1Microorganisms Causing Foodborne DiseaseGram-positive sporeformers•Clostridium perfringens•Clostridium botulinumClostridium botulinum•Strictly anaerobic•Gram-positive•Sporeformer• Causes botulism– Severe intoxication – Flaccid paralysis caused by a neurotoxin• 8 toxins described– A, B, C, C1, C2, D, E, F, G•C2is not a neurotoxin• These toxins are heat labile (80°C for 5 min)C. botulinumBotulism•1stoutbreak described in 1793 in Germany– Associated with sausage–Latin botulus= sausage• There are 3 categories of botulism– Food poisoning• Associated with home-made canned foods or preserves– Intoxication– Wound botulism• Associated with handling contaminated materials when being wounded– Toxiinfection– Infant botulism• Associated with bee honey in lactants– Toxiinfection2C. botulinumFood poisoning• Incubation period of 12 to 36 hours• Begins with gastrointestinal problems:–Nausea– Vomiting– Diarrhea• May not be caused by the toxin because not present in other illnesses• When typical botulism signs develop, constipation• First indicators of botulism:–Fatigue– Muscular weaknessC. botulinum• Followed by ocular effects:– Droopy eyelids– Sluggish response of pupils to light– Blurred and double vision• Mouth effects:–Dryness– Difficulty in speech and swallowing• Musculatures controlling limbs and respiration become progressively paralyzed• Death within 3 to 5 days (respiratory failure)• Complete recovery may require several monthsC. botulinum•Epidemiology•Most cases are caused by types A, B or E•C. botulinumType A most frequent in the U.S.–Associated with fruits and vegetables•C. botulinumType E is a psychrotroph–Associated with smoked fish•C. botulinumType B more frequent in Europe–Associated with home-canned ham or baked foods•Commercially canned foods have only been associated with few outbreaks•Sausage has not been associated with botulism in decades3Methods for isolation• Needs anaerobic environment to grow • Food suspension is heated to eliminate non-sporeformers• Enrichment in cooked meat broth at 30°C for 7 days• Plate on horse blood or egg yolk agar• Select typical colonies•Conduct toxin identificationC. perfringens•Gram positive• Sporeforming rod• Strictly anaerobic– Will survive and occasionally grow in the presence of O2•Encapsulated•NonmotileC. perfringens•Formerly C. welchii• Associated with gas gangrene• One of the most common causes of diarrhea worldwide • 5 types based on toxin production– A, B, C, D, E–Toxins ", β, ε and 4•C. perfringenstype A is responsible for food poisoning and gas gangrene– Only produces " toxin – This toxin also has lecithinase activity• Phospholipase•C. perfringenstype C– Produces " and β toxins– Cause of enteritis necroticans– Severe but rare condition associated with pork in Papua New Guinea during festivities4C. perfringens• Growth characteristics (vegetative cells)– Temperature• 12 – 50°C• Optimum 43 – 47°C– Generation time of 7.1 min at 41°C–pH• Minimum 5.0• Optimum 6.0 – 7.5–Min aw0.95 – 0.97• Resistance (spores)–D100= 0.31 – 38 minC. perfringens• Incubation period = 8-24 h • Necessary to ingest large numbers of vegetative cells (106–108cells)•Syndrome– Explosive diarrhea– Severe abdominal pain– Nausea less common– Fever and vomiting unusual– Death uncommon unless underlying illness– Symptoms subside in 12 to 24 hC. perfringens• Found in most raw, animal products– Raw meats– Raw poultry– Dehydrated soups and sauces– Raw vegetables– Food ingredients (spices)– Virtually all processed foods• Foods associated with outbreaks:– Cooked meat or poultry foods•Stews•Meat gravie•Meat pies• Roast beef • Why these foods?– High prevalence– High nutrient content (protein)– Cooking creates reduced environment suitable for growth5C. perfringens• Outbreaks frequently occurring at food-service establishments • Temperature abuse• Spores need to germinate and grow to high numbers to cause infection • Once the intestine is colonized, vegetative cells sporulate• Toxin is released at sproulationC. perfringens– Control and Prevention• FDA Food Code– Rapid, uniform cooling of food»To 21oC within 2 h, and»from 21oC to 5oC within 4 h– Reheat to internal temperature of 74oC– Hold cooked dishes ≥60oC or ≤5oC• Impossible to eliminate from foods• Prevent by proper food preparation and storage techniquesC. perfringens• Outbreaks frequently occurring at food-service establishments • Temperature abuse• Spores need to germinate and grow to high numbers to cause infection • Once the intestine is colonized, vegetative cells sporulate• Toxin is released at sproulation6C. perfringensMethods for analysis• Dilutions and plating onto tryptosesulfite cycloserin (TSC) agar• Incubate under anaerobic conditions• Select typical colonies for further identification– Reduce NO3to NO2– Non motile– Lactose fermentative– Liquefies gelatin• MPN is also used when low numbers are