2/16/2016 Learning Objectives Food 1. Use basic biochemical characteristics to identify Micrbiology Escherichia coli 0157:H7 2.Understand what conditions in foods favor the growth of E coli0157:H7 Chapter 12 AN INTRODUCTION Enterohemorrhagic 4. Choose appropriate interventions to prevent the Escherichia coli growth of E coli0157:H7 5.Identify environmental sources of E coliO157:H7 6.Understand the roles of E.coli O157:H7 toxins and virulence factors in causing foodborne illness 7.Become familiar with newly emerging Shiga toxin- produ cing E.colistrains tes with creater than i cos States with and >10 cases Outbreak Outbreak Eating raw cookie dough Dutch-style Gouda cheese Summer 2009 November 2010 65 people became infected with Escherichia coli 157:H7 after eating Nestle Toll Hou 38 people from 5 states became infected with use cookie E.coli0157:H7 after eating Dutch-style Gouda dough cheese 25 people were hospitalized Strain was isolated from unopened packages 7 developed hemolytic-uremic syndrome (HUS) found at a store in New Mexico 70%of those ill were under 19 years old ·No deaths The only outbreak of E colij0157:H7 associated 1 person developed hemolytic-uremic syndrome with cookie dough (HU5) Solution?Wait until the cookies are cooked. Any type of food can carry this pathogen 2
2/16/2016 1 Chapter 12 Enterohemorrhagic Escher ch a col ichia coli Learning Objectives 1. Use basic biochemical characteristics to identify Escherichia coli O157:H7 2. Understand what conditions in foods favor the growth fo E li . co O157 H7 O157:H7 3. Recognize, from symptoms and time of onset, a case of foodborne illness caused by E. coli O157:H7 4. Choose appropriate interventions to prevent the growth of E. coli O157:H7 5. Identify environmental sources of E. coli O157:H7 6. Understand the roles of E. coli O157:H7 toxins and virulence factors in causing foodborne illness 7. Become familiar with newly emerging Shiga toxinproducing E. coli strains Outbreak Eating raw cookie dough States with greater than 1 case Summer 2009 • 65 people became infected with Escherichia coli O157:H7 after eating Nestle Toll House cookie dough • 25 people were hospitalized • 7 d l d h l developed hemolytic-uremic d (H ) syndrome (HUS) • 70% of those ill were under 19 years old • The only outbreak of E. coli O157:H7 associated with cookie dough • Solution? Wait until the cookies are cooked. Outbreak Dutch-style Gouda cheese States with 1-9 cases and >10 cases November 2010 • 38 people from 5 states became infected with E. coli O157:H7 after eating Dutch-style Gouda cheese • Strain was isolated from unopened packages found at a store in New Mexico • No deaths • 1 person developed hemolytic-uremic syndrome (HUS) • Any type of food can carry this pathogen
2/16/2016 01 Foods or food han- Outbreak dling practices implicated or the riskacqu Fresh spinach outbreaks Summer 2006 nderkd udbeef 206 people in 25 states 4量n量中中 became infected with E.coli0157:H7 after 融高 t行cured s0hT eating fresh spinach Lettuce ·3 people died Pruc rmmd Wild pigs captured in the area where the spinach was grown tested positive for the E.coliO157:H7 Ras幼spo,af6spo strain associated with the bagged spinach. FDA emphasized the need for good agricultural ate practices. Categories of Escherichia coli Escherichia coli Diarrheagenic E.colis categorized into groups based on All categories of E.colimay be shed in the virulence factors feces of infected humans. pathogenicity -spread to other humans clinical syndromes -spread to animals -distinct O:H serotypes -spread to the environment E.coli is differentiated by three major surface antigens >O-antigens Lipopolysaccharide >H-antigens Flagella >K-antigens Capsule 2
2/16/2016 2 Outbreak Fresh spinach Summer 2006 • 206 people in 25 states became infected with E. coli O157:H7 after eating fresh spinach • 3 p p eo le died • Wild pigs captured in the area where the spinach was grown tested positive for the E. coli O157:H7 strain associated with the bagged spinach. • FDA emphasized the need for good agricultural practices. Categories of Escherichia coli • Diarrheagenic E. coli is categorized into groups based on - vil fts irulence factors - pathogenicity - clinical syndromes - distinct O:H serotypes • E. coli is differentiated by j three major surface antigens O-antigens Lipopolysaccharide H-antigens Flagella K-antigens Capsule Escherichia coli • All categories of E. coli may be shed in the feces of infected humans. - spread to other humans - spread to animals - spread to the environment
