This document provides information on various Clostridium bacteria including C. septicum, C. novyi, C. histolyticum, and C. tetani. It describes their morphological characteristics, toxins produced, diseases caused such as gas gangrene, and laboratory methods for diagnosis. Gas gangrene is defined as a rapidly spreading muscle tissue death caused by clostridial bacteria. Treatment involves surgery to remove damaged tissues, antibiotics such as metronidazole, and immunization to prevent further infection.

1. CLOSTRIDIUM

2. CLOSTRIDIUM SEPTICUM
 Pleomorphic bacillus
 Oval, central/subterminal spores
 Anaerobic. saccharolytic, abundant gas
 Four distinct toxins
 Alpha toxin is hemolytic, demonecrotic and lethal
 Gas gangrene in humans

3. CLOSTRIDIUM NOVYI
 Large, pleomorphic bacillus
 Oval, subterminal spores
 Strict anaerobe
 Type A – causes gas gangrene
 Large amounts of edema fluid, little or no observable
gas, high mortality

4. CLOSTRIDIUM HISTOLYTICUM
 Oval, subterminal, bulging spores
 Proteolytic
 Gas gangrene in humans
 Infection – exogenous/endogenous
 Exogenous – implanted foreign particles
 Endogenous – clean surgical procedures

5. ANAEROBIC WOUND INFECTIONS
 Simple wound contamination – no invasion of tissue
 Anaerobic cellulitis – invasion of fascial planes,
minimal toxin production, no invasion of muscle
tissue
 Anaerobic myositis – gas gangrene, invasion of
muscle tissue, abundant formation of exotoxins

6. GAS GANGRENE
 Rapidly spreading, edematous myonecrosis in
association with severe wounds of extensive muscle
mass
Etiology
 C. perfringens
 C. novyi
 C. septicum
 C. histolyticum

7. GAS GANGRENE
Gas gangrene

8. CLINICAL PRESENTATION
 Incubation period – 7 hours to 6 weeks
 C. perfringens – 10–48 hours
 C. septicum – 2–3 days
 C. novyi – 5–6 days
 Increasing pain, tenderness and edema over the affected
part
 Accumulation of gas – crepitus
 Untreated – profound toxemia and prostration

9. LABORATORY DIAGNOSIS
 Diagnosis on clinical grounds
 Laboratory – confirmation of diagnosis and
identification of infecting organism

10. SPECIMEN
 Films – edge of affected area, tissue from necrotic
area, exudate from deeper part of wound
 Exudate collected from depth of wound – collected
by capillary pipette or swab
 Necrotic tissue/muscle fragments

11. MICROSCOPY
 C. perfringens – Gram-positive bacilli without spores
 C. septicum – boat- or leaf-shaped pleomorphic
bacilli
 C. novyi – large bacilli with oval or subterminal
spores

12. CULTURE
 Naegler reaction
Nagler reaction

13. CULTURE
 Reverse CAMP test
Reverse CAMP test

14. TREATMENT AND PROPHYLAXIS
 Surgery – most important therapeutic and
prophylactic measure in gas gangrene
 Damaged tissue removed extensively and promptly
 Hyperbaric oxygen

15. TREATMENT AND PROPHYLAXIS
 Antibiotics – metranidazole – IV – before surgery
and repeated every 8 hours
 Mixed aerobic and anaerobic infection – combination
– metranidazole with amoxicillin and gentamicin

16. TREATMENT AND PROPHYLAXIS
 Passive prophylaxis – anti-gas gangrene serum –
containing
10,000 IU – C. perfringens
10,000 IU – C. novyi
5000 IU – C. septicum
 Given IM

17. CLOSTRIDIUM TETANI Hippocrates
 1889 – Kitasato – pure forms reproduced disease –
inoculation in animals
 Distribution: ubiqutous, soil, intestine of man,
animals, hospital dust, cotton, bandages, plaster of
Paris, catgut, talc

18. MORPHOLOGY
 4–8 x 0.5 ǔm, Gram-positive, motile
Drumstick
C.tetani: drumstick appearance

19. CULTURE
 Anaerobe
 Temperature – 14–43°C, optimum 37°C
 RCM
 Fresh blood agar
 Translucent spreading growth

20. CULTURE
 Deep agar shake culture – colonies –spherical with
filaments arranged radially
 Gelatin stab cultures – fir tree growth
 RCM – turbidity with gas formation
 Blood agar – α hemolysis, later β hemolysis

