2/16/2016 Learning Objectives Food 1.Use basic biochemical characteristics to identify Clostridium perfringens 2.Understand what conditions in foods favor the Chapter11 AN INTRODUCTION growth of C.perfringens Clostridium 3aegr228cecs caused by C.perfringens perfringens 4.Choose appropriate interventions to prevent the growth of C.perfringens 5.Identify environmental sources of C perfringens Cruel and Unusual Punishment Investigation found that roro urkey breast the day Day after Thanksgiving.half of the staff Cooked them until they"looked done" and residents of a juvenile detention Stacked them in large pots and stored them in center became ill a walk-in cooler 100%-diarrhea 90%-abdominal pain When removing the turkeys the next day,they 63%-nausea were still warm Heated and served anyway 30%-vomiting .C perfringens(10 CFU/g)was Prevention:cook turkeys adequately,cool to below 40F in less than 2h and slicing into smaller isolated from 5 of 9 stool samples pieces facilitates rapid cooling
2/16/2016 1 Chapter 11 Clostridium perfr ngens i Learning Objectives 1. Use basic biochemical characteristics to identify Clostridium perfringens 2. U d t d h t diti i f d f th Understand what conditions in foo ds favor th e growth of C. perfringens 3. Recognize, from symptoms and time of onset, and environmental conditions a case of foodborne illness caused by C. perfringens 4. Choose appropriate interventions to prevent the growth of C. perfringens 5. Identify environmental sources of C. perfringens 6. Understand the roles of spores and the sporulation cycle of C. perfringens foodborne illness Cruel and Unusual Punishment • Day after Thanksgiving, half of the staff and residents of a juvenile detention center became ill 100% - diarrhea 90% - abdominal pain 63% - nausea 30% - vomiting • C. perfringens (106 CFU/g) was isolated from 5 of 9 stool samples • Investigation found that - inmates thawed frozen turkey breast the day before the meal - Cooked them until they “looked done” - Stack d h ked them in l arge pots and d h d stored them in a walk-in cooler - Large pots of gravy were also placed in the cooler - When removing the turkeys the next day, they were still warm - Heated and served anyway • Prevention: cook turkeys adequately, cool to below 40oF in less than 2h and slicing into smaller pieces facilitates rapid cooling
2/16/2016 Incidence Occur often in institutional settings C nerfrinoens Type A Higher number of cases in summer months toxicoinfection is the third most Outbreaks are usually large 1.Institutions prepare food in advance Toxicoinfection:bacteria and hold it for later serving allowing simultaneously produce toxins and couse infections. paeoeotrecgador 2.Mild and routine symptoms of most cases- public health officials are Conservatively,250,000 cases involved when many people get sick per year in the US with an average of 7 deaths Economic losses also occur from fatal 5ona6Coetsexced240 infections in cattle,sheep,and pigs Vehicles for C.perfringens Factors Contributing to C.perfringens Illness Type A Foodborne Illness Usually results from temperature abuse during C.perfringens cannot make 13 of the 20 cooking.cooling or holding of foods amino acids required for its growth Its optimum growth temperature of 43C gives Thus,high protein foods rich in amino acids it an edge over other mesophilic pathogens are common vehicles for this microbe CDC reports for recent cases that C.perfringens illness commonly associated with meats and poultry -improper storage or holding contributed in Other high protein products subject to 100% temperature abuse are important sources -improper cooking contributed in 30% -contaminated equipment contributed in 15%
2/16/2016 2 Incidence • C. perfringens Type A toxicoinfection is the third most common foodborne disease in the US • Toxicoinfection: bacteria simultaneously produce toxins and cause infections. • Most cases are not recognized or reported • Conservatively, 250,000 cases per year in the US with an average of 7 deaths • Economic costs exceed 240 billion dollars • Occur often in institutional settings • Higher number of cases in summer months • Outbreaks are usually large 1. Institutions prepare food in advance and hold it for later serving allowing C. perfringens to grow if temperature abuse occurs 2. Mild and routine symptoms of most cases – public health