Clostridium

25
I. CLASSIFICATION
Clostridia of medical importance may be
classified on the basis of diseases they produce:
A. Tetanus Cl. tetani
B. Gas
gangrene
Established Cl. perfringens
pathogens C. septicum
CL. noUyl
C. Food poisoning
1. Gastroenteritis Cl. perfringens
2. Necrotising Cl. perfringens
enteritis
3. Botulism Cl, botulinum
D. Acute colitis Cl. difficile
Fig. 25.1 Clostridium perfringens

I. CLOSTRIDIUM PERFRINGENS
zone of complete haemolysis caused by theta
toxin_ and a mçch wider zone of incomplete
(CLOSTRIDIUM WELCHI) haemalysis due to alpha toxin. In Robertson
-Cl. perfringens is the most important and cooked meat broth, the meat pieces turn pink
common aetiological agent of gas gangrene (60%), but are not digested.
followed by Cl. noåyi (30-40%) _and C. septicum
(10-20%). Cl. perfringens also produces food ABiochemical Reactions
poisoning and necrotising enteritis in m¡n. tl. perfringens is predominantly saccha-rolytic
Cl. perfringens is a commensal in the large but also have mild ptoteolytic action (gelatin
intestines of man and animals. The spores are liquefaction). It ferments glucose, lactose,
commonly found in soil and dust. sucrose and maltose with the production of acid
and gas. It is indole negative, H,S is formed
A. Morphology abundantly. MR is positive and VP negative.
It is a large, stout, Gram positive bacillus Most strains can reduce nitrates.
measuring 4-6 um x I um with subtermibal In litmus milk, lactose fermentation leads
spore (Fig. 25.1). It is capsulated and non-motile. to formation of acid, which changes the colour
The spores are rarely seen in artifcial culture of litmus from blue to red. The acid coagulates
or in tissue and their absence is one of the the casein (acid clot) and the cloted milk is
characteristic features of Cl. perjringens. disrupted due to vigorous gas production and
B. Culture
this is known as stormy fermentation.
It grows on blood agar, cooked meatbroth(CMB) D. Resistanc
and thioglycollate broth within 24-48 hours, It
is anaerobic and grows over a pH range of 5.5- Spores are usually destroyed within îive minutes
by boiling but those ofthe food poisoning
8.0 and wide temperature range of 20°C-50°C.
strains (type Aand certain type C strains) resist
The optimum temperature for growth is 37°C. boiling for 1-3 hours. Autoclaving at 121°C for
On blood agar, colonies of most strains show
a target haemolysis, resulting from a narrow 15 minutes is lethal, Spores are resistant to
commonly used antiseptics and disinfectants.

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Unlt 1l Systernic Bacteriology

E. Classificatlon antitoxin showa no opaecity, due to specife

CI. perfringens produce at least 12 distinet neutralisntion of the alphn toxin Fig 25,2)
toxins but on the basis of productibn of four Alpha toxin (leeithinase C) splits lecithin nto
major toxins (otpha, beta, epsilon and iota). phosphorylcholine and a diglyceride (lipifThe
arotund the coloniea resulting in
these are classifed into fve tvpes A to E lipid deposite
1. Tvpe Astrains produce alpha toxin opacity
2. TypeB strains produce alpha. beta and
epsilon toxips.
3. Type C strains produce alpha and beta
No
toxins
antitoxin
4. Type D strains produce alpha and epsilon
to~ins.
5. Type E strains produce alpha and iota
toxins

reaction
Alpha (a) Toxin Fig. 25.2 Nagler
It is produçed by alltypes of Cl. perfringens but
most abundantly by type A strains. Chemically
it is a phospholipidase (lecithinase C) and Reverse CAMP Test
is responsible for profound toxaemia
in
stable, This test is similar to the CAMP test .
heat
gas gangrene. It is relatively jdentifying groupB streptococci (refer Chapt.
lethal, dermonecrotic and haemolytic. best The 20) except that Clostridium sp. is_ inocuiate
haemolysis is of the hot-cold variety i.e.
chilling in place of Staph. aureus and a known groun b
seen after incubation at 37°Cfollowed by streptococci is used. Although group Bstreptocte
at 4°C. may show some enhanced. haemolysis w
other clostridia but only Cl. perfringens exhabt.
Nagler reaction accentuated zone of haemolysis as buttert
medium containing
Cl. perfringens is grown ona of sheep blood
appearance (Fig. 25.3)
6% agar, 5%, Fildes' peptic digest
5% egg yolk in a
and 20% human serum or Other Major Toxins
may be added to the
plate. \Neomycin sulphateselectiye by inhibiting Beta (B)., epsilon ([) and ota (1) toxins hate
medium to make it more To one half lethal and necrotising properties.
and'coliforms.
aerobi spore bearers
spread on the surface.
of the plate, antitoxin is plate is incubated at Enterotoxin
The inoculated culture Some strains of type A produce enterotor:
37°C for 24 hours. Colonies on the half plate
surrounded by which causes diarrhoea and other symptoms i
without the antitoxin will be other half with food poisoning.
the
opacity while colonies on

