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Page 1: Spore Forming Bacteria

Spore forming bacteriaSpore forming bacteriaSpore forming bacteriaSpore forming bacteriaSpore forming bacteriaSpore forming bacteriaSpore forming bacteriaSpore forming bacteria

Page 2: Spore Forming Bacteria

Spore forming bacteriaSpore forming bacteria

!Spore former!Bacillus

!Clostridium

!Desulfotomaculans!Gram negative

!Converse sulfate to sulfide

!Sporolactobacillus!Rod shape, microaerophilic bacteria

!Sporpsarcina!Cocci, aerobic bacteria

Page 3: Spore Forming Bacteria

Importance of spore formerImportance of spore formerin foodin food

!Response to temperatures!Endospore formation

!Resistance

!Morphology!cortex --peptidoglycan!germ cell wall!nuclear mass

!Sporulation!Germination

Page 4: Spore Forming Bacteria

Spore cycleSpore cycle

!Control of spore forming bacteria!Prevent sporulation

!Condition that bacteria can grow: no sporulation

!Prevent activation!Dormant spore: no hazard

!Prevent initiation and outgrowth!Prevent germinate spore to grow

Vegetative cellssporulation

Germinated spore Dormant spore

Activated sporeinitiation

activation

outgrowth

Page 5: Spore Forming Bacteria

Bacillus cereusBacillus cereusBacillus cereusBacillus cereusBacillus cereusBacillus cereusBacillus cereusBacillus cereusFood poisoningFood poisoningFood poisoningFood poisoningFood poisoningFood poisoningFood poisoningFood poisoning

Page 6: Spore Forming Bacteria

Bacillus cereusBacillus cereus

!Gram positive

!Motile

!Spore forming bacteria

!Facultative aerobic bacteria

!Grows within the temperature range of 10-48 °C

!optimum temperature 28-35 ° C

!Endospores are formed freely, in almost every cells,under conditions favorable for growth

!Two forms of food poisoning

!Diarrhea syndrome and emetic syndrome

Page 7: Spore Forming Bacteria

Bacillus cereusBacillus cereus

!Unique characteristic

!Temperature 10 to 48 °C

!Produce toxins

!Diarrhea toxin: Diarrhea syndrome (toxin formed in intestine)

!Heat-labile enterotoxin

!Large MW

!Emetic toxin: Emetic (Food intoxication)

!Heat stable toxin (121 °C for 90 min)

!Low MW

!Not destroy by refrigeration and cooking

! Infective dose: high infective dose (>105 CFU/g)

Page 8: Spore Forming Bacteria

Bacillus cereus Bacillus cereus food poisoningfood poisoning

!Foodborne intoxication! Incubation period: 1-5 hrs (8-16 hrs:Diarrhea syndrome)

!Duration of illness: 6-24 hrs!Clinical signs

!Diarrhea type!Watery diarrhea!Abdominal cramps!Nausea but vomiting rarely occurs!Symptoms persist for 24 hrs

!Emetic type!Sudden onset of vomiting!Abdominal cramps!Some patients may develop diarrhea

!Usually self limiting and not severe

Page 9: Spore Forming Bacteria

Bacillus cereusBacillus cereus outbreaks outbreaks

!Most common: Rice or cereal product

!High carbohydrate food

!Starchy

!Wide variety of foods including meats, milk,vegetables and fish have been associated withdiarrheal type

!All ages are susceptible to B. cereus food poisoning

!Symptoms similar to Staphyloccus food poisoning(emetic type) and C. perfingens food poisoning(diarrheal type)

Page 10: Spore Forming Bacteria

Bacillus cereusBacillus cereus outbreak outbreak

!Emetic syndrome

!Associated with Chinese restaurant

!Fried rice

!The practices flavor to food poisoning

!The preparation of large amount of cooked rice (some B.

cereus can survive)

!Holding the cooked rice at warm temperature for long periods

(B. cereus spores can germinate and the vegetative cellsmultiply and form enterotoxin)

!The toxin can survive the stir-fry temperature

Page 11: Spore Forming Bacteria

Bacillus cereusBacillus cereus case study case study

!B. cereus food poisoning account for only 2% ofoutbreaks

!B. cereus food poisoning associated with Fried rice

!2 daycare centers:

