Kuliah toksikologi 2010 2nd meeting
TOXICOLOGY ON FISHERIES
PROCESSING– 3 (2 – 1)
DISEASE CAUSED BY MICROORGANISMS
EKO SUSANTO
Study Program of Fisheries Processing Technology
Diponegoro University
Email : eko_thp@undip.ac.id
EKO SUSANTO – DIPONEGORO UNIVERSITY
PATHOGEN BACTERIA THAT
PRODUCE TOXIN
QUESTIONS
Please mention characteristic of C.
botulinum?
What happen when butulinum toxin
ingested?
Please explain about E. coli based on
your knowledge?
Please answer those questions for 10
minutes.
CLOSTRIDIUM BOTULINUM
4
EKO
SUSANTO
– DIPONEGORO
Eko
Susanto
– DiponegoroUNIVERSITY
University(eko_thp@undip.ac.id)
THE ORGANISM
Gram (+) spore-forming
rod
Only srovar A, B, E & F
cause botulism in human.
2 types of C. Botulinum
exist : proteolytic (A, some
B & F)
Non proteolytic (E, some
B&)
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
CHARACTERISTIC OF C. botulinum
Gram positive, endospore-forming
anaerobes.
Botulism is characterized as a rare paralytic
disease caused by a nerve toxin produced by
the pathogen.
The rod-shaped organisms grow best in lowoxygen environments.
Proteolityc C. botulinum is a highly
dangerous pathogen.
BOTULINUM TOXIN TYPES
A*= vegetable, fruit, meat, fish & canned products
B*= pork meat
C = spoil vegetable, carcass, & pork liver
D = carcass
E*= fish, marine organism, & raw fish
F = liver paste
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
BOTULINOGENIC PROPERTIES OF FISH
PRODUCTS (HUSS, 1994)
Fish product
Fresh and
frozen
Pasteurized
Cold smoked
Fermented
Semipreserved
Fully
preserved
Factors adding to botulism
hazard
Vacuum packaging
Prolonged storage life
toxin produced before
putrefaction
vacuum packaging
poor hygiene
Same as above
Not cooked before being
eaten
No tradition for chill storage
Fermentation may be slow
High temperature during
fermentation
Not cooked before being
eaten
Not cooked before being
eaten
Not cooked before being
eaten
Packed in closed cans
Factors reducing
botulism hazard
Traditional chill storage
putrefaction before toxin
is produced
Chill storage (< 3oC)
Synergistic aerobic flora
eliminated
Safety of product
based on
Cooking before being
eaten
clasification
Cooking before being
eaten
Chill storage
No risk if
cooked
High risk if
not cooked
Chill storage
Salting (NaCl
concentration > 3%)
High redox-potential in
unspoiled products
Salting (NaCl
concentration 3 % in
brine)
Chill storage
Low pH
Application of salt, acid
etc
Chill storage
Autoclaving
Chill storage
Process control (raw
material, salting when
applicable)
High risk
Process control
Chill storage
High risk
Process control
Low risk
No risk
Process control
Low risk
(autoclaving, closing of
cans)
CHARACTERISTIC OF BOTULINUM NEUROTOXIN
Heat labile proteins.
Innactivation at temperature 121oC, freezing
doesn’t innactivate botulism toxins.
7 major neurotoxins (types A – G).
Neurotoxin are commonly associated with other
proteins, such as hemagluttinin & non-toxinnonhemagglutinin.
The neurotoxins are 150-kDa poteins, comprise
heavy chain (100 kDa) & light chain (50 kDa).
Neurotoxin
ingested in
food
WHAT
HAPPENS
WHEN C.
botulinum
NEUROTOXIN
IS
INGESTED ??
Neurotoxin
passes
through gut
mucosa into
bloodstream
Neurotoxin spreads
through body via
bloodstream
Toxin binds to nerve at the
nerve muscle junction
Nerve supply
to muscle
fiber
(Garbutt, 2007)
redesign by Susanto
This block the
release of
acethylcholine.
Muscle cannot to
contract, resulting
in paralysis
Muscle
fibre
Mechanism of Botulinum Toxin
Botulinum toxin ingested on human
EFFECT OF ENVIRONMENTAL FACTORS ON THE
GROWTH AND SURVIVAL OF PROTEOLYTIC C.
botulinum AND NON PROTEOLITIC
C.
botulinum B (PECK, 2010)
Factor
Proteolytic C.
botulinum
Neurotoxin formed
A,B,F
Minimum growth temperature
10-12oC
Maximum growth temperature
37oC
Minimum pH for growth
4.6
NaCl concentration preventing growth 10
(%)
Minimum water activity for growth
NaCl as humectant
0.94
Glycerol as humectant
0.93
Spore heat resistant
D121oC = 0.21 min
Spore radiation resistant
Food involved in botulism outbreak
D = 2.0 – 4.5 kGy
Home-canned
foods, faulty
commercial
processing
Heat reistance data without/with lysoyme during recovery
modified from the work of Lund & Peck (2000)
Nonproteolytic C.
