Bacterial Intoxification

Microbial IntoxicationBacillus cereusClostridium perfringens

Bacillus cereuscereus=waxG+ acerbic rodslecithinaseendosporeD100c=8min

INTRODUCTION The Genus Bacillus was established in 1872 with B. subtilis as type species. B. cereus was added fifteen years laterSeveral accounts of food poisoning attributed to members of the genus Bacillus appeared in the European literature before 1950

INTRODUCTIONAn accumulating number of reports implicate both B. subtilis and B. licheniformis as potential food poisoning agents.

OCCURRENCE IN FOOD AND ENVIRONMENT B. cereus has a wide distribution in nature, frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals It has been isolated from foods that were not involved in foodborne illness outbreaks. It is also present in the stools of 14 to 15% of healthy humansIt is frequently isolated from milk and dairy products. In milk, B. cereus causes a defect known as 'bitty' cream or sweet curdling. It is found in rice, rice products, oriental dishes and ingredients

OCCURRENCE IN FOOD AND ENVIRONMENT A variety of foods have been implicated in food-poisoning Emetic syndrome caused by B. cereus is highly associated with rice and rice products

OCCURRENCE IN FOOD AND ENVIRONMENT B. cereus was isolated from 9, 35, 14 and 48% of raw milk, pasteurized milk, Cheddar cheese and ice cream samples, respectivelyIn a local study, B. cereus occurred in 17% of fermented milks, 52% of ice creams, 35% of soft ice creams, 2% of pasteurized milks and pasteurized fruit- or nut-flavored reconstituted milks, and 29% of milk powders, mostly in fruit- or nut-flavored milk mixes (Wong et al., 1988a). B. cereus was found in 71.4% and 33.3% in spring and in autumn samples of full-fat milk in mainland China, respectively, and the average count among the positive samples was 11.7 MPN/ml (Zhou et al., 2008). Dried milk products and infant food are known to be frequently contaminated with B. cereus, 261 samples of infant food distributed in 17 countries were collected and 54% were contaminated with B. cereus reaching levels from 0.3 to 600/g (Becker et al., 1994).

OCCURRENCE IN FOOD AND ENVIRONMENT Chinese 'take-out' foods appear to be particularly vulnerable to B. cereus infection and it has been shown that suspensions (2%) of seed flours and meals from diverse botanical origins were found to be excellent sources of nutrients for growth (Beuchat and Ma-Lin, 1980)

OCCURRENCE IN FOOD AND ENVIRONMENT Of 433 honey samples collected in Argentina, 27% yielded B. cereus isolates and 14% yielded other species of Bacillus. Results showed a high degree of diversity, both phenotypic and genotypic among the isolates of B. cereus (Lopez and Alippi, 2007).

OCCURRENCE IN FOOD AND ENVIRONMENT The B. cereus isolates from food are highly toxigenic. All the isolates from local dairy products lysed rabbit erythrocytes; 98% showed verotoxicity, 68% showed cytotonic toxicity for CHO cells (Wong et al., 1988a). In another study of 136 strains of B. cereus isolated from milk and cream, 43% and 22% showed toxicity to human embryonic lung cell when the isolates were cultured in brain heart infusion and milk, respectively (Christiansson et al., 1989). In milks, B. cereus growed rapidly and produced cytotonic and cytotoxic toxins (Wong et al., 1988b). Toxin production of B. cereus in milk at low temperature was also evaluated (Christiansson et al., 1989).

OCCURRENCE IN FOOD AND ENVIRONMENT For the B. cereus isolated from seafood, 48% isolates produced both the hemolysin BL (HBL) and nonhemolytic (NHE) enterotoxins, and 94% and 50% produced NHE or HBL toxins, respectively.Only one B. cereus isolate possessed the cereulide synthetase gene, ces (Rahmati and Labbe, 2008).

OCCURRENCE IN FOOD AND ENVIRONMENT The enterotoxin genes hblA, hblC, hblD(), nheA, nheB and nheC ()occurred in B. cereus isolates from full-fat milk products with frequencies of 37.0%, 66.3%, 71.7%, 71.7%, 62.0% and 71.7% respectivelyNine B. thuringiensis isolates were also identified from six pasteurized milk samples, and most of them harbored six enterotoxic genes and the insecticidal toxin cry1A gene. The single B. mycoides isolate harbored nheA and nheC genes (Zhou et al., 2008).

CHARACTERISTICS AND TAXONOMY B. cereus is a Gram-positive, motile, facultative, aerobic sporeformer. Dimensions of vegetative cells are typically 1.0- 1.2 m by 3.0-5.0 m. The ellipsoidal spores are formed in a central or paracentral position without swelling the sporangium. The organism does not ferment mannitol and has a very active phospholipase (lecithinase) system. B. cereus is keyed as citrate(+), arabinose (-), Gram (+), aerobic sporeformer.

