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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 120MediaBrandProduct. No. Description
AC AgarSigmaA3340Supports the growth of aerobic, anaerobic and microaerophilic microorganisms.
AEA Sporulation Broth (Base), modifiedSigma-Aldrich17170For early sporulation of C. perfringens from foods.
Alternative Thioglycollate MediumSigmaA0465Recommended for sterility testing with certain biological products which are turbid or viscous.
Brain Heart Infusion AgarSigma-Aldrich70138A solid medium for the cultivation of fastidious pathogenic bacteria such as Streptococci and Neisseria.
Brewer thioglycollate mediumSigmaB2551Used for testing the sterility of biological products.
Brucella Agar with Hemin and Vitamin KSigmaB2926Used for the isolation and subculture of anaerobes.
Casein peptone Lecithin Polysorbate BrothSigma-Aldrich22089For the enumeration of samples from pharmaceutical, cosmetic industry.
Columbia AgarSigma-Aldrich27688For the cultivation of fastidious microorganisms.
Cooked Meat BrothSigma-Aldrich60865For the primary cultivation of aerobic, microaerophilic and anaerobic bacteria from clinical specimens.
Reinforced Clostridial AgarSigmaR0898Used for the cultivation and enumeration of Clostridia.
Skim Milk Agar, modifiedSigma-Aldrich17175For cultivation and enumeration of microorganisms encountered in dairy industry.
Thioglycolate BrothSigma-Aldrich70157A medium for sterility tests and the cultivation of microaerophilic and anaerobic organisms.
Thioglycolate Broth with ResazurineSigma-Aldrich90404For 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 DextroseSigmaT3938Recommended for the cultivation of anaerobes from root canals and blood.
Differential MediaBrandProduct No.Description
Blood Agar (Base)Sigma-Aldrich70133A non-selective medium for the isolation and cultivation of many pathogenic and nonpathogenic microorganisms.
Gelatin Iron AgarSigmaG0289Used for detecting gelatin liquefaction and hydrogen sulphide production.
Meat Liver AgarSigma-Aldrich46379For the cultivation of anaerobic microorganisms.
Nutrient GelatinSigma-Aldrich70151Nutrient gelatin is recommended for the determination of gelatin-liquefying microorganisms and enumeration of proteolytic organisms in water.
Selective MediaBrandProduct No.Description
Clostridium difficile Agar (Base)Sigma-Aldrich17145Used with supplement for cultivation of C. difficile from food and certain pathological specimens.
Wilkins Chalgren Anaerobic AgarSigmaW1761Used for the isolation of anaerobic bacteria.
Selective Differential MediaBrandProduct No.Description
HiCrome™ M-CP Agar BaseSigma-Aldrich75605Selective 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, modifiedSigma-Aldrich17231For the selective isolation and enumeration of C. perfringens from foods.
Table 1.Media for Clostridia
Test for Clostridia DiagnosticsBrandProduct No. Description
Aminopeptidase TestSigma-Aldrich75554For the detection of L-alanine-aminopeptidase which is found primarily in gram-negative microorganisms.
Mannitol DisksSigma-Aldrich94438Used to differentiate bacteria on the basis of mannitol fermentation.
Nitrate Reagent DisksSigma-Aldrich08086Used to detect an organism’s ability to reduce nitrate.
Tributyrin-StripsSigma-Aldrich75744The test principle is hydrolysis of tributyrin. This reaction causes color change of acidobasic indicator.
Table 2.Tests for identification and differentiation of Clostridia
Gram Stain
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Table 3. Gram staining kit and single solutions

Gram staining kit and single solutions
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Table 4. Anaerobic equipment

Materials
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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..
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