(Foodborne intoxications) x. . . . E-mail.: vpitiriga@med.uoa.gr

. . ( )

! - . , (, )

WHO Food-borne disease (FBD) disease of infectious or toxic nature caused by, or thought to be caused by, the consumption of food

4

? : ( , , ) :

. ?Estimates of the overall burden of disease from foodborne agents is challenging>250 ( ) / (Campylobacter E. coli O157, )

Global Foodborne Infections Network (GFN)

5 (Salmonella, Campylobacter, E. coli O157:H7 Shigella, caliciviruses) : $6.9 .

: , , (Super Market .) :, , , ,

? , -

(intrinsic factors) ( ) pH (aw)H (Eh)

(extrinsic factors) T 25 - 45 C ( ) 2 C2 ,

? (Foodborne Infection) - - v (Foodborne ntoxication) (Toxin-mediated infection)

. ? (Foodborne Infection) v (Foodborne ntoxication) (Toxin-mediated infection) v

: (bacterial intoxications) (Fungal intoxications) (Chemical intoxication) (Plant toxicants) (Poisonous animals)

( )

Viruses and parasites do not cause foodborne intoxication.

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15

Case study 1

Foodborne intoxication among elderly - Florida (USA) Anonymous (2007) CDC. MMWR Morb Mortal Wkly Rep: 11891191

27 2007: 18 26 2007/ () 125 : 3.4 h (: 17 h); .: 24 h (272 h)

(94%), (89%), (62%), (67%), (61%), (44%), (39%), (28%), (11%)

18 , 17 (94%) Gram + >105 cfu/gr

Staphylococcus aureus, , (coccus) Gram (+)

>50 coagulase (+/-)

Staphylococcus aureusStaphylococcus intermedius

Certain CNS strains produce enterotoxins(Vernozy-Rozand et al., 1996; Zell et al., 2008).

Reservoirs- (Human carriers): - , , , , , , ,

Staphylococci are found in varying numbers in air, , dust, water, food, feces and sewage. The primary habitat of S. aureus is the mucous membranes of the nasopharynx and skin of man and animals. The organism is found in the nose, skin, saliva, intestinal contents and in feces. Human carriers of this organism are numerous and are undoubtedly the source of a number of outbreaks.

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. : Michigan (USA) / 1884 by Vaughan and Sternberg. S. aureus241,000 ./ $1,5 . ( Bacillus cereus),

MRSA strains MRSA , , MRSA , (Normanno et al., 2007), (Van Loo et al., 2007; De Boer et al., 2008) (Kitai et al.,2005; Kwon et al., 2006). : MRSA . Jones et al. (2002): 1 community-acquired MRSA C

Staph. aureus : 7 to 48.5 C ( 37oC) pH 4.0-9.83 ( 7.4-7.6). NaCl (18%) (aw)0.86 - 0.88 0.9 . ( ) 60C 30. 121C 15 160-170C 1

23

, Cl ( )

8 A, B, C, D, E, F, G, H ( ) 26.000-28000 Da (C1-C2-C3) : .

S. aureus

[9]. Although SEs are pyrogenic toxins, high fever is absent.25

D: C: ( 350 mg/ml)D: ( ) : :

: heat stable ( 100oC x 1h) : pH (, , ) -

5

All the staphylococcal enterotoxins are heat stable (withstand heating at 100oC for one hour) and ordinary cooking procedures, pasteurization and drying do not inactivate these enterotoxins. They are insensitive to pH changes(pH stable) and resistant to most proteolysis enzymes (trypsin, chymotrypsin, renin, and pepsin). The enterotoxins are also not affected by irradiation. All the five enterotoxins have the similar potency.

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( Streptococcus pyogenes): - 20% -

: 10-48oC ( 40-45)pH >=4.2 10%

: 10 %

29

30

. 6 : /: , , . , .., , - ( ) Staph. aureus:

The prerequisite of SFP is that food or one of its ingredi-ents is contaminated with an enterotoxigenic strain ofStaphylococcus spp. Five conditions are required to induce SFPOs: (1) asource containing enterotoxin-producing staphylococci:raw materials, healthy or infected carrier, (2) transfer ofstaphylococci from source to food, e.g., unclean food prep-aration tools because of poor hygiene practices, (3) foodcomposition with favourable physicochemical characteris-tics for S. aureus growth and toxinogenesis, (4) favourabletemperature and sufcient time for bacterial growth andtoxin production, and (5) ingestion of food containing suf-cient amounts of toxin to provoke symptoms.

