Download Bacillus cereus

King Saud University
Collage of Pharmacy
Department of Pharmaceutics
Section of Microbiology
Prepared by:
Supervised by:
Duaa S. Al-Baqer
421033300
Dr. Manal Badour
283 PHT
1426 - 2005
Index
Page No.
Description
Microscopic images
Occurrence
Comparison of B. cereus Enterotoxins
Growth
Heat resistance
Nature of acute disease
Symptoms
Onset of illness
Duration of illness
Infective dose
Diagnosis of human illness
Complications
Treatment
Re-infection
Spread to others
Work and school
Susceptible individuals
Reducing the Risk
Prevention
Analysis
Lab tests
Preferences
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Description:
Bacillus cereus are Gram-positive, facultative aerobic sporeformer whose cells are
large rods and whose spores do not swell the sporangium. They can be differentiated
from other Bacillus species by their cell position and biochemical tests. The optimum
growth temperatures range from 30 to 50°C, although some psychrotrophic strains can
grow down to 4 to 5°C. They can grow at pH values of between 4.3 and 9.3, and can
grow at water activity values down to 0.912. The organism produces heat resistant spores
and these may germinate if cooling is too slow.
B. cereus. Spores appear as clear areas within the individual bacilli.
These and other characteristics, including biochemical features, are used to
differentiate and confirm the presence B. cereus, although these characteristics are shared
with B. cereus var. mycoides, B. thuringiensis and B. anthracis. Differentiation of these
organisms depends upon determination of motility (most B. cereus are motile), presence
of toxin crystals (B. thuringiensis), hemolytic activity (B. cereus and others are beta
hemolytic whereas B. anthracis is usually nonhemolytic), and rhizoid growth which is
characteristic of B. cereus var. mycoides.
Microscopic images:
1 µm wide, 5-10 µm long, arranged singly or in short chains.
Bacillus cereus Spore Stain. Stained by Shaeffer-Fulton method. Spores stained green.
Bacterial cells stained red (1000X ).
Bacteria at the top of a colony
( 2 µm )
Bacteria from the bottom of a colony
( 2 µm )
Bacteria in the form of twisted ropes ( 5 µm )
Occurrence:
1. Wide distribution in soil, dust and air.
2. Carried by humans and animals.
3. In many food products:
• Dairy products.
• Rice and cooked oriental foods.
• Spices and spice mixes.
• Dried products (flour, dry milk, pudding, soup mix).
4. Beans and bean sprouts.
5. Meats
6. Bakery products (cream-filled pastries).
Extracellular products
1.
2.
3.
4.
5.
Proteases (sweet curdling of dairy products).
Lactamases (penicillin resistance).
Phospholipases.
Hemolysins.
Toxins, which are:
• Lethal toxin: kills mice injected I.V.
• Enterotoxins: diarrheal and emetic.
Comparison of B. cereus Enterotoxins:
Property
Nature
Stabilities
pH
Enzymes
Storage
Production in foods
Lab media
Optimum temp.
Growth
Activities:
● Monkeys
● Skin permeability
● Mice
● Cell culture
● Antigenic
● Action
Diarrheal Toxin
Protein~40 kDa
Heat stable 450C, 30 min
Unstable 560C, 5 min
Most stable pH 4-11
Sensitive to pronase, trypsin
Unstable
Sometimes preformed
Complex
32-370C
Late exponential
Diarrhea in 1-3 hr
+
Lethal
Cytotoxic
+
Tissue damage, altered perm
and adenyl cyclase.
Emetic Toxin
Peptide~5 kDa
Stable 1260C, 90 min
Stable pH 2-11
resistant to pepsin, trypsin
Stable
preformed
Rice slurry
25-300C
Stationary
Vomiting in 1-5 hr
?
?
?
Growth:
Limiting conditions for pathogen growth
Pathogen
Min.
aw
(using
salt)
Min.
pH
Bacillus
Cereus
0.92
4.3
Max.
pH
Max. %
water
phase
salt
Min.
temp.
Max.
temp.
Oxygen
requirement
9.3
10
39.2°F
4°C
131°F
55°C
aerobe
Time/Temperature guidance for controlling pathogen growth and toxin
formation
Potentially
Hazardous Condition
Growth and toxin
formation by Bacillus
cereus
Maximum Cumulative
Exposure Time
Product Temperature
•
•
•
•
39.2-43°F (4-6°C)
44-50°F (7-10°C)
51-70°F (11-21°C)
Above 70°F (above
21°C)
•
•
•
•
5 days
17 hours
6 hours
3 hours
Heat resistance:
B. cereus spore heat resistance.
Temperature
(oC)
(oF)
90
95
100
194
203
212
D-Values (min.)
Medium
21-137
5-36
6.7-8.3
Water
Water
Water
Nature of acute disease:
There are two illness associated with B. cereus, emetic and diarrhoeal illness. The
emetic illness is caused by the ingestion of a heat-stable toxin produced by the
microorganisms in the food. The diarrhoeal illness is caused by the ingestion of moderate
to high number of B. cereus and their subsequent production of toxin in the stomach.
Symptoms:
The emetic type of food poisoning, with symptoms similar to that caused by
Staphylococcus aureus, is characterized by nausea and vomiting. The symptoms of B.
cereus diarrhoeal illness, similar to Clostridium perfringens food poisoning, include
watery diarrhoea, abdominal cramps, and pain. Nausea may sometimes occur, and
vomiting rarely occurs. Other Bacillus species will cause vomiting and diarrhoea.
Onset of illness:
Onset times for the emetic illness is usually ½ to 6 hours after consumption of
contaminated foods. For the diarrhoeal illness onset times are usually 6 to 15 hours, and
for other Bacillus species varies from ½ hour to 14 hours.
