit) were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40 (Table 6), and a total of 53 allelic profiles or sequence types (STs) were distinguished (Table 7). Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group (Helgason et al., 2004). 18
  19. 19. This MLST has been applied to differentiate eight strains of B. cereus isolated from bacteremia and soft tissue infections, and the results showed that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains (Barker et al., 2005). A total of 47 food-borne isolates of B. cereus were analyzed using MLST. Newly determined sequences (glpF, gmk, ilvD, pta, pur, pycA, and tpi housekeeping genes) were combined with sequences available in public data banks in order to produce the largest data set possible. Phylogenetic analysis was performed on a total of 296 strains for which MLST sequence information is available (MLST database (, and three main lineages--I, II, and III--within the B. cereus complex were identified (Fig. 3). With few exceptions, all food-borne isolates were in group I. The occurrence of horizontal gene transfer (HGT) among various strains was analyzed by several statistical methods, providing evidence of widespread lateral gene transfer within B. cereus. The occurrence of toxin-encoding genes, focusing on their evolutionary history within B. cereus was also investigated, and the results indicated a pivotal role of HGT in the evolution of toxin-encoding genes (Cardazzo et al., 2008) 19
  20. 20. 7. CONTROL Control of B. cereus during food processing has been investigated. Heated B. cereus did not grow at 10 and 50C or in a medium with pH 4.0. Decreasing pH values and increasing levels of sodium chloride decreased growth rate and 20
  21. 21. increased the lag phase of B. cereus. pH 4.5 was unable to prevent the growth of heated spores in a meat substrate with 0.5% NaCl at 12C. The combination of pH </=4.5, NaCl concentration >/=1.0% and temperatures </=12C was sufficient to inhibit B. cereus growth after heat treatment at 90 C for 10 min, for at least 50 days in nutrient broth and in meat extract (Martinez et al., 2007). The combination of mild acidification (pH 5.0) and refrigeration (</=8C) inhibited B. cereus growth for at least 60 days in vegetable substrates. Psychrotrophic strains of B. cereus were inhibited in carrot broth by heating at 90 C for 7.5 min, if the broth was refrigerated at a temperature of 8 C or lower. If the vegetable product was exposed to temperatures of mild abuse (12 C), it was necessary to implement a more drastic heat treatment (90 C for 30 min) (Valero et al., 2003). A combination of electrolyzed water and 1% citric acid exhibits synergistic effect on the inactivation of B. cereus vegetative cells and spores (Park et al., 2009). Growth and germination of B. cereus are inhibited by lactic acid bacteria and the organic acids produced by these bacteria, e.g. acetate, formate, and lactate. Spores of B. cereus are more resistant to these organic acids (Wong and Chen, 1988). In a field test, peracetic acid-based disinfectant was better than amphoteric surfactant-based disinfectant in controlling B. cereus spores (Ernst et al., 2006). B. cereus counts were decreased by 3.5 log numbers at 1.0 ppm ozone concentration for 360 min ozone treatment. Up to 2 log reductions in the number of B. cereus spores were observed above 1.0 ppm ozone concentration at the end of 360 min of ozonation (Akbas and Ozdemir, 2008). Pulsed Electric Field (PEF) technology in combination with antimicrobial cocoa powder has a synergistic effect on the B. cereus cells increasing their sensitivity to subsequent refrigerated storage (Pina-Perez et al., 2009). The initiation of B. cereus spore germination was more sensitive to pressure around 300 MPa at 20C. Increasing processing temperatures during high hydrostatic pressure processing enhanced the effect of sporulation medium pH 21
  22. 22. (i.e., environmental pH) on the inactivation of B. cereus spores (Oh and Moon, 2003) Factors inhibitory to B. cereus are: nisin, sorbic acid and sorbate, antibiotics (aureomycin, dihydrostreptomycin, terramycin, bacitracin, oxytetracycline, chloramphenicol, and gentamicin). Slight inhibition was observed with neomycin, cloxacillin, ampicillin, and penicillin (Johnson, 1984). Clinical isolates of B. cereus are most susceptible to imipenem, vancomycin, LY146032, and ciprofloxacin, and also rarely susceptible to penicillins, or cephalosporins with the exception of mezlocillin (Weber, 1988). Several antimicrobial wine recipes, each consisting of red or white wine extracts of oregano leaves with added garlic juice and oregano oil are bactericidal against B. cereus, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica (Friedman et al., 2007). Olive powder also exhibited bacteriostatic effect against B. cereus (Ferrer et al., 2008). Essences of vegetables have been assayed. Bergamot was the most inhibitory essential oil (EO) and citral and linalool mimicked its effect (P > 0.001). Citral and linalool vapours produced 6 log reductions in L. monocytogenes, Staph. aureus and B. cereus populations on cabbage leaf after 8-10 h exposure but bergamot vapour exposure, while producing a similar reduction in L. monocytogenes and B. cereus populations, had no effect on Staph. aureus (Fisher and Phillips, 2006). Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards B. cereus. A decrease of the sensitivity of B. cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations (Ultee et al., 2002). The antimicrobial effect of the broad-spectrum bacteriocin enterocin AS-48 against the toxicogenic psychrotrophic strain B. cereus has been investigated in a model food system consisting of boiled rice and in a commercial infant rice-based gruel dissolved in whole milk stored at temperatures of 37C, 15C and 6C. In food samples supplemented with enterocin AS-48 (in a concentration range of 20-35 μg/ml), viable cell counts decreased rapidly over incubation time, depending on the bacteriocin concentration, the temperature 22
  23. 23. of incubation and the food sample. Enterotoxin production at 37C was also inhibited. Heat sensitivity of endospores increased markedly in food samples supplemented with enterocin AS-48 (Grande et al., 2006). Synergistic activity of epsilon-poly-L-lysine and nisin A was observed against Gram-positive Listeria monocytogenes and B. cereus cells and spores (Badaoui et al., 2007). Kefiran, the polysaccharide produced by microorganisms present in kefir grains, is a water-soluble branched glucogalactan containing equal amounts of D-glucose and D-galactose. In the presence of kefiran concentrations ranging from 300 to 1000 mg/L, the ability of B. cereus spent culture supernatants to detach and damage cultured human enterocytes was significantly abrogated, and the mitochondrial dehydrogenase activity was higher when kefiran was present during the cell toxicity assays (Medrano et al., 2008). 8. VIRULENCE FACTORS In addition to causing foodborne illness, B. cereus is also capable of causing mastitis, systemic infection, gangrene, meningitis in immunocompromised children (Gaur et al., 2001), respiratory tract infections (Gray et al., 1999), and other clinical problems (Weber, 1988). Food-borne illness outbreaks have been reported in European countries, American, Japan, and other regions. It is generally recognized that a population of >105 B. cereus cells/g in the food sample taken is required for poisoning to occur. However, the organism apparently does not always cause illness when present in high number (Johnson, 1984). Usually, two types of B. cereus foodborne diseases occur, the diarrhoeal and the emetic types (Table 8)(Stenfors Arnesen et al., 2008). Cell culture assays measuring the cytotoxic activity of cell-free culture supernatants is now more commonly used to detect the presence of B. cereus diarrheal toxins, and these give a good indication of the cytotoxic potential of B. cereus strains. B. cereus produces a large number of secreted cytotoxins and enzymes that may contribute to diarrhoeal disease, the identity of the enterotoxin(s) is still a controversial topic. The three cytotoxins haemolysin 23
  24. 24. BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K are currently considered the aetiological agents of B. cereus diarrhoeal foodborne disease. Hbl and Nhe are related three-component toxins, while the single-component CytK belongs to the family of b-barrel pore-forming toxins. In addition, several other protein cytotoxins, haemolysins and degradative enzymes have been described that may potentially contribute to the pathogenicity of B. cereus diarrhoeal disease. These include cereolysin O, haemolysin II, haemolysin III, InhA2 (metalloprotease) and three phospholipases C (Stenfors Arnesen et al., 2008). (Stenfors Arnesen et al., 2008) 8.1. Syndromes of B. cereus Food Poisoning Diarrheal Type Outbreaks B. cereus diarrheal outbreaks have symptoms which parallel those of Clostridium perfringens. Onset of watery diarrhea and abdominal cramps and pain occur 6 to 15 h (late on-set) following consumption of contaminated foods. Nausea may accompany the diarrhea, but vomiting rarely occurs. Symptoms persist for <24 h in most instances. Foods involved are quite varied, ranging from vegetables and salads to meat dishes and casseroles. Emetic Type Outbreaks 24
