BACILLUS CEREUS

by

Tamilselvan.T51094

The word Bacillus means ‘Small rod’ and

cereus means ‘Wax- like’.

It is derived from Latin language.

The name itself reflects the morphology of B.cereus.

Scientific classification

Domain : Bacteria

Phylum : Firmicutes

Class : Bacilli

Order : Bacillales

Family : Bacillaceae

Genus : Bacillus

Species : B.cereus

Size:

Length : 3-5 µm

Width : 1-1.2 µm

Introduction

Gram positive bacteria

Spore bearing

Rod shaped

Motile

Facultative aerobe or anaerobe

Beta hemolytic

It is widespread in nature and frequently isolated from soil and

growing plants, but it is also well adapted for growth in the intestinal tract

of insects and mammals.

It is becoming one of the important causes of food poisoning in

the industrialized world.

B.cereus is the model species of “Bacillus cereus group” also

known as Bacillus cereus sensu lato, comprising five closely related

species:

1. B. anthracis

2. B. thuringiensis

3. B. mycoides

4. B. pseudomycoides

5. B. weihenstephanensis.

These all share a genetic similarity but they have

difference in both Pathogenic properties and phenotypic traits of strains.

Optimum conditions for Growth

Temperature:

Optimum : 30 - 37 ºC

Range : 4 - 55 ºC

Maximum toxin production occurs at 20 - 25 ºC

Toxin production range at 10 - 40 ºC.

pH:

Optimum : 6 – 7

Range : 4.5 – 9.5

Minimum water activity :

1. with NaCl > 0.93 and 0.95 aw.

2. with glycerol 0.93 aw.

For survival:

Temperature:

Spores more resistant to dry heat than moist heat. It is also more resistant in Oily foods.

Cooking at or below 100 ºC may allow spore survival.

pH:

Vegetative cells decline generally rapidly in stomach acid. However, some may survive depending on food and level of stomach acidity.

Spores are resistant to gastric acidity between pH 1-5.2

Water activity :

Spores survive long periods in dry foods

e.g: Population unchanged after 48 weeks in cereal 0.27 – 0.28 aw.

(Jaquette and Beuchat, 1998)

Life cycle

It is ubiquitous in nature.

B.cereus found in many types of soils, sediments, dust, plants, water

bodies, decaying organic matter, vegetables and fomites.

It also exhibits a saprophytic life cycle in the arthropods intestinal

epithelium.

Defecation or death of the host releases cells and spores into the soil,

where vegetative cells sporulate and survive until their uptake by

another host.

Generally, spores germinate when they come into contact with organic

matter or animal host.

The core of the spore is surrounded by the inner membrane,

cortex and inner and outer coats.

The B.cereus spore is refractory to extreme environmental

conditions include of heat, freezing, drying and radiation.

It has been reported to be present in stools of healthy humans at

varying levels.

The presence in environments, feed and food would ensure

B.cereus a transient presence in the mammalian gut.

The white inclusions in the rod shaped bacteria are the spores.

The spore coat is made up of proteins and small amount of

lipids and carbohydrates which contribute to its resistance to the

oxidizing agents and chemicals by blocking toxic chemicals.

The outer spore coat allows them the heat and gamma

radiation resistant.

It can metabolize a variety of compounds like

carbohydrates, proteins, peptides and amino acids for their growth

and energy.

Countries prevalent

Reports of Foodborne disease outbreaks in countries like

Norway , Spain, Sweden, UK, USA, Scotland and India.

India being a sub-tropical country known for a diverse range of

food habits provides scope for the prevalence of B.cereus.

In 1978 – First reported outbreak of B.cereus food poisoning

among the children due to the consumption of milk powders in

India .

Incidence of food poisoning in India is higher during various

cultural and religious events when food is prepared in bulk as it

becomes difficult to maintain hygiene during preparation and

storage of food.

Year Region Study

group

% Notes

2010 Sea food 29.41% 36.7% and 29.41% of

tropical seafood

samples.

2015 Uttarakhand meat 27.78 –

35%

35% of cooked meat

and 27.78% of raw

meat products.

Bacillus cereus food poisoning in Uttarakhand

(Infectious diseases in India , 2017)

B.cereus is an opportunistic pathogen cause infections like

1. Diarrhoeal syndrome

2. Emetic syndrome

Transmission is predominantly foodborne for both.

B.cereus food poisoning occurs year around without any particular

geographical distribution.

Diarrhoeal syndrome :

It is also called ‘Long- incubation’ form of B.cereus food

poisoning.

