TOXICOLOGY ON FISHERIES PROCESSING– 3 (2 – 1)

TOXICOLOGY ON FISHERIES PROCESSING– 3 (2 – 1)

DISEASE CAUSED BY MICROORGANISMS

EKO SUSANTO – DIPONEGORO UNIVERSITY

EKO SUSANTOStudy Program of Fisheries Processing Technology

Diponegoro University Email : [email protected]

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 UniversityEKO SUSANTO – DIPONEGORO UNIVERSITY ([email protected])

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 ([email protected])

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 (< 3oC)Synergistic aerobic flora eliminated

Cooking before being eaten Chill storage

No risk if cookedHigh risk if not cooked

Cold smoked Same as aboveNot cooked before being eatenNo tradition for chill storage

Chill storageSalting (NaCl concentration > 3%)High redox-potential in unspoiled products

Chill storageProcess control (raw material, salting when applicable)

High risk

Fermented Fermentation may be slowHigh temperature during fermentationNot cooked before being eaten

Salting (NaCl concentration 3 % in brine)Chill storageLow pH

Process controlChill storage

High risk

Semi-preserved

Not cooked before being eaten

Application of salt, acid etcChill storage

Process control Low risk

Fully preserved

Not cooked before being eaten Packed in closed cans

Autoclaving Process control (autoclaving, closing of cans)

Low risk

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-toxin-nonhemagglutinin.

The neurotoxins are 150-kDa poteins, comprise heavy chain (100 kDa) & light chain (50 kDa).

WHAT HAPPENS WHEN C. botulinum NEUROTOXIN IS INGESTED ??

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) redesign by Susanto

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

Nonproteolytic C. botulinum

Neurotoxin formed A,B,F B,E,FMinimum growth temperature 10-12oC 2.5-3.0oCMaximum growth temperature 37oC 25oCMinimum pH for growth 4.6 5.0NaCl concentration preventing growth (%)

10 5

Minimum water activity for growth NaCl as humectant 0.94 0.97 Glycerol as humectant 0.93 0.94Spore heat resistant D121

oC = 0.21 min D82.2

oC = 2.4/231

mina

Spore radiation resistant D = 2.0 – 4.5 kGy D = 1.0 – 2.0 kGyFood involved in botulism outbreak Home-canned

foods, faulty commercial processing

Fermented marine products, dried fish, vacuum-packed fish

a Heat reistance data without/with lysoyme during recoveryb modified from the work of Lund & Peck (2000)

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 10-8g of toxin then five of them would beexpected die.


Center for Food Security and Public Health Iowa State University 2004

Year 1982 1987 1992 1997 2002

Repo

rted

Case

s110

100

90

80

70

60

50

40

30

20

10

0

MMWR


RECORDED FOOD-BORNE BOTULISM IN DIFFERENT COUNTRIES (PECK, 2010)

Country Period No. of casesTotal Avg per yr

Belgium 1982 – 2000 32 2Canada 1971 – 2005 439 13China 1958 – 1983 4377 168Denmark 1984 – 2000 18 1France 1971 – 2003 1286 39Georgia 1980 – 2002 879 40Germany 1983 – 2000 376 22Italy 1979 – 2000 750 34Japan 1951 – 1987 479 13Poland 1971 – 2000 9219 307Spain 1971 – 1998 27 10Sweden 1969 – 2000 13 1United Kingdom

1971 – 2005 38 1

United States 1971 – 2003 934 28EKO SUSANTO – DIPONEGORO UNIVERSITY ([email protected])

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

1991

Egypt Commercial unviscerated, salte fish

E > 81 Putrefaction of fish before salting

Weber et al (1993)

1997

Fance Fish E 1 Not known Korkeala et al., 1998

1997

Germany Home-smokked, vacuum packed fish

E 4 Temperature abuse

Anonymous (1998b)

1998

France Commercial frozen, vacum packed scallop


Boyer et al (2001)

2001

Canada Hommade fermented samon roe

E 4 Unsafe process Anoymous 2002

2003

Germany Home-salted, air-dried fish


Eriksen et al (2004)

2004

Germany Commercial vacuum packed smoked salmon

E 1 Consumed after “use by date”

Dressler (2005)(Peck, 2010)

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


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


RECOMMENDED PROCEDURES TO ENSURE THE SAFETY OF MINIMALLY HEATED FOODS WITH RESPECT TO

NONPROTEOLYTIC C. botulinum

RecommendationStorage at < 3.0 oCStorage at ≤ 8 oC and shelflife of ≤ 10 oCStorage at chill temperature combined with heat treatment of 90oC for 10 min or equivalent lethality (e.g., 80oC for 129 min, 85oC for 36 minStorage of chill temperature combined with pH ≤ 5.0 throughtout the foodStorage of chill temperature combined with a salt concentration of ≥ 3.5% throughtout the foodStorage 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 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.


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

ProductsNo. of samples tested

% positive for S. aureus

No. of S. aureus CFU/g

% Positive for SE

Reference

Raw meat 139 2.8 NA 7.8 Moon et al. (2007)

Raw milk 714 7.9 NA 31.8 Moon et al. (2007)

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 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 ([email protected])

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 10-40oC with optimum tempt 37oC

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 CLASSESClass 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 countries

Watery diarrhea, abdominal cramping, fever

1-3; self limiting

Dysentry syndrome

Low

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(Beadchamp & Sofos, 2010)

MECHANISM OF PATHOGENICITY WHICH DIFFERENT E. coli

Class adhesion site

Adhesion mediator

Invassion potential

Toxins Other virulence factor

EPEC Small intestine

Intimin Moderate

Possible enterotoxin (EAST1)

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 Moderate

Stx, enterohemolysin, EAST1

LEE island, pO157, flagellin, CDT, CNF

(Beadchamp & Sofos, 2010)EKO SUSANTO – DIPONEGORO UNIVERSITY ([email protected])

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 (4oC or 40oF) 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


THANK YOU FOR ATTENTION

TOXICOLOGY ON FISHERIES PROCESSING– 3 (2 – 1)

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