Clostridium

Clostridia

Large Gram positive Straight or slightly curved rods with slightly rounded ends Anaerobic bacilli Spore bearing Saprophytes Some are commensals of the animal & human gut which invade the blood and tissue when host die and initiate the decomposition of the corpse (dead body) Causes diseases such as gas gangrene, tetanus, botulism & pseudo-membranous colitis by producing toxins which attack the neurons pathways

Clostridia of medical importance

Clostridium Causing
Tetanus e.g. Cl. tetani
Gas gangrene
Botulism e.g. Cl. botulinum
Saccharolytic e.g. Cl. perfringens &Cl. septicum
Proteolytic e.g. Cl. sporogenes
ِAntibiotic associated diarrheae.g. Cl. difficille Mixed: Cl. histolyticum

Clostridium Causing TetanusCl. tetani

Gram positive, straight, slender rod with rounded ends All species form endospore (drumstick with a large round end) Fermentative Obligate anaerobe Motile by peritrichous flagella Grows well in cooked meat broth and produces a thin spreading film when grown on enriched blood agar Spores are highly resistant to adverse conditions Iodine (1%) in water is able to kill the spores within a few hours

Toxins

Cl. tetani produces two types of toxins: Tetanolysin, which causes lysis of RBCs Tetanospasmin is neurotoxin and essential pathogenic product Tetanospasmin is toxic to humans and various animals when injected parenterally, but it is not toxic by the oral route Tetanospasmin which causes increasing excitability of spinal cord neurons and muscle spasm


Laboratory Diagnosis of Tetanus
The diagnosis of tetanus depends primarily upon the clinical manifestation of tetanus including muscle spasm and rigidity. Specimen: Wound exudates using capillary tube Culture: On blood agar and incubated anaerobically Growth appears as a fine spreading film. Gram stain is a good method for identifying Clostridium Cl. tetani is Gram positive rod motile with a round terminal spore giving a drumstick appearance

Clostridium Causing Gas Gangrene

Clostridia causing gas gangrene
Saccharolytic organisms Cl. perfringens, Cl. septicum Ferment carbohydrates Acid and gas are produced
Proteolytic organisms Cl. sporogenes Digest proteins with blackening bad smell production
Mixed saccharolytic & proteolytic Cl. histolyticum

Saccharolytic Microorganisms

Cl. perfringens Causing
Gas gangrene
Food poisoning (Enterotoxin)

Clostridium perfringens

Large Gram-positive bacilli with stubby ends Capsulated Non motile (Cl. tetani is motile) Anaerobic Grown quickly on selective media Can be identified by Nagler reaction

Toxins

The toxins of Cl. perfringens  toxin (phospholipase C, lecithinase) is the most important toxinLyses of RBCs, platelets, leucocytes and endothelial cellsIncreased vascular permeability with massive hemolysis and bleeding tissue destructionHepatic toxicity and myocardial dysfunction-toxin is responsible for necrotic lesions in necrotizing enterocolitisEnterotoxin is heat labile toxin produced in colon → food poisoning


Laboratory Diagnosis
Specimen: Histological specimen or wound exudatesHistological specimen transferred aseptically into a sterile screw-capped bottle & used immediately for microscopical examination & culture Specimens of exudates should be taken from the deeper areas of the wound where the infection seems to be most pronounced Microscopical examination (Gram, Spore stain etc)Gram-positive bacilli, non motile, capsulated & sporulated The spore is oval, sub-terminal & non bulgingSpores are rarely observedCulture: Anaerobically at 37COn Robertson's cooked meat medium → blackening of meat will observed with the production of H2S and NH3On blood agar → β-hemolytic colonies

Biochemical Tests

Cl. perfringnes characterized by: It ferments many carbohydrates with acid & gas It acidified litmus milk with stormy clot production Nagler reaction is positive

Reaction on Litmus Milk

Litmus Milk
Skimmed Milk (Without Fat)
Litmus indicator
Acid Base and Redox indicator
Lactose Sugar
Casein Protein
Contains

Reaction on Litmus Milk

Lactose Acid Pink Color (Milk Sugar)
Fermentation
Litmus Indicator
1- Acidic Reaction
2- Basic Reaction
Casein Alkaline amines Blue Color (Milk Protein)
Digestion
Litmus Indicator

Reaction on Litmus Milk

Stormy Clot Formation
Fermentation
Casein Milk Protein
Coagulation
Gas
Clot
Stormy Clot
Milk Sugar
Lactose
Acid
+

Reaction on Litmus Milk

Nagler’s Reaction This test is done to detect the lecithinase activityThe M.O is inoculated on the medium containing human serum or egg yolk (contains lecithin)The plate is incubated anaerobically at 37 C for 24 hColonies of Cl. perfringens are surrounded by zones of turbidity due to lecithinase activity and the effect is specifically inhibited if Cl. perfringens antiserum containing  antitoxin is present on the medium

Nagler Reaction

Positive Nagler Reaction
Procedure of Nagler Reaction

Anaerobic Cultivation

Removal of oxygen & replacing it with inert gas Anaerobic Jar It is especially plastic jar with a tightly fitted lid Hydrogen is introduced from commercially available hydrogen generators envelop 10 ml of water is added to envelop immediately before placing it in the jar Hydrogen and carbon dioxide will release and react with oxygen in the presence of catalyst to form water droplet Anaerobic indicator (Methylene blue) is placed in the jar Methylene blue is blue in oxidized state (Aerobic condition) while turns colorless in reduced state (Anaerobic condition)

Anaerobic Cultivation

Culture Media containing reducing agent Thioglycollate broth It contains Sodium thioglycollate (Reducing agent) Rezazurin (redox indicator) Low percentage of Agar-Agar to increase viscosity of medium Cooked Meat Medium It contains Meat particles (prepared from heart muscles) which contain hematin & glutathione that act as reducing agent

Growth on Fluid Thioglycolate

Clostridium sporogenes Growing in Thioglycolate Medium
Reducing agents in the medium absorb oxygen and allow obligate anaerobes to grow

Anaerobic Jar

Candle Jar