BACILLUS CEREUS

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BACILLUS CEREUS

• ???
• 2009

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Content

• INTRODUCTION
• OCCURRENCE IN FOOD AND ENVIRONMENT
• CHARACTERISTICS AND TAXONOMY
• SPORE AND GERMINATION
• ISOLATION AND ENUMERATION
• TYPING
• CONTROL
• VIRULENCE FACTORS
• Syndromes of B. cereus Food Poisoning
• Phospholipase and Sphingomyelinase
• Cereolysin
• Haemolysin BL
• Nonhaemolytic enterotoxin
• Enterotoxin T
• Cytotoxin K
• Detection of enterotoxins
• Emetic Toxin
• Bioassays of Enterotoxins and Emetic Toxin
• CONCLUSIONS

3
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

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INTRODUCTION

• 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.

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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
• 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. 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

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OCCURRENCE IN FOOD AND ENVIRONMENT

• A variety of foods have been implicated in
  food-poisoning
• Emetic syndrome caused by B. cereus is highly
  associated with rice and rice products

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OCCURRENCE IN FOOD AND ENVIRONMENT

• B. cereus was isolated from 9, 35, 14 and 48 of
  raw milk, pasteurized milk, Cheddar cheese and
  ice cream samples, respectively
• 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 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).

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OCCURRENCE IN FOOD AND ENVIRONMENT

• 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)

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OCCURRENCE IN FOOD AND ENVIRONMENT

• 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).

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OCCURRENCE IN FOOD AND ENVIRONMENT

• 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).

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OCCURRENCE IN FOOD AND ENVIRONMENT

• 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.
• Only one B. cereus isolate possessed the
  cereulide synthetase gene, ces (Rahmati and
  Labbe, 2008).

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OCCURRENCE IN FOOD AND ENVIRONMENT

• 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).

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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.

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CHARACTERISTICS AND TAXONOMY
15
CHARACTERISTICS AND TAXONOMY
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CHARACTERISTICS AND TAXONOMY

• 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 GC content of the DNA is reported to be
  32-33 moles (determined by Tm) and 33-37 moles
  (analysis).

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CHARACTERISTICS AND TAXONOMY

• ????

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CHARACTERISTICS AND TAXONOMY

• . 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.

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CHARACTERISTICS AND TAXONOMY

• 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)

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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.

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GERMINATION 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
• Finally, the onset of metabolic activity as
  measured by oxygen uptake.

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Role of trypsin-like enzyme

• 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).

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SPORE AND GERMINATION

• 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.
• Why?

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SPORE AND GERMINATION

• Germination of B. cereus spores is more
  extensive in rice than in trypticase soy broth at
  lt15C and is generally more extensive for
  diarrheal strains in either medium than emetic
  strains
• Germination of B. cereus spores was also
  inhibited by the growth of lactic acid bacteria
  or the organic acids produced (Wong and Chen,
  1988).

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SPORE AND GERMINATION

• B. cereus spores germinate in inosine or in
  l-alanine as sole germinants
• They 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

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Confirmation of outbreak

• B. cereus strains of the same serotype should be
  present in the epidemiologically food, feces
  and/or vomitus of the affected persons. Or
• Significant numbers (gt105 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
• Significant numbers (gt105 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.

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ISOLATION AND ENUMERATION

• 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.

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ISOLATION AND ENUMERATION

• 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).

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ISOLATION AND ENUMERATION

• 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
• authors addressed that the new chromogenic media
  represent a good alternative to the conventional
  standard media (Fricker et al., 2008).

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SEROTYPING

• Developed at the Food Hygiene Laboratory, England
  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
• In approximately 90 of outbreaks the causative
  serotypes can be established.

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SEROTYPING

• 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

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PHAGE TYPING

• 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).

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TYPING

• 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).

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amplified fragment length polymorphism (AFLP)
method

• the chromosome DNA is digested by HindIII,
  ligated to adapters (ACG GTATGC GAC AG and GAGTGC
  CATACGCTGTCTCGA
• amplified by PCR using primers
  (GGTATGCGACAGAGCTTA, GGTATGCGACAGAGCTTC, G
  GTATGCGACAGAGCTTG and GGTATGCGACAGAGCTTT)
  (Ripabelli et al., 2000).

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AFLP method
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Multilocus sequence typing (MLST)

• Primers were designed for conserved regions of
  housekeeping genes, and 330- to 504-bp internal
  fragments of seven such genes were sequenced for
  all strains.
• 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 synthetase, beta
  subunit)

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Multilocus sequence typing (MLST)

• 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 synthetase, beta subunit)
  were sequenced for all strains. The number of
  alleles at individual loci ranged from 25 to 40
  (Table 6),

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Multilocus sequence typing (MLST)
39
Multilocus sequence typing (MLST)

• The number of alleles at individual loci ranged
  from 25 to 40

dN/dS ratio nonsynonymous/synonymous
substitution rate ratios
40
Multilocus sequence typing (MLST)

• a total of 53 allelic profiles or sequence types
  (STs) were distinguished

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Multilocus sequence typing (MLST)

• 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 has a high level of resolution
  and should be an excellent tool for studying the
  population structure and epidemiology of the B.
  cereus group

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MLST

• Phylogenetic analysis was performed on a total of
  296 strains for which MLST sequence information
  is available (MLST database (http//pubmlst.org/bc
  ereus/)
• three main lineages--I, II, and III--within the
  B. cereus complex were identified
• With few exceptions, all food-borne isolates
  were in group I.