21. RESISTANCE
 Sensitivity to physical and chemical
 Varies – boiling 5–15 minutes, 3 hrs
 Iodine 1%
 Dry heat 150°C for 1 hr
 Resistant to 5% phenol

22. CLASSIFICATION
 Ten serological types – I to X
 All types produce the same type of toxin which is
neutralised by antitoxin produced against any one
type

23. TOXINS
 Two toxins – hemolysin (tetanolysin) and neurotoxin
(tetanospasmin)
 Antigenically and pharmacologically distinct

24. TETANOLYSIN
 Heat labile
 Oxygen labile hemolysin
 Produced by C. perfringens, C. novyi and Str.
pyogenes

25. TETANOSPASMIN
 Responsible – tetanus
 Heat labile – inactivated at 65°C in five minutes
 Good antigen, specifically neutralised by antitoxin
 Toxoided spontaneously, low concentration of
formaldehyde

26. TOXIN
 Monomer – toxic
 Toxin exists
 Dimer – non-toxic
 Good antigen
 Susceptibility : Horses, guinea pigs, mice, goats,
rabbits

27. TOXIN
Tetanus
 Surgical operations
 Punctured wounds
 Septic abortions
 Umblical – sepsis
 Ear piercing – unhygienic
 Circumcision – conditions

28. FAVOURABLE DISEASE CONDITIONS
1. Anaerobic
2. Presence necrotic
3. Other bacteria
4. Lack of drainage
5. Dose and toxigenicity of strain
6. Site and nature of wound
7. Immune status of host
8. Incubation 6–12 days
Mortality rate 80–90 per cent before specific
treatment was available
Tetanus neonatarum and uterine tetanus 70 per cent

29. TETANUS
Pathogenicity
 Spore – implanted wound
 Multiplies (anaerobic)
 Toxin
 CNS – via peripheral nerves
 Toxin fixed by gangliosides
 SP blocks – Syn. inhibition in spinal cord

30. TETANUS
 Ascending tetanus – up the spinal cord – opposing
hind limbs, trunk, forelimbs
 Descending tetanus – IV spasticity of head, neck,
limbs
 Naturally occurring tetanus

31. NEONATAL TETANUS

32. TETANUS IN HORSES

33. TETANUS IN HUMAN

34. LABORATORY DIAGNOSIS
Confirmation
1. Microscopy
BA half plate – polymyxin 1–2 days
2. Culture 80°C for 15 min
RCM 3 tubes 80°C for 5 min
Unheated 37°C
for 4 days

35. SEROTYPING
Ten serotypes I–X
 VI – Non-flagellate strains
Toxins and pathogenicity
 Neuro toxin develops – cultures 2–14 at 35°C

36. TOXIGENICITY TEST
In vitro
 Blood agar plates – one half tetanus antitoxin (1500
units)
 C. tetani inoculated on each half of plate
 Incubated anaerobically – two days
 Toxigenic C. tetani – hemolysis around colonies on
half without antitoxin

37. TOXIGENICITY TESTING
 In vivo
 Control
 500–1500 units
 Anti toxin 1 hr before
IP
 Test
 0.1 ml hind limb
 Ascending tetanus

38. PROPHYLAXIS
 Tetanus – preventable
 Spores – ubiquitous – wound contaminated
Prevention
1. Immunisation: Active, Passive, Combined
2. Surgical
3. Antibiotic penicillin, tetracycline

39. SPORE - ARRANGEMENT
Types of spores

40. CLOSTRIDIUM SEPTICUM
 Described – pasteur and joubert
Vibrion septque
MORPHOLOGY :
 Pleomorphic
 3.8 μ x 0.6 μm
 Oval, central, subterminal spores
 Motile
CULTURAL CHARACTERISTICS :
 Colonies – irregular & transparent
 Hemolysis – horse blood agar
 Growth - hh Glucose.
TOXINS :
 Alpha – Hemolytic
Dermonecrotic
Lethal
 Beta – Leucotoxic deoxyribonuclease
 Gamma – Hyaluronidase
 Delta – Oxygen liable hemolysin
PASTEUR

41. CLOSTRIDIUM NOVYI
MORPHOLOGY
 Gram +ve
 Large, Stout,
Pleomorphic
 Large, Oval,
Subterminal spores
 4 Types- Type A to
Type D

