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FOOD BORNE BACTERIAL TOXINS
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OVERVIEW OF PRESENTATION
INTRODUCTION CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN PATHOGENESIS BASED EXOTOXINS CLASSIFICATION EVENTS OF FOOD BORNE DISEASES FOOD BORNE ILLNESS ENTEROTOXINS PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS HEMOLYTIC EXOTOXINS NEUROTOXIN METHODS FOR DETECTION OF TOXINS. SUMMARY
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INTRODUCTION Depending on origin
Toxins are the toxicants or poisonous substance liberated or produced by living organism and generally not well defined chemically. Depending on origin Bacterial toxins Mycotoxins Zootoxins Phytotoxins
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Cont… BACTERIAL TOXIN ENDOTOXIN EXOTOXIN
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Cont… ENDOTOXIN
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CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN
PROPERTY ENDOTOXIN EXOTOXIN CHEMICAL NATURE LPS (mw = 10kDa) Protein (mw = kDa) RELATIONSHIP TO CELL Part of outer membrane Extracellular, Diffusible HEAT STABILITY Yes (600 C) Relatively No ANTIGENIC Yes FORM TOXOID No POTENCY Relatively low (100ug) Relatively high (1 ug) SPECIFICITY Low degree High degree ENZYMATIC ACTIVITY Often PYROGENICITY Occasionally SYNTHESIS CONTROL Chromosomal gene Extrachromosomal gene
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PATHOGENESIS BASED EXOTOXINS CLASSIFICATION
ENTEROTOXINS V. cholerae, E. coli, B. cereus, Cl. perfringens, Salmonella, Staph. aureus, Shigella, NAG vibrios, Aeromonas. HEMOLYTIC EXOTOXINS Staph. aureus, Streptococcus, Cl.perfringens, V. parahaemolyticus, B. cereus, Aeromonas hydrophila. NEUROTOXIN Cl. botulinum, Cl. tetani, Shigella dysenteriae. CYTOTOXIC –CYTOLYTIC EXOTOXIN Strept. Staph. aureus, Shigella , Aeromonas, V. parahaemolyticus, Cl. difficile. DIRECT MACROMOLECULAR SYNTHESIS INHIBITOR Corynebacterium diphtheriae, E. coli B.thuringensis, Yersinia pestis, Pseudomonas, Vibrio cholerae.
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EVENTS OF FOOD BORNE DISEASES Growth of pathogenic bacteria
Viral or Parasitic infection Reservoir of Pathogen Contamination of food Growth of pathogenic bacteria Food+ Live cells Food +Toxin Mycotoxin Intoxication Infection Invasive Infection Toxicoinfection
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Invasive Infection Toxicoinfection Intoxication
Cont… Invasive Infection Toxicoinfection Intoxication Bacillus cereus (Diarrhoel) Cl.botulinum ( Infant) Cl . perfringens Vibrio cholarae E. Coli (Enterotoxigenic) Staph. Aureus B.Cereus (Emetic type) Cl. botulinum Salmonella L. monocytogenes E. coli ( enteric type) Shigella Campylobacter Yersinia Vibrio parahaemolyticus Aeromonas
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BACTERIAL FOOD BORNE INFECTION FOOD BORNE BACTERIAL ILLNESS
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FOOD BORNE BACTERIAL ILLNESS
Cont…
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FOOD BORNE BACTERIAL ILLNESS
Cont…
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ENTEROTOXINS Bacterial toxins that exert some deleterious effect and host response exclusively in the small or large intestine. Alteration in intestinal cell structure or function by - A diversity of mode of action, Target cell types, Receptors. Protein associated with cell wall- Produces fluid loss in infant mice. Heat stable and rapid in action. Heat labile and delayed response :- Mol. Wt. of 25 kDa, Increases in cAMP and PGE2 levels. Stimulate secretion in 18-h Salmonella
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Cholera is caused by V. cholerae of the O1 and O139 serogroups.
