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King Saud University Collage of Pharmacy Department of Pharmaceutics Section of Microbiology Prepared by: Supervised by: Duaa S. Al-Baqer 421033300 Dr. Manal Badour 283 PHT 1426 - 2005 Index Page No. Description Microscopic images Occurrence Comparison of B. cereus Enterotoxins Growth Heat resistance Nature of acute disease Symptoms Onset of illness Duration of illness Infective dose Diagnosis of human illness Complications Treatment Re-infection Spread to others Work and school Susceptible individuals Reducing the Risk Prevention Analysis Lab tests Preferences 3 3 5 5 6 6 6 7 7 7 7 7 7 7 8 8 8 8 8 8 9 9 11 Description: Bacillus cereus are Gram-positive, facultative aerobic sporeformer whose cells are large rods and whose spores do not swell the sporangium. They can be differentiated from other Bacillus species by their cell position and biochemical tests. The optimum growth temperatures range from 30 to 50°C, although some psychrotrophic strains can grow down to 4 to 5°C. They can grow at pH values of between 4.3 and 9.3, and can grow at water activity values down to 0.912. The organism produces heat resistant spores and these may germinate if cooling is too slow. B. cereus. Spores appear as clear areas within the individual bacilli. These and other characteristics, including biochemical features, are used to differentiate and confirm the presence B. cereus, although these characteristics are shared with B. cereus var. mycoides, B. thuringiensis and B. anthracis. Differentiation of these organisms depends upon determination of motility (most B. cereus are motile), presence of toxin crystals (B. thuringiensis), hemolytic activity (B. cereus and others are beta hemolytic whereas B. anthracis is usually nonhemolytic), and rhizoid growth which is characteristic of B. cereus var. mycoides. Microscopic images: 1 µm wide, 5-10 µm long, arranged singly or in short chains. Bacillus cereus Spore Stain. Stained by Shaeffer-Fulton method. Spores stained green. Bacterial cells stained red (1000X ). Bacteria at the top of a colony ( 2 µm ) Bacteria from the bottom of a colony ( 2 µm ) Bacteria in the form of twisted ropes ( 5 µm ) Occurrence: 1. Wide distribution in soil, dust and air. 2. Carried by humans and animals. 3. In many food products: • Dairy products. • Rice and cooked oriental foods. • Spices and spice mixes. • Dried products (flour, dry milk, pudding, soup mix). 4. Beans and bean sprouts. 5. Meats 6. Bakery products (cream-filled pastries). Extracellular products 1. 2. 3. 4. 5. Proteases (sweet curdling of dairy products). Lactamases (penicillin resistance). Phospholipases. Hemolysins. Toxins, which are: • Lethal toxin: kills mice injected I.V. • Enterotoxins: diarrheal and emetic. Comparison of B. cereus Enterotoxins: Property Nature Stabilities pH Enzymes Storage Production in foods Lab media Optimum temp. Growth Activities: ● Monkeys ● Skin permeability ● Mice ● Cell culture ● Antigenic ● Action Diarrheal Toxin Protein~40 kDa Heat stable 450C, 30 min Unstable 560C, 5 min Most stable pH 4-11 Sensitive to pronase, trypsin Unstable Sometimes preformed Complex 32-370C Late exponential Diarrhea in 1-3 hr + Lethal Cytotoxic + Tissue damage, altered perm and adenyl cyclase. Emetic Toxin Peptide~5 kDa Stable 1260C, 90 min Stable pH 2-11 resistant to pepsin, trypsin Stable preformed Rice slurry 25-300C Stationary Vomiting in 1-5 hr ? ? ? Growth: Limiting conditions for pathogen growth Pathogen Min. aw (using salt) Min. pH Bacillus Cereus 0.92 4.3 Max. pH Max. % water phase salt Min. temp. Max. temp. Oxygen requirement 9.3 10 39.2°F 4°C 131°F 55°C aerobe Time/Temperature guidance for controlling pathogen growth and toxin formation Potentially Hazardous Condition Growth and toxin formation by Bacillus cereus Maximum Cumulative Exposure Time Product Temperature • • • • 39.2-43°F (4-6°C) 44-50°F (7-10°C) 51-70°F (11-21°C) Above 70°F (above 21°C) • • • • 5 days 17 hours 6 hours 3 hours Heat resistance: B. cereus spore heat resistance. Temperature (oC) (oF) 90 95 100 194 203 212 D-Values (min.) Medium 21-137 5-36 6.7-8.3 Water Water Water Nature of acute disease: There are two illness associated with B. cereus, emetic and diarrhoeal illness. The emetic illness is caused by the ingestion of a heat-stable toxin produced by the microorganisms in the food. The diarrhoeal illness is caused by the ingestion of moderate to high number of B. cereus and their subsequent production of toxin in the stomach. Symptoms: The emetic type of food poisoning, with symptoms similar to that caused by Staphylococcus aureus, is characterized by nausea and vomiting. The symptoms of B. cereus diarrhoeal illness, similar to Clostridium perfringens food poisoning, include watery diarrhoea, abdominal cramps, and pain. Nausea may sometimes occur, and vomiting rarely occurs. Other Bacillus species will cause vomiting and diarrhoea. Onset of illness: Onset times for the emetic illness is usually ½ to 6 hours after consumption of contaminated foods. For the diarrhoeal illness onset times are usually 6 to 15 hours, and for other Bacillus species varies from ½ hour to 14 hours. Duration of illness: The emetic illness usually lasts less than 24 hours, while the diarrhoeal illness usually persist for 24 hours in most instances. As with the emetic illness, illnesses caused by other Bacillus species usually last less than 24 hours. Infective dose: Foods implicated in foodborne illness caused by B. cereus and other Bacillus species usually contain at least 105 microorganisms per gram of food. Diagnosis of human illness: Confirmation of B. cereus as the cause