Clostridium perfringens Clostridium perfringens

Dean O. Cliver (materials from M. N. Hajmeer)

Outline Background • Historical background • 1890s, F.W. Andrewes and E. Klein • C. perfringens characteristics were involved in linking Clostridium • Foodborne disease estimates welchii (now C. perfringens) with food • Classification poisoning. • Pathogen prevalences • They associated eating foods • Clinical features & mechanism of disease contaminated with C. perfringens with • C. perfringens enterotoxins and spores several foodborne outbreaks. • Immunity, reservoirs, shedding, • Outbreaks were characterized with mild growth/survival, and detection to severe diarrhea and abdominal pain.

Background Background • 1892: The microorganism was found in a variety of diseases. • 1939 to 1946: Several outbreaks ¾gas gangrene observed: ¾appendicitis ¾In the U.K. during WWII ¾puerperal fever (infection of the ¾Shortage of meat led to the practice placental site following delivery or of cooking meat for later abortion) consumption. ¾enteritis

1 Background Background • 1939 to 1946: Several outbreaks observed: • 1939 to 1946: Several outbreaks ¾First warning of food poisoning observed: came from Knox and Macdonald ¾In the US, the first proven outbreak (1943). of C. perfringens was described by ¾Vehicle: gravy made the previous McClung (1945). day was heavily contaminated with anaerobic sporing bacilli including ¾Examined 4 foodborne outbreaks C. perfringens. associated with eating chicken ¾Children became ill after eating steamed 24 hrs before consumption. contaminated meal.

Background Background • 1953: Hobbs and associates • 1948: Severe necrotic gastroenteritis showed that eating food occurred in Germany. contaminated with C. ¾The vehicle for the illness was home perfringens could lead to canned rabbit. diarrheal food poisoning. ¾The associated strain was C. perfringens • Pioneered work establishing type C. C. perfringens as a cause of food poisoning.

Background Background • 1954: Experiments were conducted in US • By the 1960s and 70s, enough with human volunteers (Dack). information had accumulated to indicate ¾The strain fed was English strain of C. that C. perfringens foodborne poisoning perfringens. is caused by the release of the toxin during sporulation of the microorganism ¾Volunteers failed to develop any in the intestine of infected individuals symptoms of disease. who ate food heavily contaminated with • Public health significance was not fully C. perfringens. accepted in US until the 1960s.

2 Best estimates of annual cases and Characteristics of C. perfringens deaths for foodborne diseases, USA • Gram positive, nonmotile, encapsulated Cases Per- Deaths Per- rods with square ends. cent cent • Anaerobe but more oxygen-tolerant than C. C. perfringens 248,520 1.8 7 0.4 botulinum. Total bacterial 4,175,565 30.2 1,297 71.7 • Produces acetone, butanol, ethanol, butyric Total foodborne 13,814,924 100.0 1,809 100.0 acid, acetic acid, propionic acid, lactic acid, carbon dioxide and hydrogen. CDC, 1998–2002: 130 outbreaks, • Ferments sugars, starch and pectin. 6,724 cases, 4 deaths.

Clostridium perfringens Spores and Sporulation • Because it is a spore-former, the pathogen can survive in the environment. • Spores are seldom formed in food. • Sporulation requires a well-buffered medium rich in nutrients. • Spores are formed in the intestinal tract (spores shed in feces).

Classification of C. perfringens is Based on Toxins Produced Classification of C. perfringens is Based on Toxins Produced Toxin • The alpha toxin is a phospholipase, Type alpha beta epsilon iota the others are hemolysins or cause A + 0 0 0 necrosis. B + + + 0 • The different types cause a variety C + + 0 0 of diseases in animals, some of them D + 0 + 0 very severe. E + 0 0 +

3 Classification of C. perfringens is C. perfringens Prevalence in Foods Based on Toxins Produced Pork 0–39% • Type A is hemolytic (" and $) and non- hemolytic. Cooked Pork 45% • Killing 90% of spores takes 6 – 17 minutes Beef 22% at 100°C for non-hemolytic strains and less than one minute for beta-hemolytic strains. Chicken 0–54% Seafood 2%

Clinical Features of C. perfringens Clinical Features of C. perfringens • Severe diarrhea; no pyrexia • Large numbers of the pathogen can be (fever), shivering, headache, found in feces and food. abdominal pain, dehydration • Significant amount of enterotoxin also • Incubation time to illness is found in feces. 8-24 h. • Infective dose is high. About 108 • Duration of illness is vegetative cells need to be ingested to 12-24 h. cause symptoms.

