Bacillus cereus

1
Report on
By
Jeevan Shrestha
Roll no.: 22
Central Department of Microbiology
Tribhuwan University
Kirtipur, Kathmandu, Nepal.

i
ACKNOLEDGEMENT
I would like to acknowledge the enormous help given to me to prepare this report. For their help
and suggestion, I wish to thank all the lecturers of Central Department of Microbiology,
Tribhuvan University. The help from senior microbiologists will always be memorized. Also, I
would like to thank those friends who provided the guidelines to complete it.
Mostly, I would like to thank Ms. Shaila Basnyat and Mrs. Supriya Sharma for referring this
topic to prepare my report and providing necessary guidance to complete it.

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TABLE OF CONTENTS
ACKNOLEDGEMENT..................................................................................................................................i
CHAPTER ONE .........................................................................................................................................1
INTRODUCTION.......................................................................................................................................1
1.1 MORPHOLOGICAL CHARACTERISTICS.............................................................................................1
1.2 CULTURAL CHARACTERISTICS.........................................................................................................2
1.3 BIOCHEMICAL CHARACTERISTICS...................................................................................................4
CHAPTER TWO ........................................................................................................................................5
EPIDEMIOLOGY .......................................................................................................................................5
CHAPTER THREE ......................................................................................................................................7
VIRULENCE FACTORS...............................................................................................................................7
3.1 TOXINS ..........................................................................................................................................7
3.2 BETA-LACTAMASES AND OTHER ENZYMES.....................................................................................7
CHAPTER FOUR .......................................................................................................................................8
PATHOGENESIS........................................................................................................................................8
CHAPTER FIVE..........................................................................................................................................9
CLINICAL MANIFESTATIONS.....................................................................................................................9
CHAPTER SIX..........................................................................................................................................10
LABORATORY DIAGNOSIS ......................................................................................................................10
6.1 COLLECTION, TRANSPORTATION AND STORAGE OF SAMPLE........................................................10
6.2 SAMPLE PREPARATION ................................................................................................................10
6.3 CULTURE......................................................................................................................................10
6.4 MOST PROBABLE NUMBER (MPN) OF B. cereus ...........................................................................11
6.5 MICROSCOPY...............................................................................................................................11
6.6 BIOCHEMICAL TESTS ....................................................................................................................11
CHAPTER SEVEN ....................................................................................................................................12
ANTIBIOTIC RESISTANCE........................................................................................................................12
7.1 CAUSES OF ANTIBIOTIC RESISTANCE ............................................................................................13
CHAPTER EIGHT.....................................................................................................................................14
PREVENTION AND CONTROL .................................................................................................................14
REFERENCES..........................................................................................................................................15

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CHAPTER ONE
INTRODUCTION
Bacillus cereus is spore forming, aerobic to facultative, gram-positive, motile rod, ubiquitously
opportunistic pathogen, frequently isolated from soil and growing plants, but it is also well
adapted for growth in the intestinal tract of insects and mammals.
1.1 MORPHOLOGICAL CHARACTERISTICS
B. cereus is a Gram-positive, motile and spore former. 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 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 Ca
2+
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. The germination of B.
cereus spore is partially prevented by several inhibitors of trypsin-like enzymes (leupeptin,
antipain, 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. 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. Germination of B.
cereus spores was also inhibited by the growth of lactic acid bacteria or the organic acids
produced.
Inactivation of B. cereus spores during cooling from 90ºC occurs in two phases, one phase
occurs during cooling from 90 to 80ºC; the second occurs during cooling from 46 to 38ºC. No
inactivation occurs when spores are cooled from a maximum temperature of 80ºC. Inactivation
of spores at temperatures between 46-38C is stimulated by the higher heat treatment (80-90ºC).
Germination of spores is required for 45ºC inactivation to occur after the spores are heated to
90ºC 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 45ºC inactivation mechanism.