2/16/2016 ETEC Enterotoxigenic E.coli Major cause of infantile diarrhea in developing countries Most frequently the cause of traveler's diarrhea Colonizes the small intestine via fimbrial adhesion factors Produces heat-labile or heat-stable enterotoxin that causes fluid accumulation and diarrhea Humans are the principle reservoir of strains that cause human illness Scanning electron microscopy image showing attachment of pathogenic E.co/ito human EIEC Enteroinvasive E.coli epithelial cells Causes non-bloody diarrhea and dysentery similar to Shigella Invades and multiplies within intestinal epithelial cells causing cell death A large virulence plasmid encodes several outer membrane proteins required for invasion Site of colonization is the colon Humans are the main reservoir Main serogroup is 0124
2/16/2016 3 ETEC Enterotoxigenic E. coli • Major cause of infantile diarrhea in develop g in countries • Most frequently the cause of traveler’s diarrhea • Colonizes the small intestine via fimbrial adhesion factors • Produces heat-labile or heat-stable enterotoxin that causes fluid accumulation and diarrhea • Humans are the principle reservoir of strains that cause human illness Scanning electron microscopy image showing attachment of pathogenic E. coli to human epithelial cells EIEC Enteroinvasive E. coli • Causes non-bloody diarrhea and dysentery similar to Shigella • Invades and multiplies within intestinal epithelial cells causing cell death • A large virulence plasmid encodes several outer membrane proteins required for invasion • Site of colonization is the colon • Humans are the main reservoir • Main serogroup is O124
2/16/2016 EPEC Enteropathogenic E.coli EAEC Enteroaggregative E coli Causes severe diarrhea Associated with persistent diarrhea in in infants infants and children ·Associated with Differ from other E.coli due to their outbreaks in nurseries Humans are main aggregative adherence pattern on cells reservoir Appears like stacked bricks on cell Induces lesions in cells Bacteria are attached to surfaces to which they adhere cup-like projections at A gene probe derived from a plasmid Invade epithelial cells the cell membrane. Localized destruction of associated with this strain is available microvilli (MV) EAEC Enteroaggregative E.coli DAEC Diffusely Adhering E.coli Associated with diarrhea in young children older than infants Risk increases with age from 1-5 years ·Why is unknown Mild diarrhea without blood Adheres randomly to epithelial cells Patterns of HeLa cell adherence of EAEC strains Does not produce enterotoxins or shiga toxins (A)Negative control (X400) (B)aggregative adherence(X1000)
2/16/2016 4 EPEC Enteropathogenic E. coli • Causes severe diarrhea in infants • Associated with outbreaks in nurseries • Humans are main reservoir • I d l i i ll Induces lesions in cells to which they adhere • Invade epithelial cells - Bacteria are attached to cup-like projections at the cell membrane. - Localized destruction of microvilli (MV) EAEC Enteroaggregative E. coli • Associated with persistent diarrhea in i f t d hild infants and children • Differ from other E. coli due to their aggregative adherence pattern on cells • Appears like stacked bricks on cell surfaces • A gene probe derived from a plasmid associated with this strain is available EAEC Enteroaggregative E. coli Patterns of HeLa cell adherence of EAEC strains (A) Negative control (X400) (B) aggregative adherence (X1000) DAEC Diffusely Adhering E. coli • Associated with diarrhea in young child ld th i f t hildren older than infants • Risk increases with age from 1-5 years • Why is unknown • Mild diarrhea without blood • Adh d l i h li l ll Adheres randomly to epithelial cells • Does not produce enterotoxins or shiga toxins
2/16/2016 EHEC Enterohemorrhagic E.coli Characteristics of E coli First recognized in 1982 E.coli 0157:H7 was identified as the cause of Common microflora in the intestines of two outbreaks of bloody diarrhea. humans and warm-blooded animals Since then,other EHEC serotypes(026,0111) Most strains are harmless are associated with bloody diarrhea. Some strains cause diarrheal disease Serotype 0157:H7 is the main cause of EHEC- associated diseases .167O-antigens,53 H-antigens,and 74 K antigens Non-O157-H7 serotypes are O26:H11,0111:H8. O-and H-antigens are used to serotype E and 0157:NM coljassociated with diarrheal disease Produce verotoxins or Stx toxins(toxin similar to Shiga toxin)that are cytotoxic to cells Characteristics of EHEC 0157:H7 Characteristics of EHEC 0157:H7 Serotype O157:HZ has unique characteristics Acid tolerance compared to other E.coli strains Unlike most foodborne pathogens,serotype Shiga-toxin production 0157:H7 is tolerant of acidic pH does not grow at temperatures >44.5C Minimum pH for growth is pH 4.0 to 4.5 does not ferment sorbitol within 24 h The induced acid-tolerant state may persist does not produce B-glucuronidase and thus for a prolonged period(=28 days)at refrigerated temperatures. cannot hydrolyze MUG -Induced acid tolerance increases tolerance to carries the attaching and effacing gene (eae) other environmental stresses such as heat, carries a large plasmid radiation,and antimicrobials >Alternative sigma factor RpoS induces >200 genes in response to stress