Group B streptococci
Butterfly appearance
a
-Cl. pertringens re

br
bi
pa

Fig. 25.3 Reverse CAMP test

48
 Clostridium
25
F. Pathogenesis ^3. Animal Pathogenicity
F.
Pens produces the following humar0.1 ml of 24 hours growth in cooked
hroth is injectéd into a healthy guinea pigmeat
infections,

1. Gas Gangrene intramu8cular route. The animal dies withinby

21 hours. A control animal
antiserum prior to test /s alsoprotectedyith
Clperfringenstype Ais the predominant bacterial
agent causing gas gangrene. When awound gets included. On
contaminated by faecal matter or soil, it may autopsy, bacteria can be recovered fron reart
lead to 'simple wound contaminatipn', anaerobic añd spleen of the test animal.
celluhtis or myonecrosis (gas gangrene proper). Laboratory diagnosis of food poisoning is
It is only when muscle tissues are invaded that made by isolating heat resistant Cl. perfringens
gas gångrene results. The incubation period Type A from the faeces and food. Laboratory
varies from six hours to six weeks. methods åre same but selective media are used
for direct platipg. Cooked meat broth (CMB) is
2. Food Poisoning inoculated and heated at 100°C for 30 minutes.
After cooling, it is incubated at 37°C for 18
Some strains of type A produce food poisoning. hours and subcultured on selective medium
Incubation period varies from 8to 12 hours. A which is then incubated anaerobically at 37°C
heat labile enterotoxin is liberated in the small for 18 hours. The bacterial isolates are identified
intestine after ingestion of a contaminated food. by morphology. cultural characteristics,
usually cooked meat and poultry. biochemical reactions and Nagler reaction.
As Cl. perfringens may be present jin normal
3. Necrotising Enteritis intestines, isolation from faeces, excépt in large
A severe and fatal necrotising jejunitis is cauged numbers is not meaningful.
by type Cstrains of Cl. perfringens.
H. Prophylaxis
G. Laboratory Diagnosis 1. Surgery: As a prophylatic measure,
The diagnosis of gas gangrene must be all damaged tissue should be removed
made primarily on, clinical grounds and the promptly and the wound is irrigated
láboratory only confirms the clinical diagnosis. with antiseptic solution to remove
The specimens to be collected are exudates trom blood clots, necrotic tissue and foreign
wound, necrotic tissue and muscle fragments. materials. In established gas gangrene,
uncompromising excision of all affected
1. Direct Microscopy tissue may save life.
Gram stained smear8- gve presumptive 2. Antibiotics: Gas gangrene organisms are
diagnosis. Large number of Gram_positive susceptible to metronidazole, penicillin,
bacilli without spores is strongly suggestive of sulphonamide, tetracycline and
Cl. perfringens. amoxycillin.
3. Antitoxin: Passive immunisation with
2. Culture
anti-gas gangrene serum is used prophy
The specimens are inoculated on fresh and lactically in cases with extensively soiled
-heated blood agar and cooked meat broth wounds. Precautions must be taken
(CMB).Growth in CMB is subcultured on blood to guard against hypersensitivity to horse
agar platea after 24-48 hours. The blood agar serum.
is incubated anaerobically for 48-72 hours. 4.
Most strains produce beta haemolysis on blood Hyperbaric oxygen: Hyperbaric oxygen
is introduced in the depth of wound to
agar and few are non-haemolytic. A plate reduce anaerobiosis.
of serum or egg yolk agar is used for Nagler
reaction. The bacterial isolates are identified
by morphology, cultural characteristics, II. CLOSTRIDIUM TETANI
biochemical reactions and reverse CAMP test. Cl. tetani is the causative agent of tetanus. It is
Poxigenicity of the strain can be done by animal widely distributed in soil and intestine of
pathogenicity. man
and animals.