  25. 25. Emetic outbreaks are characterized by nausea and vomiting within 0.5 to 6 h after consumption of contaminated foods. Occasionally, abdominal cramps and/or diarrhea also occur in emetic outbreaks. Duration of symptoms is generally <24 h. These symptoms closely mimic those of Staphylococcus aureus food intoxication. Rice foods seem to be almost exclusively associated with emetic outbreaks (Gilbert and Kramer, 1986; Mortimer and McCann, 1974). Clinical and epidemiological characteristics of the emetic-type and the diarrheal-type poisoning caused by B. cereus are shown in Table 12. 8.2. Phospholipase and Sphingomyelinase Phospholipase and Sphingomyelinase were known to be toxic, but now they have been demonstrated to be nontoxic, and some of the hemolysins associated with them are marginally toxic (Beecher et al., 2000). Phospholipase (Lecithinase) Phospholipase is produced by B. cereus and is similar to the α-toxin of Clostridium perfringens. Phospholipase of B. cereus is resistant to inactivation at 45C and also resistant to trypsin inactivation. It is a small metalloprotein (MW 23,000 Da) containing two zinc atoms per molecule of enzyme and the first Zn2+ site can be replaced by lanthanides to form Gd3+ derivatives (El-sayed and Roberts, 1983). Phospholipase activity can be determined by observing zones of turbidity on agar plate containing 1% egg yolk. Production of phospholipase is regulated under the transcriptional regulator, PlcR, which controls proteins, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). These regulated proteins are related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (Gohar et al., 2008). Sphingomyelinase It is a protein of between 41,000 and 23,300 Da, depending on the method of analysis used, and requires divalent cations for activity. Sphingomyelinase 25
  26. 26. can be assayed as follows: culture filtrate is mixed with phosphate-buffered saline and TNPAL-sphingomyelin solution (N-w- trinitrophenyl aminolauryl sphingosyl phosphoryl choline). The reaction mixtures are slowly shaked at 37C for 2 h. Reaction is stopped and extracted and the absorbance of the trinitrophenylamino residue released into the organic phase is read at 330 nm. 8.3. Cereolysin Cereolysin was purified to apparent homogeneity by using ammonium sulfate fractionation, hydrophobic chromatography with AH-Sepharose, isoelectric focusing, and gel filtration. The active form of the toxin had an isoelectric point (pI) of 6.6, and the molecular weight of 55,000 (Cowell et al., 1976). Cereolysin is a cholesterol-dependant cytolysins (Brillard and Lereclus, 2007), containing two half-cystine residues. In the absence of dithriothreitol, partial spontaneous oxidation resulted in the formation of an oxidized form of the toxin. The oxidized and reduced forms of this toxin would separate during electrophoresis (Cowell et al., 1976). Therefore, cereolysin is a thiol- or SH-activated hemolysin (cytolysins) similar to the streptolysin O (produced by Streptococcus pyogenes), pneumolysin (Streptococcus pneumoniae), and listeriolysin (Listeria monocytogenes). They are apparently cross-react in neutralization and immunodiffusion tests. They are activated by thiol-reducing agents, such as cysteine, 2-mercaptoethanol, dithiothreitol, and sodium thioglycolate, and inactivated by sterols, and lethal to mice (Kreft et al., 1983). This cereolysin is also known as hemolysin I (Gilbert et al., 1981). 8.4. Haemolysin BL B. cereus produces a true enterotoxin into the cell-free filtrate and also associated with washed cells. Intraluminal growth of B. cereus did not elicit fluid accumulation (Spira and Goepfert, 1975). The B. cereus enterotoxin is capable of causing fluid accumulation in ligated rabbit ileal loops, altering the vascular permeability or rabbit and guinea-pig skin and killing mice when injected intravenously. The enterotoxin is known as diarrheagenic toxin, diarrheal agent, fluid accumulation factor, 26
  27. 27. vascular permeability factor, dermonecrotic toxin, and intestinonecrotic toxin in the literatures. Oral administration of the enterotoxin to rhesus monkeys causes diarhea (Spira and Goepfert, 1972). The enterotoxin is produced by 19 of 22 strains of B. cereus (mostly clinical isolates) and 4 of 6 strains of B. thuringiensis (Spira and Goepfert, 1972). The enterotoxin is synthesized and released during the late logarithmic growth phase of the organism at an optimum temperature of 32-37C. Cultures grown at temperatures in the range of 18C to 43C were loop active (Spira and Goepfert, 1972). The pH of the medium usually drops as the hydrolysis of carbohydrates and rises as the result of proteolytic degradation. Optimum enterotoxin is detected while the pH of the medium drops to lowest value (below 7) (Gilbert and Kramer, 1986). The growth medium employed markedly affected the ability of a given strain of B. cereus to provoke a response. Brain Heart Infusion broth proved to be best for toxin production in small scale (Gilbert and Kramer, 1986). A CA medium consisting of casamino acids, yeast extract and minerals supplemented with 1% glucose was shown to be optimum for fermenter production of B. cereus enterotoxin at 32C, controlled pH 8.0, and moderate stirring rate (Glatz and Goepfert, 1976). Characteristics of the enterotoxin in the culture filtrate are determined. It is distinct from the hemolysin and egg yolk turbidity factor. Enterotoxin has been proved to be unstable under a wide variety of conditions; ionic strength is especially critical. Enterotoxin is most stable in a pH range of 5.0 to 10.0, but lost activity rapidly outside this range. Alkylation provides some protection of enterotoxin activity. The enterotoxin interacts with intestinal receptor sites in a highly transient manner in the ileal loop system, so that flushing the challenge enterotoxin from the ileal loops prevents accumulation of fluid in those loops (Spira and Goepfert, 1975). The enterotoxin is thermolabile and susceptible to protease inactivation (Gilbert and Kramer, 1984). Activity of enterotoxin may involve stimulation of adenylate cyclase-cAMP system with probable role in non-gastrointestinal infections (Gilbert et al., 1981). 27