The food along with B.cereus is consumed and it passes the stomach

and the site of toxin production is small intestine .

Incubation time : 8-16 hours (occasionally > 24 hrs.)

Duration of illness :12 - 24 hrs. (occasionally several days)

Infectious dose : 105 – 107 cfu in total. The number required is

lower for spores compared to vegetative cells.

Toxin type : Protein : enterotoxins like Hbl, Nhe, CytK

Symptoms:

Abdominal pain , watery diarrhoea and occasionally nausea.

Foods most frequently implicated:

Proteinaceous foods, meat products, soups, vegetables,

puddings, sauces, milk and milk products.

Heat stability : Destroyed at 55 ºC after 20 minutes.

pH stability : unstable < 4 and >11

Northern Europe and Northern America are Diarrhoeal dominant

countries.

All peoples are susceptible to infection but intensity varies with

individuals.

Treatment is not usually given. Fluids are administered when diarrhoea

and vomiting are severe.

The Diarrhoeal form is diagnosed by isolation of the organism from stool

and food.

Emetic syndrome :

The toxin is vomiting form of disease following ingestion of food

with pre formed toxin.

Incubation time : 0.5 – 6 hrs.

Duration of illness : 6 – 24 hrs.

Infectious dose : 105 – 108 cells / gram is often found in implicated

foods, but live cells are not required for intoxication.

Toxin type : Cyclic peptide , emetic toxin – cereulide.

Symptoms : Nausea, vomiting, malaise and few lethal cases due to liver

damage.

Cereulide is resistant towards acidic conditions, proteolysis and

heat and also to gastric acid.

Food items which are starch-rich are most frequently implicated.

Ex: Starch rich foods , fried and cooked rice, pasta, pastry

and noodles.

The Emetic form is diagnosed by isolating the organism from

the incriminated food.

The molecular mass of emetic toxin is 1.2 kda.

Cereulide synthesis takes place ranging from 12 – 37 ºC. The

maximal production of emetic toxin occurs at 12 – 22 ºC.

Heat stability : 90 min at 121 ºC

pH stability : stable at pH 2-11.

Japan and UK are the emetic dominant countries.

Underreporting of outbreaks

Several factors contribute to the number of foodborne B.cereus

disease being largely under-reported are

The consequence of the generally short and mild course of disease ,

which does not motivate the patient to seek medical attention.

The symptoms are similar to other pathogen infections.

Ex: Emetic disease are not easily distinguished from S.aureus

intoxication.

Diarrhoeal disease shows same symptoms as C.perfringens type

A food poisoning.

Nowadays, B.cereus foodborne disease is reportedly increasing in

Industrialized countries.

For Inactivation :

Temperature:

Vegetative cells destroyed by frying, grilling, roasting and pressure cooking.

Spores ( depends on strain and food ):

D 100 ºC - 1.2 -7.5 mins. in Rice

D 120 ºC - 2.5 secs.

D 120 ºC - 3.4 mins. (e.g. soybean oil).

(van Asselt and Zwietering, 2006)

pH:

Vegetative cells inactivated in yogurt at pH 4.5 and in fruit juice at pH 3.7

Water activity :

Vegetative cells inactivated at aw < 0.91

Preservatives :

Vegetative cell growth inhibited by Sorbic acid, Benzoate, Sorbate, EDTA and polyphosphates.

Spore germination and outgrowth inhibited by Nisin.

(Nisin is not sporicidal).

Disinfectants / Sanitizers :

Most food industry sanitizers destroy vegetative cells on surfaces.

Formaldehyde, Chlorine, Iodine, Acids, Alkalis, H2O2, Peroxy acids, Ozone are all sporicidal at high concentrations with long contact times.

Phenolics, Alcohols, Organic acids, Esters and Mercurial have little sporicidal effect.

Chlorine disinfectants such as household bleach contain 5.25% sodium hypochlorite are effective against B.cereus cells but not spores.

B.cereus as Probiotic ???

It is also adapted to a lifestyle in a host , as a pathogen or

perhaps a part of intestinal flora.

The possible adaptation of B.cereus to the environment of the

animal gut could be the basis for its probiotic effect.

Such use cannot uncritically be considered safe for humans

because all B.cereus strains are able to produce at least one type of

toxins associated with diarrhoeal disease.

Certain strains producing negligible amounts of toxin at 37 ºC

have been approved for probiotic use by the European Food

Safety Authority (EFSA). (Hong et.al., 2004)

Infectious dose

Counts ranging from 200 to 109 cells /gram B.cereus have

been reported in the incriminated foods after food poisoning.

