Kuliah toksikologi 2010 2nd meeting

TOXICOLOGY ON FISHERIES

PROCESSING– 3 (2 – 1)

DISEASE CAUSED BY MICROORGANISMS

EKO SUSANTO – DIPONEGORO UNIVERSITY

EKO SUSANTO

Study Program of Fisheries Processing Technology

Diponegoro University

QUESTIONS



Please mention characteristic of

C.

botulinum

?



What happen when butulinum toxin

ingested?



Please explain about

E. coli

based on

your knowledge?

CLOSTRIDIUM BOTULINUM

4

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&)

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

low-oxygen 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

BOTULINOGENIC PROPERTIES OF FISH

PRODUCTS (HUSS, 1994)

Fish product Factors adding to botulism hazard

Factors reducing botulism hazard

Safety of product based on

clasification

Fresh and frozen

Vacuum packaging Traditional chill storage putrefaction before toxin is produced

Cooking before being eaten

No risk

Pasteurized Prolonged storage life toxin produced before putrefaction

vacuum packaging poor hygiene

Chill storage (< 3o_C)

Synergistic aerobic flora eliminated

Cooking before being eaten

Cold smoked Same as above

Not cooked before being eaten

No tradition for chill storage

Chill storage Salting (NaCl

concentration > 3%) High redox-potential in unspoiled products

Chill storage

Process control (raw material, salting when applicable)

High risk

Fermented Fermentation may be slow High temperature during fermentation

Not cooked before being eaten

Salting (NaCl

concentration 3 % in brine)

Not cooked before being eaten

Application of salt, acid etc

Chill storage

Process control Low risk

Fully preserved

Not cooked before being eaten

Packed in closed cans

Autoclaving Process control

(autoclaving, closing of cans)

CHARACTERISTIC OF BOTULINUM NEUROTOXIN



Heat labile proteins.



Innactivation at temperature 121

o

C, freezing

doesn’t innactivate botulism toxins.



7 major neurotoxins (types A – G).



Neurotoxin are commonly associated with other

proteins, such as hemagluttinin &

non-toxin-nonhemagglutinin.



The neurotoxins are 150-kDa poteins, comprise

WHAT

Nerve supply to muscle fiber

Neurotoxin spreads through body via bloodstream

Toxin binds to nerve at the

nerve muscle junction _Muscle fibre

This block the release of acethylcholine. Muscle cannot to contract, resulting in paralysis

Neurotoxin passes through gut mucosa into bloodstream Neurotoxin ingested in food

(Garbutt, 2007)



Mechanism of Botulinum Toxin

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

Nonproteolytic

C.

botulinum

Neurotoxin formed

A,B,F

B,E,F

Minimum growth temperature

10-12

o

_C

_2.5-3.0

o

_C

Maximum growth temperature

37

o

_C

₂₅

o

_C

Minimum pH for growth

4.6

5.0

NaCl concentration preventing growth

(%)

10

5

Minimum water activity for growth

NaCl as humectant

0.94

0.97

Glycerol as humectant

0.93

0.94

Spore heat resistant

D

₁₂₁o

C

= 0.21 min

D

82.2oC

= 2.4/231

min

a

Spore radiation resistant

D = 2.0 – 4.5 kGy

D = 1.0 – 2.0 kGy

Food involved in botulism outbreak

Home-canned

foods, faulty

commercial

processing

Fermented marine

products, dried

fish,

vacuum-packed 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.

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

-8

g of toxin then five of them

would beexpected die.

Center for Food Security and Public Health Iowa State University 2004

Year

RECORDED FOOD-BORNE BOTULISM IN DIFFERENT

COUNTRIES (PECK, 2010)

Country

Period

No. of cases

Total

Avg per yr

Belgium

1982 – 2000

32

2

Canada

1971 – 2005

439

13

China

1958 – 1983

4377

168

Denmark

1984 – 2000

18

1

France

1971 – 2003

1286

39

Georgia

1980 – 2002

879

40

Germany

1983 – 2000

376

22

Italy

1979 – 2000

750

34

Japan

1951 – 1987

479

13

Poland

1971 – 2000

9219

307

Spain

1971 – 1998

27

10

Sweden

1969 – 2000

13

1

United

Kingdom

1971 – 2005

38

1

United States

1971 – 2003

934

28

EXAMPLE OF RECENT INCIDENTS OF FOOD BORNE BOTULISM

INVOLVING NONPROTEOLYTIC

C. botulinum

Yr

Country

Product

Toxin

type

No. of

cases

Factor contributing

to botulism

outbreak

References

199

1

Egypt

Commercial

unviscerated, salte

fish

Fance

Fish

E

1

Not known

Korkeala et

al., 1998

199

7

Germany Home-smokked,

vacuum packed fish

E

4

Temperature

abuse

Anonymous

(1998b)

199

8

France

Commercial frozen,

vacum packed

scallop

E

1

Temperature

abuse

Boyer et al

(2001)