officials are involved when many people get sick • Economic losses also occur from fatal infections in cattle, sheep, and pigs Vehicles for C. perfringens Illness • C perfringens C. perfringens cannot make 13 of the 20 amino acids required for its growth • Thus, high protein foods rich in amino acids are common vehicles for this microbe • C. perfringens illness commonly associated with meats and poultry • Other high protein products subject to temperature abuse are important sources Factors Contributing to C. perfringens Type A Foodborne Illness • Usually results from temperature abuse during cooking cooling or holding of foods cooking, cooling or holding of foods • Its optimum growth temperature of 43oC gives it an edge over other mesophilic pathogens • CDC reports for recent cases that - improper storage or holding contributed in 100% -improper cooking contributed in 30% -contaminated equipment contributed in 15%
2/16/2016 Preventing C.perfringens Type A Identification of C.perfringens Type A Foodborne Illness Foodborne Illness Outbreaks ·Thorough cooking Incubation time and illness symptoms together -difficult with large roasts or turkeys with history of food vehicles may be used but to reach a proper internal -many similarities with other foodborne temperature pathogens Thus-a common vehicle Detection of spores or toxins in the feces is Rapidly cool and store cooked foods at more reliable temperatures where C.perfringens -<5%of environmental strains make toxin so cannot grow -below 40C and above simply identifying C.perfringens is not proof 140C CDC and FDA now use Characteristics of C.perfringens detection of enterotoxin in Type A Foodborne Illness feces of multiple persons who are sick to detect an Symptoms develop 8-16 hours after outbreak consumption of contaminated food Limited use since fecal Symptoms last about 12-24 hours samples must be collected Most only suffer severe abdominal soon after onset of cramps and diarrhea;no fever symptoms ·Death rates are low Commercial serologic kits Fatalities mostly with debilitated or are available elderly people
2/16/2016 3 Preventing C. perfringens Type A Foodborne Illness • Thorough cooking - difficult with large roasts or turkeys to reach a proper internal temperature - Thus - a common vehicle • Rapidly cool and store cooked foods at temperatures where C. perfringens cannot grow – below 40oC and above 140oC Identification of C. perfringens Type A Foodborne Illness Outbreaks • Incubation ti m ymp m g e and illness sympto ms to gether with history of food vehicles may be used but -many similarities with other foodborne pathogens • Detection of spores or toxins in the feces is more reliable - < 5% of environmental strains make toxin so simply identifying C. perfringens is not proof • CDC and FDA now use detection of enterotoxin in feces of multiple persons who are sick to detect an outb k rea k - Limited use since fecal samples must be collected soon after onset of symptoms - Commercial serologic kits are available Characteristics of C. perfringens Type A Foodborne Illness • Symptoms develop 8-16 hours after consumpti f t i t d f d tion of con tamina t ed foo d • Symptoms last about 12-24 hours • Most only suffer severe abdominal cramps and diarrhea; no fever • Death rates are low • Fatalities mostly with debilitated or elderly people
2/16/2016 Infectious Dose C.perfringens Food Poisoning Most cells are killed by stomach acid heatacthoadspore Toxicoinfection develops when heavily The mal treaterts contaminated food (>106 C.perfringens) Spores is consumed Toxin is produced in the victim Sporulation occurs in the intestines Enterotoxin binds to C Previous exposure provides no immunity Cells multiply The Organism INCLUSION BODY Gram-positive,rod-shaped,encapsulated,non- motile bacterium Causes a broad spectrum of human and animal diseases Facultative anaerobe that can be plated at the bench that is subsequently incubated anaerobically Produces numerous toxins of which two are active in the human GI tract Grows rapidly:doubles in number in 10 min with optimal growth conditions Forms heat-resistant spores that survive incompletely cooked or reheated food