  28. 28. HBL is a tripartite toxin produced by B. cereus, and it has been highly purified and established to be a diarrheal toxin by the ligated rabbit ileal loop assay. HBL is identical to the toxin purified (Thompson et al., 1984) by performing Western blots and immunodiffusion assays. The enterotoxin purified by Thompson et al. is relatively unstable. Extracellular proteins produced by B. cereus B-4ac were separated by chromatography on Amberlite CG-400, QAE-Sephadex, Sephadex G-75, and hydroxylapatite. A fraction, containing three detectable antigens, was obtained that caused fluid accumulation in ligated rabbit ileal loops, dermonecrotic to rabbit skin, cytotoxic to cultured cells, and lethality to mice. These results suggest that all of these biological activities probably are due to a single entity and that more than one component probably comprise the toxic entity. The molecular weights of these three antigens are 38,000, 39500, and 43,000 (Thompson et al., 1984). HBL is a unique and potent three component pore forming toxin composed of a binding component, B, and two lytic components, L(1) and L(2). Nucleotide and deduced amino acid sequences have been reported for all components (GenBank accession nos. L20441, U63928, AJ237785). The genes hblC (L2), hblD (L1), and hblA (B) are arranged in tandem in an operon with the promoter located upstream of hblC. Alignment of the deduced amino acid sequences of the three proteins revealed significant similarities, 20 to 24% identical to each other. Structural analysis of the HBL proteins indicates that all three components consist almost entirely of alpha-helix. Components B and L1 contain predicted transmembrane segments of 17 and 60 amino acid residues, respectively, in the same position, whereas L2 does not contain predicted transmembrane segments. These observed similarities suggest that the HBL components resulted from the duplication of a common gene. All three components in HBL are required for biological activity. HBL produces a unique discontinuous hemolysis pattern on blood agar. Hemolysis begins several millimeters from the edge of a colony or a well containing HBL, forming a ring-shaped clearing zone (discontinuous). With time, the zone moves inward toward the source. Hemolytic potency varies depending on the species of mammalian blood tested. Sheep erythrocytes do not lyse when incubated with the B component 28
  29. 29. alone. Rather, the erythrocytes become sensitized or primed and are rapidly lysed with the addition of L1 and L2. Excess concentrations of B, however, inhibit the activity of L1 on the lysis of B-primed erythrocytes, and excess L1 inhibits the priming activity of B. The L2 component is required for lysis but does not interfere with the action of B or L1. Therefore, hemolysis of erythrocytes in the blood agar plate assay occurs at the point in the diffusion gradient (away from the well) where appropriate concentrations of both B and L1 exist. In addition to its hemolytic activity, HBL is dermonecrotic, increases vascular permeability in rabbit skin, and is cytotoxic to Chinese hamster ovary cells and retinal tissue both in vitro and in vivo. It causes fluid accumulation in the rabbit ileal loop assay, and necrosis of villi, submucosal edema, interstitial lymphocytic infiltration, and variable amounts of blood were also observed in loops that were positive for fluid accumulation. HBL forms pores in eukaryotic cell membranes, with each of the components binding the membrane independently and reversibly. One hypothesis was that once bound, the components oligomerize and form transmembrane pores consisting of at least one of each component. The transmembrane segments in B and L1 may serve as mediators of oligomerization. Membrane receptors have not been identified. A high degree of molecular heterogeneity exists in HBL from different strains. In a study of 127 B. cereus isolates by Western blot analysis, four sizes of B (38, 42, 44, and 46 kDa), two L1 (38 and 41 kDa), and three L2 (43, 45, and 49 kDa) were identified (136). Individual strains produced various combinations of single or multiple bands of each component. In addition, some strains produced only one or two of the three HBL components. A total of 13 different band patterns were observed with various forms of B, L1, and L2. Commercially available kit (BCET RPLA, Oxoid) is useful for detection of L(2) component of HBL, but detection of only one component is insufficient to give comprehensive view on HBL toxin producing strains as some strains produced only one or two of the three HBL components. The conserved domains of B, L(1) and L(2) components were cloned together as single fusion gene and expressed as recombinant multidomain chimeric protein in E. 29