Cont… V. cholerae Cholera is caused by V. cholerae of the O1 and O139 serogroups. CT is the prototypic A-B subunit toxin (A/B ratio, 1:5), B is the subunit (11.6 kDa) responsible for binding of the holotoxin to its GM1 receptor A is the subunit responsible for the intracellular changes in cyclic AMP levels. C T Zot Ace
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Mechanism of Action of Cholera Toxin
Cont… Mechanism of Action of Cholera Toxin GM1
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Cont… If a strain is CT positive, it is almost always zot positive (48 KDa peptide) . The onset of action of crude Zot is immediate and reversible. Zot may contribute to diarrhea in cholera by altering the permeability of intestinal tissue. Ace kDa protein. stimulate electrogenic chloride secretion & contribute, to the pathogenicity of V. cholerae O1. Produce a 17-a.a. NAG-ST that shares 50% sequence homology with the STa of ETEC . This toxin is found only in a minority of non-O1/non-O139 V. cholerae strains. NAG Vibrio
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EAEC- EIEC – EHEC - Cont… E. coli EAST 1 Heat-labile protein
108 kDa ( Pet) Chrom.encoded 110 kDa (Pic) EAST 1 4.1 kDa protein ShET2 or EIEC enterotoxin S. flexneri Shiga toxin S.dysenteriae STx, SLT or VT Stx1, SLT-I or VTI Stx2, SLT-II or VT2 (Stx2c,2d,2e)
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Cont… ST LT ETEC STb LTI LTII STa Trypsin-sensitive protein No toxic domain Nor Intestinal receptor Sulfatid receptor No effect on cAMP or cGMP. Not Stimulate chloride sec. Bicarbonate sec. Piglets Mol wt 80,000 Bind to GM 1 Activate Adenylate cyclase Increase cAMP Increase PG Stimulate GIT Nervous system Human & Animal Absent of B subunit 2 sub unit LTIIa <IIb Bind to- Gangliosid GD1b or GD1a Increase cAMP Animal disease Cysteine-rich, 18 / 19-a.a. peptide Mol wt 2 kDa Bind to G C C- Increase cGMP- PKA- CFTR- Chloride sec.- Diarrhea Children -more effected Human & Animal
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Cont… Shigella Shiga toxin S. dysenteriae I S. flexneri 2a. ShET2 or EIEC enterotoxin ShET1 Plasmid encoded protein 63 KDa Chomo. Encoded Iron dependent 55 KDa
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Cont… C. jejuni Heat-labile enterotoxin (LT-like) 60 to 70 kDa Iron regulated. Y. enterocolitica Yst Produced at - 30o C Increased osmolarity & pH at 370 C favour Yst. Guanylate cyclase activation Increase cGMP levels Yst-II 10- to 30-kDa Protein toxin Similar to E. coli STa
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Cont… Exotoxins Staph. aureus PTS Ags Hemolysins TSST-1 SEs 18 toxins- Classical enterotoxins - SEA to SEE Recently discovered – True SEs-- SEG, SEH & SEI SEls- ( SElJ to SElP and SElU ).
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SEs are potent emetic agents whereas the other PTSAgs are not .
Cont… SEs - thermostable , resistant to GIT enzymes. Heat stability depend on the environmental factors - aw & pH (Balaban et al., 2000) SEA and SED, the most frequently involved in food poisoning (Balaban et al., 2000 ; Le et al ., 2003 ) 5% cases attributed to new enterotoxins & SEH most commonly reported (Jorgensen et al ., 2005) SEs are potent emetic agents whereas the other PTSAgs are not . The conc. of SE from 0.5 to 1.0 ng/ml and depends on the susceptibility of the individual.
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Cont… B. cereus DIARRHEAGENIC toxin EMETIC toxin Thermolabile antigenic protein Mol wt. 38, ,000 Da Susceptible to – 560 C - 30 min. Trypsin & Pronase digestion Highly stable Probably a peptide Mol. Wt. < 10,000 Withstand – 1260C hr, Extreme pH, Typsin & Pepsin exposure
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Cont… Emetic Form Diarrheal type Wide-range of foods. Large no. of the m.o. IP 8–16.5 hr. Diarrhea, abd. cramps & tenesmus. Long-incubation form. Clostridium perfringens Cooked rice & improperly refrigerated foods . Preformed toxin I P 1–5 hours Nausea & Vomiting Recovery within 24 hr Staphylococcus aureus Toxicoinfection Intoxication
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Cont… Aeromonas Several cytotonic enterotoxins 15-20 kDa heat stable protein 44 kDa heat labile protein CT-like enterotoxin .
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PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS
Salmonella, E. coli, Campylobacter , Vibrio cholerae, Aeromonas, B.cereus E.coli, Y.enterocolitica, Staph.aureus, B.cereus. PRODUCTION : In host During vegetative growth Secreted in Ileum Sub unit 6 subunits (1 A & 5 B ) TRYPSIN Proteolysis Heat stability 60 0 C(650 C -30 min.) ENZYMATIC ACTIVITY ADP ribosylate AFFECT Bind to Ganglioside receptor (epithelialCell) Alteration of trans membrane signal transduction Diarrhoea No effect. 1000 C -30 min.
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HEMOLYTIC EXOTOXINS Staph.aureus α β γ δ Streptococcus SLS SLO
Cl.perfringens θ V. parahaemolyticus TDH(1000 C -10 min) B. cereus Primary hemolysin – H-1, Cereolysin & Mouse lethal factor 2ndry – H -II A. hydrophila α hemolysin β hemolysin (Aerolysin)
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Clostridium botulinum
NEUROTOXIN :- BoNT also known as “Botox”. 150-kDa zn-binding metallo protease (holotoxin) 100-kDa heavy & 50-kDa light chain connected by a reducible disulphide bond (Schiavo et al., 2000) 100,000 times more toxic than sarin (Shapiro et al., 1998). Estimated human lethal i.v. dose 1-2 ng/kg body weight ( Arnon et al., 2001) Botulin toxin - potential bioweapon, 75 nanograms to kill a person (LD50- 1ng/kg) (Fleming., 2000) 500 grams is enough to kill half of the entire human population Shiga toxin. Clostridium botulinum Shigella dysenteriae
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METHODS FOR DETECTION OF TOXINS
BIOASSAY METHOD. IMMUNOLOGICAL ASSAYS. MOLECULAR TECHNIQUES. CELL CULTURES.
