VYSOKÉ UČENÍ TECHNICKÉ V BRNĚBRNO UNIVERSITY OF TECHNOLOGY

FAKULTA CHEMICKÁ

ÚSTAV CHEMIE POTRAVIN A BIOTECHNOLOGIÍ

FACULTY OF CHEMISTRY

INSTITUTE OF FOOD SCIENCE AND BIOTECHNOLOGY

SALMONELOVÉ INFEKCE, JEJICH ZDROJ ADIAGNOSTIKA

INFECTION OF SALMONELOSIS, THEIR SOURCE AND DIAGNOSIS.

BAKALÁŘSKÁ PRÁCEBACHELOR'S THESIS

AUTOR PRÁCE MARTIN VALKUSAUTHOR

VEDOUCÍ PRÁCE Mgr. DANA VRÁNOVÁ, Ph.D.SUPERVISOR

BRNO 2009

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Vysoké učení technické v BrněFakulta chemická

Purkyňova 464/118, 61200 Brno 12

Zadání bakalářské práce

Číslo bakalářské práce: FCH-BAK0203/2007 Akademický rok: 2008/2009

Ústav: Ústav chemie potravin a biotechnologií

Student(ka): Martin Valkus

Studijní program: Chemie a technologie potravin (B2901)

Studijní obor: Biotechnologie (2810R001)

Vedoucí bakalářské práce: Mgr. Dana Vránová, Ph.D.

Konzultanti bakalářské práce:

Název bakalářské práce:

Salmonelové infekce, jejich zdroj a diagnostika

Zadání bakalářské práce:

1. Vypracování literární rešerše na zadané téma.

2. Výběr metod pro experimentální část a jejich aplikace

3. Zpracování výsledků a jejich zhodnocení.

Termín odevzdání bakalářské práce: 29.5.2009

Bakalářská práce se odevzdává ve třech exemplářích na sekretariát ústavu a v elektronické formě

vedoucímu bakalářské práce. Toto zadání je přílohou bakalářské práce.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Martin Valkus Mgr. Dana Vránová, Ph.D. doc. Ing. Jiřina Omelková, CSc.

Student(ka) Vedoucí práce Ředitel ústavu

- - - - - - - - - - - - - - - - - - - - - - -V Brně, dne 1.12.2008 doc. Ing. Jaromír Havlica, DrSc.

Děkan fakulty

3

ABSTRAKT

rodem Salmonella, jeho

i

V

.

ABSTRACT

thesis deals with infection of salmonelosis

diagnosis. Theoretical part includes general information about Salmonella species and its

morphology. It describes pathogenesis, source of infection, clinical symptoms, treatment and

diet recommendations. It analyzes statistics of number of infection occurrences in Czech

Republic. It also deals with prevention of infection origin on schools, population

foreknowledge and climatic effects.

Experimental part of this thesis contains description of optimization procedure of

identification Enterobacteriaceae by adjusting of pH of prepared medium MIU.

Salmonel

KEYWORDS

Infection of samonelosis, prevention, incidence, source of infection, cultivation, diagnosis

4

v

hlediska obsahu majetkem

...............................................

Podpis studenta

5

OBSAH

.................................................................................................................................. 7

..................................................................................................... 8

..................................................................................................... 8

2.1.1 Morfologie ................................................................................................................. 8

2.1.1.1 Rod Salmonella .................................................................................................. 9

.................................................................................................................... 9

.............................................................................................. 10

........................................................................................ 10

2.2 Patogeneze ...................................................................................................................... 10

................................................................................................. 11

2.2.2 Postup infekce ......................................................................................................... 11

...................................................................................................... 12

2.2.3.1 Tyfus a paratyfus .............................................................................................. 12

............................................................. 12

2.2.3.3 Bakteriemi .......................................................... 12

............................................................................... 12

2.2.4 Produkce endotoxinu ............................................................................................... 13

2.3 Zdroje infekce ................................................................................................................ 14

......................................................................................................... 15

..................................................................... 15

...................... 16

2.5.1. Prevence ................................................................................................................. 17

................................................................................ 17

2.5.1.2 Prevence v ........................................................................ 17

2.5.1.3 Prevence v ......................................................................... 17

........................................................................................................ 18

................................................................ 18

............................................................................................................... 19

........................................................................... 21

potravin ..................................................................................................... 21

........................................................................................ 21

2.6.1.2 Sterilizace ......................................................................................................... 22

.............................................................................. 22

..................................................................... 23

........................................... 23

........................................... 24

..................................................................................................... 28

................................................................................................ 28

.......................................................................................... 28

............................................................................................... 29

