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1. Clostridium tetani, botulinum,
and difficile
Dr. Himanshu Khatri
Email: himanshubkhatri@yahoo.co.in

2. • C. tetani
• C. botulinum
• C. difficile

3. C. tetani
• Slender
• Gram-positive bacillus
• Round, terminal and
bulging spores
(drumstick appearance)
• Motile (except type VI
strain)
• Capsulated
• Strictly anerobic

4. Pathogenesis
• Non-invasive bacillus
• Spores of bacilli enter the skin at the site of
cuts, wounds etc.  spores germinate into
vegetative forms  vegetative forms release
toxins  toxins at the myo-neural junction are
intraaxonally transported centripetally to the
neurons of the CNS

5. Toxins
• Tetanospasmin (most important)
• Tetanolysin
• Neurotoxin

6. Tetanospasmin
• Exotoxin
• Released only after cell lysis
• Split into two chains by
endogenous protease: light
and heavy
• Heavy chain: binds to neuronal
cells
• Light chain: blocks releases of
inhibitory neurotransmitters
like gamma-aminobutyric acid
(GABA) and glycine in the
spinal cord over-excitation
and contraction of muscles

7. Tetanus toxoid
• Can be toxoided by
treatment with
formaldehyde: retains
antigenicity but loses
toxicity

8. Other toxins
• Tetanolysin: is a hemolysin
• Neurotoxin: role in pathogenesis is not yet
clear

9. Clinical syndromes
• Tetanus
• Neonatal tetanus
• Cephalic tetanus

10. Tetanus
• Incubation period: few days to several weeks
• Painful muscular spams:
1. Trismus or lock jaw (due to contraction of masseter
muscle)
2. Risus sardonicus or sardonic smile (due to contraction
of facial muscles)
3. Opisthotonus (due to contraction of back muscles)
4. Generalized rigidity as the disease progresses
5. Localized tetanus, without involvement of CNS, is an
unusual occurence

11. Risus sardonicus and trismus

13. Complications of tetanus
• Fractures
• Tendon ruptures
• Respiratory failure (due to contraction of
diaphragm)

14. Neonatal tetanus
• Caused by cutting of umbilical cord of the
neonate by unsterilized blade or knife
• Lack of immunization of mother is an
important factor
• Prevention: Immunization of mother during
pregnancy (3 doses: 1st two doses in the
second and third trimester 4-6 weeks apart,
and the third dose 4 weeks prior to delivery)

15. Epidemiology
• Found worldwide
• Affects all age groups
• Spores are present in soil, in animal feces, and
sometimes in human feces (human carriage is
rare)

16. Types of wound susceptible to tetanus
• Dead tissue
• Wound contaminated with soil or feces
• Deep punctures or crush injuries

17. Laboratory diagnosis
• Tetanus is usually a clinical diagnosis
• Specimens: bits of tissue
• Gram staining: not diagnostic
• Culture: positive in only 30% of cases
1. Swarming on agar
2. Β hemolysis in blood agar: due to tetanolysin
3. RCM medium: turbidity, gas, and blackening of
meat
4. Greenish fluorescence on MacConkey agar

18. Swarming Blackening of meat in RCM

19. Identification of C. tetani
• Confirmation by toxigenicity testing in mouse

20. Treatment
• Antibiotic therapy
• Human immunoglobulin therapy

21. Antibiotic therapy
• Prevent multiplication of bacteria
1. Metronidazole (1st line)
2. Penicillin (alternative)
3. Tetracycline (for those allergic to above two)
4. Clindamycin (other alternative)
5. Vancomycin (other alternative)

22. Human immunoglobulin therapy
1. Human tetanus immunoglobulin (TIG)
2. Equine tetanus antitoxin (ATS), if TIG is not
available
• For those at a high risk of developing tetanus
(e.g. non-immunized individuals with other
factors like contaminated wound, dead tissue,
deep punctures etc.)

23. Prevention and control
• Vaccination with tetanus toxoid (TT)
• Given separately or as triple vaccine along
with diphtheria toxoid and acellular pertussis
(DTaP)
• Booster doses of TT are required every 10
years after primary vaccination

24. C. botulinum
• Causes botulism: food poisoning  paralytic
disease

25. C. botulinum
• Gram positive bacillus
• Sub-terminal, oval and bulging spores
• Peritrichous flagella
• Non-capsulated
• Strict anerobe

26. Pathogenesis
• Non-invasive
• Pre-formed toxin is ingested in food  not
destroyed by acidity of stomach  toxin
enters blood  prevents release of
acetycholine at presynapse of neuromuscular
junction  muscle weakness (paresis) and
paralysis

27. Virulence factor
• Botulinum toxin:
1. Exotoxin: but released only after cell lysis
2. Consists of two sub-units A and B: B prevents
inactivation by stomach acid and A is the
neurotoxin responsible for paralysis

28. Clinical syndromes
• Food borne botulism
• Infant botulism
• Wound botulism

29. • Food borne botulism
• Infant botulism
• Wound botulism

30. Food borne botulism
• Short incubation period: 12 to 36 hours after
ingestion of food
• Food poisoning: nausea, vomiting, abdominal
pain etc.
• Muscle weakness: blurred vision due to
weakness of ocular muscles  descending
paralysis.
• Paralysis of diaphragm  respiratory paralysis
 death

31. Infant botulism
• Botulinum toxin is produced in vivo in the
intestine (and not preformed)

32. Wound botulism
• Due to contamination of wound by spores
• Food poisoning absent
• Longer incubation period

33. Epidemiology
• Found worldwide
• Canned food is responsible for food borne
botulism
• Honey is responsible for infant botulism

34. Laboratory diagnosis
• Specimen: feces, vomitus, gastric aspirate,
wound tissue
• Culture: is positive in 60% of cases
• Identification by glucose fermentation, gelatin
hydrolysis, and lipase production
• Reliable identification by serum toxin bioassay
in mouse

35. Prevention
• Food borne botulism: high temperature
cooking and proper canning techniques
• Wound botulism: prompt debridement of
contaminated wound

36. C. difficile
• difficile: difficult to grow
• Slender Gram positive bacillus with oval and
terminal spores

37. Pathogenesis
• Prolonged antibiotic therapy  destruction of
normal flora  overgrowth of C. difficile 
production of toxin  Clostridium difficile
associated diarrhoea (CDAD) and
pseudomembranous colitis

38. Virulence factors
• Toxins
1. Toxin A: enterotoxin responsible for
diarrhoea
2. Toxin B: cytotoxin, responsible for cell death
and subsequent pseudo-membrane
formation

39. Laboratory diagnosis
• Microscopy and culture are not useful as
carriage rates are high
• Demonstration of toxin in stool by:
1. Stool cytotoxin test: requires a tissue culture
facility, hence not done usually
2. Enzyme immunoassays

40. Treatment
• For mild cases: stoppage of antibiotic therapy
• For patients with fever, leucocytosis or severe
diarrhoea: metronidazole or vancomycin

41. Thank you