Gram positive anaerobic, spore forming bacilli. Spores are refractile, oval or spherical and usually wider than the parent cell. They may be terminal, subterminal or central within the cell. Most clostridia possess flagella and motile but C. perfringens not. In 48 hour cultures, many of the bacilli may be Gram negative.
Clostridia lack the cytochromes required for electron transport to oxygen. They contain flavoprotein enzymes that reduce oxygen to H2O2 and to superoxide, and they lack the catalase, peroxidases and superoxide dismutase that destroy these toxic products. Therefore, they can grow only under anaerobic conditions. Some are found in animal, human intestines. Many species are pathogenic, but most saprophytes found in soil and water decomposing plant and animal matter. Some are of industrial importance for the production of chemical such as acetone and butanol. They are highly pleomorphic, rod shaped, motile. They are obligate anaerobes. The optimum temperature is 370C, pH 7-7.4, growth relatively slow. The characteristic media is Robertson’s cooked meat broth- contains unsaturated fatty acids which take up oxygen. Clostridia can produce disease only when the conditions are appropriate.

The genus contains bacteria responsible for 3 major diseases of human beings-
·         Gas Gangrene
·         Food Poisoning
·         Tetanus

C. perfringens
Morphology & Cultural characteristics - It is a plump, large Gram positive bacillus of 4-6 X 1µm, usually occur in chains or small bundles. It is capsulated and nonmotile. Spores are central or subterminal. Sporulation is favored by an alkaline environment.  It is an anaerobe, also grows under microaeriphilic conditions, pH 5.5-8.0, temperature 20-500C, good growth in Robertson’s cooked meat medium. It grows best on media containing carbobydrates such as Glucose Blood Agar and forms two main types of colonies. One is round, 2-4mm, smooth, regular, convex, amorphous, greyish yellow and slightly opaque. Other is umbonate with an opaque brownish centre and a lighter, translucent, radially striated periphery with a crenated edge. It shows beta hemolysis on horse blood agar. It is a normal inhabitant of the large intestines of human beings and animals. It is found in the faeces. Spores commonly found in soil, dust, air.
Biochemical Reactions – It is actively saccarolytic and ferments glucose, maltose, sucrose, lactose and starch with the production of acid and gas. In litmus milk medium, fermentation of lactose leads to the formation of acid, which is indicated by the change in the colur of litmus from blue to red. The acid coagulates the casein and the clotted milk is disrupted due to the vigorous gas production. The cotton plug is pushed up and shreds of clot sticks to the sides of the tube. This is “stormy fermentation”(stormy clot rection). IMViC is (-+-+/-). It produces H2S. It produces phospholipase-C which gives opalescence around the colonies on egg-yolk containing medium. Spores are usually destroyed in 5 minutes boiling, resistant to antiseptics and disinfectants.
ToxinsC. perfringens produces 4 major lethal, 8 minor lethal toxins and enterotoxin. On the basis of the four major toxins C. perfringens can be divided into 5 types, A to E.
·         Alpha toxin- responsible for the profound toxaemia of gas gangrene. It is thermostable, produces a necrotic lesion when given intradermally, is a Ca++ or Mg++ dependent phospholipase, which produces opalescence in serum or egg yolk containing media by splitting phospholipid complexes. This reaction can be inhibited specific antitoxin. Nagler’s reaction - A culture plate containing 6% agar, 5% peptic digest of sheep blood and 20% human serum or 5%  egg yolk is prepared. The plate is dried. On one half of the plate, 2-3 drops of  C. perfringens antitoxin are spread and allowed to dry. The plate is then inoculated with the test organisms and incubated anaerobically at 370C for 18 hours. On the section containing no antitoxin, C. perfringens colonies show surrounding zone of opalescence, while others show no change. Neomycin sulphate may be added to inhibit aerobic spore-forming organisms and coliforms. Alpha toxin is haemolytic for red cells of most laboratory animals except horse and goat.
·         Enterotoxin- type A strains produce a potent enterotoxin. It is a heat labile, non-diffusible protein with a polypeptide chain and a mol. wt. of  34,000-35,000 daltons. It is formed in the intestine at the time of sporulation. It causes diarrhoea in humans.
·         Beta toxins- non haemolytic, lethal to mice, produces necrotic lesions if given intradermally.
·         Epsilon & iota toxins – lethal, necrotizing, non-haemolytic.
·         Gamma & eta toxins – minor lethal toxins, neither necrotizing nor haemolytic.
·         Delta toxin – lethal,actively haemolytic for the red cells of sheep, pig, cattle.
·         Theta toxin – oxygen labile haemolysin, moderately lethal.
·         Lambda toxin – non-lethal, non-necrotizing proteinase and gelatinase.
·         Kappa, mu, nu toxins – collagenase, hyaluronidase, deoxyribonuclease respectively
Pathogenicity- C. perfringens chiefly causes gas gangrene, associated with deep wounds. Spores of these obligate anaerobic organisms are introduced by injuries or surgery into tissues, where circulation is impaired, dead and anaerobic tissue is present. In body regions where O2     concentration is low, spores germinate, multiply and produce toxins and enzymes, which kill other host cells and extend the anaerobic environment. The onset of gas gangrene occurs suddenly 12-48 hours after injury. As the organisms grow and ferment muscle carbohydrates, they produce gas, mainly hydrogen, and destroy tissues. Such tissue is called crepitant tissue. The bubbles audibly crackle and pop when the patient moves. Foul odour is a prominent feature of gas gangrene. The pressure resulting from gas formation may cause restriction of the blood supply to adjoining tissues and hence more necrosis. Hyaluronidase breaks intercellular cement substance and promotes the spread of the infection. High fever, shock, massive tissue destruction and blackening of skin accompanies. If untreated slow death occurs.
            Enterotoxin producing strains of  C. perfringens are associated with a mild form of food poisoning. The incriminated food is usually meat that has been cooked hours in advance and then cooled slowly, allowed to stand at room temperature for several hours before being served. Patient develops abdominal cramps and diarrhoea with foamy and foul smelling stools 8- 12 hours after ingestion of the contaminated food. It usually subsides within 24 hours.
Laboratory Diagnosis – Edges of affected muscles, necrotic tissue, exudate from the depth of the lesion are the samples collected with a capillary tube. Gram smears are prepared, Nagler’s rection to distinguish the bacterium. Stool samples collected in case of food poisoning.
Prophylaxis & Treatment- Penicillin is administered along with metronidazole, an aminoglycoside to prevent the coexistence of coliforms, gram- positives and faecal anaerobes. Dead tissue is removed or limbs are amputated. It is common in diabetics with high blood sugar. The increased sugar levels provide an adequate carbohydrate supply in muscles for clostridia to ferment. If present in bile, gas gangrene of abdominal muscle occur. Gas gangrene prevented by adequate cleansing of wounds, delay in closing of wounds. It produces an enterotoxin which causes food poisoning.