botulinum
B,E,F
2.5-3.0oC
25oC
5.0
5
0.97
0.94
D82.2oC = 2.4/231
mina
D = 1.0 – 2.0 kGy
Fermented marine
products, dried
fish, vacuumpacked fish
SYMPTOMS BOTULISM
Nausea & vomitting
Mainly:
neuroligical-burred / blurred vission,
difficulty to swallowing (dysphagia), mouth
dryness, speech difficulties (dysphonia) &
limb & respiration become paralysed,
dizziness/vertigo, muscle weakness.
Death normally caused by respiratory &
cardiac paralysis.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
LETHAL DOSE OF TOXIN
Botulism toxins are among the most toxic
subtances.
The minimum lethal dose for mice is 0.4-2.5
ng/kg mouse tissue.
50 % lethal dose for human is 1 ng/g body
weight. ex.: 10 people weighing 80 kg each
ingested 8.0 x 10-8g of toxin then five of them
would beexpected die.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
110
100
90
Reported Cases
80
70
60
50
40
30
20
10
0
1982 1987
MMWR
1992
1997 Year 2002
Center for Food Security and Public
Health Iowa State University 2004
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
RECORDED FOOD-BORNE BOTULISM IN DIFFERENT
COUNTRIES (PECK, 2010)
Country
Period
Belgium
Canada
China
Denmark
France
Georgia
Germany
Italy
Japan
Poland
Spain
Sweden
United
Kingdom
United States
1982
1971
1958
1984
1971
1980
1983
1979
1951
1971
1971
1969
1971
–
–
–
–
–
–
–
–
–
–
–
–
–
2000
2005
1983
2000
2003
2002
2000
2000
1987
2000
1998
2000
2005
1971 – 2003
No. of cases
Total
Avg per yr
32
2
439
13
4377
168
18
1
1286
39
879
40
376
22
750
34
479
13
9219
307
27
10
13
1
38
1
934
28
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
EXAMPLE OF RECENT INCIDENTS OF FOOD BORNE BOTULISM
INVOLVING NONPROTEOLYTIC C. botulinum
Yr
Country
199
1
Egypt
199
7
199
7
199
8
Fance
Product
Commercial
unviscerated, salte
fish
Fish
Germany Home-smokked,
vacuum packed fish
France
Commercial frozen,
vacum packed
scallop
200 Canada
Hommade
1
fermented samon
roe
200 Germany Home-salted, air3
dried fish
200 Germany Commercial
4
vacuum packed
(Peck, 2010) smoked salmon
Toxin
type
No. of
cases
E
> 81
Factor contributing
to botulism
References
outbreak
Putrefaction of fish Weber et al
before salting
(1993)
E
1
Not known
Korkeala et
al., 1998
Anonymous
(1998b)
Boyer et al
(2001)
E
4
E
1
Temperature
abuse
Temperature
abuse
E
4
Unsafe process
Anoymous
2002
E
3
E
1
Temperature
abuse
Consumed after
“use by date”
Eriksen et al
(2004)
Dressler
(2005)
CONTROL OF PROTELYTIC C. Botulinum IN FOOD
PROCESSING OPERATION
Minimum growth & neurotoxin production
occur with range 10oC to 12oC
Growth of proteolytic C. Botulinum is
prevented at pH of < 4.6 or by 10 % NaCl
Minimum aW 0.94 and 0.93 with NaCl &
Glycerola.
Heat treatment at 121.1oC for 3 min has
been adopted as the minimum standard for a
botulinum cook
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
CONTROL OF NONPROTELYTIC C. Botulinum IN FOOD
PROCESSING OPERATION
Minimum growth & neurotoxin production
occur with range 3oC to 3.3oC at 5 – 7 weeks.
Growth of proteolytic C. Botulinum is
prevented at pH of < 5 or by > 5 % NaCl
Minimum aW 0.97 and 0.94 with NaCl &
Glycerola.
Alternative processing technology: high
hydrostatic-pressur-treated
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
RECOMMENDED PROCEDURES TO ENSURE THE SAFETY
OF MINIMALLY HEATED FOODS WITH RESPECT TO
NONPROTEOLYTIC C. botulinum
Recommendation
Storage at < 3.0 oC
Storage at ≤ 8 oC and shelflife of ≤ 10 oC
Storage at chill temperature combined with heat treatment of
90oC for 10 min or equivalent lethality (e.g., 80oC for 129 min,
85oC for 36 min
Storage of chill temperature combined with pH ≤ 5.0 throughtout
the food
Storage of chill temperature combined with a salt concentration
of ≥ 3.5% throughtout the food
Storage at chill temperature combined with a combination of
heat treatment and other preservative factors which can be
shown consistenly to prevent growth and neurotoxin production
by C. botulinum
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
STAHYLOCOCUS AUREUS
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
CHARACTERISTIC OF THE ORGANISM
Gram-positive cocci occuring in iregular clumps.
