Clostridium perfringens Clostridium perfringens Dean O. Cliver - PDF document

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 poisoning. • Classification • Pathogen prevalences • They associated eating foods contaminated with C. perfringens with • Clinical features & mechanism of disease several foodborne outbreaks. • C. perfringens enterotoxins and spores • Outbreaks were characterized with mild • Immunity, reservoirs, shedding, to severe diarrhea and abdominal pain. growth/survival, and detection Background Background • 1892: The microorganism was found in a variety of diseases. • 1939 to 1946: Several outbreaks � gas gangrene observed: � appendicitis � In the U.K. during WWII � puerperal fever (infection of the � Shortage of meat led to the practice placental site following delivery or of cooking meat for later abortion) consumption. � enteritis 1

Background Background • 1939 to 1946: Several outbreaks • 1939 to 1946: Several outbreaks observed: � First warning of food poisoning observed: came from Knox and Macdonald � In the US, the first proven outbreak (1943). of C. perfringens was described by � Vehicle: gravy made the previous McClung (1945). day was heavily contaminated with � Examined 4 foodborne outbreaks anaerobic sporing bacilli including C. perfringens . associated with eating chicken � Children 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. � The vehicle for the illness was home perfringens could lead to canned rabbit. diarrheal food poisoning. � The associated strain was C. perfringens • Pioneered work establishing type C. C. perfringens as a cause of food poisoning. Background Background • By the 1960s and 70s, enough • 1954: Experiments were conducted in US information had accumulated to indicate with human volunteers (Dack). that C. perfringens foodborne poisoning � The strain fed was English strain of C. is caused by the release of the toxin perfringens . during sporulation of the microorganism � Volunteers 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 acid, acetic acid, propionic acid, lactic acid, Total foodborne 13,814,924 100.0 1,809 100.0 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 Classification of C. perfringens is Based on Toxins Produced 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 very severe. D + 0 + 0 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- Cooked Pork 45% hemolytic. • Killing 90% of spores takes 6 – 17 minutes Beef 22% at 100°C for non-hemolytic strains and less Chicken 0–54% than one minute for beta-hemolytic strains. 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 10 8 • 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. 2. Ingested cells begin to • C. perfringens produces toxin- sporulate after passing mediated infection known as C. stomach. perfringens enteritis. 3. Enterotoxin produced • C. perfringens produces food in small intestine poisoning without colonization. (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 growth temperature the “primed” where pH is right and well buffered. It spores will produce rapidly growing begins in the intestines in 3 h, and vegetative cells that in turn will enterotoxin found in 10-12 h. 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 � Kill the vegetative cells in the food, � large volumes of food produced and � Prevent sporulation in the intestines • There are few if any fatalities . C. perfringens Enterotoxins C. perfringens Enterotoxins • Have similarities with V. cholerae • Simple polypeptide, molecular weight of enterotoxin 36000 ± 4000, contains 309 amino acid � Cause a transient increase in capillary residues but only one cysteine residue permeability with fluid accumulation • They are inactivated by pronase and B. and diarrhea subtilis protease but not by trypsin, � Increased secretion of water, sodium chymotrypsin, papain or bromelin. and chloride � Decreased absorption of glucose • Enterotoxin is heat labile with 90% � Enterotoxin causes desquamation of destruction in 4 minutes at 60°C. 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 • ~ 50% to 100% of normal, healthy are found in the blood but not in the humans are carriers of C. perfringens. intestines. • Carriers excrete around 10 3 spores/g • C. perfringens food poisoning does not of feces. confer immunity. • Recovering individuals shed >10 5 /g. Shedding Frequency Growth and Survival • Growth temperature is 6.5 – 47°C • Shedding frequency in animals: • Freezing cells at –18°C: only 4% � Swine 18% survived for 180 days. � Rats 41% • Freezing spores at –18°C: only 11% � Chicken 88% (60% produced toxin) survived for 180 days. � Cattle 80% (68 % produced toxin) • Storage at 5°C more lethal than freezing at –18°C. 6

Detection of C. perfringens Survival and Growth • Many different media have been • A 1.2–3.4 kGy of gamma rays kills 90% proposed of spores. –see Compendium of Methods for the • Water activity of 0.95–0.96 limits growth. 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. 7