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. (1-6 hrs) 1-5 mg , : 24-72

: 0.03% -4.4% : >105 cfu/gr [1 ng toxin/gr ]

32

:

Severity of illness depends on:Individual susceptibility to toxinAmount of contaminated food eatenAmount of toxin in food ingestedGeneral health of the victim Age of victim Nature of enterotoxin present

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>=105S. aureus S. aureus () (ELISA) (PCR, RT-PCR)

detection of staphylococcal enterotoxins in food [26], recovery of at least 105S. aureusfrom food remnants [19].S. aureusenterotoxin can be detected on the basis of three types of methods: bioassays, molecular biology, and/or immunological techniques [19,27]. Polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and RT-quantitative PCR can be carried out to evaluate the toxic potential of strain [19]. The enzyme immunoassay and enzyme-linked fluorescent assay are the most commonly used immunological methods based on the use of antienterotoxin polyclonal or monoclonal antibodies [19]. Several molecular typing methods are widely used for the genetic characterization ofS. aureussuch as multilocus sequence typing,spatyping, SCCmectyping, and Pulse-field gel electrophoresis (PFGE). These techniques provide means to trace epidemiologically related strains leading to the tracking back to the origin of contamination [28]. However, these methods have variation in their discriminating powers and can be increased by combining the methods [29]. Molecular-based methods provide information about the source of contamination (human or animal origin). The PFGE andspatyping can be used alone or in association to gather the information regarding the origin ofS. aureuscontamination [19].

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- & Coag (+) ISO 6888-1:1999/Amd 1:2003

Baird Parker Rabbit plasma brinogen agar

>=105S. aureus

- 7 200 C 1.5 h, , 6 h

??

- ?

?

- 7 204 C 1.5 h, , . 6 h .

. 0.94) >3C A o=35 C

(, , C, D, , F G) C. botulinum Clostridium baratii Clostridium butyricum BoNT, , , F

48

: : 30100 ng ( 80oc for 10 min), pH- () Botulinus

These toxins are neurotoxins, that are highly toxic, heat labile (inactivated by heating at 80oc for 10 min), unstable at alkaline pH (but stable below pH 7.0) but resistant to pepsin and acidic environment. The toxins can resist the action of the gastric and intestinal juices.Botulinus toxin is one of the most lethal poisons known. The calculated lethal dose for an adult person is 10 g.

49

Foods associated with anaerobic conditions such as spoiled canned meat, or hams and bacon stacked without air access, are particularly liable to be infective. Home made fermented foods have been incriminated, together with smoked, pickled and canned foods that are allowed to stand and then eaten without adequate cooking. Uncooked fresh foods are safe because they are eaten before the toxin has had time to develop, while, if foods are cooked, the toxin is destroyed.

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, , , , , , , , /

, , , ,, , , , ,,

51

-

: . 1 gr 1.000.000 .

Clostridium botulinum- :

(Nelson Essentials of Pediatrics, 2002 by W. B. Saunders Company, E. . . 424 Manual of Clinical Microbiology ASM 8th, ed., 2003, p. 841-2)

-

. 12-72 hrs :2 -14 . (mortality): 60-100%

( ) , , , ,

54

6 . - 1

( 2 )

:

: 10 -100

( )

Constipation (often the first sign)Floppy movements due to muscle weakness and trouble controlling the headWeak cryIrritabilityDroolingDrooping eyelidsTirednessDifficulty sucking or feedingParalysis[22]

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C. botuliniumTryptose sulfite cycloserine (TSC) growth media, , , , (ELISA)PCR-

56

( 24 ) (). (A, B, E)

: : 3 - 12

5 , , (case #2)9 , 10

,

Prosciutto,

(case 3)

(case #2)

On January 5, the family ate commercially prepared marinated seafood antipasto,bocconcini cheese, bruschetta, olives and pasta with tomatosauce made from home-canned tomatoes.

