9/18/2016 Learning Objectives Food 1.Use basic biochemical characteristics to identify Clostridium bot九ulim 2.Understand what conditions in foods favor the Chapter 10 AN INTRODUCTION growth of C.botulinum 3.Recognize.from symptoms and time of onset, case of botulism Clostridium botulinum 4.Choose appropriate interventions to prevent the growth of C.botulinum 5.Identify environmental sources of C.botu/inum 6.Understand the roles of spore,anaerobic conditions,and heat sensitivity of the toxin in causing or preventing botulism Botulism is a rare but deadly Self-Inflicted Botulism disease 116 outbreaks in botulism in US between 1999-2008 Average number of cases ·Outbreak1 reported to CDC yearly home-canned carrots and green beans 24 cases of foodborne same day 2 on ventilators,next day 1 on ventilator,1 milder symptoms,2 okay botulism ·one only ate a forkful 3 cases of wound botulism toxin isolated from patients and food -71 cases of intestinal ·Outbreak2 "infant"botulism home-canned green beans,tomatoes,pears Tasted bad but ate them anyway 40%of cases are due to ·Outbreak3 improper home canning home-canned asparagus no sign of spoilage but the lid had popped 义
9/18/2016 1 Chapter 10 Clostridium botul num i Learning Objectives 1. Use basic biochemical characteristics to identify Clostridium botulinum 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. botulinum 3. Recognize, from symptoms and time of onset, a case of botulism 4. Choose appropriate interventions to prevent the growth of C botulinum C. botulinum 5. Identify environmental sources of C. botulinum 6. Understand the roles of spore, anaerobic conditions, and heat sensitivity of the toxin in causing or preventing botulism • Botulism is a rare but deadly disease • Average number of cases reported to CDC yearly - 24 cases of foodborne botulism - 3 cases of wound botulism - 71 cases of intestinal “infant” botulism • 40% of cases are due to improper home canning Self-Inflicted Botulism 116 outbreaks in botulism in US between 1999-2008 • Outbreak 1 - home-canned carrots and green beans - same day 2 on ventilators, next day 1 on ventilator, 1 milder symptoms, 2 okay - one only ate a forkful - toxin isolated from patients and food • Outbreak 2 - home-canned green beans, tomatoes, pears - Tasted bad but ate them anyway • Outbreak 3 - home-canned asparagus - no sign of spoilage but the lid had popped
9/18/2016 Range of symptoms typical of botulism -Nausea Four Faces of Botulism ·Vomiting 1.Foodborne botulism ·Dry mouth Traditionally associated with canned foods,partic Neurological issues such as blurred vision.bilateral home-canned foods paralysis,slurred speech,fatigue,difficulty Commercial cases (9%)usually involve small companies swallowing and breathing,dizziness,change in speech 1971,FDA released a warning after learning that a New york man and his wife became seriously ill from Root of the cause botulism after eating a can of Bon Vivant vichyssoise Failure to use proper pressure cooker processes SOUD >50-yr old procedures >FDA found the company's processing practices >Neighbor's advice questionable and ordered a shutdown of the company's plant and destruction of all products >Ignored correct instruction due to a hot kitchen >5 of 324 cans were contaminated with botulinum >Ignorant of risk associated with botulism toxin >Ignored signs of spoilage Investigation revealed that workers misunderstood how to operate the canning Foodborne botulism is often associated machines with temperature abuse In response,FDA instituted good Spikes in botulism due to temp abuse manufacturing practices (GMPs)for low- acid foods (pH 4.6 and water activity 0.86 -Canning machine operators are certified Changes in processing schedules is done by processing authorifies