Causing intoxication.
Optimum growth temperature is 37oC (range of 6
to 48oC).
Growth is inhibited in the presence of 0.1%
presence acetic acid (pH 5.1) or at pH 4.8 with 5
% NaCl.
Able to grow at Aw 0.86. it is capable to grow at
Aw 0.83 in the presence of NaCl, sucrose, or
glycerol humectants.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SOURCE OF MO
Staphylococci are ubiquitous in air, dust, sewage,
water, milk, & many foods & on food equipments,
environmental surface, human & animals.
30 – 50 % of population are nasal & throat carrier.
15 % are skin carrier (hands esp. patients & staff in
hospitals having a carier rate 80%)
Skin lesson exp. boils & infection of cuts & burns.
Human faeces & clothing.
Cows & goat.
Food products with high protein is a good growth
substrates for S. aureus.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
FOOD-BORNE OUTBREAKS
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SFP CHARACTERISTIC
Caused by ingestion of food containing SE
performed by metabolically active
staphylococci.
Usually a self-limiting illness with short
incubation (1-8 h).
The severity depends on :
individual’s susceptibility to the SE,
the amount of contaminated food eaten,
the amount of in the food ingested.
The general health of victim
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SYMPTOMS
Nausea
Vomitting
Diarrhoea & abdominal pain.
Collapse & dehydration in severe cases
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
LETHAL DOSE OF TOXIN
The minimum amount of toxin required
of toxin to produce food poisoning is 1
ng/g (10-9 g/g) of food ingested
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
PREVALENCE OF S. aureus IN SEVERAL FOODS
Products
No. of
samples
tested
Raw meat
139
%
positive
for S.
aureus
2.8
Raw milk
714
7.9
NA
Frozen prawn
46
23.9
>3
Shrimp
1,468
27
>3
Ready-to-eat
fast food
3,332
8.6
NA
No. of S.
aureus
CFU/g
NA
%
Positiv
Reference
e for
SE
7.8
Moon et al.
(2007)
31.8
Moon et al.
(2007)
NA
Sanjeev et al.
(1987)
NA
Swartzentruber et
al. (1980)
47
Oh et al. (2007)
Source: Seo & Bohac, 2010
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
FOOD ASSOCIATED WITH OUTBREAK OF
S. aureus
Cooked meats & meat products
Foods containing milk of cream
Poultry meat & meat products
Pre-cooked fish & fish products
Pre-cooked crusaceans
Gelatine glazes
Canned food
pasta
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
PREVENTION OF OUTBREAK OF S.
aureus
Avoid direct handling of foods (use tongs /
gloves)
Ensure that raw materials used 4 the prodution
of high risk foods are kept refrigerated b4 used.
Ensure that high risk foods are rapidly cooled to
below 5oC after cooking.
Ensure that gigh-risk foods are refrigerate until
ready to use.
Good personal hygiene.
Cover cust / wounds with waterproof dressing
Exclude anyone coughing, sneezing / septics
cuts / boil from food handling.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
ESCHERICHIA COLI
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
THE CHARACTERISTIC OF ORGANISM
Gram negative rod.
Member family Enterobacteriaceae
family.
Able to adapt & colonize a diverse
array of environment & the
gastrointestinal (GI)
E. coli bacteria are mesophilic
organism
E. coli able to grow at temperature of
10-40oC with optimum tempt 37oC
Pathogen can replicate pH values of
4 – 10 & in the presence up t 8%
NaCl.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SOURCE OF DIARRHEAGENIC E. coli
Environment:
Water sources, compost, urban & rural
soils & landscape, sewage, animals include
beef & dairy cattle, sheep, swine, horses,
rodents, dogs, horses, rodents.
