ADVERTISEMENTS:
In this article we will discuss about Clostridium welchii (CI. perfringens) which Causes Gas Gangrene and Food Poisoning in Man:- 1. Morphology and Staining of Clostridium Welchii 2. Cultural Characteristics of Clostridium Welchii 3. Resistance 4. Antigenic Structure 5. Laboratory Diagnosis.
Contents:
- Morphology and Staining of Clostridium Welchii
- Cultural Characteristics of Clostridium Welchii
- Resistance of Clostridium Welchii
- Antigenic Structure of Clostridium Welchii
- Laboratory Diagnosis of Clostridium Welchii
1. Morphology and Staining of Clostridium Welchii:
Relatively large Gram-positive bacilli, 4-6 x 1 u, with square or rounded ends, capsulated in the tissue and are non-motile. Their spores are oval and sub terminal. The spores of CI. welchii are not readily produced in artificial culture and in animal tissues and their absence is a characteristic of CI. welchii.
ADVERTISEMENTS:
Spore formation can be induced only on special medium (Ellner medium). It appears that more toxin producing strains of CI. welchii produce few spores, and vice versa.
2. Cultural Characteristics of Clostridium Welchii:
CI. welchii are obligate anaerobes, optimum temperature 37°C, grow best on glucose agar or glucose blood agar medium. On nutrient agar medium, the colonies are large, round, smooth, regular and slightly opaque disks, On blood agar medium, CI. welchii typically produces multiple zone of haemolysis around the colonies.
In selective medium containing polymyxin B, neomycin and iron citrate (Marshal’s medium), CI. welchii produce black colonies. Biochemically, CI. welchii is actively saccharolytic, ferments many sugars with gas and acid production.
ADVERTISEMENTS:
In litmus milk medium, lactose of milk is fermented, gas and acid are produced, and casein is formed as clot which is broken up by gas producing “stormy clot” appearance. In RMC medium, the meat is reddened and no digestion occurs.
3. Resistance of Clostridium Welchii:
Spores of CI. welchii are destroyed by autoclaving at 121°C for 15 minutes; the effects of disinfectants as spores are similar to that of CI. tetani.
4. Antigenic Structure of Clostridium Welchii:
The different strains of CI. welchii produce 12 distinct toxins (Table 44.1). These toxins are four (4) major lethal toxins and 8 minor lethal or non-lethal toxins. On the basis of production of the 4 major toxins (alpha, beta, epsilon and iota),
CI. welchii are differentiated into 5 types—A, B, C, D and E:
1. Type A strains produce alpha toxin.
2. Type B strains produce alpha beta and epsilon toxins.
3. Type C strains produce alpha and epsilon toxins.
4. Type D strains produce alpha and epsilon toxins.
5. Type E strains produce alpha and iota toxins.
ADVERTISEMENTS:
Thus all strains produce alpha toxin and type A in particular produces only alpha toxin. CI. welchii type A causes gas gangrene in man while other types (B, C, D, E) are mostly related to diseases in animals. Some strains of type A of CI. welchii produce enterotoxin causing food poisoning.
A. Alpha (α) toxin and Gas Gangrene:
It is produced by all strains of CI. welchii but more abundantly by CI. welchii type A of gas gangrene. Chemically, it is phospholipidase (Lecithinase C) and most fatal of all clostridial toxins and is responsible for profound toxaemia in gas gangrene patients. It is relatively heat stable, necrotising, cytotoxic or membranolytic.
The a toxin splits lecithin which is an important constituent of the cell membrane. The specific effect of toxin is utilised in the rapid detection of CI. welchii in clinical specimens and is confirmed in Nagler reaction. This toxin also causes haemolysis of erythrocytes of animals (except horse and goat).
ADVERTISEMENTS:
Toxin, CI. welchii produces 12 different exotoxins. Of these, alpha, beta, epsilon and iota are the major lethal toxins which are lethal, necrotising, haemolytic.
The alpha toxin of CI. welchii type A is a lecithinase enzyme which is lethal as it splits lecithin (an important constituent of cell membrane) to phosphorylcholine and diglyceride, and it can be demonstrated by production of opalescent colonies on egg yolk agar which is known as “Nagler reaction.”
In this reaction, the cholesterol of egg yolk is split by lecithinase and precipitated on the colonies. DNase, hyaluronidase, and collagenase that digest collagen of subcutaneous tissue and muscle are also produced. The theta toxin has similar effects but it is not lecithinase.
Nagler Reaction:
ADVERTISEMENTS:
In presence of Ca++ and Mg++ ions, alpha toxin (Lecithinase C) splits lipoprotein of complexes in serum or egg yolk of the agar and produces opalescent colony of neutralisation of alpha toxin by specific antitoxin can be demonstrated by incorporating the antitoxin in egg yolk agar medium. If neutralized there will be no lipid deposit around the colony i.e. no opalescent colony.
