Nghiên Cứu Về Listeria Monocytogenes Trong Các Sản Phẩm Thủy Sản

Nghin cu v Listeria monocytogenes trong cc sn phm thy sn

SVTH: V Thnh Hng 1

CHNG I

GII THIU


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1.1. t vn

Cc bnh l nhim trng l nguyn nhn gy t vong hng u trn th gii.

Khng ch c nhng bnh nhim trng mi pht sinh m nhng bnh nhim trng c

gy cht ngi bit t lu cng ti xut hin. Hn na t l vi khun gy bnh

khng khng sinh ngy cng tng cao l nguy c ln cho sc khe cng ng. Nhng

bng chng gn y cho thy cc tc nhn gy bnh mc d rt khc nhau u s dng

nhng phng thc chung pht ng qu trnh nhim trng v gy bnh. Nhng c

ch ny to nn c lc (virulence) ca vi khun. Tm hiu cc c ch m vi khun s

dng xm nhp v gy bnh c ngha quan trng trong cuc chin chng li cc tc

nhn b nh ny.

Listeria monocytogenes l tc nhn gy bnh listeriosis. Vi khun ny c xp

l tc nhn gy bnh ng th ba thuc nhm B sau Streptococcus v E. coli. ng thi

l ngun chnh ly nhim bnh cho ngi trong cc sn phm bo quan lnh, vi sinh vt

ny c kh nng tn ti tng trng trong sn phm sut qu trnh bo qun lnh. i

vi vi sinh vt ng c thc phm khc, chng s pht bnh khi con ngi hp thu

liu lng, sau thi gian bnh cc triu chng lm sn biu hin. Trong Listeria

monocytogenes hin din vi s lng nh trong thc phm, khi vo c th chng

khng b o thi m tch ly ch c hi. Mc d bnh do Listeria monocytogenes gy

ra l tng s thp, 2 - 5 trng hp trn mt triu ngi mt nm, nhng t l cht do

vi khun ny l rt cao, 25 - 30% trng hp t vong trong cc ca nhim bnh.

i tng b nhim bnh do Listeria monocytogenes gy ra thng gp tr em,

tr s sinh, ngi gi, thai ph v ngi c h min dch km. Listeria monocytogenes

gy ra bnh nhim trng mu, vim mng no hoc st vim d dy rut, ng thi cng

l nguyn nhn gy ra tr cht sau khi sinh, non v sy thai ph n.

Do , vi tnh phn b rng v kh nng gy ra nhng tc hi nghim i vi

ngi b nhim L. Monocytogenes v c s chp thun ca khoa Mi trng v Cng


SVTH: V Thnh Hng 3

ngh sinh hc, ti tin hnh thc hin ti Nghin cu v Listeria monocytogenes

trong cc sn phm thy sn.

1.2. Mc ch

Cu trc v c ch gy bnh ca Listeria monocytogenes.

1.3. Ni dung nghin cu

Kho st v cu trc ca Listeria monocytogenes.

Kho st v s phn b ca Listeria monocytogenes.

Tnh hnh nhim Listeria monocytogenes trong sn phm thy sn trn th

gii v Vit Nam hin nay.

Mt s phng php xc nh Listeria monocytogenes.


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CHNG II

TNG QUAN


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2.1. Tng quan v mt s vi sinh vt nhim trong thc phm

2.1.1. Salmonella sp

Hnh 2.1: Salmonella vi khun chuyn ch bnh thng hn

2.1.1.1. Phn loi

Gii: Bacteria

Nghnh: Proteobacteria

Lp: Gamma Proteobacteria

B: Enterobacteriales

H: Enterobacteriaceae

Chi: Salmonella

Loi: Salmonella sp

2.1.1.2. c im

Salmonella l trc khun Gram m. Hu ht cc loi Salmonella u c lng

xung quanh thn (tr Salmonella gallinarum v Salmonella pullorum), vy c kh nng

di ng, khng sinh bo t. C kch thc t bo vo khong 0,5 3 m.

Salmonella l vi khun hiu kh hay hiu kh ty nghi, thch hp 370C, pH ti

u 7,2 - 7,6. mc trn cc mi trng thng thng.


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c im sinh ha:

Salmonella ln men glucose c sinh hi (tr Salmonella typhi ln men glucose

khng sinh hi) khng ln men lactose, Indol m tnh, Methyl Red dng tnh, VP m

tnh, Citrat thay i, Urease m tnh, H2S dng tnh (tr Salmonella paratyphi A: H2S

m tnh)...

Ln men sinh hi cc ng glucose, manit, sorbitol, ln men khng u

sacharose, khng ln men ng lactose, salicin, raffinose (T Minh Chu v Trn

Bch Lin, 1999).

2.1.1.3. Cu trc khng nguyn

Khng nguyn O

Mi Salmonella c th c mt hoc nhiu yu t khng nguyn. Hin nay ngi

ta bit c 67 yu t khng nguyn O. Vic xc nh cc yu t khng nguyn O l ht

sc quan trng nh nhm v nh type.

Khng nguyn H

Ch c nhng Salmonella c lng. Khng nguyn H ca Salmonella c th tn

ti di 2 pha.

Pha c hiu (phase 1): l nhng yu t c tnh cht c hiu cho

tng loi vi khun Salmonella, gm 28 khng nguyn lng c k hiu bng ch ci a,

b, c, g,.

Pha khng c hiu (phase 2): c k hiu s 1, 2, 3,.

(T Minh Chu v Trn Th Bch Lin, 2001)

Khng nguyn Vi

L khng nguyn b mt bao bn ngoi vch t bo vi khun, di dng mt

mng mng khng nhn thy c knh hin vi thng. Khng nguyn Vi ch c 2

type huyt thanh Salmonella typhi v S. paratyphi C.

Ngi ta da vo s khc nhau v cu trc khng nguyn xp loi Salmonella.


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Bng 2.1: Phn bit cc khng nguyn O, H, Vi:

Khng nguyn Tnh chu nhit Tc ng ca

alcohol 50% Formol 50%

O n nhit 2h30 1000C Khng B ngng tr ngng kt

Vi Bin nhit 1506 1000C Nhy cm Khng

H Bin nhit 2h 1000C Nhy cm Khng

Hnh 2.2: Cc khng nguyn b mt ca Salmonella.

2.1.1.4. Yu t c lc

C hai loi c t l ni c t v ngoi c t

Ngoi c t ng rut (entero toxin) c hai loi l LT v ST.

c t LT khng bn vi nhit, LT hot ha enzyme adenylcuclase trong

t bo nim mc rut, lm gia tng c-AMP (cyclo adenosine 5-monophosphate), c-AMP


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s kch thch tit Cl- v HCO3- ra khi t bo, ng thi c ch Na+ vo bn trong t bo.

Hu qu tch nc trong ng rut dn n tiu chy.

c t ST bn vi nhit, c ch tc ng tng t nh LT. ST hot ha

enzyme guanosylcyclase lm tng c-GMP (cycle guanosine 5-monophosphate) trong

t bo dn ti hin tng tiu chy.

2.1.1.5. Kh nng gy bnh

Salmonella xm nhp vo c th qua ng ming v hu ht l do n phi thc

n b nhim nh thc phm, sa, nc ung. Sau khi xuyn qua hng ro acid d dy,

vi khun di ng v pha rut non v sinh sn , tip tc chui qua 1 mng nhy v

vo thnh rut. Cc t bo Paneth ca nim mc rut tit ra mt loi peptide c tnh

chng li s xm nhp ca tc nhn gy bnh.

Salmonella nhim vo c th t hai ngun: t phn ngi hoc ng vt, t

ngi bnh. Trong phi k n tc ng ca ng vt lng v, trng v phn ca

chng lm cho vic lan truyn Salmonella d dng hn. Ngoi ra chut, mo, rui

cng l tc nhn gin tip dn n vic Salmonella lan rng ri hn khi phn ca chng

nhim vo cc thc phm khng c bo qun k. trong qu trnh git m cng cn

phng s nhim Salmonella nu khng thc hin ng quy trnh an ton thc phm.