Foods:
Cross contamination to RM, processing
water, equipments, & workers.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
GROUPS OF E. coli
Entheropathogenic E. coli
(EPEC)
Enteroinvasive E. coli (EIEC)
Enterotoxigenic E. coli
(ETEC)
Enterohaemorrhagic E. coli
(EHEC), also called
verocytotoxic E. coli (VTEC)
Causing gastroenteritis
in babies & children
Causing Travellers diarrhoea
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
MOST COMMON MODE OF TRANSMISSION, HOST,
SYMPTOMS, & CHARACTERISTICS OF ILLNESS ASSOCIATED
WITH DIFFERENT CLASSES
Class
Classic host
Symptoms
EPEC
Infants (< 6 mo);
more prevalent
in deveeloping
countries
Children; moe
prevalent in
devloping
countries
Children, more
prevalent in
developing
countries
Severe
diarrhea, fever,
vomiting
EAEC
EIEC
Watery or
bloody
diarrhea, fever
Watery
diarrhea,
abdominal
cramping,
fever
ETEC
Traveers &
Watery
infants native to diarrhea,
developing
abdominal
countries
cramping, milk
fever, nausea
EHEC
Children &
Diarrhea,
elderly
bloody diarhea,
(Beadchamp & Sofos, 2010)
abdominal
Incubation
duration
(days)
Variable
Acute / chronic
presentation
Infection dose
Chronic
diarrhea,
malnutrition
High; low in
fant
Variable
High
1-3; self
limiting
Chronic watery
diarrhea,
severe
dehydration
Dysentry
syndrome
1-3; 3-7
Cholera – like
High
1-8; 4-10
Bloody diarrhea low
(hemorrhagic
colitis), HUS,
Low
MECHANISM OF PATHOGENICITY WHICH
DIFFERENT E. coli
Class
adhesion
site
EPEC
Small
intestine
EAEC
Large &
small
intestine
Large
intestine
(colon)
EIEC
ETEC
EHEC
(Beadchamp
Adhesio
n
mediato
r
Intimin
Invassio Toxins
n
potential
Other
virulence
factor
Moderat
e
AAF
None
Possible
enterotoxin
(EAST1)
EAST1, Pet, Pic
EAF palsmid,
LEE island,
flagellin, CDT
Flagellin
Unclear
High
Enterotoxin
None
LT, ST
Cell-to-cell
spread )IcsA),
serineprotease
(SepA)
CDT
Moderat
e
Stx,
enterohemolysin,
EAST1
Small
Fibrial
intestine
CFs
Large
Intimin
intestine
&(colon)
Sofos, 2010)
LEE island,
pO157,
flagellin,
CDT, CNF
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SYMPTOMS
Diarrhoea & abdominal pain with bleeding –
blood appears in stool.
Renal failure due to blood cots in the kidney
tubules.
Internal bleeding due to resulting in brain
damage.
headache, mild fever,
Severe infections of certain serotypes include
bloody diarrhea (hemorrhagic colitis), as well as
real (kidney) malfunction and failure,
thrombocytopenia )inadequate platelet count),
microangiopathic hemolytic anemia (lysis of red
EKO SUSANTO
– DIPONEGORO
UNIVERSITY (eko_thp@undip.ac.id)
blood cells), hemolytic
uremic
syndrome
(HUS).
INTRINSIC & EXTRINSIC FACTORS INVOLVED IN
OUTBREAKS & RECALLS
Intrinsic factors:
pH, Aw, temperture,nutrients of food.
Extrinsic factors:
Higher level contamination, prevalence in
contaminated food products & frequency
of catered /picnic/outdoor gilling events.
Nationwide distribution of contaminated
products causes outbreak
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
FOOD PROCESSING CONDITION ASSOCIATED
WITH OUTBREAK
5 most significant “foodborne illness
risk factors” include:
Acquitition of products from unsafe sources
Poor personal hygiene
Contaminated processing equipments
Inadequate heat treatments
Improper handling, processing & storage
temperature
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
INFECTIVE DOSE
The infective dose to be low, possibly 10 –
100 organism.
E. coli infection strategy
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
Center for Food Security and Public
Health Iowa State University 2004
MMWR
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
ADVANCES IN DIARRHEAGENIC E. coli CONTROL
MEASURES DURING PRODUCTION & PROCESSING
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SUFFER SUPPLY CHAINS
Increased pest control & sanitation
programs.
Use of HACCP concepts, GAP, GHP,
SSOP, GDP.
Final products testing for E. coli
hide contamination and/or fish meat
contamination.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
MINIMIZING CONTAMINATION EVENTS DURING
PROCESSING
Address worker education & hygiene.
Zero tolerance policy during processing
include equipments, RM, products, &
workers.
Reduce cross contamination betwen
products & equipmentsand/or workers
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
INACTIVATION
Appropriate processing & handling
Pasteurization
Heat treatments
Low dose of irradiation (≥ 3 kGy is able
to innactivate pathogen).
Nonthermal processes: High
hydrostatic pressure, shock waves,
ultrasonication, pulsed ultraviolet
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
INHIBITION
Using antimicrobial agents/ingredients within
food products.
Using modified atmosphere packaging.