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1.2 CULTURAL CHARACTERISTICS
A variety of selective and/or differential media are available. B. cereus can be 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. Isolates retrieved from frozen glycerol
stock cultures can be streaked to tryptic soy agar (TSA) and incubated overnight at 30°C. A
single colony of each strain can also be grown overnight in brain heart infusion broth (pH 7.4)
with 1 g/L glucose (BHIG).
When grown under aerobic conditions on 5% sheep blood agar at 37°C, B. cereus colonies are
dull gray and opaque with a rough matted surface Colony perimeters are irregular and represent
the configuration of swarming from the site of initial inoculation, perhaps due to B. cereus
swarming motility. Zones of beta-hemolysis surround and conform to the colony morphology.
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 facilitate formation of spore
A Columbia base 5% blood agar surface-spread with polymyxin B is recommended by Kramer
et al. for better colonly characteristics and colony types are easily differentiated for serological
testing.
Trypticase soy polymyxin broth is recommended for the enumeration by most probable number
method.
Figure 1.2 Gray, opaque, granular, spreading colonies with irregular
(Bottone et al., 2010)
Figure 1.3 B. cereus on MYP agar

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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.
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. The Bromthymol blue is replaced with bromcresol
purple to give a new medium designated PEMPA. The advantage of this modification is
decreased incubation time (from 48 h to 22 h).
Two new chromogenic plating media (CBC and BCM) are 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.
Table 1.2 Cultural characteristics of B. cereus

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1.3 BIOCHEMICAL CHARACTERISTICS
B. cereus does not ferment mannitol and has a very active phospholipase (lecithinase) system.
They are keyed as citrate (+), arabinose (-), Gram (+), aerobic sporeformer. Biochemical
characteristics are listed below in table 1.3.
Table 1.3 Biochemical properties of B. cereus

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CHAPTER TWO
EPIDEMIOLOGY
B. cereus related food poisoning is not a notifiable disease in most countries and therefore
incidence data is extremely limited. It is recognized that there may be significant under reporting
cases of B. cereus illness due to the generally mild, short duration and self-limiting symptoms as
well as being infrequently tested for in routine laboratory analyses of stool samples.
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. A variety of foods have been
implicated in food-poisoning. Emetic syndrome caused by B. cereus is highly associated with
rice and rice products. They can be found in most common food materials such as rice, pasta
and dairy products, fish, meat, poultry, honey, vegetables, desserts and soups either in raw,
cooked or processed foods and it represents more than 68.0% of foodborne outbreaks in all
food types. In a report by Blakey and Priest, 56% of the peas, beans and cereals samples
contained B. cereus, 1x10
2
to 6x10
4
organisms/g. During normal cooking procedure, B. cereus in
the sample could increase to 10
7
cells/g.
In the European Union there were 0.04 reported cases of B. cereus foodborne illness per 100,000
population in 2011 (ranging from <0.01–0.24 per 100,000 population between countries). This
was an increase from the 2010 case rate of 0.02 cases per 100,000 population. B. cereus was
reported as a major causative agent of foodborne illness in the Netherlands in 2006 (causing
5.4% of the foodborne outbreaks) and in Norway in 2000 (causing 32% of foodborne outbreaks)
In the United States (US), B. cereus caused 0.7% of foodborne illness caused by 31 major
pathogens.
There was one reported outbreak of B. cereus foodborne illness in Australia in 2011 and one
outbreak reported in 2010. It has been estimated that B. cereus accounts for 0.5% of foodborne
illness caused by known pathogens in Australia. In New Zealand there was one foodborne B.
cereus outbreak reported in 2011, there were no outbreaks reported in 2010.

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Figure 2 Major outbreaks associated with B. cereus (www.foodstandards.gov.au)

