Download clostridium

CCl
Introductory Characteristics
 Obligate anaerobes
 Gram positive
 Capable of producing
endospores
 Rod-shaped, named after
Greek word for spindle,
kloster
 Club-shaped, as well:
endospores form club
end
 Saprophytes found in
soil, water, decomposing
matter
 Classification:
 A. Tetanus – Cl.tetani
 B. Gas gangrene –
1. Established pathogens-Cl.perfringens
Cl.septicum
Cl.novyi
2. Less pathogenic – Cl.histolyticum
Cl.fallax
3. Doubtful pathogens- Cl.bifermentans
Cl.sporogenes
 C. Food poisoning-
1. Gastroenteritis – Cl.perfringens (type A)
2. Necrotising enteritis – Cl.perfringens ( type C)
3. Botulism – Cl.botulinum
 D. Acute colitis – Cl.difficle
 Gram positive, spore forming bacilli, highly
pleomorphic
 Shape and position of spores varies in different species
and is useful in identification of clostridia
 Distribution of spores:
 1. Central or equatorial – Cl.bifermentans
 2. Sub-terminal – Cl.perfringens
 3. Oval and terminal – Cl.tertium
 4. Spherical and terminal – Cl.tetani
 Culture:
 Clostridia grow well on ordinary media under anaerobic
conditions
 Liquid media- Robertson’s cooked meat broth (RCM),
thioglycollate broth are very useful
 RCM contains unsaturated fatty acids which take up
oxygen, reaction being catalysed by hematin in meat and
sulphydryl compounds (they lower down the redox
potential)
 Thioglycollate broth contains reducing agent
thioglycollate
 More important than the absence of oxygen is the
provision of a sufficiently low redox potential in the
medium
 Reducing substances that can be used are- unsaturated
fatty acids, ascorbic acid, glutathione, cysteine,
thioglycollic acid, alkaline glucose, sulphites, metallic
iron
 Growth of clostridia results in turbid broth
 Most species produce gas
 Saccharolytic species turn the meat pink
 Proteolytic species turn the meat black and produce foul
smell
 Small concentration of CO2 enhances growth
 Inoculated plates ( eg: blood agar) are placed in anaerobic
jar and incubated at 370 C for 2-3 days
 Resistance: spores exhibit variable resistance
 Cl.tetani spores persist for years in dried earth
 Cl.perfringens spores are destroyed by boiling for less
than 5 mins except type A which takes several hours
 Cl.botulinum spores are not killed completely even at
1050C for less than 100 mins
 All spores get killed at 1210C for 20 mins (autoclaving)
 Halogens, glutaraldehyde are effective
Clostridium tetani
 Causative agent of tetanus
 Widely distributed in soil and intestine of man and
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animals
Morphology:
Gram positive slender bacilli
Spherical terminal spores giving the characteristic
drumstick appearance
Non capsulated, motile
 Culture:
 Obligate anaerobe
 Grow well on Robertson’s cooked meat broth,
thioglycollate broth, blood agar
 In RCM growth occurs as turbidity and gas formation
seen
 Meat is not digested, but becomes black on prolonged
incubation
 Bacilli produce swarming growth on blood agar ( thin
spreading film)
 Biochemical reactions :
 Cl.tetani has mild proteolytic but no saccharolytic
property
 Does not ferment any sugar
 Greenish fluorescence is produced on media containing
neutral red (MacConkey’s agar)
 Resistance:
 Spores can survive for years in soil
 Autoclaving kills all spores
 Classification:
 10 serological types (I to X) based on type specific flagellar
H antigens by agglutination test
 Toxins:
 Tetanolysin (hemolysin)
 Tetanospasmin (neurotoxin)
 Tetanolysin is heat labile oxygen labile toxin causing lysis
of RBC
 Tetanospamin is a heat labile oxygen stable powerful
neurotoxin, protein in nature, good antigen, responsible
for pathogenesis of tetanus, neutralised by antitoxin
Tetanus
 Is characterised by tonic muscular spasms, usually
commencing at the site of infection and becoming
generalised, involving the whole of somatic muscular
system