Chill temperature (4oC or 40oF) E. coli not
grow
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
PREVENTION
Do not eat raw / undercooked beef / drink
raw milk
Abattoir hygiene to prevent faecal
contamination
adequate cookin of meat & fish
Good personal hygiene & health education
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
THANK YOU FOR
ATTENTION
PROCESSING– 3 (2 – 1)
DISEASE CAUSED BY MICROORGANISMS
EKO SUSANTO
Study Program of Fisheries Processing Technology
Diponegoro University
Email : eko_thp@undip.ac.id
EKO SUSANTO – DIPONEGORO UNIVERSITY
PATHOGEN BACTERIA THAT
PRODUCE TOXIN
QUESTIONS
Please mention characteristic of C.
botulinum?
What happen when butulinum toxin
ingested?
Please explain about E. coli based on
your knowledge?
Please answer those questions for 10
minutes.
CLOSTRIDIUM BOTULINUM
4
EKO
SUSANTO
– DIPONEGORO
Eko
Susanto
– DiponegoroUNIVERSITY
University(eko_thp@undip.ac.id)
THE ORGANISM
Gram (+) spore-forming
rod
Only srovar A, B, E & F
cause botulism in human.
2 types of C. Botulinum
exist : proteolytic (A, some
B & F)
Non proteolytic (E, some
B&)
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
CHARACTERISTIC OF C. botulinum
Gram positive, endospore-forming
anaerobes.
Botulism is characterized as a rare paralytic
disease caused by a nerve toxin produced by
the pathogen.
The rod-shaped organisms grow best in lowoxygen environments.
Proteolityc C. botulinum is a highly
dangerous pathogen.
BOTULINUM TOXIN TYPES
A*= vegetable, fruit, meat, fish & canned products
B*= pork meat
C = spoil vegetable, carcass, & pork liver
D = carcass
E*= fish, marine organism, & raw fish
F = liver paste
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
BOTULINOGENIC PROPERTIES OF FISH
PRODUCTS (HUSS, 1994)
Fish product
Fresh and
frozen
Pasteurized
Cold smoked
Fermented
Semipreserved
Fully
preserved
Factors adding to botulism
hazard
Vacuum packaging
Prolonged storage life
toxin produced before
putrefaction
vacuum packaging
poor hygiene
Same as above
Not cooked before being
eaten
No tradition for chill storage
Fermentation may be slow
High temperature during
fermentation
Not cooked before being
eaten
Not cooked before being
eaten
Not cooked before being
eaten
Packed in closed cans
Factors reducing
botulism hazard
Traditional chill storage
putrefaction before toxin
is produced
Chill storage (< 3oC)
Synergistic aerobic flora
eliminated
Safety of product
based on
Cooking before being
eaten
clasification
Cooking before being
eaten
Chill storage
No risk if
cooked
High risk if
not cooked
Chill storage
Salting (NaCl
concentration > 3%)
High redox-potential in
unspoiled products
Salting (NaCl
concentration 3 % in
brine)
Chill storage
Low pH
Application of salt, acid
etc
Chill storage
Autoclaving
Chill storage
Process control (raw
material, salting when
applicable)
High risk
Process control
Chill storage
High risk
Process control
Low risk
No risk
Process control
Low risk
(autoclaving, closing of
cans)
CHARACTERISTIC OF BOTULINUM NEUROTOXIN
Heat labile proteins.
Innactivation at temperature 121oC, freezing
doesn’t innactivate botulism toxins.
7 major neurotoxins (types A – G).
Neurotoxin are commonly associated with other
proteins, such as hemagluttinin & non-toxinnonhemagglutinin.
The neurotoxins are 150-kDa poteins, comprise
heavy chain (100 kDa) & light chain (50 kDa).
Neurotoxin
ingested in
food
WHAT
HAPPENS
WHEN C.
botulinum
NEUROTOXIN
IS
INGESTED ??
Neurotoxin
passes
through gut
mucosa into
bloodstream
Neurotoxin spreads
through body via
bloodstream
Toxin binds to nerve at the
nerve muscle junction
Nerve supply
to muscle
fiber
(Garbutt, 2007)
redesign by Susanto
This block the
release of
acethylcholine.
Muscle cannot to
contract, resulting
in paralysis
Muscle
fibre
Mechanism of Botulinum Toxin
Botulinum toxin ingested on human
EFFECT OF ENVIRONMENTAL FACTORS ON THE
GROWTH AND SURVIVAL OF PROTEOLYTIC C.
botulinum AND NON PROTEOLITIC
C.
botulinum B (PECK, 2010)
Factor
Proteolytic C.
botulinum
Neurotoxin formed
A,B,F
Minimum growth temperature
10-12oC
Maximum growth temperature
37oC
Minimum pH for growth
4.6
NaCl concentration preventing growth 10
(%)
Minimum water activity for growth
NaCl as humectant
0.94
Glycerol as humectant
0.93
Spore heat resistant
D121oC = 0.21 min
Spore radiation resistant
Food involved in botulism outbreak
D = 2.0 – 4.5 kGy
Home-canned
foods, faulty
commercial
processing
Heat reistance data without/with lysoyme during recovery
modified from the work of Lund & Peck (2000)
Nonproteolytic C.
botulinum
B,E,F
2.5-3.0oC
25oC
5.0
5
0.97
0.94
D82.2oC = 2.4/231
mina
D = 1.0 – 2.0 kGy
Fermented marine
products, dried
fish, vacuumpacked fish
SYMPTOMS BOTULISM
Nausea & vomitting
Mainly:
neuroligical-burred / blurred vission,
difficulty to swallowing (dysphagia), mouth
dryness, speech difficulties (dysphonia) &
limb & respiration become paralysed,
dizziness/vertigo, muscle weakness.