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BIOASSAY METHOD WHOLE ANIMAL ASSAYS MOUSE LETHALITY
Botulinum toxin in foods Cl.perfringens enterotoxin Listeria virulence assesment
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Cont… FERRETS Staphylococcal enterotoxin B MONKEY FEEDING TEST
Staph enterotoxins ( Most sensitive )
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Cont… Staph enterotoxins Cereulide – B. cereus KITTEN / CAT TEST
SUNCUS MURINUS EMESIS MODEL Cereulide – B. cereus
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Cont… RABBIT & MOUSE DIARRHOEA E.coli Y.enterocolitica Vibrio cholerae
(enterotoxins) SUCKLING / INFANT MOUSE E. Coli enterotoxins ( STa, STb ) Y. enterocolitica (Yst)
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Cont… RABBIT SKIN TEST Vascular permeability- E. Coli
(diarrheagenic enterotoxin) GUINEA PIG SKIN TEST Erythemal activity- Cl. perfringens enterotoxin
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Cont… SERENY & ANTON TEST Listeria Guinea pig, Mouse & Rabbit Shigella
EIEC Listeria
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ANIMAL MODELS REQUIRE SURGICAL PROCEDURES
Cont… ANIMAL MODELS REQUIRE SURGICAL PROCEDURES LIGATED LOOP TECHNIQUE B.cereus, Cl perfringens, E. coli, V. parahaemolyticus RITARD MODEL ETEC V.Cholerae C. Jejuni Aeromonas
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Double antibody "sandwich" ELISA
IMMUNOLOGICAL ASSAYS ELISA Double antibody "sandwich" ELISA Staph enterotoxins A - E Bennett,.et al; 1994
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Sensitivity - 1-5 ng range E.coli STa
Cont… Radioimmunoassay Solid phase RIA : Staph. ET -: A to E Sensitivity ng range E.coli STa
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Reverse passive haemagglutination
Cont… Reverse passive haemagglutination Antibody coupled to sheep RBCs Agglutination if toxin present Sensitivity for SE–B : 1.5 ng /ml VIDAS SET Assay Staph. enterotoxins A - E Sensitivity of at least 1 ng/ml. Immunoenzymatic test. Specific for Staph enterotoxin A - E.
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Gel diffusion assay Simple gel ID tech. (Biken’s test): Cont…
Microslide gel double diffusion test: B.cereus diarrhoeal enterotoxins Cl. perfringens enterotoxins Staph. B enterotoxin Simple gel ID tech. (Biken’s test): E. coli Sensitivity : 98% Time: 3-4 days
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Electroimmunodiffusion
Cont… Electroimmunodiffusion SET-A B. cereus enterotoxin Cl.botulinum enterotoxin A to E.
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Radial immunodiffusion assay
Cont… Radial immunodiffusion assay Detection of ST- A,B Sensitivity – 0.3mcg /ml
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Reverse passive latex agglutination
Cont… Reverse passive latex agglutination Detection of soluble antigen Antibody + latex particle Presence of toxin : diffuse layer at base Negative : button formation Simple, rapid B. cereus emetic toxin.
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BoNT/A ALISSA Cont… Assay with a large immuno-sorbent surface area.
Captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. An inexpensive, simple and robust procedure that ensures high analytical specificity and attomolar sensitivity for the detection of BoNT/A in complex biological samples
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ECL ASSAYS Cont… ELECTRO CHEMI LUMINESCENCE
To detect Clostridium botulinum neurotoxins serotypes A, B, E, and F Similar to the gold standard mouse bioassay.
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MOLECULAR TECHNIQUES PCR Duplex SYBR green I real-time PCR assay- for one-step differentiation between emetic B. cereus and S. aureus (Mackay., 2004) Real-time PCR-based assays - BoNT A, B, E and F gene fragments (Akbulut ., 2005)
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TaqMan-based, real-time PCR assay –
Cont… TaqMan-based, real-time PCR assay – Provide a rapid and sensitive method for the specific detection of emetic B. cereus in food NUCLEIC ACID PROBES Cl.perfringens enterotoxin Staphylococcal enterotoxin
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Human foetal intestine
CELL CULTURES Human foetal intestine V.parahaemolyticus CHO monolayer E.coli LT V.cholerae ET Salmonella ET
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Cont… Vero cells E.coli LT Cl.perfringens ET Salmonella cytotoxin A.hydrophilla toxin Y-1 adrenal cells E.coli ET V.cholerae toxin
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SUMMARY Both Gram positive and Gram negative bactera are resposible for most of the outbreak of the food poisining. Bacterial exotoxins have enterotoxic, cytotoxic, hemolytic and neurotoxic effect . It is the bacterial enterotoxins which are responsible for production of various type of gastrointestinal manifestations like diarrhea and vomiting during different food borne bacterial illness. Some bacterial toxins are very potent and relatively easy to produce and classified As bio-threat agents. Eg.. Botulinum neurotoxins. Bioassay method, Immunological assays, Molecular techniques & Cell cultures are used to detect the bacterial toxin.
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