Bnh thng hn

Hnh 2.3: Vim d dy rut


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nc ta, bnh thng hn ch yu do S. typhi, sau n S. paratyphi A, cn

S. paratyphi B v S. paratyphi C th t gp. Bnh ly t ngi ny sang ngi khc, qua

thc n, nc ung b nhim khun. Sau khi khi bnh v mt lm sng, khong 5%

bnh nhn tr thnh ngi lnh mang vi khun ko di hng thng hoc hng nm.

h, cha Salmonella l ng mt v vi khun vn c tip tc o thi theo phn ra

ngoi cnh. Ngi lnh mang vi khun l ngun lan truyn bnh quan trng.

Sinh bnh hc: Trc khun thng hn vo c th qua ng tiu ha n rut

non th chui qua nim mc rut ri vo cc hch mc treo rut. chng nhn ln v

mt phn vi khun b dung gii, gii phng ra ni c t. Ni c t kch thch thn kinh

giao cm bng, gy thng tn mng Peyer, xut huyt tiu ha, c th gy thng

rut. Ngoi ra, ni c t theo mu ln kch thch trung tm thn kinh thc vt no

tht ba, gy ra trng thi st ko di, li b, v gy ra bin chng try tim mch... T cc

hch mc treo rut vi khun lan trn vo mu gy nn nhim khun huyt v lan i khp

c th, ri vi khun vo mt v t quay tr li rut. Vi khun theo phn ra ngoi

cnh.

Cc bnh khc

Cc bnh khng phi thng hn do Salmonella gy ra thng l mt nhim

trng gii hn ng tiu ha trong cc trng hp nhim trng nhim c thc n m

Salmonella typhimurium... l tc nhn hay gp nht, sau l Salmonella enteritidis...

Nhim trng nhim c do Salmonella c thi gian nung bnh t 10 n 48 gi. Bnh

biu hin c st, nn, tiu chy. Bnh khi sau 2 - 5 ngy, khng c bin chng.

Ngoi ra, Salmonella c th gy nn cc tn thng ngoi ng tiu ha nh

vim mng no, th nhim trng huyt n thun, nhim trng phi...

Kh nng gy bnh ca mt s loi:

Salmonella typhi : Ch gy bnh cho ngi. nc ta bnh thng hn

ch yu do S.typhi gy ra.

Salmonella paratyphi A : Ch gy bnh thng hn cho ngi v cng hay

gp nc ta sau S. typhi.


SVTH: V Thnh Hng 10

Salmonella paratyphi B : Gy bnh thng hn ch yu cho ngi, i khi

c sc vt. Bnh thng gp cc nc chu u.

Salmonella paratyphi C : Gy bnh thng hn, vim d dy rut v

nhim khun huyt. Bnh thng gp cc nc ng Nam .

Salmonella typhimurium v Salmonella enteritidis : Gy bnh cho ngi

v gia sc, gp trn ton th gii. Chng l nguyn nhn gy nhim trng, nhim c

thc n do n phi thc n nhim Salmonella.

Salmonella cholerae suis : Loi ny hay gy nhim khun huyt.

2.1.2. Campylobacter sp

Hnh 2.4: Phy khun Campylobacter

2.1.2.1. Phn loi

Gii: Bacteria



B: Eubateriales

H: campylobacteriaceae

Chi: Campylobactes

Loi: Campylobactes sp


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2.1.2.2. c im

Campylobactes l nhng vi khun b, mnh, Gram m, hnh du phy nhn hai

u, c kch thc 0,2-0,8m x 0,5-5m, rt di ng nh c 1 lng 1 u, khng sinh

bo t. Nui cy Campylobacters trn mi trng nhn to thng kh khn v vi khun

i hi iu kin vi hiu kh (micro-aerophile). Mc c 370C nhng mc tt hn

420C, khng mc 250C. Thng dng cc mi trng chn lc nh thch mu

Columbia, thch mu tryptose phn lp vi khun. Campylobacters mc chm sau 2

n 3 ngy.

Bng 2.2: c im sinh ha phn bit loi v di loi ca vi khun

Campylobacter

Loi di loi

Pht trin Tnh cht sinh ha

250

C

420

C 1%G 3.5%N Catalase Nitrate H H2S

C. fetus ss vene + - - - + - - -

C. fetus ss fetus + - + - + - - -

C. jejuni - + + - + - + -

C.coli - + + - + - - -

C. cryaerophila + - - - + + - -

C. faecalis - + + + + + - +

C. lari - + + - + + - -

C. hyointestinalis + + + - + + - +

C. spu ss spu + + - - - + - +

C. spu ss bu + + + + - + - +

C. upsaliensis - + + - - + - -

Ch thch: 1%G = 1% glycerin

3,5%N = 3,5% NaCl

H = Hippurate hydrolysis sinh H2S trn mi trng TSI (< 3 ngy)


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C. fetus ss vene = C. fetus subsp venerealis

C. fetus ss fetus = C. fetus subsp fetus

C. Spu ss Spu = C. Sputorum subsp Sputorum

C. Spu ss bu = Sputorum subsp bubulus

Vi khun dng tnh Oxydase v Catalase, phn hy nitrat, H2S dng tnh,

khng oxy ha hoc khng ln men cc loi ng.


Cc nghin cu v khng nguyn ca Campylobacter phn ln da vo cu trc

ca loi C. jejuni. n nay xc nh c 3 loi khng nguyn l:

Khng nguyn b mt LPS: c bn cht l lipopolysaccharide, gm t nht

50 serotype bn vi nhit.

Khng nguyn lng H: c bn cht l protein vi hn 36 serotype

Khng nguyn mng ngoi OMP (Outer membrane protein): l loi

protein b mt chuyn bit v duy nht sn xut vacin. (Phan Hng Din, 2005)

2.1.2.4. Yu t c lc

c t ca Campylobacter gm hai loi c t: ni c t (endotoxin) v ngoi

c t (exotoxin). Trong ngoi c t c cha c t ng rut (enterotoxin) v c t

thn kinh (cytotoxin). Vai tr tc ng ca cc loi c t ny vn cha c bit r.

Liu gy c ngi l 500 1000 vi khun. Kh nng ca vi khun

Campylobacter ngi c l do s lng ln c t (H Huy Khi, 2004).


Campylobacters l mt tc nhn gy vim rut cp tnh thng gp ngi.

Chng xm nhp vo ngi do n ung cc thc n v nc b nhim khun, do tip xc

vi ng vt, ch yu l vi gia cm, gia sc.

Thi gian bnh 2 - 11 ngy. Th lm sng thng gp l bnh cnh ca mt

vim rut cp tnh vi du hiu au bng, tiu chy d di, phn nc c khi c mu v

m. C trng hp xy ra nhim khun huyt. Tiu chy do C. jejuni ko di 5 - 10

ngy. a s trng hp khi khng cn iu tr bng khng sinh.


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C ch gy bnh cha r. Campylobacters c c t rut, nhng c kin cho

rng vi khun ny c kh nng xm nhp ging nh Shigella. Campylobacters nhn ln

ch yu hi trng v hng trng.

Bnh vim rut do C. jejuni xy ra nhiu ni trn th gii, gy tiu chy cp

ngi, c bit tr em v khch du lch t cc nc pht trin n cc nc nhit

i.


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Bng 2.3: Kh nng gy bnh ca Campylobacter.

