Transcript B. cereus

BACILLUS CEREUS
黃顯宗
Content
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INTRODUCTION
OCCURRENCE IN FOOD AND ENVIRONMENT
CHARACTERISTICS AND TAXONOMY
SPORE AND GERMINATION
ISOLATION AND ENUMERATION
TYPING
CONTROL
VIRULENCE FACTORS
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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
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
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 toxintesting 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.
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
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
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).
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)
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).
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).
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).
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).
CHARACTERISTICS AND TAXONOMY
 B. cereus is a Gram-positive, motile, facultative,
aerobic sporeformer.
 Dimensions of vegetative cells are typically 1.01.2 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 TAXONOMY
CHARACTERISTICS AND TAXONOMY
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 G+C content of the DNA is reported to
be 32-33 moles % (determined by Tm) and
33-37 moles (analysis).
CHARACTERISTICS AND TAXONOMY
分類特性
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.
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)
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 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.
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).
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?
SPORE AND GERMINATION
 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
Germination of B. cereus spores was also
inhibited by the growth of lactic acid
bacteria or the organic acids produced
(Wong and Chen, 1988).
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 lalanine as the sole germinant, with a
smaller contribution from the GerI receptor
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 (>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
 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.
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.
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).
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).
SEROTYPING
Developed at the Food Hygiene Laboratory,
England Based on the established typespecificity 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.
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
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).
http://viralzone.expasy.org/all_by_species/140.html
http://viralzone.expasy.org/all_by_species/142.html
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).
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).
AFLP method
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.
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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)
Multilocus sequence typing (MLST)
 Primers were designed (Table 5) for conserved
regions of housekeeping genes, and 330- to 504bp 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),
Multilocus sequence typing (MLST)
Multilocus sequence typing (MLST)
The number of alleles at individual loci
ranged from 25 to 40
dN/dS ratio: nonsynonymous/synonymous substitution rate ratios
Multilocus sequence typing (MLST)
a total of 53 allelic profiles or sequence
types (STs) were distinguished
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
MLST
Phylogenetic analysis was performed on a
total of 296 strains for which MLST
sequence information is available (MLST
database (http://pubmlst.org/bcereus/)
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.
MLST
horizontal gene transfer (HGT) of toxinencoding genes among various strains
determined
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 </=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).
CONTROL
 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).
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).
Electrolyzed water
http://www.ewatersystems.com.au/e-water/electrolyzedwater/how-does-it-work/
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
Essences of vegetables
Carvacrol, a natural antimicrobial
compound present in the essential oil
fraction of oregano （奧立岡）and thyme
（百里香）
http://www.stuartxchange.com/Oregano.html
http://www.bbc.co.uk/food/thyme
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
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).
VIRULENCE
 Usually, two types of B. cereus foodborne
diseases occur, the diarrhoeal and the emetic
types
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.
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
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haemolysin BL (Hbl)
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nonhaemolytic enterotoxin (Nhe)
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cytotoxin K.
 Hbl and Nhe are related three-component toxins, while the singlecomponent CytK belongs to the family of b-barrel pore-forming toxins.
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
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
 Phospholipase (Lecithinase)
 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 (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
requires divalent cations for activity
can be assayed as follows: culture filtrate is
mixed with phosphate-buffered saline and
TNPAL-sphingomyelin solution (N-wtrinitrophenyl aminolauryl sphingosyl
phosphoryl choline).
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 halfcystine 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
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
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.
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
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 nongastrointestinal infections
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.
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).
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
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.
Nonhaemolytic enterotoxin
NHE is also a multi-component toxin
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.
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
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
Nonhaemolytic enterotoxin
Vero cell assay
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A, toxin
B, control
Nonhaemolytic enterotoxin
(A)1.5 % human erythrocytes
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.
Cytotoxin K
 single-component protein toxins that are
members of the family of β-barrel poreforming
toxin
 This toxin family includes β-toxin of Clostridium
perfringens and α-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)
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

Cereulide
 It is a small ring-formed dodecadepsipeptide with
the structure [D-O-Leu-D-Ala-D-O-Val-D-Val]3
 Its genetic determinant is plasmid-borne (EhlingSchulz 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).
Detection of cereulide
 Two animal models, rhesus monkey (Macaca mulatta)
and Asian musk shrew (Suncus murinus)(臭鼩 ), 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).