3/13/2016 Learning Objectives Food 1.Use basic biochemical characteristics to identify Staphylococcus aureus 2.Understand what conditions in foods favor 5. aureus growth Chapter 16 AN INTRODUCTION 3.Recognize,from symptoms and time of onset,a case of foodborne illness caused by5.aureus Staphylococcus 4.Choose appropriate interventions(heat, aureus preservatives,and formulations)to prevent the growth of 5.aureus 5.Identify sources of 5.aureus 6.Understand the roles of staphylococcal enterotoxins in causing foodborne illness Outbreak Outbreak Flight of horror Flight of horror Flight left Alaska and passengers were served breakfast .80%of passengers had diarrhea Breakfast was an omelet with ham and .70%of passengers had vomiting cheese ·85%had nausea About 30 min after eating,a passenger The"discomfort bags"ran out rushed to the rear of the plane vomiting on passengers before reaching the toilet After landing,rectal swabs were done Most of the passengers streamed for the on some passengers and tested positive 4 available toilets for Staphylococcus aureus and its toxin 250 passengers with vomiting and diarrhea 5.aureus and toxin were also found in and only 4 toilets the ham and cheese
3/13/2016 1 Chapter 16 Staphylococcus aureus Learning Objectives 1. Use basic biochemical characteristics to identify Staphylococcus aureus 2. Understand what conditions in foods favor S. aureus growth 3. Recognize, from symptoms and time of onset, a case of foodborne illness caused by S. aureus 4. Choose appropriate interventions (heat, preservatives, and formulations) to prevent the growth of S. aureus 5. Identify sources of S. aureus 6. Understand the roles of staphylococcal enterotoxins in causing foodborne illness Outbreak Flight of horror • Flight left Alaska and passengers were served breakfast • Breakfast was an omelet with ham and cheese • About 30 min after eating, a passenger rushed to the rear of the plane vomiting on pg g assengers before reaching the toilet • Most of the passengers streamed for the 4 available toilets • 250 passengers with vomiting and diarrhea and only 4 toilets Outbreak Flight of horror • 80% of passengers had diarrhea • 70% of passengers had vomiting • 85% had nausea • The “discomfort bags” ran out • After landing, rectal swabs were done on d t t d iti some passengers and tested positive for Staphylococcus aureus and its toxin • S. aureus and toxin were also found in the ham and cheese
3/13/2016 Outbreak Outbreak Flight of horror Flesh-eating bacteria Meal had been prepared 14 h earlier Methicillin-resistant and held at room temperature 5.aureus (MRSA)are It was held at inadequate temperature usually hospital-acquired infections during flight and microwaved before These infections can also be acquired by serving food. When public health officials A family bought shredded barbecued shook hands with the head pork at a deli,heated in the microwave chef,they found a pus-filled and ate it boil on his hands 3-4 hours later,3 became ill with Guess what they found? vomiting and nausea Outbreak Outbreak Flesh-eating bacteria Flesh-eating bacteria Those who did not eat the pork did not The strain isolated was MRSA and become ill produced staphylococcal enterotoxin C Two were treated at the hospital Different and multiple isolates of 5. aureus were isolated from their stools the pork nasal swabs from three food handlers at the deli
3/13/2016 2 Outbreak Flight of horror • Meal had been prepared 14 h earlier and held at room temperature • It was held at inadequate temperature during flight and microwaved before serving • When public health officials shook hands with the head chef, they found a pus-filled boil on his hands • Guess what they found? Outbreak Flesh-eating bacteria • Methicillin-resistant S aureus S. aureus (MRSA) are usually hospital-acquired infections • These infections can also be acquired by food. • A family bought shredded barbecued pork at a deli heated in the microwave pork at a deli, heated in the microwave and ate it • 3-4 hours later, 3 became ill with vomiting and nausea Outbreak Flesh-eating bacteria • Those who did not eat the pork did not b ill become ill • Two were treated at the hospital • Different and multiple isolates of S. aureus were isolated from - their stools - the pork - nasal swabs from three food handlers at the deli Outbreak Flesh-eating bacteria • The strain isolated was MRSA and prod d t h l l duced staphylococcal ent ti ero oxin C