Loi Ngun nhim Bnh trn ngi Bnh trn ng vt

C. fetus subsp fetus Tru, b, cu

Nhim trng mu,

sy thai, vim

mng no v bnh

d day rut

Sy thai trn tru,

b, cu v ri lon

tiu ha

C. fetus subsp venerealis Tru, b Nhim trng mu Sy thai, cht phi

v v sinh

C. mucosalic Heo Khng gy bnh Vim rut v hoi

t trn heo

C. cryaerophila Tru, b, heo,

nga, cu Cha xc nh

Trong phn gia sc

khe, i khi gy

cht phi

C. jejuni subbp deylei

Gia cm, heo,

tru, b, cu,

ch, chim, cn

trng, mo v

trong mi

trng

Bnh d dy rut,

sy thai, vim

mng no,vim

khp, hi chng

cuillian-barre

Bnh d dy rut,

vim v, sy thai

vo cui giai on

ca mang thai hoc

con yu hoc

cht vi ngy sau

khi sinh, g thng

l vt mang trng

C. jejuni subsp jejuni

C. Helvetius Ch, mo Khng gy bnh Bnh d dy rut

C. laridis

Gia cm, heo,

tru, b, cu,

ch, chim, kh

Bnh d dy rut,

vim nhim trng

mu

Bnh d dy rut

C. upsaliensis Ch, mo, ngi Apse, nhim trng

mu Bnh d dy rut

(Verkens, 1996)


SVTH: V Thnh Hng 15

2.1.3. Escherichia Coli

Hnh 2.5: Escherichia Coli

2.1.3.1. Phn loi

Gii: Bacteria



B: Enterobacteriales

H: Enterobacteriaceae

Chi: Escherichia

Loi: e. Coli

2.1.3.2. c im

Hnh thi v c im nui cy

E.coli l trc khun Gram m, di ng do c lng quanh thn, mt s chng

E.coli c v polysaccharide, khng sinh bo t.

E.coli l vi khun hiu kh hoc k kh khng bt buc, pht trin d dng trn

cc mi trng nui cy thng thng, mt s c th pht trin c mi trng tng

hp n gin. Thng sng trong rut gi ca ngi v ng vt, theo phn i ra ngoi.

y l nhng vi sinh vt c kh nng pht trin nhit t 7 500C. Nhit

thch hp 370C, pH ti u l 4,4.


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Pht trin mnh trong mi trng Mac Conkey, EMB. Trn mi trng EMB, E.

coli cho khun lc c mu nh kim, trn, b u, ng knh khong ln hn 0,5m.

Trong mi trng lng, E. coli lm i mu mi trng, sinh hi hoc lm c

mi trng.

c tnh sinh ha

E.coli ln men nhiu loi ng sinh axit v sinh hi nh: Glucose, lactose,

ramnose; kh nitrat thnh nitric, gy nhy nht h hng thc phm, indol dng tnh,

methyl dng tnh, VP m tnh, citrat m tnh, urease m tnh, H2S m tnh.


Gm c cc loi khng nguyn

Khng nguyn O (somatic antigen): l khng nguyn ca thnh t bo, cu

to bi lipopolysaccharide. Cc c tnh ca khng nguyn O

Chu nhit, khng b hy 1000C trong 2 gi.

Khng b hy vi cn 50%.

B hy bi focmol 5%.

Rt c (1/20mg gy cht chut sau 24 gi)

Khng nguyn O gp khng huyt thanh tng ng s gy ra phn ng ngng kt

O. Khng nguyn O gi vai tr quan trng i vi kh nng gy bnh ca dng vi khun

c tnh cht chuyn bit cho tng loi vt ch.

Khng nguyn H (Flagellar antigen): c cu to bi protein, khi khng

nguyn H gp khng th tng ng s xy ra hin tng ngng kt H. khng nguyn H

c cc c tnh sau

Khng chu nhit.

B hy bi cn 50%.

B hy bi protease.

Khng b hy bi focmol 5%.


SVTH: V Thnh Hng 17

Khng nguyn K (Capsular antigen): loi ny ch c mt s t vi khun ng

rut, c cu to bi polysaccharide hoc protein. Nu khng nguyn K che ph hon

ton thn vi khun s ngn cng phn ng ngng kt O.

Khng nguyn tim mao F (Fimbrial antigen): c dng hnh roi, di

khong 4m, thng hay xon, ng knh 0,7 2,1m, gip vi khun bm vo t bo

nim mc rut nn rt quan trng trong kh nng gy bnh ca vi khun.

Hin nay c khong 700 tuype huyt thanh ca E. coli t s t hp ca cc nhm

khng nguyn O, H, K, F. Da vo ngi ta c th nh danh vi khun.

2.1.3.4. Yu t c lc

Ngi ta chia E.coli thnh nhiu nhm, mi nhm sinh ra cc loi c t khc

nhau, hin c 5 nhm chnh : STEC (Shiga toxin-producing E.Coli) hoc VTEC

(Verotoxigenic E.Coli) v EHEC (Enterohaemorrhagic E.Coli), EPEC

(Enteropathogenic E.Coli), ETEC (Enterotoxigenic E.Coli), EAEC (Enteroaggregative

E.Coli) v EIEC (Enteroinvasive E.Coli)

Nhm STEC

STEC sn xut c t Shiga-like toxin (Slt), cn gi l Shiga toxin (Stx) hay

Verotoxin (VT). H c t Stx gm hai nhm chnh khng phn ng cho vi nhau l

Stx1 v Stx2. Trong khi Stx1 c tnh bo tn cao th Stx2 rt thay i v trnh t, to ra

nhiu subtype nh Stx2c, Stx2hb, Stx2e, Stx2g. Mt dng STEC c th sn sinh Stx1,

Stx2 hoc c Stx1 v Stx2, v thm ch nhiu dng ca Stx2.

Yu t khc c lin quan n c lc ca STEC l vic to ra enterohaemolysin

(EHEC-Hly) v c th c c t rut chu nhit EAST1. EHEC-Hly c m ha bi

gen trn plasmid 60 MDa (pO157) m plasmid ny c tm thy gn nh tt c cc

dng O157:H7 v cng kh ph bin c nhng dng STEC non-O157 na (Nataro v

Kaper, 1998). Trn plasmid ny c s hin din ca mt operon gm 4 khung c m

(open reading frame ORF) l hlyCABD. Trong hlyA l gen cu trc khi u cho

haemolysin. c t rut chu nhit EAST1 (u tin c m t nhm EAEC l

EAEC heat-stable enterotoxin 1), cng c tm thy nhiu dng STEC. Tm quan

trng ca EAST1 i vi kh nng gy bnh ca STEC vn cha c bit, nhng c


SVTH: V Thnh Hng 18

mt vi trng hp tiu chy khng c mu m thng thy nhng ngi nhim

STEC

Nhm EPEC

Cc yu t c lc chnh bao gm gen eae m ha protein itimin cn thit cho

vic to ra tn thng dng A/E, plasmid 50 70 Mda ( EAF ) : m ha BFP ( bundle

forming pilus ), PER ( plasmid encoded regulator ) v Ler ( LEE encorded

regulator). Cc protein tit : Tir, EspA, EspB, EspD, EspF, EspG v MAP

(mitochondria associated protein), EAST 1 c kh nng ph hy t bo biu m v

CDT ( Cytolethan distending toxin )

Nhm ETEC

Nhm ETEC c hai nhm quyt nh c lc chnh l c t rut (enterotoxin)

v yu t nh v (colonization factor CF).

c t rut enterotoxin

Nhm ETEC gm nhng E. Coli to ra t nht mt trong hai loi c t ng

rut l ST v LT. ETEC gy bnh bng cch vi khun bm vo b mt mng nhy rut

non v tit ra c t rut, lm gia tng tnh trng tit dch. Nhm ETEC gy tiu chy

thng qua s tit c t ng rut LT v ST. E.Coli nhm ny c th ch tit c t

LT, hoc ch tit ST, hoc c th tit c LT v ST.

c t khng chu nhit (Heat-labile toxin - LT): c t LT ca E.Coli l

oligopeptide c lin h gn gi v mt cu trc v chc nng vi c t t (cholera toxin

- CT) do Vibrio cholerae tit ra. LT v CT ging nhau nhiu c tnh nh cu trc, trnh

t acid amin (ging nhau khong 80%), tng ng v receptor, hot tnh enzym, v tc

ng ca n trn th hay nui cy t bo.