2.6.2.4 Kultivace .......................................................................................................... 29

....................................................... 29

ST ....................................................................................... 30 ..................................................................................... 30

6

.............................................................................................................. 30

................................................................................................. 30

.................................................................................................. 30

.................................................................................................................. 30

E .......................................................................................... 32 ......................................................................................................................... 32

4.1.1 Salmonella Enteritidis ............................................................................................. 32

4.1.2 Shigella sonnei ........................................................................................................ 33

4.1.3 Escherichia coli ....................................................................................................... 33

4.1.4 Klebsiela pneumonie ............................................................................................... 34

4.1.5 Proteus mirabilis ...................................................................................................... 34

............................................................................................................... 35

.............................................................................................................................. 36

....................................................................................................... 38

........................................... 41

7

kruhu po

aj.

supe

potrav .

v

projevy.

bi .

8

2.1

Rod Salmonella Enterobacteriaceae

Salmonella enterica

poddr

[1]

Bakterii salmonely objevil roku 1885 doktor Theobald Smith z

mikrobiologie D. E. Salmona. [2]

2.1.1 Morfologie

Enterobacteriaceae ch,

, nesporu ch

rody: Citrobacter, Escherichia, Hafnia, Klebsiella, Pantoea,

Proteus, Morganella, Providentia, Salmonella, Shigella a Yersinia. [3]

Obr. 1: Bakterie Salmonelly

9

2.1.1.1 Rod Salmonella

Rod Salmonella

Kaufmann-

T fmann- [1, 25]

Skupina

A Salmonella Paratyphi A

B Salmonella Paratyphi B

Salmonella typhimurium

C Salmonella cholera-suis

Salmonella Paratyphi C

D

Salmonella Typhi

Salmonella Enteritidis

Salmonella gallinarum-pulorum

E Salmonella anatum

s je Salmonella

enterica subsp. enterica r

, aby se v - Salmonella

Enteritidis. [3]

Salmonel

:

p

ejce);

d - p [1]

V almonely hoj

st

da, kt

lze je izolovat z

ny

Salmonella Typhi a Salmonella

P je

nalezneme Salmonella Paratyphi B a z

nebo dobytka). [5]

10

Z

Salmonella cholerae-suis prase

Salmonella dublin dobytek

Salmonella gallinarum-pullorum

Salmonella Enteritidis d , dobytek

Salmonella abortus-equi

Salmonella abortus-ovis ovce

Salmonella typhimurium dobytek, holubi

tab.

velkou

produkci su

y salmonel

[5, 22]

2.1.2.2

.

s

nakazit z

p - v je krmivo vyrobeno z ;

s - ebo

( ).

je voda z

mi vodami. [8]

2.2 Patogeneze

Ke vzniku salmonelou erie

v

v

v

v 2-3

,

11

um a usmrcuje bakterie a

kvasinky.

[5]

2.2.1 infekce

K se salmonely

potv neinfikovali,

k tomu, aby se infekce rozvinula. Rozvoj infekce po expozici salmonelou j

I [5]

na vehikulu infekce. Bakterie v

ch kyselost v

je izolace

salmonel z vzor ,

em

2.2.2 Postup infekce

Jakmile se salmonel K

s

[5]

12

pii: [5]

tyfus a paratyfus;

gastroent ;

b ;

d .

. [23]

2.2.3.1 Tyfus a paratyfus

Jsou Salmonellou Typhi nebo Salmonellou Paratyphi A, B

7-

salmo do krve a ze do

20%. [5]

fekce

(symptomy jsou r d

, ch -

moc dokonce [5]

kytovat i u

osoby s mi mi , nebo osoby

Salmonelly Enteritidis. [5, 27]

Bakterie salmonely se za

paratyfu se

s , u osob nad 50 let je to

a u . [5]

13

2.2.4 Produkce endotoxinu

Endotoxiny jsou lipopolysacharidy (LPS)

specifickou tk

hydro

. U

bakt je jeho stru

metody.[10, 26]

monocyty- - monocyt cca 12-

Cirkuluje v -8%

krev . Monocyto-

( );

neutrofily

schopnost poh - jeho

jed

v

p ;

a

s

m

;

e

DIC diseminovan i

[10]

Obr. 2: St LPS Salmonelly Typhimurium [13]

14

2.3 Zdroje infekce

Salmonel

-41 -32 5- ). Pro

. Vzhledem k tomu, salmone , krom

, i

pokrmy z ;

c ;

n .

Salmonella ovoce (a to jak ze slupky, tak z ).