3/13/2016 Characteristics of the Organism Characteristics of the Organism Historical Aspects General Properties 1914,first link between 5.aureus and Golden yellow colonies foodborne illness Excretes a variety of compounds -Researchers found that drinking -Includes enterotoxins contaminated milk caused vomiting Toxins cause two human diseases and diarrhea toxic shock syndrome 1930-Dack and coworkers consumed fluids with 5.aureus that was isolated -staphylococcal food poisoning from cake and became ill with vomiting. abdominal pain and diarrhea Characteristics of the Organism Characteristics of the Organism Main agent of staphylococcal food Outbreaks are caused by foods in which poisoning the organism was killed but the toxin Caused by ingesting staphylococcal remained toxins that were previously produced in Staphylococcal toxin is not destroyed the food by heating or canning Intoxication-does not require that the bacteria infect the victim
3/13/2016 3 Characteristics of the Organism Historical Aspects • 1914 fi t li k b t 1914, first link between S . aureus and foodborne illness - Researchers found that drinking contaminated milk caused vomiting and diarrhea • 1930 – Dack and coworkers consumed fluids with S. aureus that was isolated from cake and became ill with vomiting, abdominal pain and diarrhea Characteristics of the Organism General Properties • Golden yellow colonies • Excretes a variety of compounds - Includes enterotoxins • Toxins cause two human diseases - toxic shock syndrome - staphylococcal food poisoning Characteristics of the Organism • Main agent of staphylococcal food poi i son ng • Caused by ingesting staphylococcal toxins that were previously produced in the food • Intoxication – does not require that the bacteria infect the victim Characteristics of the Organism • Outbreaks are caused by foods in which th i kill d b t th t i the organism was killed but the toxin remained • Staphylococcal toxin is not destroyed by heating or canning
3/13/2016 Characteristics of the Organism Wall teichoic acid (WTA)of to partially mediate the binding Sources of Staphylococcal Food Contamination People are the main reservoir of 5. aureus Humans are natural carriers that spread 5.aureus to food and to other people Nose is the main colonization site but also occurs on the skin Characteristics of the Organism Characteristics of the Organism ·Spreads by Direct contact Conditions associated with outbreaks -Skin fragments inadequate refrigeration -Respiratory droplets preparing foods too far in advance -poor personal hygiene Most food poisoning is traced by to those preparing the food inadequate cooking or heating of food Contamination may also occur via prolonged use of warming plates contaminated kitchen utensils and food wehen serving foods preparation equipment and surfaces
3/13/2016 4 Characteristics of the Organism Sources of Staphylococcal Food C t i ti Contamination • People are the main reservoir of S. aureus • Humans are natural carriers that spread S aureus S. aureus to food and to other people • Nose is the main colonization site but also occurs on the skin Wall teichoic acid (WTA) of S. aureus seems to partially mediate the binding Characteristics of the Organism • Spreads by - Direct contact - Skin fragments - Respiratory droplets • Most food poisoning is traced by to those prepar those prepar ng the food ing the food • Contamination may also occur via contaminated kitchen utensils and food preparation equipment and surfaces Characteristics of the Organism • Conditions associated with outbreaks - inad f equate refrigeration - preparing foods too far in advance - poor personal hygiene - inadequate cooking or heating of food - prolonged use of warming plates wehen serving foods
3/13/2016 Characteristics of the Organism Characteristics of the Organism Animals may also be sources 5.aureus is present in many food types -Mastitis(infection of cow teats)is a -Various meat products serious problem for the dairy -Various fish products industry Bovine mastitis is the single most -Assorted cream pies costly agricultural disease in the US Tuna pot pies -Mastitis is a public health concern as Deli salads bacteria can contaminate milk and Bacterial load is usually low106 CFU/g Controlled by strict hygiene Characteristics of the Organism Characteristics of the Organism Resistance to Adverse Environmental As there are so many human and animal Conditions reservoirs,controlling staphylococcal infections is challenging Unique resistance to inhibitors ·Grows with high salt Grows with low water activity of 0.86 under aerobic conditions (anaerobic 0.90) cured meats -puddings -sauces
