Detection, Identification and Differentiation of Clostridium perfringens

Clostridia are an extremely polyphyletic class of Firmicutes that include Clostridium. Their lack of aerobic respiration distinguishes Clostridia from the class of Bacilli. Because oxygen is toxic to them, Clostridia are obligate anaerobes and most of their species are gram-positive. They can be found in decaying vegetation, marine sediment, in soil and the intestinal tract of humans and other vertebrates. Clostridia form endospores that can 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, such as sugars, produces large amounts of CO₂ and H₂ as well as volatile organic compounds like acetic and butyric acid, acetone, and butanol. Clostridia also have an extended range of extracellular enzymes that degrade large biological molecules in the environment into fermentable compounds.

Most Clostridia species are non-pathogenic but some of them produce the most potent biological toxins. The three particularly lethal members of this group are C. perfringens, which is responsible for cooked meat-associated food poisoning and wound and surgical infections that lead to gas gangrene, C. tetani, which is responsible for deadly tetanus infections, and C. botulinum, which causes botulism.

Section Overview

• Clostridium perfringens Overview
• Detection and Identification of Clostridium perfringens
• Enrichment, Isolation and Enumeration of Clostridia
• ISO 11133 Compliant Culture Media for Clostridia Species
• Related Products

Clostridium perfringens – The Sulfite-Reducing Anaerobes (Clostridia)

The spores of sulfite-reducing anaerobic bacteria (clostridia) are widespread in the environment. They are present in human and animal faecal matter, in wastewater and in soil. The spores survive in water for long periods as they are more resistant than vegetative forms to the action of chemical and physical factors. They can be an indicator of ground water and drinking water contamination. Sulphite-reducing Clostridia reduce sulphite to sulphide at 37 °C within 24 hours. Clostridium perfringens is the most important organisms of this species.

Clostridium perfringens is found in undercooked or improperly sterilized canned foods (germination of endospores) and in water (surface water). The natural contamination source is human and animal feces mainly transmitted into food by water. C. perfringens produces an extensive range of invasins and exotoxins. The enterotoxins cause the undesirable, mostly meat-associated, food poisoning and wound and surgical infections that lead to gas gangrene.

C. perfringens plays a subsidiary role in water examination.⁴ Clostridia are spore builders and are resistant to heating, chlorination, and other stress factors. In contrast to vegetative cells like coliforms (E. coli, enterococci), which are less resistant, C. perfringens has the advantage of surviving for longer duration.⁶ Therefore, while a fecal contamination is detected mostly by coliforms as an indicator, which could disappear after a processing step, C. perfringens remains present. The organism is not a hazard in water; rather, it is problematic when the water comes in contact with food.

Detection and Identification of Clostridium perfringens

Detection and identification of C. perfringens is an important step towards the control and eradication of this potent pathogen. Some characteristic enzymes of C. perfringens are hemolysins (ß-hemolysis), lecithinase, extracellular proteases, lipases (phospholipase-C), collagenase, hyaluronidase, saccharolytic enzymes and enzymes to reduce sulphite to sulphide. These enzymes are also used as detection and differentiation targets. It is also notable that C. perfringens is a non-motile bacterium, and it is the most important of the sulphite-reducing clostridia. Also, C. perfringens normally grows at 44 °C, whereas some other clostridia are inhibited at this temperature. This property is used in ISO methods to give the medium more selectivity.⁴

For detection of C. perfringens, mCP and TSC agar have been recommended.^4,5 However, there are problems associated with each of these media. The mCP method is very onerous for routine screening and bacterial colonies cannot be used for further biochemical testing. In addition, the recovery of C. perfringens was rejected by ISO in favor of methods based on TSC agar.⁶ TSC detects all sulphite-reducing clostridia, however, and not only C. perfringens, in some cases, excessive blackening of the agar frustrated counting of the lower dilutions. On the other hand, if the contamination was too high, TSC did not consistently produce black colonies and, therefore, the colonies’ white color provided false negative results.