Mechanism of Disease Mechanism of Disease 1. C. perfringens in food. • C. perfringens produces toxin- 2. Ingested cells begin to mediated infection known as C. sporulate after passing perfringens enteritis. stomach. • C. perfringens produces food 3. Enterotoxin produced poisoning without colonization. in small intestine (during sporulation) 4. Symptoms

4 Mechanism of Disease Mechanism of Disease • Heat shock, 70 – 100 °C, boosts • Sporulation is poor in most foods; germination and yields of enterotoxin. therefore contain little or no enterotoxin. • If food such as meat containing spores • Sporulation is rapid in small intestines is heated and left for some time at where pH is right and well buffered. It growth temperature the “primed” begins in the intestines in 3 h, and spores will produce rapidly growing enterotoxin found in 10-12 h. vegetative cells that in turn will produce plenty of spores and enterotoxin in the small intestines.

Mechanism of Disease Mechanism of Disease • C. perfringens food poisoning • Avoid problems by boiling food outbreaks tend to be on a large immediately before eating to: scale ¾Kill the vegetative cells in the food, ¾ large volumes of food produced and ¾Prevent sporulation in the intestines • There are few if any fatalities.

C. perfringens Enterotoxins C. perfringens Enterotoxins • Simple polypeptide, molecular weight of • Have similarities with V. cholerae 36000 ± 4000, contains 309 amino acid enterotoxin residues but only one cysteine residue ¾Cause a transient increase in capillary permeability with fluid accumulation • They are inactivated by pronase and B. and diarrhea subtilis protease but not by trypsin, ¾Increased secretion of water, sodium chymotrypsin, papain or bromelin. and chloride • Enterotoxin is heat labile with 90% ¾Decreased absorption of glucose destruction in 4 minutes at 60°C. ¾Enterotoxin causes desquamation of villous epithelium.

5 C. perfringens Enterotoxins C. perfringens Spores

• About 0.2 – 36 :g toxin/g feces is • C. perfringens spores can be isolated found in patients suffering from C. from healthy persons. perfringens food poisoning. • Only toxigenic types A and C • These spores are probably formed in (seldom D) of C. perfringens produce the colon rather than in the small enterotoxin (causes gastrointestinal intestines. symptoms).

Immunity Reservoirs • C. perfringens is found in human and • After an incident of disease, circulating animal intestinal tracts, and soil. antibodies that neutralize enterotoxin are found in the blood but not in the • ~ 50% to 100% of normal, healthy intestines. humans are carriers of C. perfringens. • C. perfringens food poisoning does not • Carriers excrete around 103 spores/g confer immunity. of feces. • Recovering individuals shed >105/g.

Shedding Frequency Growth and Survival • Growth temperature is 6.5 – 47°C • Shedding frequency in animals: • Freezing cells at –18°C: only 4% ¾Swine 18% survived for 180 days. ¾Rats 41% • Freezing spores at –18°C: only 11% ¾Chicken 88% (60% produced toxin) survived for 180 days. ¾Cattle 80% (68 % produced toxin) • Storage at 5°C more lethal than freezing at –18°C.

6 Detection of C. perfringens Survival and Growth • A 1.2–3.4 kGy of gamma rays kills 90% • Many different media have been of spores. proposed • Water activity of 0.95–0.96 limits growth. –see Compendium of Methods for the Microbiological Examination of Foods • The limiting NaCl concentration is 5–8% • Sulfite cycloserine agar is convenient for • Upper redox potential permitting growth pour plates. +31 mV at pH 7.7, +230 mV at pH 6 • Neomycin blood agar for surface plating.

Detection of C. perfringens

• Most probable numbers (MPN) can be determined in cooked meat or liver medium followed by streaking on neomycin blood agar. • Enterotoxin can be detected by ELISA test.

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