2/16/2016 4 Under optimal growth conditions, C. perfringens can double in number every 10 min Large number of bacteria are consumed Temperature abuse stimulate vegetative growth Enterotoxin binds to and damages intestinal epithelial cells Cells multiply sporulate and release enterotoxin Infectious Dose • Most cells are killed by stomach acid • Toxicoinfection develops when heavily contaminated food (> 106 C. perfringens) is consumed • Toxin is produced in the victim • Sporulation occurs in the intestines • Previous exposure provides no immunity The Organism • Gram-positive, rod-shaped, encapsulated, nonmotile bacterium • Causes a broad spectrum of human and animal diseases • Facultative anaerobe that can be plated at the bench that is subsequently incubated anaerobically • Produces numerous toxins of which two are active in the human GI tract • Grows rapidly; doubles in number in < 10 min with optimal growth conditions • Forms heat-resistant spores that survive incompletely cooked or reheated food
2/16/2016 Classification:Toxin Typing Control of C.perfringens 14 different C.perfringens toxins Temperature No one bacterium encodes all toxins Heat resistance of the spore is influenced by the Thus,toxins are used to type strains environment and genetic factors Five types(A through E) -De rng for gerer Toxicoinfectious isolates have more heat resistant Epsilon toxin is a biothreat agent as it can be spores aerosolized and no vaccine exists to protect gainst it Incomplete cooking stimulates germination of the Two foodborne diseases are associated with different spore C.perfringens types -Vegetative cells have a high optimal growth Necrotic enteritis:type C strain B-toxin temperature 43-45C Type A toxicoinfection:Type A strain;enterotoxin Growth rate decreases rapidly below 15C No growth at 69C Spores are cold resistant Control of C.perfringens Control of C.perfringens Other Factors Other Factors >water activity -Heat resistance of the spore is less tolerant of low water activity influenced by the environment and genetic factors ping vegetativeghis3 >oxidation-reduction potential >water activity >oxidation-reduction potential >pH if growth is initiated it produces reducing molecules to support growth >possibly curing agents such as nitrites >pH sensitive to pH These factors inhibit outgrowth of the spores ptimally at pH6-7 but poorly at pH5 >possibly curing agents such as nitrites
2/16/2016 5 Classification: Toxin Typing • 14 different C. perfringens toxins • No one bacterium encodes all toxins • Thus, toxins are used to type strains • Five t pes (A thr u h E) Five t ypes (A through E) - Depends on the strains ability to make 4 of the 14 toxins • Epsilon toxin is a biothreat agent as it can be aerosolized and no vaccine exists to protect against it • Two foodborne diseases are associated with different C. perfringens types - Necrotic enteritis; type C strain; -toxin - Type A toxicoinfection; Type A strain; enterotoxin - Type A is more widely distributed and causes most of the illness Control of C. perfringens Temperature - Heat resistance of the spore is influenced by the environment and genetic factors - Spores of some strains survive boiling for greater than one hour - Toxicoinfectious isolates have more heat resistant spores - Incomplete cooking stimulates germination of the spore - Vegetative cells have a high optimal growth temperature 43-45oC - Growth rate decreases rapidly below 15oC - No growth at 6oC - Spores are cold resistant Control of C. perfringens Other Factors - Heat resistance of the spore is influenced b y the environment and genetic factors water activity oxidation-reduction potential pH possibly curing agents such as nitrites • These factors inhibit outgrowth of the spores Control of C. perfringens Other Factors water activity - less tolerant of low water activity - lowest aw supportin g ve getative growth is 0.93- w pp g g g 0.97 oxidation-reduction potential - does not require an extremely reduced environment - if growth is initiated it produces reducing molecules to support growth pH - sensitive to pH - grows optimally at pH 6-7 but poorly at pH 8.3 possibly curing agents such as nitrites