  30. 30. coli. The resultant protein having L(1), B and L(2) components in the form of single protein. The hyperimmune antisera raised in mice against this chimeric protein reacted with all the three components of HBL tox in of B. cereus and can be used in the development of detection method (Kumar et al., 2008). (Stenfors Arnesen et al., 2008) 8.5. Nonhaemolytic enterotoxin In addition to HBL, a nonhemolytic enterotoxin (NHE) has been characterized. NHE is also a multi-component toxin (Table 9). General 30
  31. 31. characteristics of the NHE are presented in Table 10. Various combinations of the individual NHE subunits possess some degree of biological activity, but maximal activity is achieved only when all components are present. N-terminal amino acid sequence similarity exists between L1 of HBL and the 39 kDa subunit of NHE, as well as between L2 and the 45 kDa subunit of NHE, suggesting similar functional roles in different B. cereus strains. Both NheA and NheB appear to be present in culture supernatants in two forms with slightly differing sizes, where the smallest form represents a further processed variant of the largest form. The smallest forms of NheA and NheB lack 11 and 12 N-terminal amino acids, respectively, in addition to the 26 and 30 residues of their signal peptides (Lund and Granum, 1996; Lund and Granum, 1997). Trypsin digestion of the largest form of NheA yielded a fragment with mobility identical to the smaller one (Lund and Granum, 1997). Both variants of NheA and NheB show similar biological activity (Lund and Granum, 1996; Lund and Granum, 1997). The maximal cytotoxic activity towards Vero cells was obtained when the molar ratio between NheA, NheB and NheC was about 10:10:1. Furthermore, addition of excess NheC inhibited the cytotoxic activity of Nhe against Vero cells, both in B. cereus culture supernatants and using purified proteins. Presumably, the initial lack of identification of NheC as part of the Nhe toxin was a result of NheC being produced by the bacterium in much lower concentration than NheA and NheB, in order to obtain a toxin complex with optimal ratio of components. An inverted repeat located between nheB and nheC has been suggested to mediate translational repression of nheC resulting 31
  32. 32. in lower expression of nheC compared with that of nheA and nheB. The nature of the cytotoxic activity of Nhe towards epithelial cells showed rapid disruption of the plasma membrane following exposure to Nhe, and formation of pores in planar lipid bilayers (Fig. 4)(Fagerlund et al., 2008). Fig. 4. Cytotoxicity of B. cereus NVH 0075/95 culture supernatant to Vero cells. (a) Photomicrograph taken 30 min after addition of 12.5 µl/ml NVH 0075/95 culture supernatant with dual phase-contrast and epifluorescent illumination in a bathing solution containing 5 µg PI ml–1. Note the numerous bright nuclei and clear swollen extrusions. (b) Vero cells exposed to 12.5 µl/ml culture supernatant from the nheBC mutant for 60 min. Bars, 0.1 mm (Fagerlund et al., 2008). 32
  33. 33. Nhe was also shown to have haemolytic activity towards erythrocytes from several mammalian species in suspension assays (Fig. 5) (Fagerlund et al., 2008). Fig. 5. Haemolytic activity of purified Nhe proteins. (a) Haemolytic dose–response curve of purified Nhe proteins incubated with 1.5 % human erythrocytes. Purified Nhe proteins were used at a ratio of 6 : 6 : 1 NheA : NheB : NheC, using approximately 100 ng NheA/ ml and expressed relative to this. (b) Species differences in sensitivity of erythrocytes to Nhe. Suspensions were adjusted to an OD630 equal to that of 1.5 % human blood. The amount of Nhe used corresponds to the highest concentration used in (a). The percentage of the maximal release of haemoglobin (Hb) was calculated relative to 100 % lysis of controls consisting of the same type of erythrocytes. Data represent the mean of two to five experiments for each species. 33
  34. 34. The Hbl and Nhe proteins do not show significant sequence homology towards any other known protein family. However, the crystal structure of Hbl component B determined by a structural genomics consortium (Fig. 6) showed remarkable tertiary structure resemblance with the pore-forming toxin cytolysin A (ClyA). ClyA, also known as HlyE or SheA, is a haemolytic and cytotoxic monooligomeric protein toxin of 34 kDa expressed during anaerobic growth in E. coli, Shigella flexneri and Salmonella enterica serovars Typhi and Paratypi A. The crystal structures of ClyA and Hbl B consist of long, four/five a-helix bundles that wrap around each other in left-handed supercoils, and a unique subdomain containing a hydrophobic b-hairpin flanked by two short a-helices. The main structural difference, the orientation of the subdomain (Fig. 6e), may possibly represent two different