Death normally caused by respiratory &
cardiac paralysis.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
LETHAL DOSE OF TOXIN
Botulism toxins are among the most toxic
subtances.
The minimum lethal dose for mice is 0.4-2.5
ng/kg mouse tissue.
50 % lethal dose for human is 1 ng/g body
weight. ex.: 10 people weighing 80 kg each
ingested 8.0 x 10-8g of toxin then five of them
would beexpected die.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
110
100
90
Reported Cases
80
70
60
50
40
30
20
10
0
1982 1987
MMWR
1992
1997 Year 2002
Center for Food Security and Public
Health Iowa State University 2004
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
RECORDED FOOD-BORNE BOTULISM IN DIFFERENT
COUNTRIES (PECK, 2010)
Country
Period
Belgium
Canada
China
Denmark
France
Georgia
Germany
Italy
Japan
Poland
Spain
Sweden
United
Kingdom
United States
1982
1971
1958
1984
1971
1980
1983
1979
1951
1971
1971
1969
1971
–
–
–
–
–
–
–
–
–
–
–
–
–
2000
2005
1983
2000
2003
2002
2000
2000
1987
2000
1998
2000
2005
1971 – 2003
No. of cases
Total
Avg per yr
32
2
439
13
4377
168
18
1
1286
39
879
40
376
22
750
34
479
13
9219
307
27
10
13
1
38
1
934
28
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
EXAMPLE OF RECENT INCIDENTS OF FOOD BORNE BOTULISM
INVOLVING NONPROTEOLYTIC C. botulinum
Yr
Country
199
1
Egypt
199
7
199
7
199
8
Fance
Product
Commercial
unviscerated, salte
fish
Fish
Germany Home-smokked,
vacuum packed fish
France
Commercial frozen,
vacum packed
scallop
200 Canada
Hommade
1
fermented samon
roe
200 Germany Home-salted, air3
dried fish
200 Germany Commercial
4
vacuum packed
(Peck, 2010) smoked salmon
Toxin
type
No. of
cases
E
> 81
Factor contributing
to botulism
References
outbreak
Putrefaction of fish Weber et al
before salting
(1993)
E
1
Not known
Korkeala et
al., 1998
Anonymous
(1998b)
Boyer et al
(2001)
E
4
E
1
Temperature
abuse
Temperature
abuse
E
4
Unsafe process
Anoymous
2002
E
3
E
1
Temperature
abuse
Consumed after
“use by date”
Eriksen et al
(2004)
Dressler
(2005)
CONTROL OF PROTELYTIC C. Botulinum IN FOOD
PROCESSING OPERATION
Minimum growth & neurotoxin production
occur with range 10oC to 12oC
Growth of proteolytic C. Botulinum is
prevented at pH of < 4.6 or by 10 % NaCl
Minimum aW 0.94 and 0.93 with NaCl &
Glycerola.
Heat treatment at 121.1oC for 3 min has
been adopted as the minimum standard for a
botulinum cook
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
CONTROL OF NONPROTELYTIC C. Botulinum IN FOOD
PROCESSING OPERATION
Minimum growth & neurotoxin production
occur with range 3oC to 3.3oC at 5 – 7 weeks.
Growth of proteolytic C. Botulinum is
prevented at pH of < 5 or by > 5 % NaCl
Minimum aW 0.97 and 0.94 with NaCl &
Glycerola.
Alternative processing technology: high
hydrostatic-pressur-treated
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
RECOMMENDED PROCEDURES TO ENSURE THE SAFETY
OF MINIMALLY HEATED FOODS WITH RESPECT TO
NONPROTEOLYTIC C. botulinum
Recommendation
Storage at < 3.0 oC
Storage at ≤ 8 oC and shelflife of ≤ 10 oC
Storage at chill temperature combined with heat treatment of
90oC for 10 min or equivalent lethality (e.g., 80oC for 129 min,
85oC for 36 min
Storage of chill temperature combined with pH ≤ 5.0 throughtout
the food
Storage of chill temperature combined with a salt concentration
of ≥ 3.5% throughtout the food
Storage at chill temperature combined with a combination of
heat treatment and other preservative factors which can be
shown consistenly to prevent growth and neurotoxin production
by C. botulinum
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
STAHYLOCOCUS AUREUS
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
CHARACTERISTIC OF THE ORGANISM
Gram-positive cocci occuring in iregular clumps.
