Clostridium perfringens

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Clostridium perfringens : 

V. Onur YILMAZ 130104002 Clostridium perfringens

Outline : 

Outline Definition Ecology Classification Incubation conditions Pathogenicity Epidemiology Detection methods

Definition : 

Definition Anaerobic, gram positive, spor forming, rod shaped bacteria. Under microscope, usually seen as singlets or doublets, rarely form short chains. Spores are found at subterminal or terminal position Non-motile, capsule forming. Able to fermentate many carbohydrate. Able to reduce Sulfide. Causes stormy fermentation. Forms black colonies on TSC agar

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Cl. Perfringens on TSC agar Cl. Perfringens on under light microscope

Definition : 

Definition Perfringengs food poisoning is caused by this bacteria. This disease drives slow and sometimes it doesn’t require medical attention. Poisoning cases are seen in places serving meals to plenty of people. For example, schools, hospitals, prisons. Undercooked meals, poorly stored meals are causes for poisoning. Old people are tend to catch Cl. Perfringens food poisoning.

Ecology : 

Ecology Found in guts of human, domestic pets, wild animals. Their spores exist in soil. Existance of their spores in soil indicates faecal contamination. Widespread in nature, on dust, soil, air, water, faecal.

Classification : 

Classification There are four main exotoxins. They are alpha, beta, epsilon and iota. According to 4 main exotoxins, Cl. Perfringens strains are divided to 5 sub group and they are named as A,B,C,D,E. A- alpha B- alpha, beta, epsilon C- alpha, beta D- alpha, epsilon E- alpha, iota Main type of strain causing pathogenicity is A. In some cases, strain C also founded to be guilty. Interesting point is, not all A types cause disease, but sometimes, they lost their toxin production abilities when they are cultured on medium.

Incubation conditions : 

Incubation conditions Cl. Perfringens requires 13-14 amino acids and 5-6 vitamins to grow. This requirement also indicates their prescence on foods having high proteins. Although Cl. Perfringens is anaerobic, it does not require absolute anaerobic conditions. Redox potential of Meat products are suitable for this bacteria to grow. Their optimum growth pH is between 5.5 and 8.0. optimum pH for enterotoxin production is pH 6.5-pH7.3

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It can grow between 15-50 Celcius degree. Optimum temperature is between 37-47. optimum temperature for toxin production is 37 C. Its metabolic rate decrease under 0,995 water activiy Under optimum conditions, regeneration time from spore to germination is 12 minutes. So 106 bacteria is enough for being pathogen when they enter human body to reach guts. Sharp decrease in temperature is lethal. When incubation temperature drops to 4 C from 37, it causes 96% of death ration. During 90 minutes incubation at 4 C, any drop in temperature causes 3 logs of lethality.

Pathogenicity : 

Pathogenicity Causes abdominal pain and diarrhea. No fever and no vomiting. Incubation time for disease is between 8-22 hours after pathogen uptake and its affects go off in 24 hours. There is a direct correlation between sporulation and toxin production. Inhibition of sporulation also inhibits enterotoxin production.

Pathogenicity : 

Pathogenicity Toxin protein is consist of 18 amino different amino acids. Most important ones are Serine, leucine and glutamic acid. Toxin is around 33kDa-40kDa. Isoelectric point is pH 4.3 This enteretoxin is vulnerable to temperature and at 60 C, it loses its activity in 4 minutes.

Pathogenicity : 

Pathogenicity Apart from food poisoning, Cl. Perfringens C types cause “Pig-bel” or “Nectoric perfringens”. It is lethal. After huge amount of bacteria intake, they attach to intestinal wall and start disease. If it is not treated, it causes necrosis of intestinal tissue and finally death. It first observed in Germany at 1949.

Epidemiology : 

Epidemiology Usually meals with high protein amounts cause disease. Beaf, chicken meat, chicken soup, hot-dogs, salads are main sources for disease. Especially when meals are cooked and kept at fridge for 2 or more days. In Turkey, “Sucuk” is a risky for Cl. Perfringens contamination.

Epidemiology : 

Epidemiology Cl. Perfringens poisoning was placed at 3rd in USA and was placed at 2nd in England at 1980 in category of food poisoning diseases. In 1981, in USA, 1162 cases are reported in 28 outbreaks. Annually, 10-12 outbreak and for each outbreak hundreds of cases are reported in USA.

Epidemiology : 

Epidemiology Since Cl. Perfringens is widespread in nature, protecting foods from contamination is not enough. However sanitazing should not be forgetten. Having a few numbers of bacteria is not dangerous. Inhibition of spore germination may control disease. The most effective way to protects foods contamination is consuming food just after cooking. Another way is chopping down meal to small pieces and keeping at fridges. Minimum growth temperature is 15 C. At 0 C, vegetative cells are not stable, even spores may get damaged.

Epidemiology : 

Epidemiology Vegetative cells of Cl. Perfringens may be eliminated via cooking however heat insensitive spores may get alive. Meals have been waiting in frides should be consumed after heating. Poultry meats should be heated up to 80 C.

Detection : 

Detection Sulfide reducing Clostridium are counted with cfu. With this purpose, egg yolk added Tryptose Sulfide Cycloserine Agar is used. After medium is poured to petri dishes. Non egg yolk added TSC is poured as second layer. At 37 C, 24 hour of anaerobic incubation results in black colored colonies. In a suspected meal, most important point in investigating Cl. Perfringens that Cl. Perfringens is not alive while frozen. So before analyzing, freezing meals may cause non-informative results.

Detection : 

Detection The specimen should be analyzed in 8 hours and should be kept at 10 C. Homogenisation is an important step in investigation. But it should be kept in mind that oxygen contact during homogenization damages perfringens. Black colored colonies are isolated from petri dish and they are confirmed in Lactose sulfided medium via analyzing gas and black precipitates.

Detection : 

Detection Another method for investigation is addition of MUP (4-Methylumbelliferyl phosphate). MUP is a substrate for alkaline and acid phosphotase. Acid phosphotase is highly specific to Cl. Perfringens and it degrades MUP to 4-methylumbelliferone which illuminates at 366 nm ultraviolet light.

References : 

References Juckett et. Al. W V Med J. 2008 Jul-Aug;104(4):26-7 Brynestad et al. Int J Food Microbiol. 2002 Apr 5;74(3):195-202.

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