Sau khi c t i vo ni bo, chng di chuyn trong t bo nh h thng vn

chuyn ca Golgi. ch n ca LT trong t bo l enzym adenylate cyclase nm lp

mng ngoi ca t bo biu m rut. Peptide A1 c hot tnh ADP-ribosyltransferase

chuyn phn ADP-ribosyl t NAD n ca protein lin kt GTP (GTP-binding protein)

l GS, gy hot ha enzyme adenylate cyclase, lm gia tng AMP vng (cAMP) trong t

bo. V vy enzyme cAMP-dependent protein kinase (A kinase) c hat ha dn n


SVTH: V Thnh Hng 19

s phosphoryl ha knh chloride (Cl-) mng t bo biu m vt qu mc bnh

thng. Kt qu dy chuyn l kch thch t bo bn di tit Cl- v ngn cn s hp th

NaCl bi nhng t bo c lng nhung. Hm lng ion trong lng rut gia tng ko theo

s di chuyn th ng ca nc t t bo vo lng rut, gy tiu chy (Nataro v Kaper,

1998). Mc d s kch thch ca Cl- do s gia tng lng cAMP trong t bo l cch gii

thch c in v c ch gy tiu chy ca LT v CT, ngy cng c nhiu bng chng cho

thy rng p ng tng tit i vi nhng c t ny c c ch phc tp hn. Mt c

ch tc ng khc ca c t c lin quan n nhng prostaglandin E (PGE1 v PGE2)

v yu t hot ha tiu cu. S tng hp v phng thch nhng cht chuyn ha ca acid

arachidonic nh prostaglandin v leukotriene c th kch thch s vn chuyn cc cht

in gii v kch thch nhu ng rut. C ch tc ng khc th hai c lin quan n h

thn kinh rut (enteric nervous system ENS) iu ha nhu ng v s tit ion rut.

C ch th ba l CT v LT gy p ng vim rut dng nh.

c t chu nhit (Heat-stable toxin - ST): Ngc vi LT, ST c trng

lng phn t nh v nhng cu ni disulfur ca n gii thch cho kh nng chu nhit

ca c t ny. ST c chia thnh 2 nhm l STa v STb, khc nhau v cu trc v c

ch hot ng. Gen m ha cho c 2 nhm c tm thy ch yu trn plasmid v vi

gen m ha ST cng c tm thy trn transposon. Sta (hay cn gi l ST-I) c to

bi ETEC v mt vi vi khun Gram m khc nh Yersinia enterocolitica v V.

cholerae non O1. ST ging 50% trnh t acid amin vi c t chu nhit EAST1 ca

EAEC. Gn y, cn c bo co cho rng mt vi dng ca ETEC cng c th sn sinh

c t EAST1 ngoi c t STa. Cn STb ch c tm thy ETEC.

Yu t nh v (colonization factor CF): C ch m ETEC kt

dnh v c tr trn lp mng nhy rut c nghin cu k. gy tiu chy, ETEC

u tin phi kt dnh vo t bo rut non nh vo lng trn b mt ca vi khun, gi l

yu t nh v (CF). CFA c th c phn loi da trn c tnh hnh thi. C 3 loi

chnh gm loi lng hnh que cng, lng hnh que mm c dng b, lng c cu trc

mnh mm. Gen ca CFA thng c m ha trn plasmid, cng l ni m ha cho

c t ST v/hoc LT.


SVTH: V Thnh Hng 20

Nhm EAEC

Nhng yu t c lc chnh ca EAEC bao gm cc dim bm dnh kt tp AAF

(aggregative adhesion fimbriae), yu t iu ha bm dnh kt tp aggR, protein Pet v

c t EAST 1 (enteroaggregative heat stabe toxin 1). AFF c xem l yu t

quyt nh c lc. Ba loi AAF c bit n gm AAF/I, AAF/II v AAF/III,

trong loi I v loi II c cu trc b, loi III c cu trc dng si ring bit. Cc AAF

to nn kiu bm dnh hnh chng gch ln t bo Hep 2. Yu t aggR c vai tr iu

ha s biu hin ca cc AAF. Protein Pet c tit ra mng ngoi vi khun gy ra s t

dch v gy c cho biu m tiu ha. EAST 1 c kh nng ph hy t bo biu m.

Ngoi cc yu t c lc nu trn, EAEC cn tit ra mt protein lm tan mu v lm

mt thng bng vn chuyn protein qua mng. Cc yu t c lc nu trn ca EAEC

ch yu c m ha bi cc gen nm trn plasmid c phn t lng 60 MDa. Mt s

yu t c lc c m ha trn cc gen trn nhim sc th ang c nghin cu

Nhm EYIC

EIEC gy bnh ch yu do kh nng xm nhp vo nim mc i trng. Kh

nng xm nhp c m ha bi gen trn plasmid 140 MDa. Cc gen trn plasmid ny

m ha cho cc khng nguyn xm nhp (IpaA n IpaD, Ipa : Invasion plasmid

antigen). EIEC cn c kh nng sn xut c rut ging mt s Shigella. Gen m ha

cho c t ny c tn l sen (Shigella enterotoxin), c ch gy bnh ging vi khun l.

EIEC xm nhp vo trong t bo biu m i trng, lm tiu cc ti thc bo v nhn

ln trong bo tng, ph hy t bo v xm ln sang cc t bo khc.


Kh nng gy bnh rt a dng: gy nhim khun ng tiu, vi c th yu th

gy nhim khun mu, gy vim mng no tr s sinh, gy tiu chy.

Bnh tiu chy do E.coli

Cc E.coli gy bnh tiu chy ngi gm c:

Enterotoxigenic E.coli (ETEC)

L loi E.coli sinh c t rut. ETEC l mt nguyn nhn quan trng gy tiu

chy nng ging triu chng do Vibrio cholerae 01 gy ra ngi. Bnh tiu chy do


SVTH: V Thnh Hng 21

ETEC xy ra ch yu cc x nhit i v c th gp cc la tui khc nhau, nhng

c bit tr nh thng thy bnh cnh lm sng nng d dn ti tnh trng kit nc

v ri lon in gii.

ETEC cn l mt nguyn nhn thng gy tiu chy cho khch du lch t cc

nc pht trin sang cc nc ang pht trin.

C ch gy bnh: ETEC vo rut s gn vo nim mc rut nh cc yu t bm

dnh, ng thi sn sinh ra c t rut tc ng ln t bo nim mc rut gy xut tit ra

mt s lng ln mt cht dch ng trng vi huyt tng. Bnh nhn nn, tiu chy

lin tc, phn ln c au nhc bp c, au bng v st nh.

C hai loi c t rut c nghin cu k v tnh cht sinh l, sinh ha v

tnh truyn bng plasmid l :

c t rut LT (Heat - labile)

c t rut ST (Heat - stable)

Nhng chng ETEC c th sinh ra mt hoc hai loi c t rut ty thuc vo

plasmid m chng mang.

LT loi c t rut b hy bi nhit, l mt protein gm 2 tiu phn A (Active) v

B (Binding) c chc nng ring bit. Tiu phn A c hai tiu n v A1 v A2, tiu phn

B c 5 tiu n v B1, B2, B3, B4 v B5. Cc tiu n v B c chc nng gn vi th

th ganglioside GM1 b mt t bo biu m rut v chun b m ng cho tiu phn

A m ch yu l A1 xm nhp vo bn trong t bo. Tiu n v A tc ng ti v tr

ch mt trong mng bo tng ni iu h.a enzymee adenylate cyclase. Adenylate

cyclase b hot ha v lm tng hm lng adenosine monophosphate vng (AMP

vng). Hin tng ny dn ti s tng thm ca cc in gii v nc qua mng rut,

gy tiu chy cp v kit nc, ri lon in gii.