Duration of illness:
The emetic illness usually lasts less than 24 hours, while the diarrhoeal illness
usually persist for 24 hours in most instances. As with the emetic illness, illnesses caused
by other Bacillus species usually last less than 24 hours.
Infective dose:
Foods implicated in foodborne illness caused by B. cereus and other Bacillus
species usually contain at least 105 microorganisms per gram of food.
Diagnosis of human illness:
Confirmation of B. cereus as the cause in a foodborne outbreak requires either:
Isolating the same serotype from the suspect food and faeces or vomitus of the patient
Isolating large numbers of a B. cereus serotype known to cause foodborne illness
Isolating B. cereus from suspect foods and determining their enterotoxigenicity.
The rapid onset time for the emetic illness, coupled with some food evidence, is often
sufficient to diagnose this type of food poisoning.
Complications:
No specific complications have been associated with the foodborne disease causes
by B. cereus and other Bacillus species, although dehydration may occur from the
diarrhoeal illness in the elderly and sick.
Treatment:
Generally no specific treatment is needed except for fluid replacement if necessary.
Re-infection:
As yet it is unclear if any resistance is gained against reinfection.
Spread to others:
This illness is not spread from person to person.
Work and school:
Everyone with this infection should stay off work or school while they have
symptoms especially those in High Risk groups, occupations or institutions (infants or
children in daycare, school pupils, students, food workers, child-care workers, teachers,
and health/care workers).
Susceptible individuals:
All people are believed to be susceptible to B. cereus food poisoning.
Reducing the Risk:
Bacillus cereus exists in normal bacterial and spore forms in foods. The normal
form is inactivated by cooking, but most illness is a result of the multiplication of spores
during inadequate refrigeration of moist cooked protein foods and rice. Because cooking
often kills competing bacteria and heat activates the Bacillus cereus spores, storing large
masses of cooked food between 4O C and 60O C can allow the bacteria to multiply.
Prevention:
Preventive measures that can be taken to help avoid the illness include:
Ensuring adequate temperatures are reached during cooking of food mixes such as
sauces, custards, and soups to inactivate the bacteria.
• Keeping cooked hot foods above 60O C (preferably 70O C) if not served immediately.
• Ensuring the rapid cooling of cooked food by dividing into smaller lots and
refrigerating in shallow containers (less than 10cm deep).
• Storing cold foods at or below 4O C to prevent toxin being produced.
• Avoiding storing protein-containing foods with cooked rice because this stimulates the
growth of Bacillus cereus.
• Reheating foods to 75O C or until steaming hot, as flash frying or brief rewarming is
not adequate to destroy the toxin.
• Preventing cross-contamination from raw to cooked foods (by using separate
preparation areas or sanitizing between processes).
• Thoroughly washing fruit and vegetables with clean water of drinking standard before
usage.
• Ensuring food handlers have good personal hygiene and adequate food safety training.
Analysis:
The emetic ("short incubation") form of the disease, which occurred in this
outbreak, is mediated by a highly stable toxin that survives high temperatures and
exposure to trypsin, pepsin, and pH extremes; the diarrheal syndrome is mediated by a
heat- and acid-labile enterotoxin that is sensitive to proteolytic enzymes.
The diagnosis of B. cereus food poisoning can be confirmed by the isolation of
greater than or equal to 105 B. cereus organisms per gram from epidemiologically
implicated food. Underreporting of such outbreaks is likely because illness associated
with B. cereus is usually self-limiting and not severe. In addition, findings of a recent
survey about culture practices for outbreaks of apparent foodborne illness indicate that
20% of state public health laboratories do not make B. cereus testing routinely available.
Fried rice is a leading cause of B. cereus emetic-type food poisoning in the United
States. B. cereus is frequently present in uncooked rice, and heat-resistant spores may
survive cooking. If cooked rice is subsequently held at room temperature, vegetative
forms multiply, and heat-stable toxin is produced that can survive brief heating, such as
stir frying. In the outbreak described in this report, vegetative forms of the organism
probably multiplied at the restaurant and the day care centers while the rice was held at
room temperature.
The day care staff and restaurant food handlers in this report were unaware that
cooked rice was a potentially hazardous food. This report underscores the ongoing need
to educate food handlers about basic practices for safe food handling.
Lab tests:
1. Malachite green stain:
Malachite green stain is forced into the spore by heating the cells. Vegetative cells
are then decolorized with water and stained pink with safranin counterstain.
Endospores may be located in the middle of the cells (central), at the end
(terminal), or between the end and the middle of the cells (subterminal). The endospores
themselves may be round or oval. B. cereus produces terminal endospores.
2. Growth on blood agar:
Bacillus cereus colonies on blood agar.
Colonies of Bacillus cereus on the left; colonies of Bacillus anthracis on the right. B. cereus colonies
are larger, more mucoid, and this strain exhibits a slight zone of hemolysis on blood agar.
Preferences:
Different internet sites..
1. Center for Food Safety & Applied Nutrition - Foodborne Pathogenic
Microorganisms and Natural Toxins Handbook.
http://www.cfsan.fda.gov
2. FSHN420 Food Microbiology.
http://www.ag.iastate.edu
3. Christchurch City Council.
http://www.ccc.govt.nz
4. University of Wisconsin-Madison Department of Bacteriology.
http://www.textbookofbacteriology.net
5. Microbiology @ Leicester.
http://www-micro.msb.le.ac.uk
6. Other sites:
http://www.buckman.com
http://www.safefood.net.au
http://www.seafood.ucdavis.edu