9/21/2016 Learning Objectives Food 1.Use basic biochemical characteristics to identify Bacillus cereus 2.Begin to appreciate the fluidity of bacterial nomenclature and etiology Chapter 8 AN INTRODUCTION 3.Understand what conditions in foods favor the growth of B.cereus Bacillus cereus 4.Recognize,from symptoms and time of onset,a case of foodborne illness caused by B.cereus 5.Choose appropriate interventions to prevent B. cereus from causing foodborne illness 6.Identify environmental sources of B.cereus 7.Understand the role of spores and toxins in causing foodborne illness Virginia Outbreak Virginia Outbreak ·July21,1993 B.cereus was isolated at >105/gram of Two day-care centers in Virginia vomit from one child ·Catered lunch B.cereus was isolated at >106 gram of Chicken-fried rice was implicated leftover chicken One-third of those who ate the rice Rice was cooked the night before,cooled became ill within 2 hours with nausea at room temperature,and refrigerated abdominal cramps,and diarrhea Next morning,rice and chicken were fried Median time for recovery was 4 hours with oil,delivered to the day care center, None of those who did not eat the rice held without refrigeration,and served became ill without heating
9/21/2016 1 Chapter 8 Bacillus cereus Learning Objectives 1. Use basic biochemical characteristics to identify Bacillus cereus 2. Begin to appreciate the fluidity of bacterial nomenclature and etiology 3. Understand what conditions in foods favor the growth of B. cereus 4. Recognize, from symptoms and time of onset, a case of foodborne illness caused by B. cereus 5. Choose appropriate interventions to prevent B. cereus from causing foodborne illness 6. Identify environmental sources of B. cereus 7. Understand the role of spores and toxins in causing foodborne illness Virginia Outbreak • July 21, 1993 • Two day-care centers in Virginia • Catered lunch • Chicken-fried rice was implicated • One-third of those who ate the rice became ill within 2 hours with nausea, abdominal cramps, and diarrhea • Median time for recovery was 4 hours • None of those who did not eat the rice became ill Virginia Outbreak • B. cereus was isolated at >105 / gram of vomit from one child • B. cereus was isolated at >106 / gram of leftover chicken • Rice was cooked the night before, cooled at room temperature, and refrigerated • Next morning, rice and chicken were fried with oil, delivered to the day care center, held without refrigeration, and served without heating
9/21/2016 Relevance Two Types of Toxins About 27,000 cases a year Emetic(vomiting】 Enterotoxin(diarrheal) in the US FDOD ·Produced by cells ·Produced by the .<2%of foodborne illnesses 交FOISONNG growing in the food bacteria growing in reported in the US CAN Acts fast within 0.5- the small intestines 6 hours after Prevalence in Europe is ·Acts longer,6-14 ingestion hours after ingestion 30% B.R5 ·Heat and acid stable ·Heat labile Minor foodborne pathogen Associated with Associated with Low incidence reflects that starchy foods proteinaceous foods it is not a notifiable disease Both types of illnesses are mild,brief, and self-limiting B.cereus does not compete well with other bacteria Characteristics If the food is heated,vegetative cells die and Bacillus-spore-forming,aerobic the spores germinate and grow rod-shaped bacterium If the food is heated again,the vegetative cells 16S ribosomal RNA sequences identify die and the emetic toxin remains active about 16 genera or rRNA groups of Bacillus Outbreaks are associated with meats,gravies Bacillus cereus"group"includes B. fried rice,pasta,sauces,puddings,and dairy anthracis,B.cereus,B.mycoides,B. products thuringiensis,B.pseudomycoides,and B. 17%of fish sampled identified weihenstephanensis(psychrotrophic) B.cereusand half produce the two toxins -differentiated by the toxins they produce 2
9/21/2016 2 Relevance • About 27,000 cases a year in the US • < 2% of foodborne illnesses reported in the US • Prevalence in Europe is 30% • Minor foodborne pathogen • Low incidence reflects that it is not a notifiable disease Two Types of Toxins Emetic (vomiting) • Produced by cells growing in the food • Acts fast within 0.5- 6 hours after ingestion • Heat and acid stable • Associated with starchy foods Enterotoxin (diarrheal) • Produced by the bacteria growing in the small intestines • Acts longer, 6-14 hours after ingestion • Heat labile • Associated with proteinaceous foods Both types of illnesses are mild, brief, and self-limiting • B. cereus does not compete well with other bacteria • If the food is heated, vegetative cells die and the spores germinate and grow • If the food is heated again, the vegetative cells die and the emetic toxin remains active • Outbreaks are associated with meats, gravies, fried rice, pasta, sauces, puddings, and dairy products • 17% of fish sampled identified B. cereus and half produce the two toxins Characteristics • Bacillus – spore-forming, aerobic rod-shaped bacterium • 16S ribosomal RNA sequences identify about 16 genera or rRNA groups of Bacillus • Bacillus cereus “group” includes B. anthracis, B. cereus, B. mycoides, B. thuringiensis, B. pseudomycoides, and B. weihenstephanensis (psychrotrophic) - differentiated by the toxins they produce
