Clostridium perfringens, their Properties and their Detection

Clostridia are relatively large, Gram-positive, rod-shaped bacteria that can undergo only anaerobic metabolism. Most Clostridia cannot grow under aerobic conditions and can even be killed by exposure to O₂; however, 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, such as 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. The three particularly lethal members of this group areC. 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.

Clostridium perfringens is found in undercooked or improperly sterillized 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 examination⁴. 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 longer⁶. 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.

In consideration of the aforementioned facts, it is obvious that 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 selectivity⁴.

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 agar6. TSC detects all sulphite-reducing clostridia, however, and not only C. perfringens; in some cases, exces-sive 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, as a consequence, the colonies’ white color provided false negative results.

To prevent this problem, Sigma-Aldrich has 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)

To keep Clostridium perfringens under control, 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 1. Common and new media for Colostridium perfringens (a complete list of clostridia media can be found on sigma-aldrich.com/clostridia)

Cat. No.	Description
17170	AEA Sporulation Broth (Base), modified
15997	Brewer Agar
27544	Clostridial Differential Broth
27546	Clostridial Nutrient Medium
12398	CP ChromoSelect Agar
G0289	Gelatin Iron Agar
61348	Lactose Gelatin Medium (Base)
75605	m-CP Agar Base
14305	Motility Nitrate Medium
70151	Nutrient Gelatin
39727	Perfringens Agar Base
91365	Reinforced Clostridial Agar
85627	SPS Agar
17231	SPS Agar, modified
86128	Sulphite Iron Agar
T3938	Tryptone Soya Broth without Dextrose
93745	TSC Agar
93735	TSN Agar

Table 2. Tests for identification and differentiation of Clostridia

Test for Clostridia Diagnostics>	Cat. 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.

Table 3. Gram staining kit and single solutions

Gram Stain	Cat. 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

Table 4. Anaerobic equipment

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

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(.