in a foodborne outbreak requires either: Isolating the same serotype from the suspect food and faeces or vomitus of the patient Isolating large numbers of a B. cereus serotype known to cause foodborne illness Isolating B. cereus from suspect foods and determining their enterotoxigenicity. The rapid onset time for the emetic illness, coupled with some food evidence, is often sufficient to diagnose this type of food poisoning. Complications: No specific complications have been associated with the foodborne disease causes by B. cereus and other Bacillus species, although dehydration may occur from the diarrhoeal illness in the elderly and sick. Treatment: Generally no specific treatment is needed except for fluid replacement if necessary. Re-infection: As yet it is unclear if any resistance is gained against reinfection. Spread to others: This illness is not spread from person to person. Work and school: Everyone with this infection should stay off work or school while they have symptoms especially those in High Risk groups, occupations or institutions (infants or children in daycare, school pupils, students, food workers, child-care workers, teachers, and health/care workers). Susceptible individuals: All people are believed to be susceptible to B. cereus food poisoning. Reducing the Risk: Bacillus cereus exists in normal bacterial and spore forms in foods. The normal form is inactivated by cooking, but most illness is a result of the multiplication of spores during inadequate refrigeration of moist cooked protein foods and rice. Because cooking often kills competing bacteria and heat activates the Bacillus cereus spores, storing large masses of cooked food between 4O C and 60O C can allow the bacteria to multiply. Prevention: Preventive measures that can be taken to help avoid the illness include: Ensuring adequate temperatures are reached during cooking of food mixes such as sauces, custards, and soups to inactivate the bacteria. • Keeping cooked hot foods above 60O C (preferably 70O C) if not served immediately. • Ensuring the rapid cooling of cooked food by dividing into smaller lots and refrigerating in shallow containers (less than 10cm deep). • Storing cold foods at or below 4O C to prevent toxin being produced. • Avoiding storing protein-containing foods with cooked rice because this stimulates the growth of Bacillus cereus. • Reheating foods to 75O C or until steaming hot, as flash frying or brief rewarming is not adequate to destroy the toxin. • Preventing cross-contamination from raw to cooked foods (by using separate preparation areas or sanitizing between processes). • Thoroughly washing fruit and vegetables with clean water of drinking standard before usage. • Ensuring food handlers have good personal hygiene and adequate food safety training. Analysis: The emetic ("short incubation") form of the disease, which occurred in this outbreak, is mediated by a highly stable toxin that survives high temperatures and exposure to trypsin, pepsin, and pH extremes; the diarrheal syndrome is mediated by a heat- and acid-labile enterotoxin that is sensitive to proteolytic enzymes. The diagnosis of B. cereus food poisoning can be confirmed by the isolation of greater than or equal to 105 B. cereus organisms per gram from epidemiologically implicated food. Underreporting of such outbreaks is likely because illness associated with B. cereus is usually self-limiting and not severe. In addition, findings of a recent survey about culture practices for outbreaks of apparent foodborne illness indicate that 20% of state public health laboratories do not make B. cereus testing routinely available. Fried rice is a leading cause of B. cereus emetic-type food poisoning in the United States. B. cereus is frequently present in uncooked rice, and heat-resistant spores may survive cooking. If cooked rice is subsequently held at room temperature, vegetative forms multiply, and heat-stable toxin is produced that can survive brief heating, such as stir frying. In the outbreak described in this report, vegetative forms of the organism probably multiplied at the restaurant and the day care centers while the rice was held at room temperature. The day care staff and restaurant food handlers in this report were unaware that cooked rice was a potentially hazardous food. This report underscores the ongoing need to educate food handlers about basic practices for safe food handling. Lab tests: 1. Malachite green stain: Malachite green stain is forced into the spore by heating the cells. Vegetative cells are then decolorized with water and stained pink with safranin counterstain. Endospores may be located in the middle of the cells (central), at the end (terminal), or between the end and the middle of the cells (subterminal). The endospores themselves may be round or oval. B. cereus produces terminal endospores. 2. Growth on blood agar: Bacillus cereus colonies on blood agar. Colonies of Bacillus cereus on the left; colonies of Bacillus anthracis on the right. B. cereus colonies are larger, more mucoid, and this strain exhibits a slight zone of hemolysis on blood agar. Preferences: Different internet sites.. 1. Center for Food Safety & Applied Nutrition - Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. http://www.cfsan.fda.gov 2. FSHN420 Food Microbiology. http://www.ag.iastate.edu 3. Christchurch City Council. http://www.ccc.govt.nz 4. University of Wisconsin-Madison Department of Bacteriology. http://www.textbookofbacteriology.net 5. Microbiology @ Leicester. http://www-micro.msb.le.ac.uk 6. Other sites: http://www.buckman.com http://www.safefood.net.au http://www.seafood.ucdavis.edu