42. CLOSTRIDIUM HISTOLYTICUM
Actively proteolytic
Oval, Subterminal, Bulging
spores.
Aerotolerant
5 toxin

43. GAS GANGRENE
1.ANAEROBIC
MYOSITIS
2.MALIGNANT
EDEMA
3.CLOSTRIDIAL
MYONECROSIS

44.  OAKLEY – “ Rapidly spreading, edematous myonecrosis,
occurring characteristically in association with severe wounds
of extensive muscle massel with that have been contaminated
with pathogenic clostridia particularly with cl.perfringes ”
 Clostridium – enters the wound along with implanted foreign
particles
also present on normal skin, perineum & thighs
infection may be endogenous
 MAC LENNAN – 3 TYPES
Simple wound
Anaerobic cellulites
Anaerobic myositis or gas gangrene

45. GAS GANGRENE
 Low oxygen tension
 Battle wounds
 Implanted bullets with bits
clothing & soil
 Ionised calcium salts & silicic-
necerosis
 Crushing of tissuce or tearing
of arteries – anoxia of muscle
 Reduction – blood supply
 Extravasation Hb and Myo
Hb – Reduced
 Aerobic oxidation – hatled
 Anaerobic – Pyruvate to
lactate - i Eh

46. Lecithinases → Damages cell membrane
↑ capillary permeability
Extravasation
↑ tension in affected muscles
Anoxic damage
Alpha toxin → lysis of erythrocyte
Collagenases → destroys collagen barrier
Hyaluronidases → breaks intracellular substance

47. Incubation period → 2 days to several weeks
commonly 6-12 days
Disease developes with ↑ in pain
edema
tenderness
thin watery discharge
perfuse & serosanguinous
accumulation of gas makes tissue
crepitant

48. LAB DIAGNOSIS
 Specimens – collected
1) Films – muscles – at the edge
necrotic area
exudates
2) Exudates – capillary pipette or swab
3) Necrotic tissue & muscle fragments
 GRAM STAINING
Large – regular-Cl.perfringens
“ Cirtonbodies” – irregular staining – Cl.septicum
Large bacilli – Oval subterminal spores – Cl.novyi
Slender – round terminal spore -Cl. Tetani

49. PROPHIAXIS & THERAPY
 Surgery: excision
 Drugs – Metroinidazole
 Combination-
Metroinidazole,
Gentamicin, Amoxycilin
 Passive
immunisation – anti
gas gangrene serum

50. CLOSTRIDIUM TETANI
INTRODUCTION
 Described –
HIPPOCRATES
& ARETAEUS
 Demonstrated –
Roesenbach

51. MORPHOLOGY
 Gram positive
 Slender
 4-8 μm x 0.5 μm
 Straight axis,
parallelsides
 Rounded ends
 Non capsulated
 Motile

52. CULTURAL CHARCTERISTICS
 Obligatory anaerobes
 Opt. temp 37ºC
 pH 7.4
 Grows on ordinary media
 growth increases by blood and serum
 Extremely fine transluscent

53. BIOCHEMICAL REACTIONS
 MR –ve
 VP – ve
 H2S not formed
 Indole formed
 Nitrates not reduced
 Gelatin Liquefied

54. EPIDEMIOLOGY
INDIA
 Tetanus-Important epidemic infection in
india.
 Prior-NIP-3.5 lakh children died annually-NT
 1993-94-2.8 Lakh-NT-everted
 >50%-NT-month-july-aug.-sep.
 Male infants more susceptible-no bio.
Reasons.

56. RESISTANCE
 Spores-killed-boiling 10-50 mg
 Autoclaving – 1210C – 20 min.
 Iodine & hydrogen peroxide kill – spores – few
hrs.
CLASSIFICATION
 Ten serological types (Type I to type X)
TOXIN :- Two toxin, Tetanolysin,Tetanospasmin

57. PATHOGENICITY
 Toxin – absorbed – motor nerve endings
 Transported- CNS – intraaxonally
 Toxin – fixed – gangliosides – grey matter of nervous
tissue
 Tetanus toxin – blocks synaptic inhibition in spinal
cord ( GABA)
 Uncontrolled spread of impulses in CNS
 Muscle rigidity
 Spasms- due to contraction of agonists & antagonist

58. TETANUS

59. TETANUS
 Tonic muscular spasms
 cowdung – umbilical stumps
Circumcision & earboring
 IP- 2 days to several weeks
CLINICAL MANIFESTATION OF TETANUS
 First notice - h muscle tone in masseter
TRISUMS / LOCK JAW
 Dysphagia or pain in neck, shoulder, back muscle
 Rigid abdomen & stiff proximal limb muscles
 RISUS SARDONICUS – Contraction of fascial muscles
 OPISTHO TONOS – Arched back
 Paroxysmal spasm – Cyanosis