Causing intoxication.
Optimum growth temperature is 37oC (range of 6
to 48oC).
Growth is inhibited in the presence of 0.1%
presence acetic acid (pH 5.1) or at pH 4.8 with 5
% NaCl.
Able to grow at Aw 0.86. it is capable to grow at
Aw 0.83 in the presence of NaCl, sucrose, or
glycerol humectants.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SOURCE OF MO
Staphylococci are ubiquitous in air, dust, sewage,
water, milk, & many foods & on food equipments,
environmental surface, human & animals.
30 – 50 % of population are nasal & throat carrier.
15 % are skin carrier (hands esp. patients & staff in
hospitals having a carier rate 80%)
Skin lesson exp. boils & infection of cuts & burns.
Human faeces & clothing.
Cows & goat.
Food products with high protein is a good growth
substrates for S. aureus.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
FOOD-BORNE OUTBREAKS
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SFP CHARACTERISTIC
Caused by ingestion of food containing SE
performed by metabolically active
staphylococci.
Usually a self-limiting illness with short
incubation (1-8 h).
The severity depends on :
individual’s susceptibility to the SE,
the amount of contaminated food eaten,
the amount of in the food ingested.
The general health of victim
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SYMPTOMS
Nausea
Vomitting
Diarrhoea & abdominal pain.
Collapse & dehydration in severe cases
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
LETHAL DOSE OF TOXIN
The minimum amount of toxin required
of toxin to produce food poisoning is 1
ng/g (10-9 g/g) of food ingested
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
PREVALENCE OF S. aureus IN SEVERAL FOODS
Products
No. of
samples
tested
Raw meat
139
%
positive
for S.
aureus
2.8
Raw milk
714
7.9
NA
Frozen prawn
46
23.9
>3
Shrimp
1,468
27
>3
Ready-to-eat
fast food
3,332
8.6
NA
No. of S.
aureus
CFU/g
NA
%
Positiv
Reference
e for
SE
7.8
Moon et al.
(2007)
31.8
Moon et al.
(2007)
NA
Sanjeev et al.
(1987)
NA
Swartzentruber et
al. (1980)
47
Oh et al. (2007)
Source: Seo & Bohac, 2010
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
FOOD ASSOCIATED WITH OUTBREAK OF
S. aureus
Cooked meats & meat products
Foods containing milk of cream
Poultry meat & meat products
Pre-cooked fish & fish products
Pre-cooked crusaceans
Gelatine glazes
Canned food
pasta
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
PREVENTION OF OUTBREAK OF S.
aureus
Avoid direct handling of foods (use tongs /
gloves)
Ensure that raw materials used 4 the prodution
of high risk foods are kept refrigerated b4 used.
Ensure that high risk foods are rapidly cooled to
below 5oC after cooking.
Ensure that gigh-risk foods are refrigerate until
ready to use.
Good personal hygiene.
Cover cust / wounds with waterproof dressing
Exclude anyone coughing, sneezing / septics
cuts / boil from food handling.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
ESCHERICHIA COLI
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
THE CHARACTERISTIC OF ORGANISM
Gram negative rod.
Member family Enterobacteriaceae
family.
Able to adapt & colonize a diverse
array of environment & the
gastrointestinal (GI)
E. coli bacteria are mesophilic
organism
E. coli able to grow at temperature of
10-40oC with optimum tempt 37oC
Pathogen can replicate pH values of
4 – 10 & in the presence up t 8%
NaCl.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SOURCE OF DIARRHEAGENIC E. coli
Environment:
Water sources, compost, urban & rural
soils & landscape, sewage, animals include
beef & dairy cattle, sheep, swine, horses,
rodents, dogs, horses, rodents.