149
25
Unit lf: Systemic Bacteriology

hours, However, autoclaving (at 1219(

A. Morphology minutes)
20Spores kills the spores ot mo8t strain
It is a Gram positive, slender bacillus (measuring are also killed with lodin, (1% aqueous
4-8 um x 0.5 Am) with spherical, terminal solution). hydrogen peroxide (10 volumes) and
spores giving the bacillus the. characteristic glutaraldehyde 2%) withie a few hóurs. The
drumstick appearance (Fig 254)._It is non years.
spores'may survive in soil for
capsuled and 'motile (except C1. tetani type
VI) with peritrichate fagella, E. Classification
Ten serologicaltypes, (types I to X .
based on f
CL. tetani have been recognised strain.
specific fágellar (H) antigens. Type VI
are non-flagellated.

F. Toxins
Cl. tetani producestwo distinct toxing
tetanolysin (haemolysin) and tetanospasmin
(neurotoxin).

1. Tetanolysin
Tetanolysin is a heat labile. Oxygen labile toxn
severa'
which causes lysis of erythrocytes_of
leucotoxin but
animal species. It may act as a
clear.
its pathogenic rale is not
Fig. 25.4 Clostridium tetani
2. Tetanospasmin
Tetanospasmjn is a heat labile, oxygen stable
powerful neurotoxin and rapidly gets destroved
B. Culture
It is an obligate anaerobe which grows on nature.
by proteolyticenzymes It is protein in
9rdinary media. Growth is improved by the This is responsible for clinical manifestations of
addition of blood or serum. The optimum tetanus. Tetanospasmin is a good antigen and
temperature for growth is ´37°C, and pH 7.4.
(CMB), is specifically neutralisd by the antitoxin.
It can grow well in cooked meat broth
thioglycollate brot nutrient agar)and 5lood G. Pathogenesis
agan In RCM broth Howthoccurs as turbidity Cl. tetani has little invasive power. Tetanus
and there is also some gas formation. The meat
develops following the contamination of wound
is not digested but becomes black on prolonged of infection
with Cl. tetani spores. The source
incubation. The bacilli produce aswarming (thin faeces_ett. Infection strictly
may be soil, dust,
spreading film) growth on blood agar. However,
remains localized_ in the wound. jGermination
on horse blood agar they produce<a-haemolvtic
develop into of spores and toxin production occur only
if
colonieswhich subsequently
B-haemolytid. due to_ the production of-a favourable conditions exist, such as reduced
haemolysin (tetanolysin), Q-R potential, devatilisd tissues and foreign
bodies. Pathogenic effects are mainly due to
C. Biochemical Reactions tetanospasmin (neurotoxin) of Cl. tetani..
CI. tetani has slight proteolytic, but no
saccharolytic property. Gelatin liquefaction H. Laboratory Diagnosis
occurs very slowly. It does not ferment any sugar. The diagnosis of tetanus should always be made
forms indole but is MR and VP negative. H,S clinically and laboratory tests are done only to
is not formed. Nitrates are not reduced. confirm it. Laboratorydiagnosis may be made by
demonstration of bacilli by microscopy, ulture
D. Resistance or by animal inoculation. Specimens generally
collected are wound swab, exudate or tissue
Most of the strains are killed by boiling for
10-15 minutes but some_resist boiling for three from the wound.

