Clostridium Botulinum, MORPHOLOGY, CULTURE, CLASSIFICATION, TOXIN, Mechanism of action, CLINICAL MANIFESTATIONS: Foodborne Botulism, Infant Botulism, Wound Botulism, LABORATORY DIAGNOSIS

1. DR. SIDDHARTH DUTTA (PGT)
Clostridium
botulinum

2. C.botulinum causes botulism, a paralytic disease that usually presents as
a form of food poisoning.
 The name botulism is derived (from botulus, which is Latin for sausage)
formerly associated with this type of food poisoning.
 The bacillus is widely distributed
saprophyte occurring in virgin soil, vegetables,
hay, silage, manure and sea mud.
INTRODUCTION

3. MORPHOLOGY
 It is a Gram positive, non-capsulated bacillus about 5 μm x 1 μm ,
 Motile by peritrichate flagella.
 Produces subterminal, oval, bulging spores
 Able to produce the neurotoxin during
sporulation, which can only happen in
an anaerobic environment.

4.  It is a lipase negative microorganism that grows between
pH of 4.8 and 7 and it can't use lactose as a primary carbon
source.
 Spores of the organism are highly resistant to heat,
withstanding 100 °C for several hours and for up to
10 mins at 120 °C.
Contd.

5. CULTURE
 It is a strict anaerobe and can grow on ordinary media.
 Optimum temperature is 35°C but some strains
may grow at l - 5°C.
 Commonly used media are blood agar and
cooked meat broth.
 Colonies are large, irregular, semitransparent,
with fimbriate border. On blood agar, haemolysis
around the colonies is observed.

6.  Eight types of C.botulinum strains have been identified
(A, B, C1, C2, D, E, F and G) based on the immunological
difference in the toxins produced by them.
 The toxins produced by the different types are
identical in their pharmacological activity but are
neutralised only by the homologous antiserum.
 An exception is the C2 toxin, which shows
enterotoxic activity, while the others are
neurotoxins.
CLASSIFICATION

7. TOXIN
Cl. botulinum forms a powerful exotoxin.
 The toxin differs from other exotoxins in that it is not released during the life
of the bacterium.
 Produced intracellularly and appears in the medium on autolysis of the cell.
 Botulinum toxin is the most toxic substance known so far.
 Lethal dose for humans is probably one microgram.

8. Contd.
 It is a neurotoxin and acts slowly, therefore, takes several
hours to kill.
 The toxin is relatively stable, it is inactivated only after
30-40 minutes at 80°C and 10 minutes at 100°C.
 Food suspected to be contaminated
with the botulinum toxin can be
rendered completely safe by
pressure cooking or boiling for
20 minutes.

9. Mechanism of action
 It resists digestion and is absorbed through the small
intestines in active form.
 Acts by blocking the production or release of acetylcholine
at the synapses and neuromuscular
junctions.
 Onset is marked by diplopia, dysphagia
and dysarthria due to cranial nerve
involvement.

10. CLINICAL MANIFESTATIONS
Cl. botulinum is non-invasive and its pathogenicity is due to the action
of preformed toxin and those manifestations are collectively called
botulism.
 Botulism is of three types
- foodborne
- infant and
- wound botulism.

11. Foodborne Botulism
 It is due to preformed toxin in food contaminated with
Cl. botulinum .
 Human disease is usually caused by types
A , B, E and very rarely by types C, F and G.
 The source of botulism is usually various preserved foods -
meat, fish, vegetables etc. The contaminated food usually
exhibits signs of spoilage, and cans may be inflated showing
bubbles on opening. Often the food may look normal with no
alteration in taste.

12. Contd.
 The symptoms appear 12-36 hours after ingestion of contami
nated food. Vomiting, thirst, constipation, ocular paresis, diffi
culty in swallowing, speaking and breathing are the common
symptoms. Diarrhoea is not a symptom.
 Fatality rate is 25 - 70% and death results from respiratory
failure.

13. Infant Botulism
 It affects infants, usually below 6 months.
 Occurs due to ingestion of food contaminated by spores
of Cl. botulinum.
 Honey has been incriminated as a source of infection. It
is a toxico-infection.

14. Contd
 The disease is characterised by constipation, weakness,
lethargy and cranial palsies.
 Infants exhibit flaccid paralysis, usually with a weak
sucking response, generalised loss
of tone ("the floppy baby syndrome" )
and respiratory complications.

15. Wound Botulism
 It is a very rare condition which results from wound infection
with Cl. botulinum.
 Toxin produced is absorbed into the blood and the symptoms
similar to foodborne botulism except for the gastrointestinal
components which are absent.
 Type A has been recovered from most of the cases studied.

16. Contd.

17.  Smears made from suspected food or faeces are examined by Gram's
staining which may show Gram positive sporing bacilli.
 Culture is done on blood agar or cooked meat broth. Growth on culture
media may be confirmed by Gram's staining.
 It is to be noted that presence of bacilli in food
or faeces in absence of toxin is of no
significance. Hence, toxin in culture fluid must
be demonstrated by toxigenicity test in mice.
LABORATORY DIAGNOSIS
1. Demonstration of the Organism

18. Contd.
2. Demonstration of Toxin
 Specimens (stool, food and vomitus) are macerated in
sterile saline and the filtered extract is divided into three
parts.
 One portion of extract is heated at 100°C for 10mins and
other two kept unheated.
 Two mice or guinea pigs are injected with unheated filtrate;
one of them (control) is protected with polyvalent botulinum
antitoxin prior to injection. The third animal is injected with
heated filtrate.

19. Contd.
 The test animal (unprotected) develops dyspnoea, flaccid
paralysis and dies within 24 hours whereas control animal
(protected) remains healthy.
 The animal injected with heated filtrate also remains free
of any toxic symptoms.