60. NEONATAL TETANUS
 Children born –
Inadequately immunized mother
 First 3 weeks of life
 Poor feeding, rigidity, spasms
CEPHALIC TETANUS
 Followed - Injury – head & ear infections
 Trismus, Dysfunction

62. LABORATORY DIAGNOSIS
Material- inoculated on one
half of blood agar
i 1 -2 Days
anaerobically
Swarming growth
Inoculated – root of tail of
mouse

63. PROPHYLAXSIS
 Surgical attention
 Anti biotics – Pencillin inj
Erythromycin –
500 mg, b.d. 5 days
Neomycin –
locally.
 Passive immunisation – Inj
tetanus antitoxin
 ATS – hyperimmune horse
 1500 IU – SC, IM in –
nonimmune persons
 Disadvantages – immune
elimination
Hypersensitivity

64. PROPHYLAXSIS
 Active immunisation –Inj,
toxoid 3 doses IM
4-6 weeks intervals b/w
first 2 Inj
3rd dose 6 mth later
Booster doses ten yrs
 Combined immunisation
TIG at one side
Toxoid – first dose –
contralateral
2nd, 3rd doses-monthly
intervals

65. TREATMENT
 Antibiotic :- Pencillin ( 10-12 Million units IV – 10 days)
or
Metronidazole
 Spasm control :- Diazepam of lorazepam,
Barbiturates & cholorpromazine – 2nd
line agents
 R.C:- Incubation or tracheostomy
 Autonomic dysfunction :- Labtalol
Esmolol
Clonidine
Morphinesulphate

66. MCQ’s
1.Colonies of Cl. Difficle fluoresce under Wood’s lamp to give
colour which is
A. green B. brick red
C. yellow D. pink
2. Most clostridia are motile except.
A. Cl.tetani B.Cl.novyi
C. Cl.perfringes D.Cl.septicum
3.The capsulated spieces of Clostridium is.
A.Cl.perfringens B.Cl.tetani
C.Cl.histolyticum D.Cl.sporogenes

67. 4.All following clostridia are predominantly saccharolytic
except.
A. Cl.perfringens B.Cl. Septicum
C. Cl.fallax D. Cl.histoyticu
5.Proteolytic clostridia.
A. Digest protiens B. liquefy gelatin
C. liquefy coagulated serum D. all above are true

68. 6.Lecithinase activity can be demonstrated on.
A. egg yolk agar B. MacConkey agar
C. blood agar D. phenolphthalein
7.Stromy clot is almost exclusively produced by.
A. Cl.perfringes B. Cl.tetani
C. Cl.sporogenes D. Cl.novyi

69. The Clostridium which produce swarming growth.
A. Cl. Tetani B. Cl.novyi
B. Cl.perfringens D. Cl. septicum
8.
9. All following clostridia have terminal spores except.
A. Cl.tetani B. Cl. Innocum
C. Cl. cadavers D. Cl.novyi type C

70. LONG ESSAYS:
1.Enumerate the clostridia pathogenic to
humans. Disscus the morphology,cultural characteristics
and toxins of Cl. Welchi.
2.Classify clostridia.Name the diseases produced by
Clostridium welchii. How will investigate such a case?
3.Name the clostridia pathogenic to humans.Describe
the pathogenesis of gas gangrene

71. 4.Enumerate the pathogenic clostridia .Write the steps of
laboratory diagnosis of tetanus?
5.Enumerate the organisms causing gas gangrene .Describe
the morphology of cultural characteristics & pathogenecity of
Cl welchii.add a note on lab diagnosis of gas gangrene?
6.What are gas gangrene clostridia?Describe their
morphology,cultural characteristics & lab diagnosis?
7.Name the pathogenic anaerobic bacteria.Describe the lab
diagnosis of gas gangrene?
8.Enumerate methods of anaerobiosis.Describe the
morphology & cult. Characteristics of Cl tetani.describe the
prophylaxis of tetanus?
9.Describe the morphology, cult. Characteristics of tetanus?

72. SHORT NOTES :
1.Stormy clot reaction.
2.Toxins of Cl welchii.
3.Nagler’s reaction.
4.Pathogenesis & immunoprophylaxis of tetanus.
SHORT ANSWERS :
1.Gas gangrene.
2.Name 4 clostridia causing gas gangrene.
3.Diagnosis of gas gangrene.
4.Morphology of Cl tetani.