C. botulinum – causes botulism- a paralytic disease, a form of food poisoning.
Morphology & Cultural characteristics - It is saprophytic, noncapsulated, motile, producing subterminal, oval, bulging spores. It is a straight or slightly curved Gram positive bacillus with rounded ends (5 X 1µm). It is a strict anaerobe. Optimum temperature is 350C, but some strains can grow and produce toxin at   1-50C. Surface colonies are large, irregular, semitransparent, fimbriate margin. Spores are heat and radiation resistant. C. botulinum produces an exotoxin (neurotoxin), responsible for pathogenicity.
Resistance – Spores of  C. botulinum are highly resistant, survive boiling for several hours, and some can even survive at 1210C for 20 minutes. Insufficient heating in the process of preserving food is an important factor of botulism. They are also resistant to radiation, UV light, alcohols and phenolic quaternary ammonium compounds. They are relatively susceptible to hypochlorite, ethylene oxide and formaldehyde.
Antigenic TypesOn the basis of the type of toxin produced, the genus has been divided into 7 serologically distinct types, A to G.
Toxin – Botulinum toxin is a small protein with 19 aminoacids and a molecular weight of about 150,000 daltons. Minimum lethal dose for humans may be 1µg. It acts slowly taking several hours to kill. It is a neurotoxin which acts by inhibiting the release of acetylcholine from the motor nerve endings of the parasympathetic system. The toxin is relatively stable being inactivated at 800C for 30-40 minutes. It resists digestion in the intestine and is absorbed through intestinal mucosa in an active form. It is a good antigen and is specifically neutralized by the antitoxin.
Pathogenicity - C. botulinum is widely distributed in soil and decaying vegetation, thus meat and vegetables may become contaminated with this bacilli. It is noninvasive and the pathogenicity is entirely due to the toxin produced by it. Botulism, the disease occur in 3  forms - Food Borne, Infant, Wound..