 Etiology: Clostridium tetani
 Route of entry of pathogen: following any injury,
puncture wounds, surgical operations with a lapse in
asepsis, local suppuration, Otitis media, dental
procedures, septic abortion, application of cow dung on
umbilical stump or rituals like ear boring, circumcision
 Incubation period:
 2 days to several weeks, commonly 6-12 days
 depending on factors like site and nature of wound,
dose and toxigenicity of the strain, immune status of
the patient
 IP is of prognostic significance, grave prognosis when
it is short IP
 Tetanus was a serious disease with high mortality rate in
the past
 Even with proper treatment case fatality rate varies from
15-50%
 Tetanus neonatorum and uterine tetanus are grave
conditions with very high fatality rate
 Tetanus is more common in developing countries, where
climate is warm, rural areas where soil is fertile, where
human and animal populations live in close association,
where unhygienic practices are common and medical
facilities are poor
 In rural India, neonatal tetanus was common
 Pathogenesis:
 Infection strictly remains localised in the wound
 Germination of Cl.tetani spores and toxin production
occur only if favourable conditions exist like:
 Reduced O-R potential, devitalised tissues and foreign
bodies
 Toxin that is produced locally is absorbed by the motor
nerve endings and transported to the central nervous
system intra-axonally
 The toxin is specifically and avidly fixed by gangliosides of
grey matter of nervous system
 Pathogenic effects are mainly due to tetanospasmin
 It resembles strychnine in its effects
 It specifically blocks synaptic inhibition in the spinal
cord, presumably at the inhibitory terminals that use
glycine and GABA as neurotransmitters
 The abolition of spinal inhibition causes uncontrolled
spread of impulses initiated anywhere in the CNS
 This results in muscle rigidity and spasms due to
simultaneous contraction of agonists and antagonists,
in the absence of reciprocal inhibition
 Lock Jaw- initial symptom
 The convulsion pattern is determined by the most
powerful muscles at a given point
 In most it is characterised by tonic extension of the
body and of all limbs
Muscular spasms leading to Opisthotonos in tetanus
 Laboratory diagnosis:
 Clinical diagnosis by history, symptoms and signs
 1. Microscopy:
 Gram’s staining show Gram positive bacilli with
drumstick appearance
 2. Culture:
 Blood agar incubated at 370C for 24-48 hours under
anaerobic conditions
 3. Toxigenicity test:
 Animal testing
 Prophylaxis for tetanus:
 1. Surgical
 2. Antibiotics
 3. Immunisation
 1. Surgical:
 Removal of foreign body, blood clots etc to prevent
favourable anaerobic conditions
 Simple cleaning of the wound to radical excision
 2. Antibiotics:
 Penicillin, erythromycin
 3. Immunisation:
 Tetanus is a preventable disease
 A) Active immunisation:
 Most effective method of prophylaxis
 Tetanus toxoid (formol toxoid) either as ‘plain toxoid’ or
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adsorbed on aluminium hydroxide( better antigen)
Dosage: 0.5 ml
Doses: 3 doses with an interval of 4 to 6 weeks
Given as triple vaccine along with Diphtheria toxoid and
pertussis vaccine
Booster doses
If wounding occurs 3 years or more after full course,
booster dose given
 B) Passive immunisation:
 Antitetanus serum (ATS) prepared by immunising
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horses with toxoid
Dose is 1500 IU by intramuscular route immediately
after the person is wounded
Risk of hypersensitivity reaction
Homologous serum prepared from humans (HTIG) is
with less risk of hypersensitivity
Dose 250 units
 C) Combined prophylaxis:
 In non-immune person
 Combining active with passive immunisation
 First dose of TT in one arm along with administration
of ATS or HTIG in another arm, followed by 2nd and
3rd doses of TT