conformational states that both molecules may adopt, with the subdomain and the main helix bundle being connected by a hinge region. This is supported by the observation that the two structures represent different crystallization states, as Hbl B was crystallized as a monomer while ClyA was a dimmer in a head-to-tail conformation, where the subdomain containing the b-hairpin was buried against a second hydrophobic surface patch on the opposite end of the protein structure. NheB and NheC show sufficient sequence identity towards Hbl B for generation of 3D homology models based on the Hbl B structural template. Interestingly, as observed for Hbl B and ClyA, the hydrophobic segments of NheB and NheC correlate with the predicted b-hairpin in the homology models. Despite limited sequence identities, the strong structural and functional similarities suggested that the Hbl/Nhe family and the ClyA family of toxins constitute a new superfamily of toxins (Fagerlund et al., 2008). 34
  35. 35. Fig. 6. Structural comparison of Hbl, Nhe and ClyA. (a) Structure of Hbl component B as determined by X-ray crystallography (PDB ID: 2nrj). (b) Homology model of NheB. (c) Homology model of NheC. The models in (b) and (c) were created on the basis of the Hbl B crystal structure. The first 29 and 34 residues of the mature sequences of NheB and NheC, respectively, and the last 26 residues of NheB were not present in the models obtained. (d) Crystal structure of E. coli ClyA (PDB ID: 1qoy). Protein structures in (a–d) are shown in ribbon format, with the β-hairpins in blue, and drawn using MOLMOL (Koradi et al., 1996). (e) Structural alignment visualized as a 3D superimposition of Hbl B (blue) and ClyA (grey) obtained using DaliLite, viewed as a C-trace (Fagerlund et al., 2008). 8.6. Enterotoxin T A third toxin, enterotoxin T, has been described recently as a single component enterotoxin and appears to possess biological activity similar to HBL and NHE. The authors were able to detect the enterotoxin T gene, bceT, in all ten strains tested by PCR, including some environmental isolates. However, no evidence exists that the 41 kDa enterotoxin T has been involved in any cases of B. cereus food poisoning. Later, bceT was detectable in only 40% of 95 strains of B. cereus screened by PCR. Nevertheless, when this bceT clone was expressed in E. coli no biological activity was found in either supernatants or cell extracts. Cell extracts from the bceT positive B. cereus strains were also negative on Vero cells suggesting that this toxin can probably not contribute to food poisoning (Choma and Granum, 2002). 35
  36. 36. 8.7. Cytotoxin K B. cereus produces two single-component protein toxins that are members of the family of β-barrel poreforming toxins, namely Cytotoxin K (CytK) (Lund et al., 2000) and Hemolysin II (Hly II). Hly II is a thermolabile antigenic protein, MW 29-34,000, pI 4.92, also susceptibel to pronase, pepsin and trypsin. In vitro activity unaffected by thiols, cholesterol and anti-streptolysin O (Gilbert et al., 1981). This toxin family includes β-toxin of Clostridium perfringens (Steinthorsdottir et al., 2000) and α-haemolysin of S. aureus. These toxins are secreted as water-soluble monomers that associate into oligomeric prepores at the target cell surface, which subsequently insert their pore-forming regions into the cell membrane forming a transmembrane pore. CytK is a 34-kDa protein with dermonecrotic, cytotoxic and haemolytic activities, and shows similar cytotoxic potency towards cell cultures as Hbl and Nhe (Lund et al., 2000). CytK was implicated as the toxin responsible for the severe symptoms and uncharacteristic bloody diarrhoea presenting in this outbreak. However, because genes encoding nhe were later identified in this strain, contribution by Nhe to the pathogenicity of B. cereus NVH 391/98 cannot be excluded. Characterization of the two variants of CytK showed that the CytK protein from NVH 391/98 had fivefold greater cytotoxic activity towards Caco-2 and Vero cells than the most common CytK variant, represented by CytK from B. cereus NVH 1230/88, which was initially named CytK-2. 8.8. Detection of enterotoxins B. cereus enterotoxin production can be measured with the B. cereus reversed passive latex agglutination (RPLA) test kit (Unipath-Oxoid, Columbia, Md.), which detects the L2 component of the tripartite toxin HBL, and with the Bacillus diarrheal enterotoxin visual immunoassay (Tecra Bioenterprises, Pty, Ltd., Roseville, Australia), which measures the NheA antigen from the Nhe complex. Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific 36