58

?

3 ?

5 , , bruschetta, 9 , 10

,

prosciutto,

(case 3)

(case #2)

On January 5, the family ate commercially prepared marinated seafood antipasto,bocconcini cheese, bruschetta, olives and pasta with tomatosauce made from home-canned tomatoes.

60

case 3 6C 1 h, pH= 6

pH >4.6 > 4C

?

= ( ). ( pH)>10% (NaNO2) 10. 29 - ()2-5% .

- - / 14-16% 1969 1 //

: 30-50C, (4 - 5C) pH 4,3 - 9,3, aw 0.912-0.950 biofilm

Bacillus cereus - 3 - . BL (HBL), (NHE), K

Bacillus cereus 56C 5 ( 90 126C)

-

:

- : - . : 8-16 :24 : 105107 cfu/gr

- : . .: 2 - 5 . : 24 :105108 cfu/gr

75

:

,

,

,

,

1. - . : Clostridium perfringens Staph aureus

2. B. cereus

,

B. cereus : 105 cfu/g

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(Cell Cytotoxicity Assays) Polymerase Chain Reaction TECRA Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDE-VIA) latex (Reverse Passive Latex Agglutination)OXOID Bacillus Enterotoxin - Reverse Passive Latex Agglutination (BCET-RPLA) 3. B. cereus

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Case study Analysis: 0 10.30 ..

80

? ? ?

1. 2. 3. . 4.

82

, . : B. cereus - : ( ) . = > 140F

(mycotoxins)

1952: mycotoxicosis, . 1960/ UK: - 100,000 ( ) Aspergillus spPenicillium sp, Fusarium, sp Claviceps Alternaria sp. >100 : , ,

,

( 1.) , Aspergillus spPenicillium sp, Fusarium, sp Claviceps Alternaria sp. . ( , , ), .

85

: , , , ,

:, , ( ) (25-35OC) (>80%) ( )pH ( 4-4,6) ( )

4 : -: : , : DNA : :

(aflatoxins)

Aspergillus flavus, only B aflatoxins (B1 and B2),Aspergillus parasiticus G aflatoxins (G1 and G2),Aspergillus nomius B and G aflatoxins, : , ( ), , ,

(aflatoxins) 1C17H12O6 Aspergillus flavus Aspergillus parasiticus. (425 nm). .2C17H14O6 Aspergillus flavus Aspergillus parasiticus. (425 nm).G1C17H12O7 Aspergillus parasiticus. (450 nm).G2C17H14O7 Aspergillus parasiticus. (450 nm).M1C17H12O7 B1 . ng.M2C17H14O7 B2 . ( ) B2.B2aC17H14O7 ( ) B1. (425 nm).G2aC17H14O8 ( ) G1. (450 nm).

.. B (B1, B2, B2a): (B: blue) , G (G1, G2, G2a): (G: green)

: , 100,000/

. :

1: : , ,

- - - ' . ( )

Enzyme-linked immunosorbent assay (ELISA) (HPLC) (TLC) - (LC-MS) PCRRT-PCR (mRNAs)

in-field rapid diagnostic strips such as AgriStrips used in rapid test enzyme-linked immunosorbent assay (ELISA) Aflatoxins possess significant ultraviolet (UV) absorption and fluorescence properties, and chromatographic methodseither high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) with UV or fluorescence detectionare widely used. Such methods require sample extraction and extract clean-up by solid-phase extraction (SPE) or immunoaffinity chromatography (IAC) followed by chromatographic separation and aflatoxin detection. Total aflatoxins can also be measured by direct fluorescence measurements of these purified extracts (for example, VICAM).Liquid chromatography-mass spectrometry (LC-MS) technology offers the advantage of dilute and shoot techniques where simple sample extracts are analyzed without clean-up, and with the added advantage of multi-mycotoxin analysis whereby a range of mycotoxins can be analyzed in the same sample analysis run (Sulyok et al. 2006).

94

Aspergillus sp. Sabouraud dextrose UVMethyl-b-cyclodextrin 36 h- 3 .

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