 Adsorbed toxoid should be used here, plain toxoid
may be interfered with ATS
 Treatment:
 Treated in special isolation unit in-order to protect from
noise and light which may provoke convulsions
 Person to person transmission does not occur
 Controlling spasms, maintaining airway by tracheostomy
 Antitoxin, antibiotic therapy penicillin
 Recovering patients should receive full course of active
immunisation, an attack of disease does not confer
immunity
Nature of wound
Immune person
Partially immune
Non-immune
Clean wound (
wound cleaning
done within 6
hours)
Toxoid 1 dose
Toxoid 1 dose
toxoid 3 doses
Contaminated (Soil
or other foreign
material
Toxoid 1 dose
Toxoid 1 dose, TIG,
antibiotics
Toxoid 3 doses, TIG,
antibiotics
Infected wound
Toxoid 1 dose,
antibiotics
Toxoid 1 dose, TIG,
antibiotics
Toxoid 3 doses, TIG,
antibiotics
Clostridium perfringens
 Also called Cl.welchii
 Is a commensal in large intestine of man and animals
 Spores are commonly found in soil and dust
 Morphology:
 Large, stout, Gram positive bacillus with sub-terminal
spore
 Capsulated, non-motile
 Spores are rarely seen in ordinary culture media or in
tissue (Characteristic feature), induced only in special
media
 Culture: RCM, thioglycollate broth, blood agar
 Biochemical reactions:
 Predominantly saccharolytic but also have mild
proteolytic action
 In litmus milk, lactose fermentation leads to
formation of acid, which changes the colour of litmus
from blue to red
 The acid coagulates the casein(acid clot) and the
clotted milk is disrupted due to vigorous gas
production
 This is known as Stormy fermentation
 Classification:
 Cl.perfringens produce 12 toxins
 Classification is based on production of 4 major
toxins: alpha, beta, epsilon and iota
 1. type A strains produce alpha toxin
 2. type B strains produce alpha, beta and epsilon
toxins
 3. type C strains produce alpha and beta toxins
 4. type D strains produce alpha and epsilon toxins
 5. type E strains produce alpha and iota toxins
 Alpha (α ) toxin:
 Produced by all types of cl.perfringens but most
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abundantly by type A strains
Chemically it is lecithinase C
Is responsible for profound toxaemia in gas gangrene
Relatively heat stable, lethal, dermonecrotic,
hemolytic
It splits lecithin, an important constituent of
mammalian cell membrane
This special effect is utilised for rapid detection of
Cl.perfringens in clinical specimen (Nagler reaction)
 Nagler reaction:
 Cl.perfringens is grown on a medium containing 6% agar,
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5% Filde’s peptic digest of sheep blood and 20% human
serum or 5 % egg yolk
Antitoxin is spread on one half of the plate and incubated
at 370C for 24 hours
Colonies on the half of plate without antitoxin will be
surrounded by opacity while colonies on the other half
with antitoxin shows no opacity
This is due t0 specific neutralisation of the alpha toxin
Alpha toxin splits lecithin into phosphoryl choline and a
diglyceride (lipid)
This lipid deposits around the colonies resulting in
opacity
 Reverse CAMP test:
 Is similar to the CAMP test for identifying group B
streptococci
 Here Clostridium species is inoculated in place of
Staphylococcus aureus and a known group B
streptococci is used
 Only Cl.perfringens exhibits accentuated zone of
hemolysis as butterfly appearance
 Other major toxins:
 Beta, epsilon and iota toxins have lethal and necrotising
properties
 Minor toxins:
 Gamma, eta have only minor lethal actions
 Delta toxin is lethal and hemolytic
 Theta toxin is oxygen labile
 Kappa toxin is a collagenase
 Lambda toxin is a proteinase and gelatinase
 Mu toxin is a hyaluronidase
 Nu toxin is a deoxyribonuclease
 Enterotoxin:
 Some strains of type A cause food poisoning and
diarrhoea by producing this toxin
 Other soluble substances:
 Haemagglutinins, neuraminidase, fibrinolysin,