150
 25
Costridium

) Antibiotics
stainingmavshow Gram
1. Microscopy positive bacilli CAntibiotics dest roy or inhibit tetanus bacilli and
Withdrumstickappeatance. other pyogenic bacteria in wounds and thus the
Gram
production of toxin is prevented. Long actipg
2. Culture inoculated on freshly prepared
penicillin injection or erythromycin may be
given.
Specimen is
and incubated at 37°C for 24-48
blood sgar anaerobic conditions, Cæ. tetani
hours
under (3) Immunisation
produces a swarming growth. The specimen is Tetanus is apreventable disease
inoculated in three tubes of cooked meat
also
broth (CMB). One of these tubes is heated at (a) Active immunisation
soC for 15 minùtes, the second tube for fiye It is the most effective method of prophylaxis.
minutes and the thirdlleft unheated. Heating for Tetanus oxoid (formol toxoid), which is
diferent periods js to kill vegetative bacteria, Ayailble eitheY as 'plain toxoid', or adsorbed
while leaying tetanus spores undamaged. These . on aluminium hydroxid or phosphate (APT). is
cooked meat broth_ are incubated at 37°C and commonly used for active immunisation. Three
doses of 0.5 ml tetanus toxoid (APT) each are
subcultured on blood agar plates daily for upto
four days. given intramuscülarly, with an interval of 4,to 6
Grami Stained smear from culture shows weeks between first two doses and 6-12 months
full course
tvpical Gram positive bacilli with drumstick between the second and third dose. A
appearance. of three doses confers immunity for a period of
t least 10 years, Abooster dose' of toxoid is
. Toxigenicity Test recommended after 10 years.
Pathogenicity of. the jsolated organism is Tetanus toxoid is given along with diphtheria
established with demonstration of toxin toxoid and pertusis vaccine (DPT) in children.
"broduction. It is best tested in animals. 0.2 ml Pertusis vaccine acts as adjuvant. Three doses
of 2to 4 days old cooked meat culture is injected are given intramuscularly at interval of 44-6
into. the rootof the tail of one mouse (test weeks, starting at age as early as_6 weeks.
animal), same amount is injected into another. Booster doses are given at age of 18 months and
animal (control) that has received tetanus then at five years.
antitoxin (1000 units) an hour earier.
In positive case, the test animal develops (b) Passive immunisation
symptoms within 12-24 hours, beginning with Antitetanus serum (ATS), prepared by
stifness of the tail. Rigidity proceeds_to the leg immunising horses with toxoid, has been
on the inoculated side, the another leg, trunkand used for preventing tetanus. The dose is 1500
forelimbs, in that particular order. Death occurs IU by intramuscular route immediately after
within two days, but may be killed earlier. as wounding. Being ahorse serum, it carries the
the ascending tet¯nus is very much diagnostic. risk of hypersensitivity reaction.
The control animal does not show any change Homologous serum prepared from humanS,
due to neutralisation oftoxin by antitoxin. human antitetanus immunoglobulins (HTIG),
is now being used without the risk of
I. Prophylaxis hypersensitivity. Dose of 250 units is used in
The available methods are prophylaxis.
(1) Surgical (2) Antibiotiçs (3) Immunisation
(c) Combined prophylaxis
(1) Surgical In non-immune person, it is ideal to immunisè
It aims at removal of foreign body, blood clots with first dose of tetanus toxoid in one site
etc., in order to prevent anaerobie- conditions along with administration of ATS or HTIG
favourable for the bacillus. Depending on the in another arm, followed by the second and
type of wound, surgical prophylaxis- may vary third doses of tetanus toxoid at monthly
from simple cleansing to radical excision. interval.

151
 25
Unit ll: Systemic Bacteiology
The toxin has
IV.CLOSTRIDIUM BOTULINUM medium on autolysis of the cell. protein with
Clostridium botulinup causes a sevbre form of been isolated as a pure crystalline
A MW. 70.000. It has a lethal
dose for mics
food poisoning named botulism. It js a widely dose
0.000,000,033 mg and a lethalIt ig
for hunmans
distributed saprophyte and is found in goil. microgram. a neurotoxin
animal mänure, vegetables and sea mud. is probably one takes several hoursto
and acts glowly, therefore,
substanea
kil). Botulinum toxin is the most toxic
A. Morphology known so far.,
It is a Gram positive, non-capsulated bacillis Preformed toxin in food is destroved bv.
about 5um x lum, motile by peritrichate flagella boiling for 20 minutes. It 1s, a good antigen and
and produces subterminal, oval, bulging spores can be neutralised specificlly by its antitoxin
(Fig. 25.5). The toxin acts by bloçking the production
acetylcholine at syriapses and
or release of
neuromuscular junctions. Death occurs due to
loyespiratoryfailure.
F. Pathogenesis
C. botulinum. is non-invasive and its
pathogenicity is due to the action of preformed
toxin, the manifestations of which are eolBeetively
called botuljsm., Botulism is of three types
foodborne, infant and wound botulism.
1. Foodborne Botulism
Fig. 25.5 Clostridium
botulinum It is due to preformed toxin in food contaminated
with Cl. botulinum. The source of botulism is
B. Culture usually various preserved foods -meat, fish.
anaerobe and can grow
It is a strict temperature is vegetables etc.
on ordinary media. Optimum and The symptoms appear 12-36 hours after
35°C._Commonly used media are blood agar food. Vomiting.
cooked meat broth (CMB). Colonies are large,
ingestion of contaminatd
fmbriate thirst, constipation,_ oculr paresi_, difficulty
iregular, semitransparent,with the in swallowing, speaking and breathing are
border. On blood agar, haemolysis around the common symptoms. Diarrhoea is not a
colonies is observed.
symptom.
C. Resistance
2. Infant Botulism
The spores are highly resistant, and can
withstand heat for several hours at 100°C and It affects infants, usually below 6months. It is
for upto 10 minutes-at 120°C. They are also
a disease due to ingèstion of food contaminated
resistant to radiation. by spores, of_CI. botulinum. The disease is
characterised by constipation, weakness,
lethargyAnd (cranial palsiesnfants exhibit
D. Classification
FandG have
Eight types-A, B, C1, C2, D, E, antigenically flaccid paralysis usually with a weak sucking
response,_generalised loss of tone ("the flopps
been identiñed on the basis of their
distinct toxins. baby syndrome") and respiratory complications.
Severity of illness 'varies from mild illness to
E. Toxin fatal disease
exotoxin. The
Cl. botulinum forms a powerful
exotoxins in that it is 3. Wound Botulism
toxin differs from other
not teteased during the life of the
bacterium. It It is a very rare condition which results from
is Predutced intracellularly and appears in the wound infection with Cl. botulinum.

