Food borne bacterial toxins classification, properties and detection methods

1. FOOD BORNE
BACTERIAL TOXINS

2.  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
OVERVIEW OF PRESENTATION

3.  Toxins are the toxicants or poisonous substance liberated or produced by
living organism and generally not well defined chemically.
INTRODUCTION
Depending on origin
Bacterial toxins Mycotoxins Zootoxins Phytotoxins

4. BACTERIAL TOXIN
ENDOTOXIN EXOTOXIN
Cont…

5. ENDOTOXIN
Cont…

6. PROPERTY ENDOTOXIN EXOTOXIN
CHEMICAL NATURE LPS (mw = 10kDa) Protein (mw = 50-1000kDa)
RELATIONSHIP TO CELL Part of outer membrane Extracellular, Diffusible
HEAT STABILITY Yes (600 C) Relatively No
ANTIGENIC Yes Yes
FORM TOXOID No Yes
POTENCY Relatively low (100ug) Relatively high (1 ug)
SPECIFICITY Low degree High degree
ENZYMATIC ACTIVITY No Often
PYROGENICITY Yes Occasionally
SYNTHESIS CONTROL Chromosomal gene Extrachromosomal gene
CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN

7. 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.
PATHOGENESIS BASED EXOTOXINS CLASSIFICATION

8. Reservoir of Pathogen Contamination of food
Viral or Parasitic
infection
Growth of pathogenic bacteria
Infection Intoxication
Toxicoinfection
Food+ Live cells Food +Toxin
Invasive Infection
EVENTS OF FOOD BORNE DISEASES
Mycotoxin

9. Invasive Infection IntoxicationToxicoinfection
o Salmonella
o L. monocytogenes
o E. coli
( enteric type)
o Shigella
o Campylobacter
o Yersinia
o Vibrio parahaemolyticus
o Aeromonas
o Staph. Aureus
o B.Cereus
(Emetic type)
o Cl. botulinum
o Bacillus cereus
(Diarrhoel)
o Cl.botulinum
( Infant)
o Cl . perfringens
o Vibrio cholarae
o E. Coli
(Enterotoxigenic)
Cont…

10. BACTERIAL FOOD BORNE INFECTIONFOOD BORNE BACTERIAL ILLNESS

11. FOOD BORNE BACTERIAL ILLNESSCont…

12. FOOD BORNE BACTERIAL ILLNESSCont…

13.  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 -
o A diversity of mode of action,
o Target cell types,
o Receptors.
 Protein associated with cell wall-
o Produces fluid loss in infant mice.
 Heat stable and rapid in action.
 Heat labile and delayed response :-
o Mol. Wt. of 25 kDa,
o Increases in cAMP and PGE2 levels.
o Stimulate secretion in 18-h
Salmonella
ENTEROTOXINS

14.  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),
o B is the subunit (11.6 kDa) responsible for binding of the
holotoxin to its GM1 receptor
o A is the subunit responsible for the intracellular
changes in cyclic AMP levels.
V. cholerae
C T Zot Ace
Cont…

15. Mechanism of Action of Cholera Toxin
Cont…
GM1

16.  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
Cont…

17.  EAEC-
 EIEC –
 EHEC -
E. coli
Stx1, SLT-I or VTI
ShET2 or
EIEC enterotoxin
EAST 1
4.1 kDa protein
Heat-labile protein
108 kDa ( Pet)
S. flexneri
Stx2, SLT-II or VT2
(Stx2c,2d,2e)
Cont…
Chrom.encoded
110 kDa (Pic)
STx, SLT or VT
S.dysenteriae
Shiga toxin

18. ST LT
STbSTa LTI LTII
o Cysteine-rich,
o 18 / 19-a.a. peptide
o Mol wt 2 kDa
o Bind to G C C-
Increase cGMP-
PKA-
CFTR-
Chloride sec.-
o Diarrhea
o Children -more
effected
o Human & Animal
o Trypsin-sensitive
protein
o No toxic domain
o Nor Intestinal
receptor
o Sulfatid receptor
o No effect on cAMP
or cGMP.
o Not Stimulate
chloride sec.
o Bicarbonate sec.
o Piglets
o Mol wt 80,000
o Bind to GM 1
o Activate
Adenylate cyclase
o Increase cAMP
o Increase PG
o Stimulate GIT
Nervous system
o Human
& Animal
o Absent of B
subunit
o 2 sub unit
LTIIa &LTIIb
o Bind to-
Gangliosid
GD1b or GD1a
o Increase cAMP
o Animal disease
 ETEC
Cont…

19. Shigella
S. flexneri 2a.
S. dysenteriae I Shiga toxin
ShET2 or
EIEC enterotoxinShET1
Cont…
o Chomo. Encoded
o Iron dependent
o 55 KDa
o Plasmid encoded
protein
o 63 KDa

20. Y. enterocolitica
C. jejuni
o Heat-labile enterotoxin (LT-like)
o 60 to 70 kDa
o Iron regulated.
o Yst
o Produced at -
o 30o C
o Increased osmolarity
& pH at 370 C favour Yst.
o Guanylate cyclase activation
o Increase cGMP levels
o Yst-II
o 10- to 30-kDa
o Protein toxin
o Similar to E. coli STa
Cont…

21. Staph. aureus
Exotoxins
PTS Ags Hemolysins
SEsTSST-1
o 18 toxins-
o Classical enterotoxins -
SEA to SEE
o Recently discovered –
o True SEs-- SEG, SEH & SEI
o SEls-
( SElJ to SElP and SElU ).
Cont…

22.  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.
Cont…

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

24. o Wide-range of foods.
o Large no. of the m.o.
o IP 8–16.5 hr.
o Diarrhea, abd. cramps
& tenesmus.
o Long-incubation form.
o Clostridium perfringens
Diarrheal type
Toxicoinfection
Cont…
o Cooked rice & improperly
refrigerated foods .
o Preformed toxin
o I P 1–5 hours
o Nausea & Vomiting
o Recovery within 24 hr
o Staphylococcus aureus
Emetic Form
Intoxication

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

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.
o PRODUCTION :
o In host
o During vegetative growth
o Secreted in Ileum
o Sub unit 6 subunits (1 A & 5 B )
o TRYPSIN
o Proteolysis
o Heat stability
o 60 0 C(650 C -30 min.)
o ENZYMATIC ACTIVITY
o ADP ribosylate
o AFFECT
o Bind to Ganglioside
receptor (epithelialCell)
o Alteration of trans membrane
signal transduction
o Diarrhoea
1000 C -30 min.
No effect.

27. Staph.aureus α β γ δ
Streptococcus SLS SLO
Cl.perfringens α δ 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)
HEMOLYTIC EXOTOXINS

28. :-
 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.
NEUROTOXIN
Clostridium botulinum
Shigella dysenteriae

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

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

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

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

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

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

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

36. LIGATED LOOP TECHNIQUE
o B.cereus,
o Cl perfringens,
o E. coli,
o V. parahaemolyticus
RITARD MODEL
o ETEC
o V.Cholerae
o C. Jejuni
o Aeromonas
ANIMAL MODELS REQUIRE SURGICAL PROCEDURES
Cont…

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

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

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

40.  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
Gel diffusion assay
Cont…

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

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

43.  Detection of soluble antigen
 Antibody + latex particle
 Presence of toxin : diffuse layer
at base
 Negative : button formation
 Simple, rapid
 B. cereus emetic toxin.
Reverse passive latex agglutination
Cont…

44.  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
BoNT/A ALISSA
Cont…

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

46.  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)
PCR
MOLECULAR TECHNIQUES

47. 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
Cont…

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

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

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.