200

1

Canada

Hommade

fermented samon

roe

E

4

Unsafe process

Anoymous

2002

200

3

Germany Home-salted,

air-dried fish

E

3

Temperature

abuse

Eriksen et al

(2004)

200

4

Germany Commercial

vacuum packed

smoked salmon

E

1

Consumed after

“use by date”

Dressler

(2005)

CONTROL OF PROTELYTIC

C. Botulinum

IN FOOD

PROCESSING OPERATION



Minimum growth & neurotoxin production

occur with range 10

o

C to 12

o

C



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 121.1

o

C for 3 min has

been adopted as the minimum standard for a

botulinum cook



CONTROL OF NONPROTELYTIC C. Botulinum IN FOOD

PROCESSING OPERATION



Minimum growth & neurotoxin production

occur with range 3

o

C to 3.3

o

C 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



RECOMMENDED PROCEDURES TO ENSURE THE SAFETY

OF MINIMALLY HEATED FOODS WITH RESPECT TO

NONPROTEOLYTIC

C. botulinum

Recommendation

Storage at < 3.0

o

C

Storage at ≤ 8

o

C and shelflife of ≤ 10

o

C

Storage at chill temperature combined with heat treatment of

90

o

_{C for 10 min or equivalent lethality (e.g., 80}

o

_{C for 129 min,}

85

o

_{C 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

STAHYLOCOCUS AUREUS

CHARACTERISTIC OF THE ORGANISM



Gram-positive cocci occuring in iregular clumps.



Causing intoxication.



Optimum growth temperature is 37

o

C (range of 6

to 48

o

C).



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.

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.

FOOD-BORNE OUTBREAKS

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

SYMPTOMS



Nausea



Vomitting



Diarrhoea & abdominal pain.



Collapse & dehydration in severe cases

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

PREVALENCE OF

S. aureus

IN SEVERAL FOODS

Frozen prawn

46

23.9

>3

NA

Sanjeev et al.

(1987)

Shrimp

1,468

27

>3

NA

Swartzentruber et

al. (1980)

Ready-to-eat

fast food

3,332

8.6

NA

47

Oh et al. (2007)

Source: Seo & Bohac, 2010

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

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 5

o

C 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.

ESCHERICHIA COLI

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

10-40

o

C with optimum tempt 37

o

C



Pathogen can replicate pH values of

4 – 10 & in the presence up t 8%

NaCl.



Most strains of

E.coli

are not human

pathogen

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.

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

MOST COMMON MODE OF TRANSMISSION, HOST,

SYMPTOMS, & CHARACTERISTICS OF ILLNESS ASSOCIATED

WITH DIFFERENT CLASSES

Class

Classic host

Symptoms

Incubation

duration

(days)

Acute / chronic

presentation

Infection dose

EPEC

Infants (< 6 mo);

more prevalent

in deveeloping

countries

Severe

diarrhea, fever,

vomiting

Variable

Chronic

diarrhea,

malnutrition

High; low in

fant

EAEC

Children; moe

prevalent in

devloping

countries

Watery or

bloody

diarrhea, fever

Variable

Chronic watery

diarrhea,

severe

dehydration

High

EIEC

Children, more

prevalent in

developing

ETEC

Traveers &

infants native to

developing

countries

Watery

diarrhea,

abdominal

cramping, milk

fever, nausea

1-3; 3-7

Cholera – like

High

EHEC

Children &

elderly

Diarrhea,

bloody diarhea,

abdominal

pain, vomiting

1-8; 4-10

Bloody diarrhea

(hemorrhagic

colitis), HUS,

kidney failure

low

MECHANISM OF PATHOGENICITY WHICH

DIFFERENT

E. coli

Class

adhesion

site

Toxins

Other

virulence

factor

EPEC

Small

intestine

EAF palsmid,

LEE island,

flagellin, CDT

EAEC

Large &

small

intestine

AAF

None

EAST1, Pet, Pic

Flagellin

EIEC

Large

intestine

(colon)

Unclear

High

Enterotoxin

Cell-to-cell

spread )IcsA),

serine-protease

(SepA)

ETEC

Small

intestine

Fibrial

CFs

None

LT, ST

CDT

EHEC

Large

intestine

(colon)

Intimin

Moderat

e

Stx,

enterohemolysin,

EAST1

LEE island,

pO157,

flagellin,

CDT, CNF

(Beadchamp & Sofos, 2010)

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

blood cells), hemolytic uremic syndrome (HUS).

Seizure stroke, herniated bowel, chronic rhenal

malfuction.

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

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

INFECTIVE DOSE



The infective dose to be low, possibly 10 –

100 organism.



E. coli infection strategy

Center for Food Security and Public Health Iowa State University 2004

MMWR

ADVANCES IN DIARRHEAGENIC

E. coli

CONTROL

MEASURES DURING PRODUCTION & PROCESSING

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.



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

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

INHIBITION



Using antimicrobial agents/ingredients within

food products.



Using modified atmosphere packaging.



Chill temperature (4

o

C or 40

o

F) 

E. coli

not

grow

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