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CHAPTER THREE
VIRULENCE FACTORS
3.1 TOXINS
B. cereus produces toxins that are supposed to be important factors for its pathogenicity.They
produces three enterotoxins: non-hemolytic enterotoxin (NHE), hemolysin BL (HBL) and
cytotoxin K1 (CytK1) are potentially responsible for diarrhea whereas cereulide is responsible
for emesis. The NHE is composed of three subunits A, B and C whereas the HBL is composed of
other three subunits L1, L2 and B.
HBL is composed of a binding component, B (35 kDa), and two lytic components, L1 and L2
(36 and 45 kDa) and it requires all three components for maximal activity of hemolytic,
cytotoxic, dermonecrotic and vascular permeability as well as fluid. NHE is also the three-
component enterotoxin complex (39, 45, and 105 kDa) associated with the diarrheal food
poisoning.
The cereulide toxin is cyclic dodecadepsipeptide which does not lose its activity at 121 °C, it
tolerates extreme pH values between pH 2 and pH 11 and it is also stable to pepsin and trypsin
treatment. It has been assumed that cereulide is not a genetic product but is synthesized
enzymatically in the growth medium. It is also responsible for the inhibition of hepatic
mitochondrial fatty-acid oxidation and has been reported to cause liver failure.
3.2 BETA-LACTAMASES AND OTHER ENZYMES
Beta-lactamases are a clinically important cause of beta-lactam antibiotic resistance. Three
different forms of beta-lactamases have been reported among different strains of B. cereus.
ß-lactamase I belongs to the class A ß-lactamases and is an extracellular penicillinase with a
serine in the active site. Beta-lactamase II, a class B ß-lactamase, is activated by binding Zn(II)
and Co(II) ions. Betalactamase III is a class A membrane bound lipoprotein also having a
secreted form .
Collagenase, purified from an oral isolate of B. cereus degrades soluble and insoluble collagens,
gelatin and bradykinin. Collagenolytic proteases produced by dental plaque bacteria may affect
the disruption of the crevicular basement membrane, although in periodontal infections host-
derived proteinases appear to play a major role in collagen degradation. Bacterial collagenases
cleave collagen to small peptides, which have been suggested to attract neutrophils by
chemotaxis. The production of extracellular collagenase was reduced when the growth of the
bacterial cells was inhibited by salivary peroxidase systems. Proteases have been suggested to
have a role in nongastrointestinal infections caused by B. cereus. A neutral protease purified
from a virulent strain of B. cereus was indicated as having an ability to hydrolyze hemoglobin,
albumin and casein. Sphingomyelinase, a 34-kDa hemolytic protein, binds to sphingomyelin on
erythrocytes. Phospholipase C is supposed to contribute to tissue damage by inducing the
degranulation of human neutrophils. B. cereus produces three types of phospholipase C:
phosphatidylinositol hydrolase, phosphatidylcholine hydrolase and the hemolytic
sphingomyelinase.

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CHAPTER FOUR
PATHOGENESIS
The pathogenicity of B. cereus is closely associated with tissue-destructive exoenzyme
production. Among these secreted toxins are four hemolysins, three distinct phospholipases, an
emesis-inducing toxin, and three pore-forming enterotoxins: hemolysin BL (HBL), nonhemolytic
enterotoxin (NHE), and cytotoxin.
Vegetative cells ingested into the gastrointestinal tract secrete an enterotoxin and induce a
diarrheal syndrome. In rabbit ligated ileal-loop assays, culture filtrates of enterotoxigenic strains
induce fluid accumulation and hemolytic, cytotoxic, dermonecrotic and vascular permeability
activities in rabbit skin. This tripartite enterotoxin is composed of a binding component (B) and
two hemolytic components, designated HBL. Also diarrheagenic in the gastrointestinal tract is a
nonhemolytic three-component enterotoxin, designated NHE.
The cereulide is a preformed toxin produced in food products which is synthesized in the
contaminated food product represent a metabolic product of growth. It causes nausea and
vomiting.
Inhibition of NK cell cytotoxicity has been seen with cereulide concentrations from 20 ng/ml,
which is less that cause emesis. Mitochondria of NK cells are found to be swollen after
treatment with cereulide. Cereulide inhibit the modest IFN-g production of NK cells induced by
IL-12 or IL-15. The lytic activity of monocytes is also slightly inhibited by the toxin.
Figure 4 Fluorescence microscopy showing continuous degenaration of NK cells (Paananen et al., 2002)