!Acute gastroenteritis

!Following lunch

!Lunch served to 82 children, 9 adults

!Nausea, vomit, abdominal cramps and diarrhea

!Median incubation period: 2 hrs

!Symptom resolved: 4 hrs after onset

Page 12: Spore Forming Bacteria

Bacillus cereusBacillus cereus case study case study

! Interview cases

!Chicken fried rice!Prepare at a local restaurant

!29% of persons who ate fried rice compare to 0% whodid not eat

!Leftover chicken fried rice

!B. cereus > 106 CFU/gm

!Source of contamination?!Rice had been cooked at night

!Cooled at room temperature before refrigeration

! In the morning rice was pan-fried and delivery to the daycare

!Foods was served at noon

Page 13: Spore Forming Bacteria

Bacillus cereusBacillus cereus

!Bacillus cereus prevention and control

!Education

!Control cooking temperature, storage temperature

!Proper temperature

!< 5 ° C or > 60 °C

!Reheating before serving

!Limit condition for B. cereus

!pH < 5

!Temperature < 5

!Water activity < 0.95

Page 14: Spore Forming Bacteria

Isolation ofIsolation of Bacillus cereus Bacillus cereus

!Suspected food, vomitus and feces of patients

!Selective media for B. cereus

!Polymyxin B: Inhibit Gram negative bacteria

!Egg yolk: Bacteria produce lecithinase

!Manitol: B. cereus fermented manitol

!pH indicator-bromthymol blue

!Colony: Distinctive blue color surrounded by precipitation ofegg yolk blue zone

!Serology typing for B. cereus enterotoxin isdeveloped

!Vomiting type toxin can be detected by animalmodels

Page 15: Spore Forming Bacteria

BotulisumBotulisumBotulisumBotulisumBotulisumBotulisumBotulisumBotulisum

Page 16: Spore Forming Bacteria

Clostridium botulinumClostridium botulinum

!Gram positive

!Rod shape

!Spore forming bacteria

!Anaerobic bacteria

!The spores are heat resistant and can survive infoods

!Seven types (A, B, C, D, E, F and G) of botulisum arerecognized

!based on the antigenic specificity of the toxin

!Types A, B, E and F: human botulism

!Types C and D: botulism in animals (Horse, cattle, poultry)

Page 17: Spore Forming Bacteria

Clostridium botulinumClostridium botulinum

!Foodborne botulism (Foodborne intoxication)

!Distinct from wound botulism and infant botulism

!Severe type of food poisoning

! Ingestion of foods containing the potent neurotoxin

!Neurotoxin

!Heat labile and can be destroyed if heated at 80 °C for 10

minutes or longer.

!The incidence of the disease is low

!Considerable concern because of its high mortality rate

Page 18: Spore Forming Bacteria

Clostridium botulinumClostridium botulinum

!Unique characteristic

!Strict anaerobic bacteria

!Heat labile toxin

!Produce neurotoxin

! Infective dose: Low infective dose

!A very small amount (a few nanograms) of toxin can cause

illness

Page 19: Spore Forming Bacteria

BotulisumBotulisum

!Foodborne intoxication

! Incubation period: 12-19 hrs (Usually 18-36 hrs)

!Duration of illness: 1-8 days (or slow recovery over 6-8 mts)

!Four types of botulism!Foodborne

! Infant

!Wound (not related to food)

!A form of botulism whose classification is as yet undetermined

!Neurotoxin cause food intoxication

!High mortality rate

Page 20: Spore Forming Bacteria

BotulisumBotulisum!Clinical signs of intoxication

!Marked fatigue

!weakness and dizziness

!usually followed by double vision

!Progressive difficulty in speaking and swallowing

!Difficulty in breathing, weakness of other muscles,abdominal distention

!Clinical symptoms of infant botulism!Constipation

!Poor feeding, lethargy, weakness, pooled oral secretions,and altered cry

!Loss of head control

!Recommended treatment is primarily supportive care

!Antimicrobial therapy is not recommended

! Infant botulism is diagnosed by demonstrating botulinaltoxins and the organism in the infants' stools.