  37. 37. of emetic toxin producers was also investigated using PCR Portions of the hblA, hblC, hblD, nheA, nheB, nheC, entFM, bceT, and cytK genes and the emetic-specific sequence (em) were amplified using the set of primers (Yang et al., 2005) with the 16S to 23S rRNA internal transcribed sequence (ITS) used as an internal control. 8.9. Emetic Toxin Emetic toxin, cereulide, produced by B. cereus has been purified. Optimum production occurs in a rice culture slurry incubated at 25-30C during the stationary growth phase of the organism. From about 12 to 37C, although maximal production of emetic toxin appears to occur between 12 and 22C. However, two isolates belonging to the psychrotolerant species B. weihenstephanensis were recently shown to produce cereulide at 8C. Cereulide production was influenced by the composition of the formula, with a combination of dairy and cereal ingredients giving higher levels of cereulide production than rice and nondairy ingredients. In contrast, boiled rice and farinaceous (rich, containing starch) foods could sustain production of high levels of cereulide (Shaheen et al., 2006). Strains carried cereulide synthase gene, ces, on a megaplasmid of ca. 200 kb, grew up to 48-50C, but produced cereulide only up to 39C. On tryptic soy agar five strain produced highest amounts of cereulide at 23 to 28C. On oatmeal agar only one strain produced major amounts of cereulide. On skim milk agar, raw milk agar, and MacConkey agar most strains grew well but produced only low amounts of cereulide. Three media components, the ratio [K+]:[Na+], contents of glycine and [Na+], appeared of significance for predicting cereulide production. Increase of [K+]:[Na+] (focal variable) predicted (P < 0.001) high cereulide provided that the contents of glycine and [Na+] (additional variables) were kept constant. The results show that growth medium and temperature up and downregulate cereulide production by emetic B. cereus in a complex manner (Apetroaie-Constantin et al., 2008). Potato puree and penne pasta were inoculated with cereulide producing B. cereus. Static incubation at 28C proved these two foods to be a better substrate for higher cereulide production (4,080 ng/g in puree and 3,200 ng/g in penne were produced by B. cereus 5964a during 48 h of incubation) 37
  38. 38. compared with boiled rice (2,000 ng/g). This difference occurred despite B. cereus counts of more than 10(8) CFU/g in all three products. Aeration of cultures had a negative effect on cereulide production (Rajkovic et al., 2006). It can be extracted by chloroform and nine peaks eluted off Sephadex G-15, two or three have emetic activity. It is stable at 126C for 90 min, also stable at pH 2-11, resistant to pepsin and trypsin (Gilbert and Kramer, 1984; Gilbert and Kramer, 1986; Gorina et al., 1975; Johnson, 1984). Therefore, it will not be destroyed by gastric acid, the proteolytic enzymes of the intestinal tract or by reheating foods. Highly alkaline pH plus heating above 100C is needed to achieve inactivation (Rajkovic et al., 2008). Cereulide is a small ring-formed dodecadepsipeptide with the structure [D-O-Leu-D-Ala-D-O-Val-D-Val]3, and its genetic determinant is plasmid-borne (Ehling-Schulz et al., 2006). The peptide is 1,165 Da with a predicted pI of 5.52. Cereulide is hydrophobic and not easily solubilized in aqueous solutions and may be delivered to its target cells bound to or dissolved in carriers found in food (Schoeni and Wong, 2005). Cereulide is produced by a nonribosomal peptide synthetase, encoded by the 24-kb cereulide synthetase (ces) gene cluster, which is located on a megaplasmid. The mechanism by which cereulide causes emesis in humans has not been definitely determined. Following release from the stomach into the duodenum, cereulide binds to the 5-HT3 receptor, and stimulation of the vagus afferent causes vomiting in Suncus murinus (錢鼠), an animal model. The toxin also acts as a cation ionopore, like valinomycin, and is therefore able to inhibit mitochondrial activity by inhibition of fatty acid oxidation. This effect of cereulide was the reason for the liver failure in two lethal cases of emetic food poisoning (Dierick et al., 2005). In an experiment mice model, intraperitoneally injection with high doses of synthetic cereulide, massive degeneration of hepatocytes occurred. General recovery from the pathological changes, and regeneration of hepatocytes, were observed after 4 weeks. Cereulide has also been shown to cause cellular damage and inhibit human natural killer cells of the immune system. Detection of cereulide Two animal models, rhesus monkey (Macaca mulatta) and Asian musk 38
  39. 39. shrew (Suncus murinus), have been used for cereulide assays. These assays have received limited use because they can be difficult and expensive and because cell culture assays have been developed and improved. The HEp-2 vacuolation assay with colorimetric modifications is commonly used to test for the emetic toxin. In this assay, the mitochondrial swelling caused by cereulide appears as cytoplasmic vacuoles in HEp-2 cells. Paralysis of boar spermatozoa and changes in oxidation rates in isolated rat liver mitochondria have also been used as indicators of cereulide-induced toxicity. Measurement of oxygen consumption in these assays indicates that cereulide acts by uncoupling mitochondrial oxidative phosphorylation (Schoeni and Wong, 2005). 