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CHAPTER FIVE
CLINICAL MANIFESTATIONS
B. cereus can cause two distinct types of illnesses:
1) a diarrheal illness with an incubation time of approximately 10 to 16 hours. The symptoms
include abdominal cramps and watery diarrhea.
2) an emetic (vomiting) illness with an incubation time of one to six hours. The symptoms
include nausea and vomiting.
B. cereus also causes a number of systemic and local infections in both immunologically
compromised and immunocompetent individuals. Among those most commonly infected are
neonates, intravenous drug abusers, patients sustaining traumatic or surgical wounds, and those
with indwelling catheters. The spectrum of infections includes fulminant bacteremia, central
nervous system (CNS) involvement (meningitis and brain abscesses), endophthalmitis,
pneumonia, and gas gangrene-like cutaneous infections. Endocarditis, osteomyelitis and urinary
tract infections are rare cases due to B. cereus.
Due to the widespread distribution of Bacillus spores in soil, dust, water, and the hospital
environment, B. cereus is usually considered a contaminant when isolated from clinical
specimens of various origins (blood, wounds, and sputum etc.). B. cereus are gaining notoriety as
causing definitive nosocomial outbreaks among immunosuppressed hospitalized patients.
Figure 5 Haemorrhagic necrosis of brain due to B. cereus invasion (Bottone et al., 2010)

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CHAPTER SIX
LABORATORY DIAGNOSIS
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 (>10
5
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 (>10
5
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.
6.1 COLLECTION, TRANSPORTATION AND STORAGE OF SAMPLE
The specimens for diagnosis can be the contaminated food, pans used for cooking, stools and
vomits. Clinical specimens and food samples are collected, stored temporary at refrigerating
temperature if necessary, diluted if necessary and plated on selective agar media. If the food is
a powder or consists of small discrete particles, then it should be thoroughly mixed before taking
samples. Transport samples promptly in insulated shipping containers with enough gel-type
refrigerants to maintain them at 6°C or below. Upon receipt in the laboratory, store the samples
at 4°C and analyze as soon as possible. If analysis cannot be started within 4 days after
collection, freeze samples promptly and store at -20°C until examined. Thaw at room
temperature and proceed with analysis as usual. Maintain frozen samples at -20°C until
examined. Shipping should be carried on dry ice to avoid thawing. Dehydrated foods may be
stored at room temperature and shipped without refrigeration.
6.2 Sample preparation
Weigh 50 g of sample into sterile blender jar. Add 450 mL Butterfield's phosphate-buffered
dilution water (1:10 dilution) and blend for 2 min at high speed (10,000-12,000 rpm). Prepare
serial dilutions from 10-2
to 10-6
by transferring 10 ml homogenized sample (1:10 dilution) to 90
ml dilution blank, mixing well with vigorous shaking, and continuing until 10-6
dilution is
reached.
6.3 CULTURE
Inoculate MYP agar plates with each dilution of sample (including 1:10) by spreading 0.1 ml
evenly onto surface of each plate with sterile glass spreading rod. Incubate plates 18-24 h at
30°C and observe for colonies surrounded by precipitate zone, which indicates that lecithinase is
produced. B. cereus colonies are usually pink on MYP and may become more intense after
additional incubation. Pick at least 5 presumptive positive colonies from MYP plates and transfer
one colony to BHI with 0.1% glucose for enterotoxin studies and a nutrient agar slant for
storage. Typical colonies grown on MYP must be confirmed with biochemical testing as
described.