1. Food Borne Botulism – by ingestion of neurotoxin from improperly home canned non-acid foods. It is an intoxication, the organisms do not infect tissues. The ability of C. botulinum to form toxin depends on infection with a bacteriophage. This phage carries the information for botulism toxin production. The toxin is produced inside the cytoplasm of Clostridium  and released only upon death and autolysis of the cell. It is activated by proteolytic enzymes(trypsin). The toxin is colourless, odourless, tasteless.. If endospores are not destroyed, they germinate in food during storage under anaerobic conditions and release large quantities of toxin. Food responsible for botulism is usually abnormal in appearance and odour, and bulging of tins and the presence of gas bubbles indicates its presence. Eventhough endospores are highly resistant, the toxin inactivated by a few minutes boiling. Boiling home canned foods vigorously before serving would eliminate most cases. Botulism is a neuroparalytic disease with sudden and rapid paralysis. Symptoms usually begin 18-36 hours after ingestion of food and may include nausea, vomiting, thirst, constipation, double vision, difficulty in swallowing, speaking and breathing. This may be followed by muscular weakness, blurred vision. It ends in death from respiratory arrest (25-70% fatality rate). The toxin acts at junctions between neurons and muscle cells and prevents the release of acetylcholine, the chemical that neurons release to cause muscles to contract. The toxin thus paralyses muscles in a relaxed state, from eye muscles, larynx, pharynx, respiratory muscles. This causes double vision, difficulty in speaking and swallowing and difficulty in breathing and causes gastrointestinal disturbances. The toxin is demonstrated in faeces, serum or food remains. Help in maintaining respiration is important. Antitoxin tried for the treatment.
2. Infant Botulism – is associated with feeding honey to infants. Endospores in the honey when      consumed, germinate and grow in the immature digestive tact of infants. As toxin is produced and  absorbed by the intestine, the   infant loses the ability to suck and  swallow, constipation, loss of head control, so the disease often known as “floppy baby syndrome” and develops respiratory arrest. Patient excretes toxin and spores in the faeces.
3. Wound Botulism -  is the least common form of botulism. It occurs in deep, crushing wounds. Tissue damage impairs circulation and creates anaerobic conditions, so endospores germinate, multiply and produce toxin, which enters the blood and is distributed throughout the body. It reaches junctions between neurons and muscle cells about a week after injury and cause progressive paralysis.

Laboratory Diagnosis – the specimens that can be collected are faeces, food, vomitus, gastric fluid, serum, environmental samples, wound exudates. The best method of diagnosis is the specific neutralization of toxin in the patient serum by toxin-antitoxin neutralization test in mice. Also detected by direct immunofluorescence. For the isolation, the specimen is inoculated on Egg Yolk Agar, Blood agar and three bottles of Cooked Meat Broth. Hold one of these 3 bottles in a water bath at 800C for 10 minutes and another for 20 minutes and the third is unheated. This selects heat-resistant spores and also allows heat-sensitive spores to grow in unheated CMB. The culture is incubated anaerobically at 300C for 3-5 days. Cultures in CMB are screened at intervals for toxin production. Absence of toxin production upto 5 days rules out botulism.

Prophylaxis & Treatment – the toxin in the stomach should be removed by lavage with 2-5% bicarbonate solution. Saline enemas given to remove toxin from the colon. The antitoxin given as soon as possible, the antitoxin cannot reverse the effect of toxin already affecting the nerves but will neutralize unfixed toxin.
Since botulism follows consumption of inadequately canned or preserved food, it can be prevented by proper canning and preservation. When an outbreak occurs, polyvalent antitoxin should be given intramuscularly .