9/21/2016 Plasmid-mediated toxins Bacillus cereus B.cereus has 3 toxins Most strains are unable to grow below B.anthracis has 1 toxin 10C B.thuringiensis has 1 insecticide Some are motile by peritrichous Plasmid-mediated toxin production flagella creates an unstable classification Plasmid may be lost or mobilized to another species con时ugation: 心p →安 one Environmental Source Environmental Source ·Widespread in nature Easily spread from environment to foods Associated with decaying matter Problematic in dairy products Isolated from soil and plants spores present in the soil 105/g spread to the cow udder then to the raw milk In soil spores are hydrophobic and spores germinate upon attach to pipelines and solid favorable conditions surfaces in the dairy converge to filamentous spores survive pasteurization multicellular forms that swarm psychrotrophic strains can grow at in a coordinated direction refrigeration
9/21/2016 3 • Plasmid-mediated toxins - B. cereus has 3 toxins - B. anthracis has 1 toxin - B. thuringiensis has 1 insecticide • Plasmid-mediated toxin production creates an unstable classification • Plasmid may be lost or mobilized to another species Bacillus cereus • Most strains are unable to grow below 10 oC • Some are motile by peritrichous flagella Environmental Source • Widespread in nature • Associated with decaying matter • Isolated from soil and plants In soil - spores germinate upon favorable conditions - converge to filamentous multicellular forms that swarm in a coordinated direction Environmental Source • Easily spread from environment to foods • Problematic in dairy products - spores present in the soil 105 / g - spread to the cow udder - then to the raw milk - spores are hydrophobic and attach to pipelines and solid surfaces in the dairy - spores survive pasteurization - psychrotrophic strains can grow at refrigeration
9/21/2016 Foodborne Outbreaks Foodborne Outbreaks Highly underestimated Incidences of outbreaks vary greatly short duration of the illness (24 h) Vary in countries from 1%-47% similarity of symptoms caused by other Highest number of cases are in Iceland. foodborne pathogens Norway,and the Netherlands number of cases are too small to In the Netherlands,B.cereus caused register as an outbreak 27%of the outbreaks for which an dosage-103-104organisms organism was identified spoilage due to proteases which give off- However,B.cereus caused only 2.8% flavors of foodborne cases since most had an unknown etiology Disease Characteristics Dose Steinar Hauge Some strains make both toxins 1947-1949 -first diarrheal outbreaks of Table Character ofthe two types ofneby B.cereus occurred in Oslo,Norway from Characteristic Diarrheal syndrome Emetic syndrome vanilla sauce Dose cauing illn线 10-1cells ota) Taxin produced ntheln Dessert was prepared and stored at room Type o toxin Proteinr) temperature until it was eaten the next day 1950,Steinar Hauge proved that B.cereus is a human Duration of ilinesn 6-24h pathogen causing food poisoning -corn starch contained ~10 B.cereusspores per gram -demonstrated Koch's postulates >grew B.cereus to 4x106 cells per ml >drank 200 ml of bacterial suspension >13 hrs later-symptoms of food poisoning started 4
9/21/2016 4 Foodborne Outbreaks Highly underestimated • short duration of the illness (<24 h) • similarity of symptoms caused by other foodborne pathogens • number of cases are too small to register as an outbreak • dosage – 103 – 104 organisms • spoilage due to proteases which give offflavors Foodborne Outbreaks Incidences of outbreaks vary greatly • Vary in countries from 1% - 47% • Highest number of cases are in Iceland, Norway, and the Netherlands • In the Netherlands, B. cereus caused 27% of the outbreaks for which an organism was identified - However, B. cereus caused only 2.8% of foodborne cases since most had an unknown etiology Disease Characteristics Some strains make both toxins Dose • 1947-1949 -first diarrheal outbreaks of B. cereus occurred in Oslo, Norway from vanilla sauce • Dessert was prepared and stored at room temperature until it was eaten the next day • 1950, Steinar Hauge proved that B. cereus is a human pathogen causing food poisoning - corn starch contained ~104 B. cereus spores per gram - demonstrated Koch’s postulates grew B. cereus to 4×106 cells per ml drank 200 ml of bacterial suspension 13 hrs later - symptoms of food poisoning started
