1. FE- Food Microbiology Spring 2015 LECTURE FOODBORNE TOXICOINFECTIONS

2. outline Introduction Main characteristics of foodborne toxicoinfections Type of Foodborne Toxicoinfections – Aeromonas hydrophila – Bacillus cereus – Clostridium perfringens – Clostridium difficile – Plesiomonas shigelloides – Vibrio Cholera (Cholera) – Enterotoxigenic and Enteropathogenic E. coli

3. Foodborne Toxicoinfection It is the combination of food intoxication and infection. The microorganism grows to very high number in a food and continues its growth in the intestinal tract and cause ilness. Generally, toxicoinfections involve either sporulation or death of bacterial cells and release toxins in the gastrointestinal tract.

4. Some characteristics of Foodborne toxicoinfections For spore formers, ingestion of large numbers of vegetative cells is required. Vegetative cells do not multiply in the digestive tract, but some sporulate and release toxins. For Gram-negative non spore forming bacteria, the cells can be ingested in moderate numbers. The cells rapidly multiply in the intestinal tract and many cells are died and died cells release toxins Toxins o both groups produce gastroenteritis symptoms. Food borne toxicoinfections are caused by Aeromonas hydrophila,Bacillus cereus, Clostridium perfringens,Clostridium difficile,Plesiomonas shigelloides,Vibrio cholerae (Cholera),Enterotoxigenic and Enteropathogenic E. coli

5. 1. Aeromonas hydrophila Characteristics of organism Members of Vibrionaceae family Present in salt and freshwater and estaurine water environments Gram negative Facultative anearobic Catalase and oxidase-positive Can be motile and non motile Bacilli shaped (1-3.5 µm length) usually presents in single or short chains Mesophilic or psychrophilic. Optimum temp. 28 ˚C. It can grow temp range from 5 to 42 ˚C İs killed by heating 60 C in 10 min It is fairly sensitive to low pH(below 5.5) It does not grow at high salt concentration(above 5%NaCl)

6. 1. Aeromonas hydrophila - Nature and Food Associations They present primarily aquatic environment. Common in sea water, well water, sink water, sewage and wide range of seafood. In fresh meat and poultry they are in small numbers but can increase during storage at chill temperatures Wide variety of vegetables can contain this organism from contaminated water

7. 1 Aeromonas hydrophila -Disease and Symptoms Can cause wide range of localized, wound and systemic illnesses. The symptoms are dependent on bacterium, Host, inoculum size, and infection site. The most common infection is gastroenteritis (infants, children under five years old, elderly and immunocompromised people) Infective dose ranges from 10 4 to 10 10 cells. The main virulence factors associate with the production adhesions, exotoxins, heat stable and heat label shigella like toxin, extracellular enzymes and endotoxins.

8. 1. Aeromonas hydrophila -Prevention and Control Use additional preservation method for foods stored at low temp( below 5 °C) Sufficiently cooking seafoods and disinfection of drinking water. Organic and inorganic acid can be effective. Polyphosphate and NaNO2 can also control the control the growth. Can be controlled by heat treatment, preventing post heat contamination and by using one or more growth limiting factor such as pH ad water activity. Are easily destroyed by heating at 56 ˚C for 10 min.

9. 1. Aeromonas hydrophila -Isolation and Identification When low numbers are present, enrichment media such as alkaline peptone water are used to increase number of A. hydrophila but when high numbers direct plating is sufficint. Many strains ferment lactose and can not ferment xylose and these are useful distinguishing factors in media. As well as bile salts, ampicillin is used as a selective agent in media such as starch ampicillin agar and blood ampicillin agar.