3/13/2016 5 Characteristics of the Organism • Animals may also be sources - Mastitis (infection of cow teats) is a seri bl f th d i ious problem for the dairy industry - Bovine mastitis is the single most costly agricultural disease in the US - Mastitis is a public health concern as bacteria can contaminate milk and dairy products - Controlled by strict hygiene Characteristics of the Organism • S. aureus is present in many food types - Various meat products - Various fish products - Assorted cream pies - Tuna pot pies - Deli salads • Bacterial load is usually low 106 CFU / g Characteristics of the Organism • As there are so many human and animal reservoi t lli t h l l irs, controlling staphylococcal infections is challenging Characteristics of the Organism Resistance to Adverse Environmental Conditions • Unique resistance to inhibitors • Grows with high salt • Grows with low water activity of 0.86 under aerobic conditions (anaerobic 0.90) Mannitol Salt Agar - cured meats - puddings - sauces Mannitol Salt Agar
3/13/2016 Halotolerant Halophile Extreme Example: le 8t2gococeus io fischer salinarum -3% osmophile 15-30% extreme halophile mos bacte ia osmotolecant halotolerant, Nonhalophile 1.00 0.90 0.80 0.70 Example: Water Activity Aw NaCI (% Characteristics of the Organism Characteristics of the Organism Compatible solutes are small molecules that act as osmolytes and help organisms survive Glycine betaine and L-proline are the extreme osmotic stress main osmoprotectants for 5.aureus OH do not interfere with biochemical reactions -accumulate to very high levels in response to low water activity adjust water activity inside the cell lower the intracellular water synthesized or obtained from the growth activity to match that of the L-Prolin medium environment Osmoprotectants for growth at low water Transport into the cell requires activity energy Compatible solutes accumulate in the cell and Toxin may not be produced under enhance growth under osmotic pressure extreme conditions Glycine Betaine 6
3/13/2016 6 1-3% 15-30% Characteristics of the Organism • Compatible solutes are small molecules that act as osmolytes and help organisms survive extreme osmotic stress - do not interfere with biochemical reactions - adjust water activity inside the cell - synthesized or obtained from the growth medium • Osmoprotectants for growth at low water activity • Compatible solutes accumulate in the cell and enhance growth under osmotic pressure Characteristics of the Organism • Glycine betaine and L-proline are the main osmoprotectants for S. aureus - accumulate to very high levels in response to low water activity - lower the intracellular water activity to match that of the environment • Transport into the cell requires energy • Toxin may not be produced under extreme conditions
3/13/2016 Model: Stabilization of proteins by solutes is accomplished by compatible-solute 1-indirect by perturbation of uptake and DpuA Opuc 1 the solvent synthesis in 2-direct by protein-solute Bacillus subtilis. interactions proline Prol ProH GbsA ProA partitioning of water araund a glutamate protein into three hydration layers Foodborne Outbreaks water Incidence of Staphylococcal Food Poisoning Not usually reported,1-5%of cases are Recovery is within 24-48h rying with National surveillance occurs Not a reportable disease 14%of the total foodborne outbreaks in US ~25 outbreaks annually Gel Phase >
3/13/2016 7 Model: compatible-solute uptake and synthesis in Bacillus subtilis. Stabilization of proteins by solutes is accomplished by 1-indirect by perturbation of the solvent 2-direct by protein-solute interactions partitioning of water around a protein into three hydration layers Roche C J et al. J. Biol. Chem. 2006;281:38757-38768 Foodborne Outbreaks Incidence of Staphylococcal Food Poisoning • Not usually p re orted, 1-5% of cases are • Recovery is within 24-48h • National surveillance occurs • Not a reportable disease • 14% of the total foodborne outbreaks in US • ~25 outbreaks annually
3/13/2016 Foodborne Outbreaks Foodborne Outbreaks Seasonal occurrence in late summer Typical Large Staphylococcal Food Second peak in November-December Poisoning Outbreak due to mishandling of holiday foods Traced to one meal fed to 5,824 school Leading cause of foodborne illness children at 16 sites in Texas worldwide 1,364 children developed staphylococcal food poisoning 95%had eaten chicken salad contaminated with 5.aureus Foodborne Outbreaks Foodborne Outbreaks Meal was prepared in a central kitchen ·What went wrong? the day before -Chicken likely contaminated when Frozen chicken was boiled for 3 h, deboned by food handlers deboned,cooled to room temperature, -Storage in deep pans did not allow ground into small pieces and placed into for quick cooling deep pans and stored overnight at 5- -Holding the food at room temp 70C provided additional time for growth Next day,other ingredients were and toxin production added,trucked to the schools and kept at room temperature until served