V roce 2001 propukla infekce

sestry. Bylo

z

V y

(racci a holubi)

15

Jednou z batole

organismu (dehydr

m

(gastroenteritidou) p ale naopak

p .

bakteriemie. [6]

v m

v [7]

Na 1

hodinu.

P d

1

ho

cukru.

B

B

.

N .

N .

V ,

y nebo

pomno

16

2.5 tu

Salmonella v

2008 pod v grafu 1:

Tab.3:

Rok salmonelami

1999 44845

2000 40233

2001 33594

2002 27964

2003 26899

2004 30724

2005 32927

2006 25102

2007 18204

2008 11009

Graf 1: salmonelami v letech

2008

0

10000

20000

30000

40000

50000

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Roky

1999 - 2008

P klesl

17

2.5.1. Prevence

oblasti

prevenci v

kniha) a v V

se v [18]

2.5.1.1

formou

v

ovanost

2.5.1.2 Prevence v

V

2.5.1.3 Prevence v

podkapitoly:

-

-

18

v

2.5.1.4 Vliv m

ozhlas a

v

V

kontrolovalo zda:

n

a

muse

HACCP slo

, a mohou

stanice. [19, 29]

19

2.5.1.6

i

almonely v po

, Toto riziko

roste potravin z

22 meteorolo

z S

do tabulky (Tab.4 grafu (Graf 2).

Tab.4: [14]

Rok

1999 8,45

2000 9,22

2001 7,88

2002 8,79

2003 8,52

2004 8,09

2005 7,89

2006 8,45

2007 9,22

2008 8,96

Graf 2:

7,00

7,50

8,00

8,50

9,00

9,50

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Rok

20

letech

letech

letech,

21

2.6 Diagnostika rodu Salmonella

Salmonella

Roli hraje i aktivita vody

pod [15]

potravin

[15]:

p ;

p

tetrathionanem a novobiocinem);

v

(

p

to, aby se nerozp

y ve

nabobtna

tuhost

o 2,5-

[15]

22

2.6.1.2 Sterilizace

(je uvedeno v popisu

na obal

po dobu 30-ti

minut ve varu

deionizovanou vodu a nav

v [15]

2.6.1.3 F

na 45

p

v ,

po rozli

zkumavek lze d

petriho misky p j ipraveny

k je

zkumavky

inou v [15]

23

o

.

v

Pokud

, a nelze ji po

m

k

.

.

.

[15]

vzorku

inkubujeme v

dobu

du Salmonella

-

redukuje

24

Tab.5.: Biochemi

Mikrob pohyb plyn z

Utilizace

Tvorba

H2S

Tvorba

indolu

Salmonella

Typhi + - - - - + -

Salmonella

spp. + + - - + + -

Shigella spp. - - - - - - V

Escherichia

coli + + + - - - +

Citrobacter

spp. + + + - + V V

Klebsiella spp. - + + + + - V

Proteus spp. + V - + V V V

-

Pohyb n h testovat na

pohyb,

na 24 hodin do termostatu, a oblasti

vpichu nebo v

Plyn z bakterie rodu Salmonella

nitol 2vrstvami MIU a agaru podle Haj

se realizuje v tzv. -

2:1.

Salmonelly Salmonelly

Indii, kde byly 2 % z

. [28]

Klebsielly nebo Protea a projevuje se

n Toto bylo

-

25

Tvorba H2S j

- (Kligler iron

agar

- Manitol

agar obr. 3 - obr. 4 Lysin

obr. 5)

[16]

26

nteritidis na XLD [16]

zkumavce s

Na obr. 6 Salmonella Enteritidis a

Escherichia coli.

Escherichia coli. Je

je

tvorba plynu.

Salmonellou En

Obr. 6: Kombinace agaru podle Hajna a MIU

27

Tvorba indolu

Na obr. 7 byl proveden test na tvorbu indolu u Escherichia coli a Salmonelly

Enteritidis. V ce Escherichie coli a v

Salmonelly Enteritidis.

Obr. 7

28

[5]:

s

dle Stuarta,

Blaira).

Tampon se do zkumavky z [5]

patogen, , 20]

Transport

Kultivace, izolace

29

2.6.2.3

, a to jak pro pacienta, tak

z [5]

2.6.2.4 Kultivace

v kapitole a.

je spojeno s , proto t ch

24 hodin. Pacienti s

a [5]

Na obr. 8 je

Obr.

30

ST

east extrakt (IMUNA

Pharm a.s., SR), Gluk ), K2HPO4 (M

- - 121 V

(Chirana, o),

pH metr

pipeta, Petriho misky

10 %) vu pH.

roztok

2HPO4

a 7,5 g agaru b 5

vody bylo

a

31

vypnuto a Roztok se 20 minut.