ST loi c t rut khng nhit, l mt phn t c trng lng thp nht v khng

c tnh khng nguyn. Th th dnh cho ST khc vi th th LT. Sau khi c gn vi

th th, ST s hot ha guanylate cyclase trong t bo nim mc rut. Hin tng ny

dn ti s tng guanosine monophosphate vng (GMP vng) v do xy ra tnh trng

tng tit dch rut.


SVTH: V Thnh Hng 22

Enteropathogenic E.coli (EPEC)

EPEC hin nay c bit gm mt s type huyt thanh thng gy bnh tiu

chy cp (bnh vim d dy - rut) tr em la tui nh (tr di mt tui), c th gy

thnh dch. Cc v dch do EPEC thng hay gp trong bnh vin, c ch gy bnh ca

EPEC cha c bit r. Cc EPEC phn lp t cc v dch thng l thuc cc typee

huyt thanh:

O26 : B6 O111 : B4 O126 : B16

O55 : B5 O119 : B4 O127 : B18

O86 : B7 O125 : B15 O128 : B12

Enteroinvasive E.coli (EIEC)

L loi E.coli gy bnh bng c ch xm nhp t bo biu m nim mc rut, gy

tiu chy ngi ln v tr em vi nhng triu chng bnh l. ging Shigella: ngha l

au bng qun, mt rn, i tiu nhiu ln, phn c nhiu mi nhy v mu.

Ngi ta chng minh c rng kh nng xm nhp t chc rut ca EIEC

c chi phi bi plasmid. EIEC c th khng ln men lactose, khng di ng v ging

Shigella v nhiu mt k c cu trc khng nguyn. Do vy nhng v dch tiu chy do

EIEC gy nn d b ln ln vi tiu chy do Shigella.

Enteroadherent E.coli (EAEC)

L loi E.coli bm dnh ng rut gy bnh do bm vo nim mc v lm tn

thng chc nng rut.

Enterohemorrhagic E.coli (EHEC)

EHEC l mt trong nhng tc nhn gy tiu chy c th dn ti vim i trng

xut huyt v hi chng tan mu - ure huyt. EHEC l nhng chng E. coli c kh nng

sn xut mt c t gy c t bo Vero (Verocytotoxin), gi l VT.

Cc nhim khun khc do E.coli

E. coli c th gy nn nhim khun ng tit niu: s ng nc tiu do si,

thai nghn... to iu kin thun li cho bnh nhim khun ng tit niu d xy ra .

Mt khc, khi thng niu o, ngi ta c th gy ra nhim khun ngc dng.


SVTH: V Thnh Hng 23

E. coli c th gy ra nhim khun ng sinh dc, nhim khun gan mt, vim

mng no tr cn b, nhim khun huyt ...

2.2. Gii thiu v Listeria monocytogenes

2.2.1. Lch s pht hin

L. monocytogenes ln u tin c m t bi E.G.D Murray vo nm 1926 da

trn su trng hp t t tr. Murray gii thiu Vi khun monocytogenes trc khi

JH Harvey Pirie thay i tn chi l Listeria nm 1940. Mc d L. monocyotogenes

nhim c ng vt v con ngi c ni n vo nhng nm 1920, nhng cho n

nm 1952 ti ng c cng nhn L. monocyotogenes l mt nguyn nhn quan

trng gy ra nhim trng v vim mng no ca tr s sinh. ngi ln mc bnh do vi

khun ny nguyn nhn l do tn thng h thng min dch, chng hn nh ngi lm

dng thuc c ch min dch v Corticosteroid cho khi u c tnh hoc cy ghp ni

tng, v nhng ngi nhim HIV.

Mi cho n nm 1981 L. monocytogenes c xc nh l mt nguyn nhn

gy bnh t thc phm, lin quan n 41 trng hp mc bnh v 18 ca t vong, ch

yu ph n mang thai v tr s sinh. T mt s trng hp nhim Listeria t thc

phm c bo co, v L. monocytogenes c cng nhn rng ri nh l mt

mi nguy him quan trng trong ngnh cng nghip thc phm.

Listeria c xem l vn nghim trng nhim vo cc sn phm tht v tht gia

cm vo nhng nm 1980. Nhng nm 1990, B Nng nghip M cho bit c mt trn

dch bnh ny t bnh m kp xc xch nng v c th l cc ca hng bn tht ngon dn

n t nht 101 ngi b bnh khin 15 ngi ln t vong v 6 thai nhi b sinh non.

Nm 1999, loi L. monocytogenes c bit cc c tin trin bo ng cc

vin chc y t v h buc nhng nh sn xut thc phm phi gii quyt vn . Mc d

quy nh tm thi i vi cc sn phm tht v tht gia cm ch bin sn pht hnh

nm 2003 gip kim sot s bng pht vi khun, nhng r rng khng th loi b

c bnh hon ton. Theo Trung tm y t d phng CDC, c tnh 2.500 ngi b

bnh listeriosis nng mi nm, trong s c 500 ngi t vong. Ngi c nguy c cao

c th mc bnh ny sau khi n thc phm thm ch ch nhim vi con vi khun.


SVTH: V Thnh Hng 24

Nm 2006, trong mt n lc bo v cng ng, C quan lng thc v dc

phm FDA ph chun tn thnh vic s dng thuc xt kh trng gip gim nhim

khun cc sn phm tht v tht gia cm ch bin sn. Thuc phun bao gm 1 hn hp

gm 6 loi virus v hi dit khun L. monocytogenes.

2.2.2. Phn loi

Theo George M. Garrity, Julia A.Bell v Timothy G.Lilburn, phn loi hc ca

Listeria spp. Trong gii vi sinh vt nh sau:

Gii: Bacteria

Ngnh: Fitmicutes

Lp: Bacilli

B: Bacillales

H: Listeriaceae

Ging: Listeria

Lc u Listeria ch c bit n vi mt hay hai loi ch yu l Listeria

monocytogenes, v sau th xc nh c 6 loi gm:

Listeria monocytogenes

Listeria innocua

Listeria seeligeri

Listeria ivanovii

Listeria grayi

Listeria welshimeri

Cc loi c tnh tng ng v di truyn nn gip gii thch c kiu hnh tng

t nhau gi cc loi. Kh nng tan huyt l c tnh nhm phn bit gi cc loi.

n nay ch c loi L. monocytogenes cho thy l tc nhn chnh gy bnh cho

ngi v ng vt, khi L. invanovii chi gy bnh cho ng vt c bit l th.


SVTH: V Thnh Hng 25

2.2.3. c im

Hnh 2.6: L. monocytogenes.

2.2.3.1. c im chung

L. monocytogenes l trc khun Gram dng, k kh ty nghi pht trin nhit

t 1 - 45oC, khng to bo t nhng c chuyn ng in hnh khi c cy nhit

20 - 25oC v c th pht trin trong t bo .

L vi khun hnh que mnh, chiu ngang khong 0,5m, chiu di khong 1-

2m. Phn ng catalase dng tnh. Sau khi nhum Gram ta thy nhng t bo hnh que

ring l hoc nhng t bo ni vi nhau thnh mt chui t bo.

Vi khun ny c th tn ti pH rng t 4,3 - 9,6.

Hnh 2.7: T bo vi khun L. monocytogenes Gram(+)


SVTH: V Thnh Hng 26

Hnh 2.8: Lng roi ca L. monocytogenes

Vi khun L. monocytogenes sng rt dai v khc vi a s vi khun khc L.

monocytogenes c th tng trng chm trong t lnh nhit 4C.