2/16/2016 Reservoirs for C.perfringens Type A Reservoirs for C.perfringens Type A Natural environments Wide spread distribution is linked to its Soil 103-104 CFU per gram frequent occurrence in foodborne illness Foods,contained in 50%of raw or ·However,‘5%of all isolates contain the frozen meat cpe gene,which encodes the entertoxin -Dust required for illness -Intestinal tracts of humans and Thus,the ubiquitous nature of this domestic animals microbe is not relevant for understanding -Human feces contains 104-106 CFU per the reservoir of C.perfringens gram Many questions are still to be answered Virulence Factors Contributing to C. Virulence Factors Contributing to C perfringens perfringens Foodborne Illness Foodborne Illness ·Heat Resistance ·Enterotoxin -Most Cperfringens toxicoinfections are caused by isolates carrying a Induces loss of fluid from the chromosomal cpe gene intestinal tract -Isolates carrying chromosomal or plasmid cpe genes make the same amount of toxin Oddly,strains carrying a chromosomal hpeehn ran
2/16/2016 6 Reservoirs for C. perfringens Type A • Natural environments S il 103 – - Soil 103 – 104 CFU p m CFU per gram - Foods, contained in 50% of raw or frozen meat - Dust - Intestinal tracts of humans and domestic animals - Human feces contains 104 – 106 CFU per gram Reservoirs for C. perfringens Type A • Wide spread distribution is linked to its frequent occurrence in foodborne illness • However, < 5% of all isolates contain the cpe gene, which encodes the entertoxin required for illness • Thus, the ub hus, the ub qu tous nature of th s iquitous nature of this microbe is not relevant for understanding the reservoir of C. perfringens • Many questions are still to be answered Virulence Factors Contributing to C. perfringens Foodborne Illness • Heat Resistance - Most C perfringens C. perfringens toxicoinfections toxicoinfections are caused by isolates carrying a chromosomal cpe gene - Isolates carrying chromosomal or plasmid cpe genes make the same amount of toxin - Oddly, strains carrying a chromosomal cpe gene are more heat resistant than those with a plasmid-based gene Virulence Factors Contributing to C. perfringens Foodborne Illness • Enterotoxin - Induces loss of fluid from the intestinal tract
2/16/2016 Virulence Factors Contributing to C.perfringens Foodborne Illness Virulence Factors Contributing to C.perfringens Epidemiological evidence for enterotoxin Foodborne Illness causing foodborne illness ·Enterotoxin 1.Presence of C perfringens enterotoxin in Koch's postulate on a molecular basis was victim's feces fulfilled 2.Toxin causes serious intestinal effects in Sporulating but not vegetative cultures of animal models cpe-positive isolates induced fluid 3.Human volunteers feed enterotoxin get accumulation in animals sick with symptoms of C perfringens Expression of toxin is sporulation associated foodborne illness .Strains carrying a cpemutation did not cause 4.Intestinal inflammation caused by toxin illness producing strains can be neutralized with Complementation of the mutation restored virulence toxin-specific antiserum Virulence Factors Contributing to C.perfringens Virulence Factors Contributing to C.perfringens Foodborne Illness Foodborne Illness ·Enterotoxin Expression and release of enterotoxin -C.perfringens type A causes non-foodborne GI illnesses 1.Toxin is made only by sporulating >antibiotic-associated diarrhea vegetative cells >sporadic diarrhea 2.Toxin is not secreted by sporulating >veterinary diarrheas cells but is released when the cpe-positive strains causing foodborne toxicoinfections are genetically distinct mother cell lyses from those causing non-foodborne illness 3.Isolates make extremely large 、 cpe is located on the chromosome for toxicoinfections and on the plasmid for amounts of toxin other illnesses
2/16/2016 7 Virulence Factors Contributing to C. perfringens Foodborne Illness • Epidemiological evidence for enterotoxin causing foodborne illness 1. Presence of Presence of C perfringens C. perfringens enterotoxin enterotoxin in victim’s feces 2. Toxin causes serious intestinal effects in animal models 3. Human volunteers feed enterotoxin get sick h fwit symptoms of C. f per ringens foodborne illness 4. Intestinal inflammation caused by toxin producing strains can be neutralized with toxin-specific antiserum Virulence Factors Contributing to C. perfringens Foodborne Illness • Enterotoxin - Koch s postulate on a ’s postulate on a molecular basis was fulfilled - Sporulating but not vegetative cultures of cpe-positive isolates induced fluid accumulation in animals - Expression of toxin is sporulation associated - Strains carrying a cpe mutation did not cause illness - Complementation of the mutation