8.10. Bioassays of Enterotoxins and Emetic Toxin Vascular Permeability Reaction (VPR). For the VPR test, 0.05 ml volumes of the culture filtrates in quadruplicate are injected intradermally into the depilated backs of adult (2-3 kg) rabbits. At 3 h post-inoculation, 4 ml of 2% w/v Evans Blue dye in 0.85% (w/v) saline is injected intravenously into each rabbit. After a further hour, diameters of the zones of blueing and, when present, necrosis, are measured. Total VPR is calculated as the sum of the mean radii of each of these zones (Kramer et al., 1982). This method is preferred for screening of isolates. Ligated Rabbit Ileal Loop Test. Young New Zealand white rabbits (1-1.5 kg) are fasted for 48 h prior to externalizing the ileum (Spira and Goepfert, 1972). Intraluminal injections of 1.0 ml of test preparation in about 5 cm loops up to a maximum of 12 loops per rabbit, or 2 ml in 10 cm loops to a maximum of 6 loops should provide optimum reliability. Loops are separated from one another by about 5-cm blank loops. Generally, if the ratio of the volume of accumulated fluid to loop length (V/L) is >0.5, this is regarded as evidence that net fluid secretion into the lumen has occurred (Fig. 8, Table 15) (Kramer et al., 1982). Rhesus Monkey Feeding Test. 39
  40. 40. Monkey feeding is currently the only available method of detecting emetic activity in a test preparation. For obvious reasons this test can only be carried out in a very few laboratories in the world and not on any kind of routine basis (Kramer et al., 1982). Mouse-lethality Assay. Mouse-lethal toxin activity is determined by the injection of 0.1 ml of material in phosphate buffer (0.02 M, pH 7.0) into the caudal vein of two ICR mice. Death within 30 min is considered to be a positive response (Thompson et al., 1984). The mouse-lethal dose (MLD/ml) is expressed as the reciprocal of the highest dilution which is lethal for both mice within 30 min (Spira and Goepfert, 1975). Cytotoxicity Assay. Cytotoxic activity is assayed by the use of Vero cells. Cells are maintained in tissue culture medium and split into 24-well plates 3 days before the test. Cell density at the time of the test is about 105 cells per cm2 . Test samples are sterilized by filtration and 100 μl is added to each well. Phosphate buffer controls are run concurrently with all tests. Cells are incubated in 5% CO2 at 36C and observed at intervals up to 12 h for disruption of the cell monolayer. Cytotoxicity is expressed as the reciprocal of the dilution that resulted in complete loss of activity (Thompson et al., 1984). Aggregate-Hemagglutination Technique. Antiserum against purified B. cereus enterotoxin is obtained. The washed erythrocytes are treated with glutaraldehyde for 3 h at 37C, washed and resuspended in saline plus 0.25% glutaraldehyde. The antiserum is treated with glutaraldehyde for 1 h at 37C. The erythrocytes are then added to the aggregated antiserum for sensitization (incubate for 90 min at 56C and then 30 min at room temperature). The sensitized erythrocytes in saline are preserved with merthiolate (1:10,000). Agglutinations are performed by microtitration procedure with 0.2% normal rabbit serum as diluent. A 0.004 μg quantity of enterotoxin per ml can de detected by this method (Gorina et al., 1975). 40
  41. 41. 9. CONCLUSIONS The widespread distribution of B. cereus and the ability of the spores to survive long-term storage in dried products, and the thermal resistance of spores help to explain the wide variety of foods that have been implicated in B. cereus foodborne illness outbreaks. Therefore, one should assume that B. cereus is present and should take preventative measures to prevent growth during food handling. Such measures include: (a) keeping the holding time of a food between preparation and consumption to a minimum and (b) where storage is necessary, using rapid and adequate cooling to a temperature below 10C to prevent multiplication of the organism. So, investigation should be done on the survival, growth and germination of vegetative cells and spores of B. cereus in different food processing systems. Besides being a foodborne pathogen, B. cereus has been applied in controlling Salmonella. Toyocerin® is a zootechnical additive composed of spores of a strain of Bacillus cereus mixed with maize flour and calcium carbonate. The product has already been granted permanent or provisional authorisation for use with most livestock species and categories. Feeding Toyocerin significantly reduces the prevalence of Salmonella in poultry (Vila et al., 2009). B. cereus and Lactobacillus are used as probiotics in animal feed (Li et al., 2009). 10. REFERENCES Ahmed,A.A.H., Moustafa,M.K., Marth,E.H. 1983. Incidence of Bacillus cereus in milk and some milk products. J. Food Prot. 46, 126-128. Ahmed,R., Sankar-Mistry,P., Jackson,S., Ackermann,H.W., Kasatiya,S.S. 1995. Bacillus cereus phage typing as an epidemiological tool in outbreaks of food poisoning. J. Clin. Microbiol. 33, 636-640. 41
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