Thus any food containing more than 103 cells /gram cannot

be completely safe for consumption.

Both the Diarrhoeal and emetic syndromes are generally

mild and self limiting. Treatment is not usually needed for both.

Acid stress resistance

It is especially important for B.cereus that must survive the

acidic pH of the stomach which is 1.5 in fasting state and rises to 3-5

after ingestion of food.

Before entering the small intestine or colon it have to cross acidic

condition of stomach.

The acid stress in many foods by means of food preservatives.

Thus, bacteria have evolved resistance mechanisms to prevent

cell damage due to acid stress, this are called as acid tolerance

responses (ATR)

The ATR’s are

1. Production of alkali

2. Homeostasis of Internal pH

3. Decarboxylation of amino acids

4. Buffering ability of cytoplasm

5. Cell density

Transfer from soil to food

B.cereus isolated from rice, dairy products , vegetables,

spices and sometimes it can cause cross contamination to other

foods like meat products also.

At harvest, B.cereus cells or spores may accompany plant

material into food production areas and establish on food

processing equipment.

Bacillus cereus is a common contaminant of milk.

Spores or cells of it can contaminate udders of cows during

grazing or enter diary farm through bedding material or feed.

The endospores have adhering capacity which are not removed

by regular cleaning of surfaces.

It has the ability to form Biofilms is important for its persistence

in food industry equipment such as diary pipelines.

The biofilms protect spores and vegetative cells against

inactivation by sanitizers.

B.cereus is not a competitive microorganism, but it

grows well after cooking and cooling (<48 ºC) . This heat treatment

will cause spore germination and in the absence of competing flora

it grows well.

Toxins

B.cereus produces one type of emetic toxin and three types of

enterotoxin.

Three pore forming enterotoxin responsible for diarrhoeal syndrome

are 1. Hemolysin bl (Hbl)

2. Non – Hemolytic enterotoxin (Nhe)

3. Cytotoxin K (cyt K)

Hbl consists three different protein components L1, L2, B.

Nhe contain three different protein constituents namely

NheA, NheB, NheC. and Cyt K is a single component toxin

The emetic toxin is (dodecadepsipeptide) cereulide and

having a ring shaped structure of three repeats of four amino acids .

Laboratory diagnosis

When grown under aerobic conditions on 5% sheep blood

agar at 37 ºC, B.cereus colonies are dull, grey and opaque with

a rough matted surface.

Grey, opaque, granular, spreading colonies with irregular

perimeters growing on 5% sheep blood agar.

No type of food with pH > 4.8 can be excluded as a possible

vehicle or as representing a risk of food spoilage or food borne

disease.

Failures by consumers to follow basic food preparation rules

i.e., slow or inadequate cooling, storage at ambient temperature or

pro longed heat keeping at <60 ºC may allow growth of B.cereus.

It occurs as a contaminant during the pre-processing and

post- processing operations in the food chain.

Prevention :

According to NIH, NIAID and NFPA the suggestions below

are good examples of how to destroy B.cereus.

steaming under pressure, roasting, frying and grilling foods can

destroy the vegetative cells and spores.

Foods infested with diarrheal toxin can be inactivated by heating

for 5 mins at 133 ºF

Foods infested with emetic toxin can be inactivated by heating to

259 ºF for more than 90 mins.

In meat processing facilities to prevent toxin and contamination:

1. Assure current GMP being used in slaughter houses and

processing units.

2. Apply approved treatments of carcasses to remove fecal bacteria.

3. Use proper cleaning and disinfection of food contact surfaces with

hypochlorite or other approved sanitizers.

4. Keep hot foods above 60 ºC and cold foods below 4 ºC to prevent

formation of spores.

5. Cook beef and beef products thoroughly. Properly refrigerate left

overs.

References:

1. Academic press. 2011. Foodborne infection and intoxication. 564p.

2. GIDEON informatics. 2017. Infectious diseases in India.36p.

3. Vijaykumar, D.S. 2010. DNA based detection of food isolates of Bacillus cereus and its behavioral pattern in selected foods, Ph.D. CFTRI, Mysore .25p.

4. Arnesen, L. and fagerlund, A. From soil to gut: Bacillus cereus and its food poisoning toxins. Norwegian school of veterinary science, Norway.

5. Granum, P. and Lund, T. Bacillus cereus and its food poisoning toxins. FEMS microbiology letters. 157 (223-228).

6. Bottone, E. Bacillus cereus: a volatile human pathogen. Clinical microbiology reviews. 2010, 382-398p.