Tab. 6:

Erlenmayerova

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

6,00 6,20 6,40 6,60 6,80 7,00 7,20 7,40 7,60 7,80 8,00

6,02 6,21 6,42 6,61 6,81 7,01 7,20 7,42 7,61 7,82 8,01

pH po sterilizaci 5,96 6,15 6,38 6,57 6,76 6,95 7,16 7,37 7,58 7,79 7,98

6,01 6,19 6,41 6,59 6,81 7,01 7,20 7,39 7,61 7,80 8,01

V takto

za a

Tab. 6.

jeho

etriho misku a

a n

22 zkumavek tak, aby sloupec

5 cm.

Salmonellou Enteritidis, Shigelou sonnei, Eschericia coli, Klepsielou pneumoniae

a Proteem mirabilis

32

E

Salmonella Enterobacteriaceae a to

bioch . :

XLD

MAL

DCA

Agar podle Hajna

Bizmut

TSI

detekci salmonel :

-

ONPG test

Jednou z

je komb . P

ohyb na hodnotu pH

Enteritidis v

Enterobacteriaceae.

4.1.1 Salmonella Enteritidis

Tab. 7: u Salmonelly Enteritidis

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Pohyb - +/- + +/- + + + +/- +/- +/- +/-

Indol - - - - - - - - - - -

Urea - - - - - - - - - - -

+

-

+/-

33

v V

roz 7,2. Po

s

4.1.2 Shigella sonnei

Tab. 8: u Shigelly sonnei

Ozn

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Pohyb +/- +/- +/- - +/- - - - - +/- +/-

Indol - - - - - - - - - - -

Urea - - - - - - - - - - -

+

-

+/-

se

Shigelly roz

7,0 7,6. P s

4.1.3 Escherichia coli

Tab. 9 Escherichia coli

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Pohyb + + + + + + + + + + +

Indol + + + + + + + + + + +

Urea - - - - - - - - - - -

+

-

+/-

U Escherichie coli

34

V

bublinek tvorba plynu z

4.1.4 Klebsiela pneumonie

Tab. 10 Klebsiely pneumonie

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Pohyb +/- +/- - - - - - +/- +/- - +/-

Indol - - - - - - - - - - -

Urea V V V V V V V V V V V

+

-

+/-

V

U Klebsiely pneumonie

v termos

Klebsiela pneumonie n 7,2.

Tvorba indolu byla negativ

4.1.5 Proteus mirabilis

Protea mirabilis

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Pohyb + + + + + + + + + + +

Indol - - - - - - - - - - -

Urea + + + + + + + + + + +

Poz +

-

+/-

U Protea mirabilis

35

Tab. 11: Roz Enterobacteriaceae

Bakterie Pohyb Indol Urea

Salmonella Enteritidis + - -

Shigella sonnei - - -

Escherichia coli + + -

Klepsiela pneumonie - - +

Proteus mirabilis + - +

+

-

Tab. 12: ,

Bakterie

Pohyb Indol Urea

Salmonella Enteritidis 6,8-7,2 6,0-8,0 6,0-8,0

Shigella sonnei 7,0-7,6 6,0-8,0 6,0-8,0

Escherichia coli 6,0-8,0 6,0-8,0 6,0-8,0

Klepsiela pneumonie 6,4-7,2 6,0-8,0 6,0-8,0

Proteus mirabilis 6,0-8,0 6,0-8,0 6,0-8,0

Klepsielu

pneumonie (viz. kap. 4.1.4 Klebsiela pneumonie) .

Salmonelly Enteritidis, Shigelly sonnei a Klebsiely

pneumonie. v 7,2 (viz. Tab. 12)

Enterobacteriaceae Tab. 11.

36

V byla zhodno teristika Salmonella, jeho

klin

Byly

kupovat v dispozici.

letech 1999

V

dle a 561/2004 Sb. v

k

prodejen.

letech 1999-2008 z

Salmonella z potravin a

Jsou uvedeny

Salmonella.

V byl proveden pokus o hodnoty

7,6

37

a pH byla stanovena na 7,0 7,2

o na

minimum.

38

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s. 53-62. ISBN 80-902896-6-5

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41

ATB Antibiotika

BGA Brilliant Green Agar

DCA -

DIC

H2S

HACCP Hazard Analysis Critical Control Points (

)

HCl

HPCP Fenol-chloroform-benzol extrakce

ISO International Organization for Standardization

K2HPO4

KIA Kligler iron agar

LPS Lipopolysacharid

MAL Manitol- -

MIU Pohyb-urea-

NaOH

PS

Sb.

SBP

TSI Triple sugar iron agar

XLD r