2/16/2016 5 EHEC Enterohemorrhagic E. coli • First recognized in 1982 • E. coli O157:H7 was identified as the cause of two outbreaks of bloody diarrhea. • Since then, other EHEC serotypes (O26, O111) are associated with bloody diarrhea. • Serotype O157:H7 is the main cause of EHECassociated diseases • Non-O157-H7 serotypes are O26:H11, O111:H8, and O157:NM • Produce verotoxins or Stx toxins (toxin similar to Shiga toxin) that are cytotoxic to cells Characteristics of E. coli • Common microflora in the intestines of humans and warm-blooded animals • Most strains are harmless • Some strains cause diarrheal disease • 167 O-antigens, 53 H-antigens, and 74 K antigens • O- and H-antigens are used to serotype E. coli associated with diarrheal disease Characteristics of EHEC O157:H7 • Serotype O157:H7 has unique characteristics compared to other E. coli strains - Shiga-toxin production - does not grow at temperatures >44.5oC - does not ferment sorbitol within 24 h - does not produce -glucuronidase and thus cannot hydrolyze MUG - carries the attaching and effacing gene (eae) - carries a large plasmid Characteristics of EHEC O157:H7 • Acid tolerance - Unlike most foodborne pathogens, serotype O157:H7 is tolerant of acidic pH - Minimum pH for growth is pH 4.0 to 4.5 - The induced acid-tolerant state may persist for a prolonged period ( ≥ 28 days) at refrigerated temperatures. - I d d id t l I n duced acid t olerance i tl t increases t olerance t o other environmental stresses such as heat, radiation, and antimicrobials Alternative sigma factor RpoS induces >200 genes in response to stress
2/16/2016 Characteristics of EHEC O157:H7 Characteristics of EHEC 0157:H7 Antibiotic resistance Now,it is resistant to multiple antibiotics Inactivation by heat Non-0157:H7 strains are also resistant to Not more heat resistant than other many antibiotics used in human and animal pathogens medicine Fat in foods(hamburger meat)protects Antibiotic-resistant strains possess a growth 0157:H7 from heat advantage over other bacteria that colonize Critical Control Point to inactivate 0157:H7 the intestines of animals that are treated >heat food to an internal temperature of at therapeutically or sub-therapeutically. least 68.3C for several seconds Antibiotic-resistant strains may become the predominant strains during antibiotic therapy. Characteristics of EHEC 0157:H7 Reservoirs of E.coliO157:H7 Inactivation by irradiation Reservoir of infection:Any person,animal,plant, -Irradiation of food is approved to eliminate soil or substance in which an infectious agent normally lives and multiplies. foodborne pathogens Irradiation eliminates pathogens while -The reservoir typically harbors the maintaining the raw character of foods infectious agent without injury to itself and serves as a source from which other -Irradiation is approved to use on refrigerated individuals can be infected. and frozen raw ground beef The infectious agent primarily depends on the reservoir for its survival. Radura,symbol required on all food exposed to UV -The reservoir transmits the infectious radiation during processing substance to a human or another susceptible host. 6
2/16/2016 6 Characteristics of EHEC O157:H7 • Antibiotic resistance - Now, it is resistant to multiple antibiotics - Non-O157 H7 t i l i t t t O157:H7 strains are also resistant to many antibiotics used in human and animal medicine - Antibiotic-resistant strains possess a growth advantage over other bacteria that colonize the intestines of animals that are treated therapeutically or sub-therapeutically. - Antibiotic-resistant strains may become the predominant strains during antibiotic therapy. Characteristics of EHEC O157:H7 • Inactivation by heat - Not more heat resistant than other pathogens - Fat in foods (hamburger meat) protects O157:H7 from heat - Critical Control Point to inactivate O157:H7 heat food to an internal temperature of at least 68.3oC for several seconds Characteristics of EHEC O157:H7 • Inactivation by irradiation - Irradiation of food is approved to eliminate foodborne pathogens - Irradiation eliminates pathogens while maintaining the raw character of foods - Irradiation is approved to use on refrigerated and frozen raw ground beef Radura, a symbol required on all food exposed to UV radiation during processing Reservoirs of E. coli O157:H7 Reservoir of infection: Any person, animal, plant, soil or substance in which an infectious agent normally lives and multiplies normally lives and multiplies. - The reservoir typically harbors the infectious agent without injury to itself and serves as a source from which other individuals can be infected. - Th i f ti t i il d d The infectious agent primarily depends on the reservoir for its survival. - The reservoir transmits the infectious substance to a human or another susceptible host