Hin nay cc nh khoa hc cho rng con ng nhim L. monocytogenes ph bin nht

l qua thc phm.

Trong s by loi Listeria c bit n, ch c L. monocytogenes mi l tc 11

chng huyt thanh (srotypes) trong 90% trng hp bnh Listeriosis ngi u do

cc serotype 1a, 1b v 4b gy nn. Trong ba nhm va k trn, th 4b l serotype c hi

nht.

2.2.3.2. c im sinh ha

Ty tng loi hay tng kiu huyt thanh m Listeria. spp c cc biu hin sinh

ha khc nhau. c im sinh ha chung ca Listeria. spp c th hin nh sau.


SVTH: V Thnh Hng 27

Bng 2.4: c im sinh ha ca mt s loi Listeria

Loi

Acid sn sinh t

-Hemolysis1 Manitol Rhamnose Xylose c tnh2

L. monocytogenes + - + - +

L. ivanovii3

+ - - + +

L. innocua - - V

4 - -

L. welshimeri - - V

4 + -

L. seeligeri + - - + -

L. grayi5

- + V4 - -

1 Th nghim trn mi trng thch mu cu.

2 Th nghim trn chut.

3 Nhng chng ln men ribose c phn thnh L. ivanovii subsp. Invanovii v khng

ln men nh L. ivanovii. Lomdiniesis.

V4: biotypes thay i

5 Bao gm hai loi ph L. grayi. Murrayi bin i nitrate v L. gayi subsp. Grayi khng

bin i nitrate


SVTH: V Thnh Hng 28

2.2.4. Cu trc

2.2.4.1. Cu trc t bo

Hnh 2.9: Cu to t bo L. monocytogenes.

2.2..4.2. Cu trc phn t

Kch thc b gen ca loi L. monocytogenes khong 3,0 Mb

(Ganbank/EMBL_accession number AL591824). Nhng gen qui nh tnh gy c nm

trn b nhim sc th ca chng Listeria sn xut protein cho php to yu t iu ha

gip chng thch nghi vi iu kin mi trng khc nhau v iu kin c tnh gy

c.

Bng 2.5: B gen ca loi L. monocytogenes

L. monocytogenes

Kch thc nhim sc th (kb) 2.944.528

T l G+C (%) 39

T l G+C ca gen m ha protein 38

Tng s lng gen m ha protein 2.853

Lng Nhn

Tin mao

Ribosomes

Mng t bo

V t bo

Bao v


SVTH: V Thnh Hng 29

Mt khc, cc loi nh L.innocua thiu gen qui nh cho mt protein to c t

nn khng th m ha cho protein ng vai tr quan trng cho vic xm nhp vo t bo

ch. iu ny c th gii thch c kh nng gy bnh gia cc loi khc nhau thuc

cng ging Listeria. C L. monocytogenes v L. ivanovii u c mt t hp gen qui nh

c tnh vi chiu di 8,2 Kb trn b gen, c iu ha bi yu t PrfA (Positive

regulatory factor A).T hp gen ny nm gi gen prs v gen ldh trn nhim sc th.

2.2.5. Yu t c lc

PrfA

Hnh 2.10: V tr t hp gen qui nh c tnh trong L. monocytogenes.

Phn ln t hp gen ny tham gia trong vic xm nhp vo t bo ch v tin

hnh chu k xm nhim bn trong cc t bo ca tc nhn gy bnh. T hp ny m ha

bao gm 6 gen plcB, hly, mpl, actA, plcB, prfA v c 3 khung c m X, Y, Z.

Trong gen prfA rt cn thit cho tnh c ca loi L. monocytogenes, c vai tr

nh yu t iu ha chnh nhm n nh hot ng ca t hp gen ny. Bn cnh ,

gen hly m ha protein to sn phm listeriolysin O (LLO), plcA m ha to

phosphatidylinositol-specific phospholipase C (PI-PLC), plcB m ha to

phosphatidulcholine-spefic phospholipase C (PC-PLC).

Tng t nh LLO, cc phospholipase C ny dung gii t bo ch bng cch to

mt l nh trn t bo v ph v lipid mng t bo. Vi khun sinh ra mt protease mpl

ph thuc Zn+ c hot tnh nh mt vi ngoi c t.

Z X plcB Y mpl actA hly plcA prfA


SVTH: V Thnh Hng 30

Ngoi ra c mt vi gen c qui nh tnh gy c nhng t hp gen ni trn bao

gm inlA, inlB, inlC m ha ln lt cho internalin A, B v C. Cc gen ny ng vai tr

quan trng nhm ni b ha t bo ch do tc nhn gy bnh. Tuy nhin tt c cc gen

trong t hp gen gy c v tt c cc gen nm ngoi t hp gen ny vn hon ton ph

thuc vo hot ng ca yu t prfA.

Bng 2.6: Chc nng ca cc gen trong hot ng to c t ca L. monocytogenes.

Gen Chc nng

prfA Positive regulotory factor A Hot ha cho qu trnh ly lan tronh t bo

ch ca vi khun.

plcA Phosphatidylinositol-specific

phopholipase C Dung gii mng khng bo ca th thc bo.

plcB Phosphatidylcholine-specific

phospholipase C Dung gii mng khng bo ca th thc bo.

hlyA Listeriolysin O Gii phng vi khun ra khi khng bo.

Mpl Zinc-dependent metalloprotease Tham gia trong qu trnh hot ng ca

plcB.

actA Actin-polymerizing protein Gip ly nhim t t bo ny sang t bo

khc.

inlA Internalin A Ni b ha t bo ch.

inlB Internalin B Ni b ha t bo ch.

2.2.5.1. Listeriolysin O

LLO l mt yu t c lc chnh c sinh ra bi L. monocytogenes gip cho L.

monocytogenes thot khi cc khng bo thc bo u tin. N l mt protein c trng

lng phn t 58,5 kDa c to thnh bi mt chui n polypeptide gm 529 aa.

Trnh t u N c c im ca trnh t du hiu in hnh vi khun Gram dng:

phn u c tnh i nc v mang in tch dng c theo sau bi 20 u k nc.

Trnh t ny m ha trc tip cho protein v c phn ct sau v tr lysine 25. Kt qu


SVTH: V Thnh Hng 31

l LLO c bt u t amino acid 26, l mt phn t protein di 504 aa v c trng

lng phn t 55,8 kDa.

LLO thuc v h c t gn cholesterol c hot bi nhm thiol c to ra bi

hu ht cc vi khun Gram dng. Mt s c t thuc h ny nh Streptolysin O t

Streptococcus pyogenes, pneumolysin O t Streptococcus pneumoniae, perfringolysin O

t Clostridium perfringens, cereolysin O t Bacillus cereus, alveolysin O t Bacillus

alvei Hot tnh ly gii ca cc c t ny c tng cng bi cc tc nhn c ch v

b km hm bi s oxy ha v biu hin khi c cholesterol hoc khng th khng

streptolysin O. Cytolysin c hot ha bi thiol c mt cystein n vng u C lm

cho chng d b bt hot bi s oxy ha mc d cystein ny c th c thay th bng

alanine m khng lm mt chc nng ly gii. Chng to nn nhng l khng lin tc vi

ng knh bn trong ln n 30 nm trn nhng mng cha cholesterol. C ch hot

ng ca chng da vo s tng tc vi cholesterol vi chc nng khng ch l v tr

gn kt m cn nh mt tc nhn lm thay i hot ng ca enzyme dn n qu trnh

oligomer ha ca khong 20 80 toxin monomer thnh cu trc vng hoc ging vng

cung.

LLO cho thy s tng tc k l vi cholesterol trong dung dch nh sau: protein

b bt hot bi cholesterol nhng, thay v ging cc c t khc, duy tr kh nng gn

trn mng. 1 HU (hemolytic unit) LLO c th bt hot 0,2 ng cholesterol trong khi cc

sterol khc nh epicholesterol v dehydroepiandrosterone l nhng cht c ch yu.