Certain lecithinase producing bacteria (CI. oedematiens), some vibrio’s and spore bearers may produce opalescence in egg yolk medium but it is not neutralized by specific anti-toxin of CI. welchii except CI. bi-fermentans which produces a lecithinase serologically related to that of CI. welchii.
Other Soluble Substances:
1. Haemagglutinins variety of RBCs (man, sheep, cattle, pigs) are produced by CI. welchii type A and D.
ADVERTISEMENTS:
2. Neuraminidase produced by CI. welchii destroys Myxovirus receptors on red blood cells.
3. Histamine, haemolysin, fibrinolysin are also produced.
Some strains of CI. welchii type A produces a powerful enterotoxin that induces “Food poisoning” with its characteristic intense diarrhoea, abdominal cramps, fever, vomiting in 6-18 hours after ingestion of toxin. This illness is self-limited.
Pathogenicity and Clinical Features:
Spores of CI. welchii which contaminated the wounds with free Ca and Mg ions germinate, multiply, ferment carbohydrates of the tissue and produce gas. The spread of infection is favoured by the distension of tissue and interference with blood supply together with the toxin causing tissue necrosis suitable for bacterial growth— haemolytic anaemia, severe toxaemia and death may ensue finally.
ADVERTISEMENTS:
When a wound gets contaminated by soil with faecal matter containing spores of CI. welchii, there may be 3 types of anaerobic wound infection:
1. Simple Contamination of Wound:
There are some Clostridia of low virulence which cannot multiply due to lack of proper anaerobic environment. They do not invade the tissue but their presence delays healing of wound.
2. Anaerobic Cellulitis:
This type of lesion is caused by Clostridia of low virulence and poor toxigenicity. The spores germinate in necrotic tissue and invade facial plane with minimal toxin production without invasion of muscle tissue. There is gas production but little toxaemia and prognosis is good. It tends to be a self-limiting lesion which is common in war causalities.
3. Anaerobic Myelitis (Gas Gangrene):
ADVERTISEMENTS:
CI. welchii type A is the predominant causal agent of anaerobic myelitis (gas gangrene). The condition is always associated with the formation of excessive amount of exotoxin (alpha toxin) and is very serious. Clinically, it is a spreading gangrene of the muscles with profound toxaemia and shock.
There is increasing pain, tenderness; oedema with blackening of tissues and foul smelling of serous exudate. Crepitus due to accumulation of gas bubbles is mostly detected under the skin. Its incubation period is from 6 hours to 6 weeks.
The spores of CI. welchii germinate and thrive in dead tissue; oxygen tension is low. The infection may complicate wound with extensive destruction of tissue (accidental injury), pelvic infection of women.
The gas bubbles may cause distension of sarcolemmal sheath of muscle cells and ischemia. Destruction of cell membrane of muscle fibre and alteration of vascular permeability caused by the alpha toxin of CI. welchii may result into necrosis of muscle tissue which favours the bacterial growth.
Other toxins and enzymes (hyaluronidase, collagenase, leucocidin, fibrinolysin) and haemolytic toxins may help the spread of infection. There is rapid development of anaemia and, in terminal stage, the bacterial dissemination may lead to bacteriaemia, shock and death.
ADVERTISEMENTS:
B. Food Poisoning:
It is caused by some streams of CI. welchii type A. When meat is cooked in bulk, heat resistant spores survive due to slow penetration of heat (100°C for 30 minutes). These spores germinate into vegetative forms during cooling after cooking in anaerobic condition.
After consumption of large dose of organism along with contaminated cooked meat or poultry, a heat labile enterotoxin is liberated in the small intestine. This toxin acts on the intestinal mucosa like enterotoxin of E. coli causing dehydration and symptoms of food poisoning (abdominal pain, diarrhoea and nausea for 1-2 days). The incubation period is 8-12 hours. It is self-limited disease.
C. Other diseases:
Gangrenous appendicitis, urogenital infection, puerperal infection, brain abscess, meningitis and panophthalmitis are also due to incrimination of CI. welchii.
D. Necrotising enteritis:
It is a severe and often fatal necrotising jejunitis caused by type C of CI. welchii reported in New Guinea, Germany, Thailand, Nepal and East Africa. Protection against this disease is offered by active immunization with toxoid of CI. welchii type C.
5. Laboratory Diagnosis of Clostridium Welchii:
Specimens:
Wound, pus, tissue.
Smears:
The presence of large Gram-positive, spore forming bacillus suggests gas gangrene, they may be capsulated in the tissue, but the spores are present in culture.
Culture:
Material is inoculated into RCM and on blood agar medium and incubated anerobically. The growth is inoculated into litmus milk medium and if stormy clot appears it is identified as CI. welchii and its lecithinase activity is shown by Nagler reaction. Final identification rests on toxin production and neutralisation of toxin by specific antitoxin in mice and guinea pigs.
Treatment:
It is by surgical removal of all devitalized tissue with administration of penicillin. Hyperbaric oxygen may help to detoxify patients rapidly. Polyvalent antitoxin may be used. Toxoids are not in practical use. Food poisoning due to CI. welchii requires only symptomatic care.