2/16/2016 Reservoirs of E.coliO157:H7 Reservoirs of E.coli O157:H7 Cattle-On the Farm Cattle-On the Farm Undercooked beef and unpasteurized Young,weaned animals carry 0157:H7 milk have been the source of many more often than adult cattle O157:H7 outbreaks An increase in cattle that test positive Rates of EHEC carriage in herds is occurs during summer months commonly 10-25%but can be up to 60% Agrees with the seasonal variation in Non-0157:H7 isolated more often than human disease 0157:H7 Farm-To-Fork Livestock E.coli Route of Transmission Reservoirs of E.coli O157:H7 Factors associated with Bovine carriage Association between fecal shedding and feed or environmental factors Feed that contains whole cottonseed is linked with a reduction of 0157:H7 An increase in 0157:H7 is linked with keeping calves in groups before weaning. sharing of feeding utensils without Dairy sanitation,early feeding of grain
2/16/2016 7 Reservoirs of E. coli O157:H7 Cattle – On the Farm • Undercooked beef and unpasteurized milk have been the source of many O157:H7 outbreaks • Rates of EHEC carriage in herds is commonly 10-25% but can be up to 60% • Non-O157:H7 isolated more often than O157:H7 Reservoirs of E. coli O157:H7 Cattle – On the Farm • Young, weaned animals carry O157:H7 more often than adult cattle • An increase in cattle that test positive occurs during summer months • Agrees with the seasonal variation in human disease Reservoirs of E. coli O157:H7 Factors associated with Bovine carriage • A i i b f l h ddi d Association between fecal shedding and feed or environmental factors - Feed that contains whole cottonseed is linked with a reduction of O157:H7 - An increase in O157:H7 is linked with keeping calves in groups before weaning, sharing of feeding utensils without sanitation, early feeding of grain
2/16/2016 Reservoirs of E.coliO157:H7 Reservoirs of E.coliO157:H7 Factors associated with Bovine carriage Factors associated with Bovine carriage Susceptibility of cattle to intestinal colonization Water,feed sources,and farm management likely play an important role is a function of age -0157:H7 is frequently found in water young animals are more likely to be positive than older animals in the same herd troughs -0157:H7 can survive for weeks or 0157:H7 is transiently carried in the months in bovine feces and water intestines and is intermittently excreted for a few weeks to months by young calves and heifers Cows can carry more than one strain Reservoirs of E.coliO157:H7 Cattle model for infection by 0157:H7 0157:H7 is not a pathogen of weaned calves and adult cattle But,0157:H7 can cause diarrhea and lesions in newborn calves
2/16/2016 8 Reservoirs of E. coli O157:H7 Factors associated with Bovine carriage • Water, feed sources, and farm management likely play an important role - O157:H7 is frequently found in water troughs - O157:H7 can survive for weeks or months in bovine feces and water Reservoirs of E. coli O157:H7 Factors associated with Bovine carriage • Suscept Suscept b l ty of cattle to ntest nal colon zat on ibility of cattle to intestinal colonization is a function of age - Young animals are more likely to be positive than older animals in the same herd - O157:H7 is transiently carried in the intestines and is intermittently excreted for a few weeks to months by young calves and heifers - Cows can carry more than one strain Reservoirs of E. coli O157:H7 Cattle model for infection by O157:H7 • O157 H7 i h f O157:H7 is not a pathogen of weaned calves and adult cattle • But, O157:H7 can cause diarrhea and lesions in newborn calves
2/16/2016 Reservoirs of E.coli O157:H7 Reservoirs of E.coli O157:H7 Domestic animals and wildlife Humans EHEC strains have been isolated from Fecal shedding of 0157:H7 by patients other domestic animals and wildlife with hemorrhagic colitis or HUS lasts 13- 21 days or even longer -Sheep,goat,deer,dog,horse,swine, An asymptomatic carrier state in humans cat,seagull,and rat has not been identified Prevalence in sheep is higher than other Person to person transmission animals Fewer than 100 cells is needed to cause 0157:H7 in feral animals contaminates illness food crops in the field or home gardens Poor personal hygiene can transmit the bacteria to others Disease Outbreaks Disease Outbreaks Geographic distribution Geographic distribution 30 countries on 6 continents have reported ·CDC estimates 0157:H7 outbreaks -0157:H7 causes 63,153 illnesses and 26 In the US,350 outbreaks were documented in deaths annually in the US 49 states from 1982-2002 Non-0157 STEC strains account for an Number of outbreaks has increased but the size additional 127,757 illnesses and 20 deaths has decreased annually ·improved detection 75%of these cases are due to foodborne notifiable disease since 1994 transmission