3/13/2016 8 Foodborne Outbreaks • Seasonal occurrence in late summer • Second peak in November-December due to mishandling of holiday foods • Leading cause of foodborne illness worldwide Foodborne Outbreaks Typical Large Staphylococcal Food Poisoning Outbreak • Traced to one meal fed to 5,824 school children at 16 sites in Texas • 1,364 children developed staphylococcal food p g oisoning • 95% had eaten chicken salad contaminated with S. aureus Foodborne Outbreaks • Meal was prepared in a central kitchen the day before • Frozen chicken was boiled for 3 h, deboned, cooled to room temperature, ground into small pieces and placed into deepp g ans and stored overnight at 5- 7OC • Next day, other ingredients were added, trucked to the schools and kept at room temperature until served Foodborne Outbreaks • What went wrong? - Chicken likely contaminated when deboned by food handlers - Storage in deep pans did not allow for quick cooling - Holding the food at room temp provided additional time for growth and toxin production
3/13/2016 Characteristics of Disease Characteristics of Disease Staphylococcal food poisoning is a self- limiting illness Symptoms develop within 6 hours after Vomiting occurs after an unusually short eafing time of onset,as little as 30 min Other common symptoms Average incubation time is 4.4 hours nausea Illness can appear within 30 min -cramps Death is rare,ranges from 0.03-4.4% diarrhea headaches ·1o%seek medical help Lack of fever is consistent with the illness ·Treatment is minimal being caused by a toxin and not an infection Toxic Dose Mechanism of Staphylococcal Enterotoxins-vomit Analyzed using recovered food from outbreaks Very toxic,1 ng of enterotoxin per gr of 2.Stimulation of food will cause illness vagus nerve Enterotoxin levels ingested range from 3.Transmits 0 signal to the 1-5g vomitting .aureus levels >105 cells/g may produce center in the brain the level of toxin required to cause illness intestinal epithelium 9
3/13/2016 9 Characteristics of Disease • Staphylococcal food poisoning is a selflimiting illness • V iti ft ll h t Vomiting occurs after an unusually short time of onset, as little as 30 min • Other common symptoms - nausea - cramps - diarrhea - headaches • Lack of fever is consistent with the illness being caused by a toxin and not an infection Characteristics of Disease • Symptoms develop within 6 hours after eating • Average incubation time is 4.4 hours • Illness can appear within 30 min • Death is rare, ranges from 0.03-4.4% • 10% seek medical help • Treatment is minimal Toxic Dose • Analyzed using recovered food from outbreaks • Very toxic, 1 ng of enterotoxin per gr of food will cause illness • Enterotoxin levels ingested range from 1-5 g • S. aureus levels >105 cells/g may produce the level of toxin required to cause illness
3/13/2016 Microbiology,Toxins,and Microbiology,Toxins,and Mix culture Pathogenicity Pathogenicity filtrate with fibrin Characteristics Coagulase test confirms identification ·Staphylococci Fibrinogen(soluble)to small,spherical fibrin (insoluble) gram-positive associated with toxin -0.5×1.5μm production -23 species -FDA notes this is not a Catalase positive-breaks down reliable test hydrogen peroxide other Staphylococcus strains can produce enterotoxins Microbiology,Toxins,and Staphylococcal Enterotoxins Pathogenicity Toxin classification Genetic techniques used in investigating Current classification based on antigenicity outbreaks 5.aureus make multiple toxins that can be -Pulse-field gel electrophoresis differentiated immunologically -Randomly amplified polymorphic DNA Staphylococcal enterotoxins A-E are the Polymerase chain reaction major types Enterotoxins G,H,I,and J are recently discovered Toxic shock syndrome enterotoxin(F)does not cause vomiting 10
3/13/2016 10 Microbiology, Toxins, and Pathogenicity Characteristics • Staphylococci - small, spherical - gram-positive - 0.5 x 1.5 m - 23 species • Catalase positive – breaks down hydrogen peroxide Mix culture filtrate with fibrin Microbiology, Toxins, and Pathogenicity • Coagulase test - confirms identification - Fibrinogen (soluble) to fibrin (insoluble) - associated with toxin production - FDA t thi i t FDA no tes thi s i s not a reliable test - other Staphylococcus strains can produce enterotoxins Microbiology, Toxins, and Pathogenicity • Genetic techniques used in investigating outb k rea k s - Pulse-field gel electrophoresis - Randomly amplified polymorphic DNA - Polymerase chain reaction Staphylococcal Enterotoxins Toxin classification • Current classification based on anti g y enicit y • S. aureus make multiple toxins that can be differentiated immunologically • Staphylococcal enterotoxins A-E are the major types • Enterotoxins G, H, I, and J are recently discovered • Toxic shock syndrome enterotoxin (F) does not cause vomiting