CHARACTERISTICS AND TAXONOMY

CHARACTERISTICS AND TAXONOMYPlasmids have been identified in B. cereus. Plasmids of molecular weight ranged from 1.6 to 105 MDa. Bacteriocin production could be attributed to a 45 MDa plasmid (pBC7), and tetracycline resistance to a 2.8 MDa plasmid (pBC16)

SPORE AND GERMINATIONB. cereus produces elliptical shaped endospore with dominant central position, no distended sporangium. The spore when liberated from the sporangium is encased in a loose fitting exosporium. On germination the spore coat undergoes rapid lysis while the vegetative cell is emerging. Since spores of B. cereus may survive heat processing, spore germination is important in B. cereus study.

GERMINATION STEPSOnce the initial 'trigger reaction' has been activated, germination continues in the absence of the inducer. After the 'trigger' steps, the various spore properties are changed sequentially in the following order: loss of heat resistance, release of dipicolinic acid (DPA) and Ca2+ into the medium, increase in spore stainability, beginning of phase darkening and decrease of the optical density of spore suspension as cortex peptidoglycan is hydrolyzed and the products released to the mediumFinally, the onset of metabolic activity as measured by oxygen uptake.

Role of trypsin-like enzymeThe germination of B. cereus spore is partially prevented by several inhibitors of trypsin-like enzymes (leupeptin, antipain, and tosyl-lysine-chloromethyl ketone)A synthetic substrate of trypsin also inhibited germination (Boschwitz et al., 1983).

SPORE AND GERMINATION Inactivation of B. cereus spores during cooling from 90C occurs in two phases, one phase occurs during cooling from 90 to 80C; the second occurs during cooling from 46 to 38C. No inactivation occurs when spores are cooled from a maximum temperature of 80C.

SPORE AND GERMINATION Germination of B. cereus spores is more extensive in rice than in trypticase soy broth at

SPORE AND GERMINATIONB. cereus spores germinate in inosine or in l-alanine as sole germinants

Confirmation of outbreak B. cereus strains of the same serotype should be present in the epidemiologically food, feces and/or vomitus of the affected persons. OrSignificant numbers (>105 CFU/g) of B. cereus of an established food poisoning serotype should be isolated from the incriminated food, or feces, or vomitus of the affected persons. or Significant numbers (>105 CFU/g) of B. cereus should be isolated from the incriminated food, together with detection of the organism in the feces and/or vomitus of the affected persons.

Bacillus cereus Diarrheal type Emetic type Intermediate type1~18

Bacillus cereus HH1 H

Bacillus cereus cereolysinemetic toxin cereulidepeptideenterotoxin

Enterotoxinshemolysin BL (HBL) enterotoxinsnonhemolytic (NHE) enterotoxins

Bacillus cereuslecithinase reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and the Duopath Cereus Enterotoxins (Merck) gene

Detecting Cereulidecereulide synthetase gene (ces)liquid chromatography-tandem mass spectrometry analysisHEp-2 cell vacuolation testmitochondrial respiratory uncoupling activitysperm micro assay(boar spermatozoa )

Staphylococcus aureus Aw = 0.8610% NaCl45toxic shock syndrome

Staphylococcus aureus1~8 12~15

Staphylococcus aureusCoagulasecoagulate citrate plasmaAlpha-exotoxinBeta-toxina hot-cold FS toxinHyaluronidasespreading factorToxic shock syndrome toxins toxin, TSSTEnterotoxins

Enterotoxins ABC1C2C3DEFGSEGSElX(SER, SES, SET)staphylococcus enterotoxin-like toxin (SEL)SElX

Enterotoxins ABA 60 20minB 100 5minpepsintrypsinchymotrypsinrenninpapain

Staphylococcus aureus Enterotoxinhttp://seafood.ucdavis.edu/HACCP/Compendium/chapt19.htmhttp://textbookofbacteriology.net/staph.html

SEB

Baird-Parker MediumTSA

(7.5-10.0%)(Potassium telluritsK2TeO3)(0.01-0.05%)0.2-0.5%(Lithin chlorideLiCl)(0.12-1.26%)Glycinepolymycin(40ug/ml)(1)egg yolkglycine(2)K2TeO3

PCRPCR-ELISAmicroarray

Clostridium botulinumType A,B,EAEType C,DType FType G

Clostridium botulinumG+ rodsanaerobicpH 4.87pH 5.01pH 4.515~48

Clostridium botulinum

Botulism100trypsin

Botulinum Toxins

Botulinum Toxin A

Action of Botulinum Toxinneuromuscular junctions peripheral automatic synapsesacetylcholoine18~360.1~1.0 g

Action of Botulinum ToxinAyres et al., 1980

Prevention of Outhbreaksgassy (swollen)

Infant Botulism

Infant Botulism1970in vivo6floraweaknesslack of suckling lose of head control

Trypsin treatment. Toxins of nonproteolytic types, if present, may need trypsin activation to be detectedConduct parallel tests with trypsin-treated materials and untreated duplicatesInject each of separate pairs of mice intraperitoneally (i.p.) with 0.5 ml untreated undiluted fluid and 0.5 ml of each dilution of untreated test sample

Typing of toxin. USE monovalent antitoxinsBAM http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070879.htm

*Emetic toxin MW=2000*Emetic toxin MW=2000*Coagulase *

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Bacterial Intoxification