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FOOD BORNE BACTERIAL TOXINS

FOOD BORNE BACTERIAL TOXINS. OVERVIEW OF PRESENTATION. INTRODUCTION CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN PATHOGENESIS BASED EXOTOXINS CLASSIFICATION EVENTS OF FOOD BORNE DISEASES FOOD BORNE ILLNESS ENTEROTOXINS

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FOOD BORNE BACTERIAL TOXINS

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  1. FOOD BORNE BACTERIAL TOXINS

  2. 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

  3. INTRODUCTION • 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

  4. Cont… BACTERIAL TOXIN ENDOTOXIN EXOTOXIN

  5. Cont… ENDOTOXIN

  6. CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN

  7. PATHOGENESIS BASED EXOTOXINS CLASSIFICATION

  8. EVENTS OF FOOD BORNE DISEASES 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

  9. 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

  10. BACTERIAL FOOD BORNE INFECTION FOOD BORNE BACTERIAL ILLNESS

  11. Cont… FOOD BORNE BACTERIALILLNESS

  12. Cont… FOOD BORNE BACTERIAL ILLNESS

  13. 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

  14. 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

  15. Cont… Mechanism of Action of Cholera Toxin GM1

  16. 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 11.3-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

  17. Cont… E. coli • EAEC- • EIEC – • EHEC - 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)

  18. 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 &LTIIb • 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

  19. 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

  20. 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

  21. 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 ).

  22. 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.

  23. Cont… B. cereus DIARRHEAGENIC toxin EMETIC toxin • Thermolabile antigenic protein • Mol wt. 38,000 - 57,000 Da • Susceptible to – • 560 C - 30 min. • Trypsin & Pronase digestion • Highly stable • Probably a peptide • Mol. Wt. < 10,000 • Withstand – • 1260C - 1.5 hr, • Extreme pH, • Typsin & Pepsin exposure

  24. 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

  25. Cont… Aeromonas • Several cytotonic enterotoxins • 15-20 kDa heat stable protein • 44 kDa heat labile protein • CT-like enterotoxin .

  26. PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS Heat Labile Heat Stable 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.

  27. HEMOLYTIC EXOTOXINS

  28. NEUROTOXIN Clostridium botulinum :- • 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. Shigella dysenteriae

  29. METHODS FOR DETECTION OF TOXINS • BIOASSAY METHOD. • IMMUNOLOGICAL ASSAYS. • MOLECULAR TECHNIQUES. • CELL CULTURES.

  30. BIOASSAY METHOD WHOLE ANIMAL ASSAYS MOUSE LETHALITY • Botulinum toxin in foods • Cl.perfringens enterotoxin • Listeria virulence assesment

  31. Cont… FERRETS • Staphylococcal enterotoxin B MONKEY FEEDING TEST • Staph enterotoxins ( Most sensitive )

  32. Cont… KITTEN / CAT TEST • Staph enterotoxins SUNCUS MURINUS EMESIS MODEL • Cereulide – B. cereus

  33. Cont… RABBIT & MOUSE DIARRHOEA • E.coli • Y.enterocolitica • Vibrio cholerae (enterotoxins) SUCKLING / INFANT MOUSE • E. Coli enterotoxins ( STa, STb ) • Y. enterocolitica (Yst)

  34. Cont… RABBIT SKIN TEST Vascular permeability- • E. Coli (diarrheagenic enterotoxin) GUINEA PIG SKIN TEST Erythemal activity- • Cl. perfringens enterotoxin

  35. Cont… SERENY & ANTON TEST Guinea pig, Mouse & Rabbit • Shigella • EIEC • Listeria

  36. 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

  37. IMMUNOLOGICAL ASSAYS ELISA Double antibody "sandwich" ELISA • Staph enterotoxins A - E Bennett,.et al; 1994

  38. Cont… Radioimmunoassay Solid phase RIA : Staph. ET -: A to E Sensitivity - 1-5 ng range E.coli STa

  39. Cont… Reverse passive haemagglutination VIDAS SET Assay • Staph. enterotoxins A - E • Sensitivity of at least 1 ng/ml. Immunoenzymatic test. • Specific for Staph enterotoxin A - E. Antibody coupled to sheep RBCs Agglutination if toxin present Sensitivity forSE–B: 1.5 ng /ml

  40. Cont… Gel diffusion assay • 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

  41. Cont… Electroimmunodiffusion SET-A B. cereus enterotoxin Cl.botulinum enterotoxin A to E.

  42. Cont… Radial immunodiffusion assay Detection of ST- A,B Sensitivity – 0.3mcg /ml

  43. 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.

  44. Cont… BoNT/A ALISSA • 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

  45. Cont… ECL ASSAYS • ELECTRO CHEMI LUMINESCENCE • To detect Clostridium botulinum neurotoxins serotypes A, B, E, and F • Similar to the gold standard mouse bioassay.

  46. 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)

  47. NUCLEIC ACID PROBES Cont… • TaqMan-based, real-time PCR assay – • Provide a rapid and sensitive method for the specific detection of emetic B. cereus in food • Cl.perfringens enterotoxin • Staphylococcal enterotoxin

  48. CELL CULTURES Human foetal intestine • V.parahaemolyticus CHO monolayer E.coli LT V.cholerae ET Salmonella ET

  49. Cont… Vero cells • E.coli LT • Cl.perfringens ET • Salmonella cytotoxin • A.hydrophilla toxin Y-1 adrenal cells • E.coli ET • V.cholerae toxin

  50. 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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