10. 2. Bacillus cereus Characteristics of organism Gram positive Rod shaped Motile and areobic It produces central to term,nal allipsoidal or cylindrical endosspore and do not cause swelling in the sporangium Temp range 10 to 50 ˚C pH range 4.9-9.3 Salt concentrations below 10% Due to the spores it can be isolated from soil, water and vegetables. Can grow in past. milk and produces a protease to cause off flavors. Spores are relatively heat resistant

11. 2. Bacillus cereus - Nature and Food Associations Widespread in nature and commonly in soil, dust and growing plants. Meat, eggs, rice, pasta and dairy products are the most common food vehicles for B. cereus. this organism from contaminated water

12. 2. Bacillus cereus - Nature and Food Associations Heat processing will select the spore formers and a high number of B. cereus spores survive at pasteurized and other heat processing of foods. In most of these cases the numbers are low. When pasteurized milk or cream is stored at inadequate chill temperatures, B.cereus can grow and cause spoilage known as ‘’Sweet Curdling’’ or ‘’bitty cream’’.

13. 2. Bacillus cereus –Foodborne outbreaks B. cereus causes two types of food poisoning: The emetic type The diarrheal type.

14. CharacteristicDiaarheal syndromeEmetic syndrome Infectıve dose10 5 -10 7 10 5 -10 8 cells/g food Toxin producedIn the small intestine Performed in the food Type of toxinProtein Cyclic peptides Incubation period18-24 h( approx. 12 h) 1-6 h Duration of ilness12-24 h 8-24 h SymptomsAbdominal pain, watery diarrhea (occasionally bloody diarrhea) Nausea, vomiting, malaise, diarrhea Foods of most frequently associate implicated Meat products, soups, vegetables, puddings,sauces,milk and milk products Fried and cooked rice, pasta, pizza, pastry, noodles

15. 2. Bacillus cereus –Prevention and control The important factors in the B. cereus disease are improper holding temperature, contaminated equipment, inadequate cooking and poor personal hygiene. The food should be kept at low or high temperature to prevent spore germination. Quick chilling of the food to 4 °C or holding the food above 60 °C can achieve this. Strict temperature control must be exercised in the cooling and holding of cooked foods. The strong adhesion problem of B. cereus can be overcomed with use of hypochlorite(0.2% at pH 7-8)

16. 2. Bacillus cereus –Isolation and Identification In outbreaks of B. cereus food poisoning, foods will contain large numbers (above 105/g) so enrichment techniques are not required. Selective agar medium(Bacillus cereus agar)contains mannitol egg yolk and polymyxin as a selective agent to suppress Gram negative bacterium. B. cereus can be identified after 24 h incubation at 35 °C by its characteristic colonial morphology. Hemolysing producing B. cereus gives hemolytic colonies. Colonies can be further confirmed by microscopic procedure with spore staining.

17. 3. Clostridium perfringens- Characteristics of the organism Gram positive Large rod shaped (2-4 µm in length) Anaerobic Spore forming Swelling the sporangium with spore Capsulated Non motile Mesophilic Catalase negative It produces H 2 S Growth occurs over the temperature range 12-52 °C with optimum 6.0-7.5 The vegetative cells are sensitive to low heat treatment but their spores are extremely heat resistant and some can survive at boiling temperature for several hours.

18. 3. Clostridium perfringens- Characteristics of the organism It is probably the most widespread of all pathogenic bacteria. There are several toxigenic types :A,B,C,D and E based on the production of four enterotoxins: alpha,beta, epsilon and iota. C. Perfringens type A:produces only alpha toxin C. Perfringens type B:produces alpha,beta, epsilon C. Perfringens type C:produces alpha,beta. C. Perfringens type D:produces alpha, epsilon C. Perfringens type E:produces alpha and iota C. Perfringens type A is responisble of food poisoning and gas gangrene on human.

19. 3. Clostridium perfringens-Nature and Food Associations It is extremely widespread in nature; dust, air and raw foods. It is a common inhabitant of the intestinal tract of heathy animals and human beings. Spores of organism can survive at many food processing procedures Due to the its ability to grow over a wide temperature range, it is often implicated in human food poisoning. Clostridium perfringens most closely associates with gangrene and wound infections. Foods commonly associating with C. Perfringens are meat, poultry, fish, shellfish, vegetables, dairy products, eggs and pepper

20. 3. Clostridium perfringens-Disease and symptoms Human food poisoning is caused by the ingestion of food containing a large number of vegetative cells. The enterotoxin causes gastroenteritis. The symptoms appear 8-24 h following ingestion of a large number of viable cells(about half million cells/g). Symptoms include diarrhea, abdominal pain, nausea, vomiting and fever. The toxin changes the permeability of intestinal cells. Fatality is rare, but it can occur in very young, elderly and sick. Symptoms disappear within 24 h. This disease is considered a mild one.