hemolysin, histamine
 Bursting factor has specific action on muscle tissue
and is responsible for the characteristic muscle lesions
in gas gangrene
 Pathogenesis:
 1. Gas gangrene:
 Cl.perfringens type A is the predominant bacterial
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agent causing gas gangrene
When a wound gets contaminated by faecal matter or
soil, it may lead to
simple wound contamination,
anaerobic cellulitis or myonecrosis
When the muscle tissue is invaded it results in gas
gangrene
IP- 6 hours to 6 weeks
 2. Food poisoning:
 Some strains of Type A produce enterotoxin
 IP 8-12 hours
 Cause: ingestion of cold or warmed up meat dish
 When contaminated meat is cooked, the spores in
the interior may survive
 During storage or warming they germinate and
multiply in the anaerobic environment in cooked meat
 Large numbers of clostridia is consumed which
escapes gastric acid due to high protein in meal
 Reaches intestine and produces enterotoxin
 Symptoms:
 Pain abdomen, diarrhoea, vomiting
 Self-limited illness
 Recovery in 24-48 hours
 Diagnosis by isolating heat resistant Cl.perfringens
type A from stool and food
 3. Necrotising enteritis:
 Type C strains cause a severe and fatal necrotising
jejunitis
 4. Gangrenous appendicitis:
 Type A strains and occasionally type D strains
 5. Other diseases:
 Urogenital infections, brain abscess, meningitis,
panophthalmitis and puerperal infection
Gas gangrene
 Is a rapidly spreading, edematous myonecrosis
 Occurs in association with severe wounds of extensive
muscle masses that have been contaminated with
pathogenic clostridia
Etiology: 1. Established pathogens-Cl.perfringens
Cl.septicum
Cl.novyi
2. Less pathogenic – Cl.histolyticum
Cl.fallax
3. Doubtful pathogens- Cl.bifermentans
Cl.sporogenes
Others: anaerobic streptococci, E.coli, Proteus,
Staphylococci
 Route of entry of pathogen:
 Along with implanted foreign s like soil, road dust, bits
of clothing etc
 Pathogenesis:
 MacLennon’s description of 3 types of anaerobic
wound infections:
 Simple wound contamination
 Anaerobic cellulitis
 Anaerobic myositis or gas gangrene
 Favourable conditions for development of gas
gangrene:
 crushing tissue or tearing of arteries produce anoxia of
the muscle
 The Eh and pH of damaged tissues fall, breakdown of
carbohydrates and muscle proteins provides a
nourishing environment for anaerobes
 S/S:
 Increasing pain, tenderness, edema of the affected part
along with systemic signs of toxaemia
 Thin watery discharge from the wound, later becomes
profuse and sero-sanguinous
 Accumulation of gas makes the tissues crepitant
 Death occurs due to circulatory failure
 Lab diagnosis of gas gangrene:
 Specimen: exudates from wound, necrotic tissue and
muscle fragments
 Direct microscopy:
 Gram’s staining of the specimen
 Culture:
 Aerobic and anaerobic culture
 Bacterial isolates are identified by morphology,
cultural characteistics, biochemical reactions and
reverse CAMP test
 Animal pathogenicity:
 To determine the toxigenicity of the strain
 Prophylaxis:
 Surgery: all damaged tissue should be removed
promptly and wound is irrigated with antiseptic
solution
 Antibiotics: metronidazole, penicillin, tetracycline etc
 Antitoxin: passive immunisation by anti-gas gangrene
serum
Clostridium botulinum
 Causes a severe form of food poisoning named
botulism
 Widely distributed saprophyte, found in soil, animal
manure, vegetables, sea mud
 Morphology:
 Gram positive, non-capsulated bacilli, motile
 Sub-terminal, oval bulging spores
 Culture:
 Obligate anaerobe
 Blood agar, cooked meat broth at 350 C under anaerobic
atmosphere
 Hemolysis around colonies in blood agar
 Resistance:
 Spores are resistant and can withstand heat for several
hours at 1000C and for up to 10 minutes at 1200C
 Classification;
 Eight types based on antigenically distinct toxins
 Types A, B, C1, C2, D, E, F and G