9/18/2016 2 • Range of symptoms typical of botulism - Nausea - Vomiting - Dry mouth - Neurological issues such as blurred vision, bilateral paralysis, slurred speech, fatigue, difficulty swallowing and breathing, dizziness, change in speech • Root of the cause - Failure to use proper pressure cooker processes 50-yr ld d o procedures Neighbor’s advice Ignored correct instruction due to a hot kitchen Ignorant of risk associated with botulism Ignored signs of spoilage Four Faces of Botulism 1. Foodborne botulism Traditionally associated with canned foods, particularly home-canned foods - Commercial cases (9%) usually involve small companies 1971, FDA released a warning after learning that a New York man and his wife became seriously ill from botulism after eating a can of Bon Vivant vichyssoise soup FDA found the company FDA found the company s processing practices 's processing practices questionable and ordered a shutdown of the company's plant and destruction of all products 5 of 324 cans were contaminated with botulinum toxin - Investigation revealed that workers misunderstood how to operate the canning machines - In response, FDA instituted good manuf i i (GMP ) f l facturing practices (GMPs) for lowacid foods (pH > 4.6 and water activity > 0.86 - Canning machine op f erators are certified - Changes in processing schedules is done by processing authorities • Foodborne botulism is often associated with temperature abuse - Spikes in botulism due to temp abuse
9/18/2016 Large outbreak in Clovis,New Mexico 2.Infant botulism/intestinal botulism >1978,40 people who ate at a local salad bar Infants are not thriving,cannot lift began showing various signs of paralysis themselves up,and have poor muscle tone >Potato salad implicated after FDA investigation -Deaths resemble sudden death syndrome >Leftover potatoes stored in a box at room temp Associated with eating raw agricultural products like honey >When enough accumulate,they are used in potato salad Infants have a less diverse intestinal microbiota that protects adults from without heating consumed spores >Perfect scenario Immunocompromised or antibiotic o Spores found on potatoes treatment lead to intestinal botulism in o Heating kills vegetative cells and drives off adults oxygen Recommended that children under the age o Spores germinate and toxin is produced of 2 not be feed raw agricultural products 3.Wound botulism is not foodborne but 4.Agent of bioterrorism can still be fatal .2003,spore-laden letter sent to politicians that contained Bacillus Spores are introduced into the body anthracis tissue below the skin Drug users injecting with ."Select agents"are now under strict governmental regulation contaminated heroin =a ) 言三三化。&惑 29500
9/18/2016 3 - Large outbreak in Clovis, New Mexico 1978, 40 people who ate at a local salad bar began showing various signs of paralysis Potato salad implicated after FDA investigation Leftover potatoes stored in a box at room temp When enough accumulate, they are used in potato salad without heating Perfect scenario o Spores found on potatoes o Heating kills vegetative cells and drives off oxygen o Spores germinate and toxin is produced 2. Infant botulism / intestinal botulism - Infants are not thriving, cannot lift themselves up, and have poor muscle tone - Deaths resemble sudden death syndrome - A i t d ith ti i lt l Associated with eating raw agricultural products like honey - Infants have a less diverse intestinal microbiota that protects adults from consumed spores - Immunocompromised or antibiotic treatment lead to intestinal botulism in adults - Recommended that children under the age of 2 not be feed raw agricultural products 3. Wound botulism is not foodborne but can still be fatal • Spores are introduced into the body tissue below the skin • Drug users injecting with contaminated heroin 4. Agent of bioterrorism • 2003, spore-laden letter sent to politicians that contained Bacillus anthracis • “Select agents” are now under strict governmental regulation
9/18/2016 ·Select agents must be Biosafety Levels -inventoried Biosafety levels range from BSL-1 to BSL-4 -kept locked in a room assessable only microbes do not consimimol poreease rd tn me m。po in healtl to people cleared by the Dept of Justice BSL-2-microbes pose moderate hazards to lab -handled using BSL-3 level work workers and the environment and are associated practices High Riak Mlernes with diseases of varying severity BSL-3-microbes cause serious or potentially lethal disease through respiratory transmission BSL-4-microbes are dangerous and exotic,posing 85L2 BSL-1 freatment or vaccines. Disease Characteristics Mild to serious disease that can kill within a day Symptoms 12-36 h but up to 14 days following ingestion of the neurotoxin Earlier the symptoms the more severe the disease First-nausea and vomiting -Then-neurological symptoms >double vision,drooping eyelids >loss of normal mouth and throat function >general fatigue >lack of muscle coordination >respiratory impairment-main cause of death