21. 3. Clostridium perfringens-Disease and symptoms Gas gangrene is a bacterial infection that produces gas within tissue in gangrene. It is caused by exotoxin-producing clostridial species(most often C. perfringens).The exotoxin is commonly present in C. perfringens type A species and is known as alpha toxin. These toxins destroy nearby tissue, generating gas at the same time.

22. 3. Clostridium perfringens-Prevention and Control Proper sanitation in all phases of food preparation and handling. Food should be cooked to the highest temperature recommended to kill the cells and as many spores as possible. The food should be cooled quickly and uniformly To refrigerated temperature. If a food is stored for a long time, it is important to reheat it quickly and uniformly and keeping it hot while being served.

23. 3. Clostridium perfringens-Isolation and Identification Selective agar medium is used in ısolation. In the examination of foods, total count for C. perfringens (vegetative cells plus spores) is determined with fecal sample, a spore count is obtained after heating at 80 °C for 10 min. The most common selective medium used to enumerate C. perfringens is perfringens agar and incubated anaerobically at 35 °C for 24 h. Characteristic colonies can be confirmed by the absence of motility, ability to reduce nitrate to nitrite, lactose fermentation, gelatin liquefaction and Nagler reaction

24. 4. Clostridium difficile(pseudomembranous colitis) C. difficile causes antibiotic associated diarrhea(AAD) and more serious intestinal conditions such as colitis and pseudomemranous colitis in humans. People in good health usually do not get C. difficile disease and individuals who have other conditions that require prolonged use of antibiotics and elderly are at greater risk of disease

25. 4. Clostridium difficile- symptoms and prevention İt produces two toxins: Toxin A is referred to as an enterotoxin because it causes fluid accumulation in the bowel. Toxin B is an extremely lethal(cytopathic) toxin Prevention: Avoid broad spectrum antibiotic use. Probiotics may be considered in recurrent cases. Practice good hygiene(hand washing, disinfect surfaces such as bleach, alkaline glutaldehyde and ethylene oxide)

26. 5.Plesiomonas shigelloides- Characteristics of organism It is an enteric pathogen and causing foodborne outbreaks. It present in the family of Plesiomonadeceae Gram negative Catalase and oxidase positive Mesophilic, rod-shaped non-halophilic It grows temp. Range from 8 to 45 °C with optimum 35 °C, at pH 5 to 8 but susceptible to low pH. Aerobic or facultative anaerobic Non spore forming rod(3.0µm in length) or filamentous Motile with lophotrichous 2-5 flagella Produce H2S

27. 5.Plesiomonas shigelloides-Nature and Food Associations Most infections occur in the summer months and correlate with environmental contamination of freshwater(rivers, streams, ponds, etc) The usual route of transmission of the organism is by ingestion of contaminated water or uncooked raw fish and shellfish. The most common foods associating with P. Shigelloides outbreak are oyster, chicken,fish and shrimp.

28. 5.Plesiomonas shigelloides-Disease and symptoms It can cause wound infections, septicemia and gastroenteritis. The symptoms appear within 24 h following ingestion of contaminated food or water with viable cells Virulence factors of P. shigelloides are extracellular enzymes(e.g.elastase), heat stable enterotoxins,cytotoxins,) hemolysin, exotoxin,invasions (invasion of HeLa cells )and adhesions (glycocalyx) P. shigelloides is gastroenteritis is usually a mild self- limiting disease with fever, nausea, abdominal pain, dehydration, vomitting and chill.