Page 21: Spore Forming Bacteria

Infant Infant botulisumbotulisum

! Infant botulism, first recognized in 1976, affects infants under 12months of age

! Caused by the ingestion of C. botulinum spores which colonizeand produce toxin in the intestinal tract of infants (intestinaltoxemia botulism)

! Various potential environmental sources such as soil, cisternwater, dust and foods

! Honey is the one dietary reservoir of C. botulinum spores thus fardefinitively linked to infant botulism by both laboratory andepidemiological studies

! The number of confirmed infant botulism cases has increasedsignificantly

! I t is now internationally recognized, with cases being reported ofinfant botulisum related to consumption of honey in infant

Page 22: Spore Forming Bacteria

BotulisumBotulisum

!Botulinum toxin causes flaccid paralysis!Blocking motor nerve terminals at the myoneural junction

!The paralysis progresses symmetrically downward

!the eyes and face, to the throat, chest and extremities

!the diaphragm and chest muscles

! Inhibit respiration and death from asphyxia results

!Recommended treatment for foodborne botulism includesearly administration of botulinal antitoxin

! Intensive supportive care (including mechanical breathingassistance).

!The incidence of the disease: low,

!The mortality rate is high if not treated immediatelyand properly.

!Rare in the United States (< 50 cases per year) (34 casesin 1994)

Page 23: Spore Forming Bacteria

Botulism Botulism outbreakoutbreak

!Clostridium botulinum has been associated with

! Inadequately processed, home-canned foods,

!Occasionally commercially produced foods have beeninvolved in outbreaks

!Sausages, meat products, canned vegetables and seafoodproducts have been the most frequent vehicles for humanbotulism

!Any food that is conducive to outgrowth and toxinproduction, that when processed allows sporesurvival, and is not subsequently heated beforeconsumption can be associated with botulism

!Almost any type of food that is not very acidic (pHabove 4.6) can support growth and toxin productionby C. botulinum

Page 24: Spore Forming Bacteria

BotulisumBotulisum outbreaks outbreaks

! In 1987, 8 cases of type E botulism occurred

!2 in New York City and 6 in Israel

!All 8 patients had consumed an uneviscerated, dry-salted,air-dried, whole whitefish

!The product was made in New York City and some of it wastransported by individuals to Israel

!All 8 patients with botulism developed symptoms within 36hours of consuming the food

!One died, 2 required breathing assistance, 3 were treatedtherapeutically with antitoxin, and 3 recoveredspontaneously

!The food involved in this outbreak contained high levels oftype E botulinal toxin despite salt levels that exceeded thosesufficient to inhibit C. botulinum type E outgrowth

Page 25: Spore Forming Bacteria

BotulismBotulism case study case study

!Foodborne botulism due to the consumption ofimproperly stored beef stew

!A 47-year-old patient

!Subacute onset of progressive dizziness, blurred vision,slurred speech, difficulty swallowing, and nausea

!Examination included facial paralysis, palatal weakness, andimpaired reflex

!The patient developed respiratory compromise and requiredmechanical ventilation

!Clinical signs:

!Why differential and tentative diagnosis includedfoodborne illness ?

Page 26: Spore Forming Bacteria

BotulismBotulism case study case study

!Diagnosis and treatment

!Anaerobic culture of a stool sample

!Detected type A toxin

!Culture of stool yielded C. botulinum

!Differential diagnoses included wound and foodbornebotulism

!Botulism antitoxin

!Electromyography demonstrated an incremental response torapid repetitive stimulation consistent with botulism

!The patient was hospitalized for 49 days, including 42days on mechanical ventilation, before beingdischarged

Page 27: Spore Forming Bacteria

BotulismBotulism case study case study

!Source of contamination

!During the 24 hours before onset of symptoms

!Patient had eaten home-canned green beans and a stewcontaining roast beef and potatoes

!Although analysis of the leftover green beans was negativefor botulism toxin

! type A toxin was detected in the stew

!The stew had been cooked, covered with a heavy lid, andleft on the stove for 3 days before being eaten withoutreheating

!No other persons had eaten the stew

Page 28: Spore Forming Bacteria

Clostridium botulinumClostridium botulinum

!C. botulinum prevention and control

!Education!Heat treatment

!Temperatures necessary to kill botulism spores (> 100 °C for10 minutes)

!Boiling food for 10 minutes before eating destroys any toxinpresent

!Proper storage!Refrigeration

!Cooked foods should not be held at room temperatures forhours

!Preservation!Low acid food

!Chemical: Na nitrite: prevent germination

!Jams and jellies: high sugar content

Page 29: Spore Forming Bacteria

Isolation ofIsolation of Clostridium botulinum Clostridium botulinum

!To confirm the clinical diagnosis of botulism

!Demonstrate the presence of toxin in the serum or feces ofthe patient or in the food

!Mouse neutralization test: 48 hours

!Culturing of specimens: 5-7 days

!Selective media

!Strict anaerobic condition

! Incubate at 30-35 C for 7 days

!Biochemical confirmation