9/18/2016 4 • Select agents must be - inventoried - kept locked in a room assessable only to people cleared by the Dept of Justice - handled using BSL-3 level work practices Biosafety Levels Biosafety levels range from BSL-1 to BSL-4 • BSL-1 - microbes do not consistently cause disease in healthy adults and present minimal potential hazard to lab workers and the environment • BSL-2 – microbes pose moderate hazards to lab workers and the environment and are associated with diseases of varying severity • BSL-3 – microbes cause serious or potentially lethal disease through respiratory transmission • BSL-4 – microbes are dangerous and exotic, posing a high risk of aerosol-transmitted infections. Infections are frequently fatal and without treatment or vaccines. Disease Characteristics • Mild to serious disease that can kill within a day • Symptoms - 12-36 h but up to 14 days following ingestion of the neurotoxin - Earlier the symptoms the more severe the disease - First – nausea and vomiting - Then – neurological symptoms double vision, droopgy in e elids loss of normal mouth and throat function general fatigue lack of muscle coordination respiratory impairment – main cause of death
9/18/2016 Disease Characteristics Often incorrectly diagnosed as another Infant botulism starts with abdominal pain disease,most commonly Guillain-Barre -diarrhea syndrome constipation 女女 Botulinum toxin as agent of biowarfare ·Fatality rates -not enough ventilators or antitoxin -early1900s>50% nationwide Present day about 10% ·Treatment >if attack involves 40,000 antisera targeting the >only 4,000 ventilators available toxin respiratory support systems Botulinum Toxin as a Weapon Treatment Initial treatment involves inactivation of the Favoring Use as a Weapon Limiting Use as a Weapon neurotoxin ·Available in nature ·Not contagious 1.Neutralizing circulating toxin with antiserum ·Spread as an aerosol ·Production takes 2 Enemas to remove residual neurotoxin from the skill bowel Broken down by 3.Gastric lavage(stomach pump)or treatment ·Highly lethal heat and sunlight with emetics to remove remaining food ·Labile in normal Antiserum is most effective in the early stages atmosphere (1%per Iraq admitted to producing 3x Subsequent treatment is mainly ventilators to min) the amount of toxin required to counteract paralysis of the respiratory muscles kill the entire human population Infant botulism-mainly high-quality supportive care 9
9/18/2016 5 Disease Characteristics • Infant botulism starts with - abdominal pain - diarrhea - constipation • Fatality rates - early 1900s > 50% - Present day about 10% • Treatment - antisera targeting the toxin - respiratory support systems - 3-4 months on a ventilator and years of therapy to fully recover • Often incorrectly diagnosed as another disease, most commonly Guillain-Barré syndrome • Botulinum toxin as agent of biowarfare - not enough ventilators or antitoxin nationwide if attack involves 40,000 onl 4 000 l l bl ly 4,000 ventilators availabl e Botulinum Toxin as a Weapon Favoring Use as a Weapon • Available in nature Limiting Use as a Weapon • Not conta gious • Spread as an aerosol • Spread in food or water • Highly lethal g • Production takes skill • Broken down by heat and sunlight • Labile in normal atmosph ( % here (1% per min) Iraq admitted to producing 3x the amount of toxin required to kill the entire human population Treatment • Initial treatment involves inactivation of the neurotoxin 1. Neutralizing g circulating toxin with antiserum 2. Enemas to remove residual neurotoxin from the bowel 3. Gastric lavage (stomach pump) or treatment with emetics to remove remaining food • Antiserum is most effective in the early stages • Subsequent treatment is mainly ventilators to counteract paralysis of the respiratory muscles • Infant botulism – mainly high-quality supportive care
