Clostridia Diagnostic

Jvo Siegrist

AnalytiX Volume 7 Article 2

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.

Non-selective 120Media	Brand	Product. No.	Description
AC Agar	Sigma	A3340	Supports the growth of aerobic, anaerobic and microaerophilic microorganisms.
AEA Sporulation Broth (Base), modified	Sigma-Aldrich	17170	For early sporulation of C. perfringens from foods.
Alternative Thioglycollate Medium	Sigma	A0465	Recommended for sterility testing with certain biological products which are turbid or viscous.
Brain Heart Infusion Agar	Sigma-Aldrich	70138	A solid medium for the cultivation of fastidious pathogenic bacteria such as Streptococci and Neisseria.
Brewer thioglycollate medium	Sigma	B2551	Used for testing the sterility of biological products.
Brucella Agar with Hemin and Vitamin K	Sigma	B2926	Used for the isolation and subculture of anaerobes.
Casein peptone Lecithin Polysorbate Broth	Sigma-Aldrich	22089	For the enumeration of samples from pharmaceutical, cosmetic industry.
Columbia Agar	Sigma-Aldrich	27688	For the cultivation of fastidious microorganisms.
Cooked Meat Broth	Sigma-Aldrich	60865	For the primary cultivation of aerobic, microaerophilic and anaerobic bacteria from clinical specimens.
Reinforced Clostridial Agar	Sigma	R0898	Used for the cultivation and enumeration of Clostridia.
Skim Milk Agar, modified	Sigma-Aldrich	17175	For cultivation and enumeration of microorganisms encountered in dairy industry.
Thioglycolate Broth	Sigma-Aldrich	70157	A medium for sterility tests and the cultivation of microaerophilic and anaerobic organisms.
Thioglycolate Broth with Resazurine	Sigma-Aldrich	90404	For cultivation of aerobic and anaerobic organisms, and for sterility testing. Any increase in the oxygen content is indicated by a color change of redox indicator resazurin.
Tryptone Soya Broth without Dextrose	Sigma	T3938	Recommended for the cultivation of anaerobes from root canals and blood.

Differential Media	Brand	Product No.	Description
Blood Agar (Base)	Sigma-Aldrich	70133	A non-selective medium for the isolation and cultivation of many pathogenic and nonpathogenic microorganisms.
Gelatin Iron Agar	Sigma	G0289	Used for detecting gelatin liquefaction and hydrogen sulphide production.
Meat Liver Agar	Sigma-Aldrich	46379	For the cultivation of anaerobic microorganisms.
Nutrient Gelatin	Sigma-Aldrich	70151	Nutrient gelatin is recommended for the determination of gelatin-liquefying microorganisms and enumeration of proteolytic organisms in water.

Selective Media	Brand	Product No.	Description
Clostridium difficile Agar (Base)	Sigma-Aldrich	17145	Used with supplement for cultivation of C. difficile from food and certain pathological specimens.
Wilkins Chalgren Anaerobic Agar	Sigma	W1761	Used for the isolation of anaerobic bacteria.

Table 1. Media for Clostridia

Selective Differential Media	Brand	Product No.	Description
HiCrome™ M-CP Agar Base	Sigma-Aldrich	75605	Selective chromogenic media recommended by the Directive of the Council of the European Union 98/83/EC for isolation and enumeration of C. perfringens from water (see Figure 1).
SPS Agar, modified	Sigma-Aldrich	17231	For the selective isolation and enumeration of C. perfringens from foods.

Table 2. Tests for identification and differentiation of Clostridia

Test for Clostridia Diagnostics	Brand	Product No.	Description
Aminopeptidase Test	Sigma-Aldrich	75554	For the detection of L-alanine-aminopeptidase which is found primarily in gram-negative microorganisms.
Mannitol Disks	Sigma-Aldrich	94438	Used to differentiate bacteria on the basis of mannitol fermentation.
Nitrate Reagent Disks	Sigma-Aldrich	08086	Used to detect an organism’s ability to reduce nitrate.
Tributyrin-Strips	Sigma-Aldrich	75744	The test principle is hydrolysis of tributyrin. This reaction causes color change of acidobasic indicator.

Table 3. Gram staining kit and single solutions

Table 4. Anaerobic equipment

References

1. Selmer T, Andrei PI. 2001. p-Hydroxyphenylacetate decarboxylase fromClostridium difficile. 268(5):1363-1372. https://doi.org/10.1046/j.1432-1327.2001.02001.x

2. Wells, C. L..; Wilkins, T. D. Botulism and Clostridium botulinum. 1996. In Baron‘s Medical Microbiology, 4th ed., S. Baron Editor, Univ. of Texas Medical Branch..

3. K.J. Ryan, ed., C.G. Ray. Sherris Medical Microbiology, 4thand Editors, McGraw Hill, (2004)..

4. Wells, C. L..; Wilkins, T. D., Clostridia. 1996. Sporeforming Anaerobic Bacilli, In Baron‘s Medical Microbiology, 4th ed., S. Baron Editor, Univ. of Texas Medical Branch..

5. Elmer Koneman, W. et al . 1997. Color Atlas and Textbook of Diagnostic Microbiology, 5th ed., Lippincott Williams & Wilkins..