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6.4 MOST PROBABLE NUMBER (MPN) OF B. cereus
The most probable number (MPN) method is recommended for routine surveillance of products
in which small numbers of B. cereus are expected. This method is also effective in testing foods
that may contain a large population of competing species or in dehydrated food products in
which the potential spores would outnumber vegetative cells and require additional nutrients to
germinate. Inoculate 3-tube MPN series in trypticase soy-polymyxin broth, using 1 m; inoculum
of 10-1
, 10-2
, and 10-3
dilutions of sample with 3 tubes at each dilution. (Additional dilutions
should also be tested if B. cereus population is expected to exceed 103
/g.) Incubate tubes 48 ± 2 h
at 30 + 2°C and observe for turbid growth, which is typical of B. cereus. Streak cultures from
turbid, positive tubes onto separate agar plates (MYP) and incubate plates 18-24 h at 30°C. Pick
one or more pink (MYP), lecithinase-positive colonies from each agar plate and transfer to BHI
with 0.1% glucose for enterotoxin studies and nutrient agar slants for storage. Typical colonies
grown on MYP must be confirmed with biochemical testing as described.
6.5 MICROSCOPY
Prepare Gram-stained smears from slants and examine microscopically. B. cereus will appear as
large Gram-positive bacilli in short-to-long chains; spores are ellipsoidal, central to subterminal,
and do not swell the sporangium.
6.6 BIOCHEMICAL TESTS
Various biochemical tests can be 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. recommended the following biochemical tests
for confirmation of B. cereus: Glucose +, mannitol -, xylose -, and arabinose -. Serotyping is
recommended by a number of investigators.

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CHAPTER SEVEN
ANTIBIOTIC SUSCEPTIBILITY
Almost all isolates of B. cereus collected displayed low susceptibility to β-lactam antibiotics.
Local strains are more resistant than standard strain (ATCC 11778). Variations in the resistance
may be due to the differences in the concentrations of antimicrobial agents used, differences in
the source of isolates, drug resistance transfer and the overall wide spread use of the antibiotics
in the environment. The isolates of B. cereus from chicken meat were highest resistance to
Penicillin, Cloxacilin, Cephalexin (100%) and the least in Ampicillin (13%). B. cereus strains
isolated from infections have usually been resistant to beta-lactam antibiotics, including
thirdgeneration cephalosporins, but have been shown to be susceptible to chloramphenicol,
clindamycin, vancomycin, ciprofloxacin, erythromycin, gentamycin and streptomycin.
Table 7.1 Antimicrobial susceptibility test (Tahmasebi et al, 2014 )
Table 2 Antimicrobial susceptibility test (Tahmasebi et al, 2014 )

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7.1 CAUSES OF ANTIBIOTIC RESISTANCE
1. A common cause of antibiotic resistance in bacteria is an increased abundance of B-
lactamases. This can be the result of genetic engineering.
2. These chromosomal B-lactamases do not generally provide effective antibiotic
resistance in wild-type bacilli but the genes are not completely silenced. Under
antibiotic selection pressure, however, a number of strains show increased resistance,
suggesting mutation-induced upregulation of –lactamase expression.
3. Reasons of β–lactam resistance are they have altered bacterial targets, penicillin-binding
proteins (PBPs or transpeptidase) or Β-lactamases bind to the antibiotics and cleave the
β-lactam ring.
4. Tetracycline resistance gene on a plasmid pBC16 is demonstrated and sequenced. It is
also demonstrated that resistance to antimicrobial substances methicillin, gentamicin,
kanamycin and tetracycline can be acquired by the transfer of the relevant plasmid.
5. Numbers of antibiotics are added to feed for rapidly growth of chicken, improve feed
efficiency cause the antibiotic resistance.
6. Streptomycin is a constituent of a number antimicrobial preparations used for the
treatment of mastitis in cows and also for the protection of plants.

14
CHAPTER EIGHT
PREVENTION AND CONTROL
1. Long-term storage must be at temperatures below 8 °C.
2. Rapid cooling and proper reheating of cooked food are very essential if the food is not
consumed immediately.
3. Steaming under pressure, roasting, frying and grilling foods can destroy the vegetative
cells and spores.
4. Foods infested with the diarrheal toxin can be inactivated by heating for 5 min at 133 °F.
5. Foods infested with the emetic toxin need to be heated to 259 °F for more than 90 min.
6. The udder and the teats should be cleaned to reduce the contamination of raw milk
7. Educate food handlers about their responsibilities for food safety and train them on
personal hygiene policies and basic practices for safe food handlings
Figure 8 Preventive measures of food poisoning caused by B. cereus (http://www.health.gov.nl.ca)

15
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18. www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070875.htm
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20. www.health.gov.nl.ca/health/publichealth/envhealth/bacillus_cereus_2011.pdf

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