9/18/2016 Toxic Doses Characteristics of C.botulinum Little is known about the minimal Classification toxic dose or the efficacy of immunization Gram-positive,rod-shaped,obligate anaerobe (oxygen kills it) -Cultivation requires anaerobic chambers ·Botulinum toxin Forms spores in stationary-phase cultures Most toxic natural substance known 50%lethal dose in monkeys is 0.4 ng kg of body weight For a 150 Ib human,this mears 0.000000001 ounce is enough Iraq had produced 50 gallons of concentrated botulinum but no weapons were ever found Characteristics of C.botulinum Botulinum Toxin Types,Target Sites,Discoverers,and Year Classification Discovered ·7 types of toxins A-G Type Target Discoverer Year SNAP-25 Landman 1904 -human botulism due to types A,B.E, VAMP Ermengem 1897 and rarely F C1 Syntaxin Bergston and Seldon 1922 -types C and D cause botulism in 0 VAMP Robinson 1929 animals E SNAP-25 Gunnison 1936 type G is not linked to disease VAMP Moller and Scheibel 1960 VAMP Gimenez and Ciccarelli 1970 6
9/18/2016 6 Toxic Doses • Little is known about the minimal toxic dose or the efficacy of immunization • Food safety perspective – no tolerance for any neurotoxin or organism producing such • Botulinum toxin - Most toxic natural substance known - 50% lethal dose in monkeys is 0.4 ng / kg y of body weight - For a 150 lb human, this means 0.000000001 ounce is enough • Iraq had produced 50 gallons of concentrated botulinum but no weapons were ever found Characteristics of C. botulinum Classification • Gram-positive, rod-shaped, obligate anaerobe (oxygen kills it) - Cultivation requires anaerobic chambers • Forms spores in stationary-phase cultures Characteristics of C. botulinum Classification • 7 types of toxins A – G - human botulism due to types A,B,E, and rarely F - types C and D cause botulism in animals - type G is not linked to disease Botulinum Toxin Types, Target Sites, Discoverers, and Year Discovered Type Target Discoverer Year A SNAP-25 Landman 1904 B VAMP Ermengem 1897 C1 Syntaxin Bengston and Seldon 1922 D VAMP Robinson 1929 E SNAP-25 G nnis n Gunnison 1936 F VAMP Moller and Scheibel 1960 G VAMP Gimenez and Ciccarelli 1970
9/18/2016 Grouping based on physiological differences Characteristics of C.botulinum Group I strains-proteolytic Tolerance to preservation methods produce high levels of toxin spores have high heat resistance Main factors controlling growth -Temperature Group II strains-nonproteolytic -pH Both cause human disease -Water activity 他0tm图ed5ra+oWt中aCn -Oxidation-reduction potential Preservatives -Competing microbes Used alone are rarely effective Used in synergy have additive effects Multiple hurdle effect Characteristics of C.botulinum Characteristics of C.botulinum Low Temperature Thermal Inactivation ·Thermal processing Refrigeration prevents growth ·inactivates spores lower limit for Group I-10C common way to produce shelf-stable foods -lower limit for Group II-30C ·6 roup I spores depends on other growth conditions very heat tolerant Neurotoxin production requires Di2rc values 0.1-0.2 min weeks at the low temp limits ·Optimum growth temp Industry standard is 0.2 min at 121C Group I-35C-40C ·Group II spores Group II-
9/18/2016 7 Grouping based on physiological differences • Group I strains – proteolytic - produce high levels of toxin - s p g ores have hi gh heat resistance • Group II strains – nonproteolytic • Both cause human disease Characteristics of C. botulinum Tolerance to preservation methods • Main factors controlling growth - Temperature - pH - Water activity - Oxidation-reduction potential - Preservatives - Competing b micro bes • Used alone are rarely effective • Used in synergy have additive effects • Multiple hurdle effect Characteristics of C. botulinum Low Temperature • Refrigeration prevents growth - l limit f G p I lower limit for Group I – 10oC - lower limit for Group II - 3oC - depends on other growth conditions • Neurotoxin production requires weeks at the low tem p limits • Optimum growth temp - Group I – 35oC - 40oC - Group II - < 25oC Characteristics of C. botulinum Thermal Inactivation • Thermal processing - inactivates spores - common way to produce shelf-stable foods • Group I spores - very heat tolerant - D121o C values 0.1 - 0.2 min - main target for sterilization of canning lowacid f d oo ds - Industry standard is 0.2 min at 121oC • Group II spores - inactivation at moderate temperatures 40- 50oC when combined with high pressure