29. 5.Plesiomonas shigelloides-Prevention and control It is killed at pasteurization temperature. Adequately cooked foods can not contain viable cells and proper refrigeration controls its growth. 5.Plesiomonas shigelloides-Isolation and control It is an enteric pathogen. Alkaline peptone water and tetrathionate broth can be used as an enrichment media. Selective medium used for isolation of organisms is Plesiomonas agar containing inositol, brillant green and bile salts.

30. 6.Vibrio cholerae (Cholera)- Characteristics of the organisms It is a member of family Vibrionaceae is a Gram-negative motile with monotrichous flagellum, comma-shaped, facultative anaerobic. Two serogroups of V. cholerae: 01 and 0139 they cause outbreaks but 01 serogroup causes the majority of cases. V. Cholerae 01 has 2 biotypes; Classical and El Tor Each biotype has three serotypes; Inaba, Ogawa and Hikojima. Non 01 serotype strains are isolated from coastal waters.

31. 6.Vibrio cholerae (Cholera)- Nature and food associations It is widely distributed in aquatic environments. Marine environments may serve as long-term reservoirs. Both serotypes, especially non-01, have been isolated from water. The disease results from ingestion of infective doses of V. cholerae( about one million cells) through foods and water contaminated with feces of humans suffering from the disease The handling of food by a person suffering from the disease can also contaminate food, because of poor personal hygiene. Food originating from natural reservoirs of the causative bacteria can be contaminated and spread cholera. The natural reservoirs include marine and brackish water environments. Among the types of contaminated foods are raw oysters, Crab, fish and shrimp and cooked rice.

32. 6.Vibrio cholerae (Cholera)- Disease and symptoms Illness of cholera is due primarily to action of cholera enterotoxin. Cholera is a disease characterized by severe, dehydrating diarrhea Infectious dose is about 10 6 cells or more per gram of food. Incubation period for cholera varies from several h to days. Toxin stimulates adenyl cyclase in the intestinal epithelial cells to produce cyclic AMP which causes massive secretion of water along with chloride, potassium, and bicarbonate in the lining of the intestine therefore toxin disrupts ion transport. Initially the stool is brown with fecal matter, than becomes a pale gray color with slightly fish odor; mucus in the stool imparts the characteristics «rice water» appearance. The symptoms fallow with the sudden onset of rice watery diarrhea, severe prostration, painful muscle cramps, clouded mental status, loss of fluids, dehydration of intestinal cells and circulatory collapse.

33. 6.Vibrio cholerae (Cholera)- Prevention Adopting proper hygienic measures. Provision of properly treated municipal water. Decontamination of suspected water by boiling. Proper disposal of sewage. Treating of infected persons with antibiotics along with replacing body fluids. Vaccination of unexposed people. Seafoods should not be eaten raw.

34. 6.Vibrio cholerae (Cholera)- Isolation and Identification Isolation of V. cholerae from a sample is achieved by an initial pre enrichment in alkaline peptone water, followed by streaking on a selective agar medium plate such as thiosulfate citrate bile salt sucrose agar. Suspected colonies(yellow) are biochemically and serologically tested for confirmation. The toxin is detected by immunoassay.

35. 7. a.Enterotoxigenic Escherichia coli(ETEC) Humans are the principle reservoir of enterotoxigenic E. coli (ETEC) strains that cause human diarrhea. Human carriers can spread the ETEC strains directly and indirectly. Foods (such as cheese) and water associated with human ETEC outbreaks. ETEC strains are major cause of diarrhea among travelers and infants for poor sanitation. Large numbers of cells(106-109 cells/g) should be ıngested for developing the symptoms. Diarrhea is usually fallow by low grade fever, abdominal pain, malaise and nausea

36. 7. b.Enteropathogenic Escherichia coli Humans are an important reservoir of enteropathogenic E. coli (EPEC).Many types of foods(meat products, fish, milk, milk products, vegetables, baked products and water canbe contaminated with EPEC. EPEC strains are importantt in infant diarrhea worldwide and have high mortality rate, especially in places with poor sanitation. High numbers of cells (106-109 cells/g)should be ingested to develop symptoms