To prevent this issue, we have introduced a new medium called CP ChromoSelect Agar, a chromogenic media from the new generation. Studies have shown that this medium produces counts that are not significantly different from those obtained from other media. The identification of typical and atypical colonies isolated from all media has demonstrated that CP ChromoSelect Agar (see Figure 1) was more specific to C. perfringens in water samples than mCP (see Figure 2) and TSCF agar.

Figure 1. Scanning electron micrograph of C. perfringens grown on a silicon wafer (source: S. Melville, Department of Biological Sciences, Virginia Tech University)

Figure 2. CP ChromoSelect Agar (C. perfringens appears as green colonies)

Enrichment, Isolation and Enumeration of Clostridia

To keep Clostridium perfringens under control, we have 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.

Tests for Identification and Differentiation of Clostridia

Test for Clostridia Diagnostics	Product No.	Description
Aminopeptidase Test	75554	For the detection of L-alanine-aminopeptidase which is found primarily in Gram-negative microorganisms.
Kovac’s Reagent for indoles (isoamylic alcohol as solvent)	67309	For the differentiation of clostridia. In the presence of oxygen, some bacteria are able to split tryptophan into indole and
Kovac’s Reagent for indoles (butanol as solvent)	60983	a-aminopropionic acid. Clostridium perfringens is, for example, an indole-negative bacterium.
Mannitol Disks	94438	Used to differentiate bacteria on the basis of mannitol fermentation.
Nitrate Reagent Disks	08086	Used to detect an organism’s ability to reduce nitrate.
Tributyrin-Strips	75744	The test principle is hydrolysis of tributyrin. This reaction causes color change of acidobasic indicator.

Gram Staining Kit and Single Solutions

Gram Stain	Product No.
Gram Staining Kit	77730
Gram’s Crystal Violet Solution	94448
Gram’s Decolorizer Solution	75482
Gram’s Fuchsin Solution	87794
Gram’s Iodine Solution	90107
Gram’s Safranin Solution	94635

Anaerobic Equipment

Anaerobic Equipment	Product No.
Anaerobe atmosphere generation bags	68061
Anaerobe indicator test	59886
Anaerobic jar	28029

ISO 11133 Compliant Culture media for Clostridia species

Our high quality and consistent culture media are available in a variety of formulations as dehydrated culture media or in the form of granules. The granulated culture media are not only convenient but also safe, meeting the highest industry performance standards as described in ISO 11133. Granules cause significantly less dust than powdered media, leading to less inhalation of hazardous media components that cause allergic responses. In addition, they minimize contamination of the work environment. All our media are quality controlled according to stringent standards. This guarantees the consistent high quality of our products.

References

1. Sherris Medical Microbiology, 4th ed., K.J. Ryan and C.G. Ray Editors, McGraw Hill, 2004. SMM4eKRaCREMH2.

2. Wells, C. L..; Wilkins, T. D., Clostridia: Sporeforming Anaerobic Bacilli, In Baron’s Medical Microbiology, 4th ed., S. Baron Editor, Univ. of Texas Medical Branch, 1996. WCLWTDCSABIBMM4eSBEUoTMB1.

3. Elmer Koneman, W. et al Color Atlas and Textbook of Diagnostic Microbiology, 5th ed., Lippincott Williams & Wilkins, 1997. EKWeaCAaToDM5eLW&W1.

4. ISO 6461 Part 2. Water Qualily – detection and enumeration of Clostridium perfringens. (Revision of ISO 6461-2: 1986). I6P2WQ–daeoCp(oI61.

5. Council Directive 98/83/EC, Relating to the Quality of Water Intended for Human Consumption, Official Journal of the European Communities, p.L330-32-L330/53 (1998). CD9RttQoWIfHCOJotECp(.

6. National Standard Method, Enumeration of Clostridium perfringens by membrane filtration, issue no 3.1, reference no W5i3.1 (2005). NSMEoCpbmfin3rnW(.