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MLST

• horizontal gene transfer (HGT) of toxin-encoding
  genes among various strains determined

44
CONTROL

• 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
  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 lt/4.5, NaCl concentration
  gt/1.0 and temperatures lt/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).

45
CONTROL

• The combination of mild acidification (pH 5.0)
  and refrigeration (lt/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).

46
CONTROL

• 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).

47
CONTROL

• A combination of electrolyzed water and 1 citric
  acid
• peracetic acid-based disinfectant
• ozone
• Pulsed Electric Field (PEF) technology
• High pressure around 300 MPa
• Several antimicrobial wine recipes, each
  consisting of red or white wine extracts of
  oregano leaves with added garlic juice and
  oregano oil are bactericidal

48
Essences of vegetables

• Carvacrol, a natural antimicrobial compound
  present in the essential oil fraction of oregano
  (???)and thyme(???)

49
bacteriocin

• enterocin AS-48 ( 20-35 µg/ml )against the
  toxicogenic psychrotrophic strain B. cereus
• 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

50
VIRULENCE

• 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).

51
VIRULENCE

• Usually, two types of B. cereus foodborne
  diseases occur, the diarrhoeal and the emetic
  types

52
Cell culture assays

• 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.

53
VIRULENCE FACTORS

• 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 are currently considered the
  aetiological agents of B. cereus diarrhoeal
  foodborne disease
• haemolysin BL (Hbl)
• nonhaemolytic enterotoxin (Nhe)
• cytotoxin K.
• Hbl and Nhe are related three-component toxins,
  while the single-component CytK belongs to the
  family of b-barrel pore-forming toxins.

54
VIRULENCE FACTORS

• 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

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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)

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Phospholipase

• Phospholipase (Lecithinase)
• similar to the a-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
  (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)

57
Sphingomyelinase

• It is a protein of between 41,000 and 23,300 Da,
  depending on the method of analysis used
• requires divalent cations for activity
• can be assayed as follows culture filtrate is
  mixed with phosphate-buffered saline and
  TNPAL-sphingomyelin solution (N-w- trinitrophenyl
  aminolauryl sphingosyl phosphoryl choline).

58
Cereolysin

• 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
• Cereolysin is a cholesterol-dependant cytolysins,
  containing two half-cystine residues.
• In the absence of dithriothreitol, partial
  spontaneous oxidation resulted in the formation
  of an oxidized form of the toxin.
• 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

59
Haemolysin BL

• 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

60
Haemolysin BL

• 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 synthesized and released
  during the late logarithmic growth phase of the
  organism at an optimum temperature of 32-37C.

61
Haemolysin BL

• 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
• 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

62
Haemolysin BL

• The enterotoxin is thermolabile and susceptible
  to protease inactivation
• Activity of enterotoxin may involve stimulation
  of adenylate cyclase-cAMP system with probable
  role in non-gastrointestinal infections

63
Haemolysin BL

• 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.

64
Haemolysin BL

• Only Components B and L1 contain predicted
  transmembrane segments of 17 and 60 amino acid
  residues, respectively
• 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).

65
Haemolysin BL

• 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
• HBL forms pores in eukaryotic cell membranes,
  with each of the components binding the membrane
  independently and reversibly
• A high degree of molecular heterogeneity exists
  in HBL from different strains

66
Haemolysin BL

• 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.

67
Nonhaemolytic enterotoxin

• NHE is also a multi-component toxin

68
Nonhaemolytic enterotoxin

• 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.

69
Nonhaemolytic enterotoxin

• 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

70
Nonhaemolytic enterotoxin

• 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

71
Nonhaemolytic enterotoxin

• Vero cell assay
• A, toxin
• B, control

72
Nonhaemolytic enterotoxin

• (A)1.5 human erythrocytes

73
Enterotoxin T

• 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.

74
Cytotoxin K

• single-component protein toxins that are members
  of the family of ß-barrel poreforming toxin
• This toxin family includes ß-toxin of Clostridium
  perfringens and a-haemolysin of S. aureus
• 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)

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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

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Cereulide

• It is a small ring-formed dodecadepsipeptide with
  the structure D-O-Leu-D-Ala-D-O-Val-D-Val3
• 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).

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Detection of cereulide

• Two animal models, rhesus monkey (Macaca mulatta)
  and Asian musk shrew (Suncus murinus)(?? ), have
  been used for cereulide assays
• The HEp-2 vacuolation assay with colorimetric
  modifications is commonly used. 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).