152
 Clostridium
25

G. Laboratory Diagnosis V. CLOSTRIDIUM DIFFICILE

Diagnosis may be confrmed by demonstration Cl. difficile was first isolated from the faeces
of the bacillus or the toxin in suspected residual of newborn infants, It was s0 named due to
food or in faeces. unusual difficulties involved in its isolation.
1. Demonstration of the Organism A. Morphology
Smears made from suspected food or faeces are It is a long, slender, Gram positive bacillus
examined by Gram's staining which may show containing oval and terminal spores.
Gram positive sporing bacilli. Culture is done on
blood agar or cooked meat broth (CMB). Growth B. Toxins
on culture media may be confirmed by Gram's
It produces two toxins, an enterotoxin (toxin A)
staining. Presence of bacilli in food or faeces in and a cytotoxin (toxin B). The enterotoxin is
absence of toxin is of no significance. Hence, primarily responsible for diarrhoea whereas
toxin in culture fluid must be demonstrated by
cytotoxin is capable of producing cytopathogenic
toxigenicity test in mice. effects in several tissue culture cell lines.
2. Demonstration of Toxin C. Pathogenesis
Specimens (stool. food and vomitus) are The pathogenic role of Cl. diffcile is established
macerated in sterile saling and the filtered in 1977, when it was found to be responsible for
extract is divided into three_parts, One portion antibiotic associated colitis. Many antibiotics
of extract is heated at 100°C for i0 minutes and have been incriminated but lincomycin and
other two kept unheated. Two mice or guinea clindamycin are particularly prone to cause
pigs are injected with unheated filtrate; one pseudomembranous colitis. If the condition is
of them (control) is protected withLpolyvalent not recognised early and properly treated, it
botulinum antitoxin prior to injection. The terminates fatally.
third animal is injected with heated iltrate.
The test
animal-(unprotected) develops D. Laboratory Diagnosis
dyspnoea, flaccid aralysis> and- dies within 1. Demonstration of Toxin
24 hours whereas control animal (protected) Toxin can be demonstrated in the faeces by
rmains healthy. The animal injected with its characteristic effect on human diploid cells
heated filtrate also remains free of any and HEp-2. ELISA can also be used for the
toxic symptoms. demonstration of toxin. The toxin is specifically
neutralised by the Cl. sordelli antitoxin.
H. Prophylaxis and Treatment
As botulism follows consumption of canned 2. Isolation of Bacilli
or preserved food, control may be achieved by Cl. difficile can be grown from the faeces of
proper canning and preservation. By the time patients with subsequent test for toxigenicity.
symptoms appear, the toxin is fixed to nervous
tissue and it can no longer be inactivated by E. Treatment
antitoxin. Intensive aupportive therapy may Cl. difficile are generally resistant to most
bring down the mortality rate. However, if antibiotics, but these are susceptible to
outbreak occurs, prophylactic dose of antitoxin vancomycin. Clindamycin and lincomycin
should be given intramuscularly to all who have should be avoided. Metronidazole is the drug of
consumed the suspected food. choice.