9/18/2016 Characteristics of C.botulinum Characteristics of C.botulinum pH Salt and water activity ·Minimum pH Sodium chloride acts by decreasing Group I-pH4.6 the water activity Group II-pH5 Concentration of salt in the water Most fruits and vegetables are acidic phase called brine concentration is enough to inhibit C.botulinum critical Other growth conditions affect the NaCl,KCl,glucose and sucrose give pH growth response similar effects Molds and yeasts may raise the pH so Acidity and preservatives may also that C.botulinum growth is allowed affect the water activity Characteristics of C.botulinum UltraZapXtendaPak Carbon dioxide is used in Active pad contains modified-atmosphere Oxygen and redox potential additives that pro ce Amount of oxygen is not the critical variable carbon dioxide,which has packaging Oxygen-reduction potential is the critical variable bacteriostatic properties Strict anaerobes are generally active at negative Moisture from the packaged protein is redox values absorbed by the Most foods are low enough to permit growth UltraZapXtendaPak Little data-difficult to measure cellulose pad When foods are heated. A steady stream of CO2 gas is produced inside the -dissolved oxygen is driven out package oxygen diffuses slowly back in Pressurized CO2stimulates food remains anaerobic for hours C.botulinum arowth
9/18/2016 8 Characteristics of C. botulinum pH • Minimum pH - Group I – pH 4.6 - Group II – pH 5 • Most fruits and vegetables are acidic enough to inhibit C. botulinum • Oth th diti ff t th Other growth conditions affect th e pH growth response • Molds and yeasts may raise the pH so that C. botulinum growth is allowed Characteristics of C. botulinum Salt and water activity • Sodium chloride acts by decreasing th i i h e water activity • Concentration of salt in the water phase called brine concentration is critical • NaCl , KCl, glucose and sucrose give glucose and sucrose give similar effects • Acidity and preservatives may also affect the water activity Characteristics of C. botulinum Oxygen and redox potential • Amount of oxygen is not the critical variable • Oxygen-reduction potential is the critical variable • Strict anaerobes are generally active at negative redox values • Most foods are low enough to permit growth • Little data – difficult to measure • When foods are heated, - dissolved oxygen is driven out - oxygen diffuses slowly back in - food remains anaerobic for hours UltraZapXtendaPak • Active pad contains additives that produce carbon dioxide, which has bacteriostatic properties Carbon dioxide is used in modified-atmosphere packaging p p • Moisture from the packaged protein is absorbed by the UltraZapXtendaPak cellulose pad • A steady stream of CO gas 2 is produced inside the package • Pressurized CO2 stimulates C. botulinum growth
9/18/2016 Characteristics of C.botulinum Characteristics of C.botulinum Preservatives C美NG Competitive and Growth-Enhancing Microbes Nitrite used in cured meats to inhibit C. botulinum Promote growth of Inhibit growth of C. C.botulinum bo九lUm Effectiveness depends upon pH.NaCl, heat,time,and temperature of storage, ·Acid-tolerant ·Microbes lower the and composition of food molds increase the pH of foods pH of acidic foods ·Microbes produce Inhibits by more than one mechanism bacteriocins such as reacting with iron-sulfur Cladosporium, ·Lactobacillus proteins needed for energy production Penicillium Streptococcus. Numerous other compounds used as food Pediococcus preservatives also inhibit growth Wisconsin Process Characteristics of C.botulinum Developed to produce bacon as antibotulinal as regular bacon but with lower sodium nitrite Inactivation by Irradiation Uses 40-80 ppm sodium nitrite(120 ppm C.botulinum spores are the most regular bacon),Pediococcus acidilactici and 0.7%sucrose irradiation resistant spores of public health concern When temperature abused >P.acidilactici(probiotic)grows Industrial canning is designed to reduce o ferments sucrose producing acid the number of viable C botulinum spores o produces bacteriocins Spores in the presence of oxygen, >C.botulinum growth is inhibited preservatives,and temperatures>20C are usually more irradiation sensitive
