Clostridia Diagnostic

Detection, Identification and Differentiation of Clostridia Species

Clostridia are relatively large, gram-positive, rod-shaped bacteria that can undergo only anaerobic metabolism. Most Clostridia cannot grow under aerobic conditions and even can be killed by exposure to O₂, but they form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. The natural sources of Clostridia are anaerobic habitats with organic nutrients, particularly soils, aquatic sediments and the intestinal tracts of animals. Their fermentation of organic compounds, like sugars, produces large amounts of CO₂ and H₂ as well as volatile organic compounds like acetic and butyric acid, acetone and butanol. Metabolism of substrates like amino acids and fatty acids results in foul-smelling degradation products. Clostridia also have an extended range of extracellular enzymes that degrade large biological molecules in the environment into fermentable compounds. Although there are non-pathogenic Clostridia, this genus produces some of the most potent biological toxins. Three particularly bad actors in this group are C. perfringens, which is responsible for cooked meat-associated food poisoning and wound and surgical infections that lead to gas gangrene, and C. tetani, which is responsible for deadly tetanus infections, and C. botulinum, which causes botulism.

Below are the most well-known pathogenic Clostridia species with their typical properties and occurrence:

Clostridium Perfringens

• produces a huge range of invasins and exotoxins
• enzymes: hemolysins (ß-hemolysis), lecithinase, extracellular proteases, lipases (phospholipase-C), collagenase, hyaluronidase, saccharolytic enzymes and is able to reduce sulphite to sulphide
• enterotoxins causes food poisoning
• found in improperly sterilized canned foods (germination of endospores) and water
• nonmotile

Clostridium Difficile

• produces two enterotoxins toxin A and toxin B (lethal cytopathic toxin)
• enzymes: hydrolytic enzymes, p-hydroxyphenylacetate decarboxylase, ferments mannitol
• formation of p-cresol as the main fermentation product of tyrosin

Clostridium tetani

• toxin: tetanospasmin (causative tetanus)
• obligate anaerobe (sensitive to oxygen)
• sensitive to heat
• flagella give limited motility
• terminal spore (resistant to heat and most antiseptics)
• typical gram-positive, may stain gram-negative or gram-variable, especially in older cells

Clostridium botulinum

• seven subtypes (A-G) produces different botulinum toxin (types C and D are not pathogenic)
• grow best in low-oxygen conditions
• subterminal endospores (resistant to boiling without pressure)
• occurrence: soil, aquatic sediments, decaying vegetation, found in improperly sterilized canned foods (germination of endospores)
• acidity, high concentration of sugar, very low levels of moisture or high levels of oxygen inhibits the growth
• enzyme: lipase production on egg yolk agars

Identification of Clostridia is an important first step toward the control and eradication of this potent pathogen. To aid in the diagnosis, Sigma-Aldrich has developed a broad range of selective media (Table 1), tests (Tables 2 and 3) and anaerobic equipment (Table 4) for the detection, identification and differentiation of Clostridia.

Table 3. Gram staining kit and single solutions

Table 4. Anaerobic equipment