 Toxin:
 Powerful exotoxin
 Differs from other exotoxins in that it is not released
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during the life of bacterium
Produced intracellularly and is released on autolysis of
the cell
Synthesised as non-toxic protoxin which is converted
to active toxin by trypsin, other proteolytic enzymes
Action: neurotoxin, acts slowly, takes several hours
to kill
Type A toxin is the most potent
 Preformed toxin in food is destroyed by boiling for 20
minutes
 Is a good antigen and can be neutralised specifically by
its antitoxin
 The toxin acts by blocking the production or release of
acetylcholine at synapses and neuromuscular
junctions
 Death occurs due to respiratory failure
Botulism
 Etiology : Clostridium botulinum
 Pathogenicity is due to the action of preformed toxin
 Botulism is of 3 types:
 1. Foodborne botulism
 2. Infant botulism
 3. Wound botulism
 1. Foodborne botulism:
 Due to preformed toxin in food contaminated with
Cl.botulinum
 Types A,B,E are usually associated with human disease,
rarely types C,F and G
 Source of infection: preserved foods- meat, fish,
vegetables
 Contaminated food usually shows signs of spoilage
 Cans with preserved food may be inflated and show
bubbles on opening
 There may be no alteration in taste
 2. Infant botulism:
 Seen in infants below 6 months
 Disease due to ingestion of food contaminated by
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spores of Cl.botulinum
Source of infection: honey
S/S : constipation, poor feeding, weakness, lethargy,
pooled oral secretions, weak or altered cry, floppiness
and loss of head control, respiratory complications
“floppy baby syndrome”
Patient excretes toxin and spores in faeces
Severity- mild illness to fatal disease
 3. Wound botulism:
 Rare condition
 Wound infection by Cl.botulinum
 Toxin produced is absorbed into blood
 Symptoms similar to foodborne botulism except for
gastrointestinal components
 Type A is more common
 Laboratory diagnosis:
 Confirmation of clinical diagnosis by demonstration of
the bacillus or the toxin in faeces or left over food
 1. Microscopy:
 Gram’s staining shows Gram positive sporing bacilli
 2.Culture: Blood agar, RCM
 3. Demonstration of toxin:
 Animal testing
 Prophylaxis:
 Proper canning and preservation of food
 By the time symptoms develop, toxin is fixed to nervous
tissue and it can no longer be inactivated by antitoxin
 Intensive supportive therapy, maintenance of respiration
needed
 Prophylactic dose of antitoxin is given to all others who
have consumed the food which was the source of
infection
 Immunisation: 2 doses of toxoid given at an interval of 10
weeks followed by booster after a year
 Can be used for lab workers exposed to risk
Clostridium difficle
 Morphology:
 Long, slender, Gram positive bacillus with oval and
terminal spores
 Toxins:
 Produces 2 toxins, an enterotoxin (toxin A) and a
cytotoxin (toxin B)
 Enterotoxin is responsible for diarrhoea and cytotoxin
produces cytopathogenic effects
 Pathogenesis:
 Causes antibiotic associated colitis /
pseudomembranous colitis
 Patients on long term oral antibiotic therapy can go in
for acute colitis with or without membrane formation
 Lincomycin and clindamycin are particularly prone to
cause pseudomembranous colitis
 Active multiplication of Cl.difficle and the production
of enterotoxin and cytotoxin causes antibiotic
associated colitis
 If the condition is not recognised early and properly
treated, it can be fatal
 Laboratory diagnosis:
 1. Isolation of bacilli:
 From the faeces of the patient
 Cycloserine-cefoxitin-fructose agar (CCFA) is a
selective medium for primary isolation of Cl.difficle
 2. demonstration of toxin:
 Toxin can be demonstrated in the faeces by its effect
on human diploid cells and Hep-2 cells, ELISA
 Treatment:
 Metronidazole
 Clindamycin and lincomycin should be avoided