9/21/2016 Dose Virulence Factors and Mechanisms of Pathogenicity ·Total dose ranges from5×104-5×10n Emetic toxin(cereulide)causes vomiting and cells nausea Total number of B.cereus required to cyclic dodecadepsipeptide contains three repeats of four amino acids produce enough toxin to cause illness is and or oxyacids:D-Oxy-Leu-D-Ala-L- Oxy-Val-L-Val Food containing 103 bacteria gram is potent cytotoxin that destroys mitochondria not safe to eat -ionophore with a high affinity to potassium cations -causes loss of the membrane potential and uncoupling of oxidative phosphorylation in the mitochondria Virulence Factors and Mechanisms of Cereulide Pathogenicity Enterotoxins cause diarrhea Table82 Properties of the emetic tooin cereulide -5 different proteins possible Trait (genome sequencing) 2 Antigenic point Detected through microslide diffusion tests Cytotaxic No Heat stobility 90 min at 121C pH stability Stablea p出2-11 In food (rice and milk)at 25-32C 5
9/21/2016 5 Dose • Total dose ranges from 5 x 104 - 5 x 1011 cells • Total number of B. cereus required to produce enough toxin to cause illness is 105 - 108 • Food containing > 103 bacteria / gram is not safe to eat Virulence Factors and Mechanisms of Pathogenicity Emetic toxin (cereulide) causes vomiting and nausea - cyclic dodecadepsipeptide - contains three repeats of four amino acids and / or oxyacids: D-Oxy-Leu—D-Ala—LOxy-Val—L-Val - potent cytotoxin that destroys mitochondria - ionophore with a high affinity to potassium cations - causes loss of the membrane potential and uncoupling of oxidative phosphorylation in the mitochondria Cereulide Virulence Factors and Mechanisms of Pathogenicity Enterotoxins cause diarrhea - 5 different proteins possible (genome sequencing) - Antigenic - Detected through microslide diffusion tests
9/21/2016 Three enterotoxins studied B.cereus causes disease other than One toxin CytK is a 34-kDa protein foodborne Produces:4 hemolysins,3 phospholipases,3 pore- B-barrel pore-forming toxin . dermonecrotic,cytotoxic and forming enterotoxins,emetic toxin,non-hemolytic haemolytic activities enterotoxin,and cytotoxin B cytotoxic to cell cultures Two enterotoxins are multicomponent and related lines Characterislic ·Hemolysin Hbl1 Respiralory infection consists of three proteins dermonecrotic and vascular permeability causes fluid accumulation in the rabbit ileal loop Vision-the model-diarrhea Non-hemolytic enterotoxin Nhe Consists of three proteins,all required for activity Gas gangrene Sepeis,exudate,tissue destructon Its Spore Summary Similar to spores in other microbes except: Bacillus cereus is a sporeformer that is More hydrophobic especially problematic in dairy foods Adhere to surfaces Cross section shows a It is a normal inhabitant of soil and is better central dark endospore isolated from a variety of foods Contains appendages or Two different toxins cause an emetic or pili involved in adhesion a diarrheal type of foodborne illness to surfaces or cells Difficult to remove and Desserts,meat dishes,and dairy la) target for disinfection products are frequently associated with Shadowed image of spore- diarrheal illness appendages and exosporium 6
9/21/2016 6 • One toxin CytK is a 34-kDa protein -barrel pore-forming toxin - dermonecrotic, cytotoxic and haemolytic activities - cytotoxic to cell cultures • Two enterotoxins are multicomponent and related • Hemolysin Hbl1 - consists of three proteins - dermonecrotic and vascular permeability - causes fluid accumulation in the rabbit ileal loop model – diarrhea • Non-hemolytic enterotoxin Nhe - Consists of three proteins, all required for activity Three enterotoxins studied B. cereus causes disease other than foodborne • Produces: 4 hemolysins, 3 phospholipases, 3 poreforming enterotoxins, emetic toxin, non-hemolytic enterotoxin, and cytotoxin B Its Spore Similar to spores in other microbes except: - More hydrophobic - Adhere to surfaces better - Contains appendages or pili involved in adhesion to surfaces or cells - Difficult to remove and target for disinfection Cross section shows a central dark endospore Shadowed image of spore - appendages and exosporium Summary • Bacillus cereus is a sporeformer that is especially problematic in dairy foods • It is a normal inhabitant of soil and is isolated from a variety of foods • Two different toxins cause an emetic or a diarrheal type of foodborne illness • Desserts, meat dishes, and dairy products are frequently associated with diarrheal illness
9/21/2016 Summary Rice and pasta are the most common vehicles of emetic illness Some strains of the B.cereus group grow at refrigeration temperatures Bacillus cereus foodborne illness is probably highly underreported 7
9/21/2016 7 Summary • Rice and pasta are the most common vehicles of emetic illness • Some strains of the B. cereus group grow at refrigeration temperatures • Bacillus cereus foodborne illness is probably highly underreported