C. tetani – is the causative organism of tetanus.
Morphology & Cultural Characteristics - It is widely distributed in soil and in the intestines of human and animals. It is Gram positive, slender, 2-5 X 0.4-0.5 µm, rounded end, occur singly or in chains, tends to be pleomorphic and filamentous. Spores are spherical, terminal and bulging, give the “drum-stick appearance”. It is non-capsulated and motile. It is an obligate anaerobe, optimum temperature 370C, pH 7.4. Growth improved by blood and serum. Swarming growth, but fine, translucent film of growth. Grows in Robertson’s cooked meat broth, colonies are irregularly round, 2-5mm in diameter with fine branching projections. On horse blood agar, the colonies are surrounded by a zone of ά hemolysis which subsequently develops into β hemolysis due to the production of an oxygen labile haemolysin known as tetanolysin. On egg yolk agar no opalescence, grows well in CMB.
Biochemical reactions – slightly proteolytic, slow gelatin liquefaction, forms indole, MR-VP negative, no H2S, nitrates not reduced.
Resistance-  Spores killed by boiling for 10-15 minutes, resistant to antiseptics, but sensitive to 1% iodine, H2O2. .  In the soil spores can survive for years.
Antigenic Structure – Flagellar (H), somatic (O) and spore antigens. On the basis of agglutination and complement fixation tests, the strains of the organism are of ten types.
  1. Tetanolysin – oxygen-labile hemolysin
  2. Tetanospasmin – heat-labile protein that may be inactivated by heating to 600C for 20 minutes. It is a single polypeptide chain with a molecular weight of 150,000 daltons. As it releases from the organism , bacterial protese cleaves to yield 2 chains that remain linked by a disulphide bond. The large chain binds the toxin to neuronal gangliosides, while smaller chain exert the biologic effect of the toxin. It is extremely powerful toxin second to botulinum toxin. It is a good antigen and specifically neutralized by the antitoxin.
Pathogenicity - The organisms are part of the normal bowel microflora of horses and cattles and 25% humans, therefore handling bedpans, diapers, other objects contaminated with faeces can transmit organisms to persons who have any breaks in their skin. If spores are deposited deep in tissues, where O2 is unavailable, through deep cuts and puncture wounds, infection occurs. Once inside the host, the noninvasive tetanus organisms stay at the wound site and release a powerful exotoxin, tetanospasmin. It is absorbed fro  the site of its production and ascends to the central nervous system. The forst symptoms appear in head and neck because of the shorter length of the cranial nerves. After 4- 10 days’ incubation, symptoms with generalised muscle stiffness followed by spasms that affect every muscle. An arched back, clenched fists and jaws are classic symptoms. Spasms can be violent enough to break bones. Eventually, respiratory muscles become paralysed, heart function disturbed, patient dies. The mortality rate is 80-90% with out proper treatment, and even with proper treatment it is 15-50%.
Laboratory Diagnosis – Pus or wound scrappings should be plated on one half of the blood agar plate and three CMB bottles. Blood agar plated incubated anaerobically at 370C, it produces swarming growth spreaded to the other half.. Hold one of the 3 CMB bottles in a water bath at 800C for 10 minutes and another for 20 minutes and the third is unheated. This selects heat-resistant spores and also allows heat-sensitive spores to grow in unheated CMB. The culture is incubated anaerobically at 370C.
Prophylaxis - Tetanus toxoid vaccine given prior to injuries protected against toxin. Antitoxins and antibiotics given to nonimmunised patients.
Active immunisation : All persons should be actively immunised against tetanus in infancy and their immunity maintained by booster doses. DPT and OPV given at the age of 6 weeks, 10 weeks, 14 weeks and 16-24 months followed by DT vaccine at the age of 5-6 years. Thereafter, booster doses of  TT are given at the age of 10 and 16 years. Immunity can be maintained by booster doses of toxoid every 10 years.
Passive immunisation : Tetanus antitoxin or antitetanus serum (ATS) can be given immediately after wounding. Administration of ATS may lead to hypersensitivity reactions like fatal anaphylaxis and serum sickness.

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