9/18/2016 9 Characteristics of C. botulinum Preservatives • Nitrite used in cured meats to inhibit C. botulinum • Effectiveness depends upon pH, NaCl, heat, time, and temperature of storage, and composition of food • Inhibits by more than one mechanism such as reacting with iron-sulfur proteins needed for energy production • Numerous other compounds used as food preservatives also inhibit growth Characteristics of C. botulinum Competitive and Growth-Enhancing Microbes P h f Promote growth of I hibit th f C C. botulinum • Acid-tolerant molds increase the pH of acidic foods I nhibit growth of C. botulinum • Microbes lower the pH of foods • Microbes produce bacteriocins • Cladosporium, Penicillium • Lactobacillus, Streptococcus, Pediococcus Wisconsin Process • Developed to produce bacon as antibotulinal as regular bacon but with lower sodium nitrite • Uses 40 Uses 40-80 ppm sodium nitrite (120 ppm regular bacon), Pediococcus acidilactici and 0.7% sucrose • When temperature abused P. acidilactici (probiotic) grows o f t d i id ferments sucrose pro ducing acid o produces bacteriocins C. botulinum growth is inhibited Characteristics of C. botulinum Inactivation by Irradiation • C botulinum C. botulinum spores are the most irradiation resistant spores of public health concern • Industrial canning is designed to reduce the nu m f ber o f viable C. botulinum s pores • Spores in the presence of oxygen, preservatives, and temperatures > 20oC are usually more irradiation sensitive
9/18/2016 Sources of C.botulinum Sources of C.botulinum Occurrence in the environment Occurrence in food -Common in soils and sediments -Food surveys focus mostly on meats,fish Number and types vary with locations and infant foods MPN-most probable rumber MPN-most probable rumber 0000000 Sources of C.botulinum Virulence Factors and Mechanisms of ·Occurrence in food Pathogenicity Spores can contaminate fruits and vegetables particularly those that come into contact with Botulinum toxin is called the most poisonous the soil poison known Overall incidence of C botulinum in precut .C.botulinum produces 8 antigenically distinct vegetables is low -0.36% toxins Mushrooms are of concern A,B.C1.C2.D.E,F,and G >grown in compost All are neurotoxins except C2 >favorable to botulinal growth All are similar in structure and mode of >Contain 2,100 type B spores per kg action >Packaging has holes to allow oxygen flow All are heat sensitive >Packaging uses films that allow high oxygen Inactivated by-heating a sample to 80C diffusion for 10 min or bring to a boil completely 9
9/18/2016 10 Sources of C. botulinum • Occurrence in the environment - Common in soils and sediments - Number and types vary with locations MPN – most probable number Sources of C. botulinum • Occurrence in food - Food surveys focus mostly on meats, fish, and infant foods MPN – most probable number Sources of C. botulinum • Occurrence in food - Spores can contaminate fruits and vegetables particularly those that come into contact with th il e so - Overall incidence of C. botulinum in precut vegetables is low – 0.36% - Mushrooms are of concern grown in compost f bl t b t li l th favorable to botulinal growth Contain 2,100 type B spores per kg Packaging has holes to allow oxygen flow Packaging uses films that allow high oxygen diffusion Virulence Factors and Mechanisms of Pathogenicity • Botulinum toxin is called the most poisonous poison known • C. botulinum produces 8 antigenically distinct toxins - A, B, C1, C2, D, E, F, and G - All are neurotoxins except C2 - All are similar in structure and mode of action - All are heat sensitive - Inactivated by - heating a sample to 80oC for 10 min or bring to a boil completely