Cht oxy ha HgCl2 v p chloromercuribenzoate bt hot c t nng 1 mM. S

c ch bi cc hp cht thy ngn b o ngc bi dithiothreitol 2 mM hoc cystein,

iodacetic acid 2 mM, iodacetamide 2 mM, 1 mM tosyllysine chloromethyl ketone v 1

mM tosylphenylalanine chloromethyl ketone khng c ch.

Hot tnh dung huyt ca LLO c lin quan n pH ca mi trng: n hot ng

cao nht pH 5,5 v gn nh b bt hot pH 7,0. Tri li, cc c t c hot ha

bng thiol c ph pH hot ng rng hn nh pH 6.0 i vi pneumolysin, pH 6.5 i

vi perfringolysin O v alveolysin, pH 7.0 i vi streptolysin O. S pht hin ny c

th gii thch vai tr ca LLO trong qu trnh xm nhim ca L. monocytogenes. Sau


SVTH: V Thnh Hng 32

qu trnh thc bo, mi trng acid ca khng bo cha t bo vi khun s hot ha

LLO. Chnh c t ny s xc tin qu trnh ly gii khng bo v t bo vi khun xm

nhp vo t bo cht trong khi , mi trng pH cao hn s lm gim hot tnh ca

LLO. S h hi ca mng t bo ch cng b ngn cn bi s phn hy nhanh ca LLO

trong t bo cht do s nhn bit ca mt trnh t ging PEST. Mt trnh t amino acid

giu Proline (P), glutamic acid (E), serine (S) v threonine (T) c gi l PEST v

protein ch phn hy. V u N ca LLO nm vng 19 aa (t 32 50) chng

minh c im ny. Mt iu th v khi loi b motif ging PEST ny khng ch nng

cao c lc ca LLO m cn sa i cc yu t c lc ca vi khun. L. monocytogenes

b t bin, thiu trnh t PEST LLO, c chng minh l vic thot ra khi khng bo

thc bo theo mt con ng rt kh khn. iu ny chng t rng vng ny cng rt

quan trng cho qu trnh ph hy mng.

2.2.5.2. Vai tr ca cc yu t c lc trong qu trnh xm nhim ca

L. monocytogenes

L. monocytogenes xm nhim vo t bo ca c th vt ch thng qua c ch

thc bo. Nu L. monocytogenes vn cn sng sau giai on u xm nhim ny, n

c c th vt ch tip nhn nh protein b mt c gi l internalin, ng ch l

internalin A (InlA), internalin B (InlB). InlA l protenin 88 kDa c m ha bi gen

inlA tng tc vi E Carherin to iu kin cho L. monocytogenes i vo t bo biu

m, InlB l protein 65 kDa c m ha bi gen inlB, nhn din Clq R (hoc Met) cho

php L. monocytogenes xm nhp vo trong nhiu loi t bo ca vt ch nh t bo

gan, nguyn si bo v cc t bo biu m.

Tc xm nhim ca t bo vt ch nhanh cho php L. monocytogenes trnh

khi s kim sot ca chc nng min dch. Sau khi xm nhim c vo t bo vt

ch, L. monocytogenes ch yu nm mng n ca khng bo, phn gii mng n s

cp ca khng bo, nh vo s kt hp ca hai phn t gy c: listeriolysin O (LLO) l

protein 58 kDa c m ha bi hly, to ra cc l trn mng t bo, hot gii c tnh

lu hunh l yu t cn thit cho tnh c ca Lm. Phosphatidylinositol-phpspholipase

C (PI-PLC) l protein 33 kDa c m ha bi gen plcA, kt hp vi


SVTH: V Thnh Hng 33

Phosphatidylcholine-phospholipase C (PC-PLC, protein 29 kDa c m ha bi plcB),

gip LLO phn gii mng n s cp ca khng bo.

Sau khi phn gii mng n s cp ca khng bo, L. monocytogenes c

phng thch vo cytosol, ti y chng pht trin v nhn ln, ni bo chng di chuyn

t t bo ny sang t bo khc, s di chuyn ny i hi phi c protein b mt khc

nh ActA (protein 67kDa c m ha bi gen ActA), ng sao chp vi PC PLC v

iu chnh s thnh lp ca protein cu thnh lng ui b phn c nhm a L.

monocytogenes vo mng t bo cht

Ti y, L. monocytogenes tr thnh dng v bao trong cu trc ging nh chn

gi dng si, xm nhim vo cc t bo cn k v phn gii mng i ca khng bo th

cp. khi mng i b phn gii l du hiu bt u 1 chu trnh xm nhim mi, n ph

thuc vo hot ng ca PC-PLC v Mpl (l metallprotease 60 kDa c m ha bi

gen mpl).

2.2.6. Con ng xm nhim

Hnh 2.11: Qu trnh xm nhim v tc hi ca L. monocytogenes.


SVTH: V Thnh Hng 34

Sau khi xm nhim vo c th qua ng tiu ha, L. monocytogenes tip tc di

chuyn qua d dy ri i vo cc m rut non ca vt ch, bao gm cc t bo biu

m v cc i thc bo ca mng Peyer. Trong sut qu trnh di chuyn chng s khng

gy ra nhng thng tn m hc nu liu lng nhim khng ln. Trong dch

lympho hoc mu th L. monocytogenes di chuyn rt nhanh n hch bch huyt, l

lch v gan ri nhanh chng i su vo khng bo, hu ht vi khun xm nhp v ph

hy khong bo nh kt hp vi pH thp, cc enzyme thy phn v oxi ha trong khng

bo.

Listeriolysin dung gii Lan truyn t t bo ny sang t bo khc

i thc bo

hnh 2.12: Qu trnh xm nhim v ly lan ca Listeria monocytogenes

T khng bo chng s i vo t bo cht v nhn ln trong vng 30 pht sau khi

xm nhim, t 40-60 pht trong t bo gan v i thc bo. Qu trnh xm nhim vo

c quan hon thanh sau 5-7 ngy. Tuy nhin nu khng t c p ng min dch t


SVTH: V Thnh Hng 35

bo bch huyt CD8+ v gamma, interferon gin tip hot ha i thc bo. Nu t bo

ca vt ch khng to ra p ng min dch th Listeria monocytogenes s di chuyn

n cc v tr khc trong c th nh no v nhau thai.

S pht tn ni bo v gian bo ca L. monocytogenes

Hnh 2.13: Cch xm nhim ni bo ca L. monocytogenes.

a)L. monocytogenes xm nhp vo t bo. b)Vi khun pht trin ni ti khng

bo. c,d). Mng ca khng bo bi ph v bi hai phospholipases: PicA, PicB v hnh

thnh cc c t listeriolysin O. Vi khun di chuyn vo trong t bo cht, ni chng

nhn ln v bt u tng hp actin. e)ui Actin cho php vi khun i vo mt t bo

ln cn bng cch hnh thnh ch nh ra trn mng huyt tng. f)Vi khun ph v t

bo thot ra ngoi v ko di chu k.


SVTH: V Thnh Hng 36

Tm li s xm nhim ca L. monocytogenes c m t theo cc bc nht nh

sau:

S xm nhim v tip thu mm bnh

Thot khi th thc bo ca khng bo

Nhn ln trong t bo cht ca vt ch

nh hng di chuyn trong t bo cht

2.2.7. Kh nng gy bnh

2.2.7.1. Trn ngi

Bnh do Listeria gy ra l mt bnh him gp ngi nhng v cc triu chng

rt nguy him v gy t l cht kh cao do cn bnh ny nhn c s quan tm

ca cc cp chnh quyn a phng v vn sc khe. Phn ln ngi b nhim u

c cc du hiu cn lm sng nh. Thng thng i vi ngi khe mnh, s tiu th

thc phm b nhim l nguyn nhn gy ra vim d dy rut, triu chng st. Tri li,

L. monocytogenes l tc nhn gy cht c bit l tr em di 1 thng tui, ph n

mang thai, nhng ngi nhn m cy ghp v nhng bnh nhn c h min dch km.