restored virulence Virulence Factors Contributing to C. perfringens Foodborne Illness • Enterotoxin - C. perfringens type A causes non-foodborne GI illnesses antibiotic-associated diarrhea sporadic diarrhea veterinary diarrheas - cpe-positive strains causing foodborne toxi if i co nfections are geneti ll di i ically distinct from those causing non-foodborne illness - cpe is located on the chromosome for toxicoinfections and on the plasmid for other illnesses Virulence Factors Contributing to C. perfringens Foodborne Illness • Expression and release of enterotoxin 1. Toxin is made onl yyp g b y s porulatin g vegetative cells 2. Toxin is not secreted by sporulating cells but is released when the mother cell lyses 3. Isolates make extremely large amounts of toxin
2/16/2016 Virulence Factors Contributing to C.perfringens Virulence Factors Contributing to C perfringens Foodborne Illness Foodborne Illness Expression of enterotoxin Release of enterotoxin Synthesis begins when cells sporulate Toxin accumulates in cells during synthesis Increases for 6-8 hours High concentrations form Toxin can make up 30%of total cell paracrystalline inclusion bodies protein Toxin is released when sporulation is The better an isolate sporulates the completed and the mother cell lysis more toxin it produces Explains in part why symptoms develop 8-24 hours after ingestion Cells must grow in the intestines and complete sporulation Virulence Factors Contributing to C.perfringens Biochemistry of enterotoxin Foodborne Illness 3.5 kDa protein -Damages intestinal tissues within 15-30 mins Biochemistry of enterotoxin Acts via a multistep process -Heat sensitive 1.Binds a specific receptor on the intestinal cells Biological activity is inactivated by heating for 5 min at 60C 2.Enterotoxin inserts into the cell membrane 3.Ions and water are released pH sensitive Resistant to some proteases Human intestinal proteases may activate the toxin
2/16/2016 8 Virulence Factors Contributing to C. perfringens Foodborne Illness • Expression of enterotoxin - Synth i b i h ll hesis begins when cells sporulate - Increases for 6-8 hours - Toxin can make up 30% of total cell protein - The better an isolate sporulates the more toxin it produces Virulence Factors Contributing to C. perfringens Foodborne Illness • Release of enterotoxin - Toxin accumulates in cells during synthesis - High concentrations form paracrystalline inclusion bodies - Toxin is released when sporulation is completed and the mother cell lysis - Explains in part why symptoms develop 8-24 hours after ingestion - Cells must grow in the intestines and complete sporulation Virulence Factors Contributing to C. perfringens Foodborne Illness • Biochemistry of enterotoxin - H ii eat sensitive - Biological activity is inactivated by heating for 5 min at 60oC - pH sensitive - Resistant to some proteases - Human intestinal proteases may activate the toxin • Biochemistry of enterotoxin - 3.5 kDa protein - Damages intestinal tissues within 15-30 mins - Acts via a multistep process 1. Binds a specific receptor on the intestinal cells 2. Enterotoxin inserts into the cell membrane 3. Ions and water are released
2/16/2016 Summary Summary C.perfringens is an anaerobic spore- The cpe gene encodes the enterotoxin forming bacterium commonly found on The cpe gene is located chromosomally proteinaceous foods in toxicoinfectious isolates but on Toxin is released as a paracrystalline plasmids of isolates not associated with protein when vegetative cells release foods their spores in the gut Victims with toxicoinfections with C. Improper heating or cooling is the major perfringens usually only suffer diarrhea cause of C.perfringens typeA and severe abdominal cramps toxicoinfection
2/16/2016 9 Summary • C. perfringens is an anaerobic sporef i b t i l f d forming bac terium commonly found on proteinaceous foods • Toxin is released as a paracrystalline protein when vegetative cells release the r spores n the gut ir spores in the gut • Improper heating or cooling is the major cause of C. perfringens typeA toxicoinfection Summary • The cpe gene encodes the enterotoxin • The cpe gene is located chromosomally in toxicoinfectious isolates but on plasmids of isolates not associated with foods • Victims with toxicoinfections with C . perfringens usually only suffer diarrhea and severe abdominal cramps