Foods:
Cross contamination to RM, processing
water, equipments, & workers.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
GROUPS OF E. coli
Entheropathogenic E. coli
(EPEC)
Enteroinvasive E. coli (EIEC)
Enterotoxigenic E. coli
(ETEC)
Enterohaemorrhagic E. coli
(EHEC), also called
verocytotoxic E. coli (VTEC)
Causing gastroenteritis
in babies & children
Causing Travellers diarrhoea
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
MOST COMMON MODE OF TRANSMISSION, HOST,
SYMPTOMS, & CHARACTERISTICS OF ILLNESS ASSOCIATED
WITH DIFFERENT CLASSES
Class
Classic host
Symptoms
EPEC
Infants (< 6 mo);
more prevalent
in deveeloping
countries
Children; moe
prevalent in
devloping
countries
Children, more
prevalent in
developing
countries
Severe
diarrhea, fever,
vomiting
EAEC
EIEC
Watery or
bloody
diarrhea, fever
Watery
diarrhea,
abdominal
cramping,
fever
ETEC
Traveers &
Watery
infants native to diarrhea,
developing
abdominal
countries
cramping, milk
fever, nausea
EHEC
Children &
Diarrhea,
elderly
bloody diarhea,
(Beadchamp & Sofos, 2010)
abdominal
Incubation
duration
(days)
Variable
Acute / chronic
presentation
Infection dose
Chronic
diarrhea,
malnutrition
High; low in
fant
Variable
High
1-3; self
limiting
Chronic watery
diarrhea,
severe
dehydration
Dysentry
syndrome
1-3; 3-7
Cholera – like
High
1-8; 4-10
Bloody diarrhea low
(hemorrhagic
colitis), HUS,
Low
MECHANISM OF PATHOGENICITY WHICH
DIFFERENT E. coli
Class
adhesion
site
EPEC
Small
intestine
EAEC
Large &
small
intestine
Large
intestine
(colon)
EIEC
ETEC
EHEC
(Beadchamp
Adhesio
n
mediato
r
Intimin
Invassio Toxins
n
potential
Other
virulence
factor
Moderat
e
AAF
None
Possible
enterotoxin
(EAST1)
EAST1, Pet, Pic
EAF palsmid,
LEE island,
flagellin, CDT
Flagellin
Unclear
High
Enterotoxin
None
LT, ST
Cell-to-cell
spread )IcsA),
serineprotease
(SepA)
CDT
Moderat
e
Stx,
enterohemolysin,
EAST1
Small
Fibrial
intestine
CFs
Large
Intimin
intestine
&(colon)
Sofos, 2010)
LEE island,
pO157,
flagellin,
CDT, CNF
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SYMPTOMS
Diarrhoea & abdominal pain with bleeding –
blood appears in stool.
Renal failure due to blood cots in the kidney
tubules.
Internal bleeding due to resulting in brain
damage.
headache, mild fever,
Severe infections of certain serotypes include
bloody diarrhea (hemorrhagic colitis), as well as
real (kidney) malfunction and failure,
thrombocytopenia )inadequate platelet count),
microangiopathic hemolytic anemia (lysis of red
EKO SUSANTO
– DIPONEGORO
UNIVERSITY (eko_thp@undip.ac.id)
blood cells), hemolytic
uremic
syndrome
(HUS).
INTRINSIC & EXTRINSIC FACTORS INVOLVED IN
OUTBREAKS & RECALLS
Intrinsic factors:
pH, Aw, temperture,nutrients of food.
Extrinsic factors:
Higher level contamination, prevalence in
contaminated food products & frequency
of catered /picnic/outdoor gilling events.
Nationwide distribution of contaminated
products causes outbreak
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
FOOD PROCESSING CONDITION ASSOCIATED
WITH OUTBREAK
5 most significant “foodborne illness
risk factors” include:
Acquitition of products from unsafe sources
Poor personal hygiene
Contaminated processing equipments
Inadequate heat treatments
Improper handling, processing & storage
temperature
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
INFECTIVE DOSE
The infective dose to be low, possibly 10 –
100 organism.
E. coli infection strategy
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
Center for Food Security and Public
Health Iowa State University 2004
MMWR
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
ADVANCES IN DIARRHEAGENIC E. coli CONTROL
MEASURES DURING PRODUCTION & PROCESSING
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
SUFFER SUPPLY CHAINS
Increased pest control & sanitation
programs.
Use of HACCP concepts, GAP, GHP,
SSOP, GDP.
Final products testing for E. coli
hide contamination and/or fish meat
contamination.
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
MINIMIZING CONTAMINATION EVENTS DURING
PROCESSING
Address worker education & hygiene.
Zero tolerance policy during processing
include equipments, RM, products, &
workers.
Reduce cross contamination betwen
products & equipmentsand/or workers
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
INACTIVATION
Appropriate processing & handling
Pasteurization
Heat treatments
Low dose of irradiation (≥ 3 kGy is able
to innactivate pathogen).
Nonthermal processes: High
hydrostatic pressure, shock waves,
ultrasonication, pulsed ultraviolet
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
INHIBITION
Using antimicrobial agents/ingredients within
food products.
Using modified atmosphere packaging.
Chill temperature (4oC or 40oF) E. coli not
grow
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
PREVENTION
Do not eat raw / undercooked beef / drink
raw milk
Abattoir hygiene to prevent faecal
contamination
adequate cookin of meat & fish
Good personal hygiene & health education
EKO SUSANTO – DIPONEGORO UNIVERSITY (eko_thp@undip.ac.id)
THANK YOU FOR
ATTENTION