2/16/2016 9 Reservoirs of E. coli O157:H7 Domestic animals and wildlife • EHEC strains have been isolated from other domestic animals and wildlife - Sheep, goat, deer, dog, horse, swine, cat, seagull, and rat • Prevalence in sheep is higher than other animals • O157:H7 in feral animals contaminates food crops in the field or home gardens Reservoirs of E. coli O157:H7 Humans • Fecal shedding yp of O157:H7 by patients with hemorrhagic colitis or HUS lasts 13- 21 days or even longer • An asymptomatic carrier state in humans has not been identified • Person to person transmission • Fewer than 100 cells is needed to cause illness • Poor personal hygiene can transmit the bacteria to others Disease Outbreaks Geographic distribution • 30 countries on 6 continents have reported O157:H7 outbreaks • In the US, 350 outbreaks were documented in 49 states from 1982-2002 • Number of outbreaks has increased but the size h d d as ecreased - improved detection - notifiable disease since 1994 Disease Outbreaks Geographic distribution • CDC estimates - O157:H7 causes 63,153 illnesses and 26 deaths annually in the US - Non-O157 STEC strains account for an additional 127,757 illnesses and 20 deaths annually - 75% of these cases are due to foodborne transmission
2/16/2016 Disease Outbreaks Disease Outbreaks Age of patients Seasonality of 0157:H7 infection All ages are susceptible Outbreaks and clusters of infection peak during Very young and elderly have severe illness with the summer months complications 89%of outbreaks occur between May-November HUS occurs in children Reasons are unknown but may include Thrombotic thrombocytopenic purpura(TTP) occurs in adults increased prevalence in cattle Children 2-10 years of age have a higher rate of greater exposure to ground beef or other infection contaminated foods undeveloped hygiene skills increased temperature abuse and cross- undeveloped immune system contamination increased exposure increased person-person contact Disease Outbreaks Tbe Foods or food han- Disease Outbreaks dling practices implicated or Transmission of 0157:H7 suspected of being associated Example of a foodborne outbreak with Escherichia coli0157:H7 Many foods are vehicles First documented outbreak occurred in Oregon outbreaks of transmission in1982 Ground beef is the most Undercooke ground beef 26 cases and 19 hospitalized frequent Bloody diarrhea with severe abdominal pain Contact of food with meat Eating undercooked hamburgers from a fast- Dry cured sabm or feces is the likely food chain source of cross- Lettuce Procemi 0157:H7 was recovered from the stools of contamination patients Outbreaks attributed to Second outbreak occurred 3-months later at the person-person same fast-food chain in Michigan from frozen transmission and hamburger patties waterborne transmission 10
2/16/2016 10 Disease Outbreaks Seasonality of O157:H7 infection • Outbreaks and clusters of infection p g eak durin g the summer months • 89% of outbreaks occur between May-November • Reasons are unknown but may include - increased prevalence in cattle - greater exposure to ground beef or other contaminated foods - increased temperature abuse and crosscontamination Disease Outbreaks Age of patients • All ages are susceptible • Very young and elderly have severe illness with complications • HUS occurs in children • Thrombotic thrombocytopenic purpura (TTP) occurs in adults • Children 2- 10 yg g ears of a ge have a hi gher rate of infection - undeveloped hygiene skills - undeveloped immune system - increased exposure - increased person-person contact Disease Outbreaks Transmission of O157:H7 • Many foods are vehicles of transmission • Ground beef is the most frequent • Contact of food with meat or feces is the likely source of crosscontamination • Outbreaks attributed to person-person transmission and waterborne transmission Disease Outbreaks Example of a foodborne outbreak • First documented outbreak occurred in Oregon in 1982 • 26 cases and 19 hospitalized • Bloody diarrhea with severe abdominal pain • Eating undercooked hamburgers from a fastfood chain • O157:H7 was recovered from the stools of patients • Second outbreak occurred 3-months later at the same fast-food chain in Michigan from frozen hamburger patties