LM hng s kch thch vo h thn kinh trung tm v c cc du hiu lm sng l vim

mng no v hnh thnh nn p xe. S nhim trng l mt dng bnh khc do listeria

gy ra tc ng n cc bnh nhn mang bnh v min dch. Ngoi cc bnh ni chung

ny, vi khun ny cng gy nhim nh v nh vim mng kt, nhim trn da, bnh ca

hch bch huyt. ph n mang thai, khi ngi m b nhim Listeria th khng c triu

chng r rng hoc c nhng triu chng ging nh b cm cm trong khi kt qu bo

thai v thai nhi b nh hng nghim trng hn bao gm sy thai, cht non, vim mng

no tr s sinh hay nhim trng.

2.2.7.2. Trn ng vt

Trong s cc ng vt, bnh do listeria tc ng chuyn bit trn gia sc, cu v

d vi thi gian bnh t 2 n 6 tun. ng vt b nhim Listeria khi n phi thc n

nhim, qua ng h hp hoc do tip xc trc tip. S pht bnh xy ra km theo s

suy gim min dch i vi tc nhn gy bnh do cc yu t gy stress (mang thai, s


SVTH: V Thnh Hng 37

ng c, s vn chuyn, iu kin mi trng km). ng vt b nhim bnh do

listeria thng st, bing n, suy nhc v mt phng hng

S biu hin bnh c th hnh thnh nn cc du hiu lm sng nh sau:

Thn kinh (bnh quay vng) nh bnh vim no v vim mng no. Du

hiu lm sng bao gm s n n, u quay v mt bn, i vng vng, hc u vo vt

rn. Nhng s biu hin ny do s hnh thnh cc vi p xe trong ty xng, xng

sng v trong tiu no.

Ni tng nh nhim trng mu. Ban u n thng xy ra chim, bo thai

hoc ng vt khong 1 thng tui. S nhim ny xy ra gy hoi t gan vi cc vng

tn thng mu trng xm v c th lan n l lch.

S sinh sn nh sy thai, non. iu ny rt hay xy ra ng vt n c.

Vi khun xm nhim vo t cung thng qua mch mu v s sy thai thng xy ra t 5

- 12 ngy sau khi vi khun tng trng trong nhau.

S nhim v nh chng vim v. Triu chng ny thng do s nhim

cn lm sng m khng c bng chng sng v. Sa do sinh vt tit ra lm tng ri ro

nhim trong sn phm sa.

Vim mng mt l triu chng thng t gp.

2.2.8. Phn b

Listeria monocytogenes phn b rng ri trong mi trng t, ct, nc. C th

truyn bnh qua ng ming v phn, v phn lp c t tht nu cha chn, gia

cm, sa ti cha thanh trng, phomat v hi sn.

L. monocytogenes l vi khun c mt khp ni trong rau c c th nhim khun

t t hoc t phn bn. Sc vt nng tri c th nhim khun m khng c triu

chng g v thc phm t ng vt nh tht, tht gia cm v cc sn phm sa c th b

nhim khun.

Trong khi vi khun c th tm thy cc loi thc phm ti sng nh tht cha

nu chn, rau c qu, sa cha tit trng v thc phm t sa ti, thc phm ch

bin sn nh rau qu ct lt c th nhim khun sau quy trnh sn xut. Cc thc phm


SVTH: V Thnh Hng 38

khc nh c th nhim khun L. monocytogenes khi thc phm thng mi lm nhim

khun ny trn b mt bp v thc phm khc hoc thng qua nc p qu ng hp.

Vi khun ny rt bn. Chng khng nhit (mc du nhit khng cao), mui,

nitrite v acid, sng st iu kin lnh ng v thm ch c th sinh si pht trin

chm chp trong t lnh. Do , con ngi c nguy c nhim khun cc k cao lu

cc loi thc phm cha nu chn k trc khi n c th c rt nhiu L. monocytogenes.

Theo S gim st v an ton thc phm thuc B nng nghip, nhng trng hp

bnh listeriosis M u lin quan n thc phm xc xch nh xng khi, tht hp ti

ngon, pat lnh, xc xch , pho pht mm ca Php, pho mt mm kiu Mhic, tm,

b, rau qu ti v sa cha tit trng. Trn dch bnh bng pht c th lin quan n s

hn tm cua, c ti, thy sn xng khi, li heo, kem, x lch go, x lch ci bp v

pat tht.

Vi khun c th nhim vo chng ta qua cc vt dng nh bp nh dao, tht bn

hoc t tay b nhim trng.

Nu nng thc phm v hp kh trng sa u dit c vi khun. Tuy nhin,

i vi mt s thc n lm sn (ready to eat products) nh tht g, cua v tht ngui nh

hot dog, deli meats, luncheon meats, v.vchng cng c th b nhim vo sau giai on

nu nng v trc khi c cho vo bao.

Chng ta cng c th b nhim khun L. monocytogenes nu c s tip xc trc

tip vi th c mang vi khun ny.

2.2.9. Tnh hnh nhim Listeria monocytogenes trong sn phm thy sn

trn th gii v Vit Nam hin nay

T l nhim bnh thanh nin khe mnh l rt thp, khong 0.7 trng hp trn

100.000 ngi. Tuy nhin, kh nng nhim tr em thng cao hn khong trn 10

trng hp trn 100.000 ngi v ngi gi t l ny l 1.4 trng hp trn 100.000

ngi. Ph n mang thai d dng b nhim bnh gp 17 ln so vi thanh nin khe mnh

2.2.9.1. Tnh hnh th gii

Nhiu nc trn th gii nh M, Thy S, nht l Php, t l nhim vi khun

ny trong thc phm nh sa ti v cc sn phm ch bin t sa, tht c ti sng v


SVTH: V Thnh Hng 39

cc sn phm ca tht c, rau xanh ... l rt cao. Nhim trng do Listeria monocytogenes

cng xy ra tri rc Chu u, Chu M v Chu Phi cng nh trong khu vc Chu

Ti M c khong 76 triu trng hp b ng c thc phm hng nm, trong

t l nhim do Listeria ch khong 2.5000 trng hp nhng c 500 trng hp t vong.

Mc d con s cc ca nhim bnh thp nhng t l cht cao chim t 20 30 % cc ca

nhim bt chp c s dng thuc khng vi sinh, cho thy mc nguy him ca s c

mt cc loi Listeria trong thc phm.t l t vong cao hn ng k so vi cc tc nhn

gy ng c thc phm khc nh Escherichia coli O157:H7 (E.coli),

Campylobacter.spp v Samonella.

Bng 2.7: So snh t l t vong gy ra gia L. monocytogenes v cc tc nhn

gy bnh khc

Tc nhn gy bnh T l

t vong (%)

T l

nhp vin (%)

Tng s

ca nhim bnh

Campylobacter.spp


SVTH: V Thnh Hng 40

Gn y nht, 12/10/2002, dch Listeriosis ti cac ban Ty Bc nc M c 23

ngi cht v 120 ngi ng c phi vo bnh vin, nguuyn nhn l tht g ca hng

Pilgrims Pride nhim Listeria monocytogenes. Tht g khng ch gy ng c Listeria

m cn nhiu vi khun khc. Nc di g v rut g c sn nhng vi khun gy bnh

nh Salmonella, Shigella, Campylobacter. Nhng khun ny khng gy bnh g m

gy bnh ngi (au bng, tiu chy, thng hn).

Bng 2.8: T l pht hin s c mt ca L. monocytogenes trong tht v tht gia

sc ca cc nghin cu mt s quc gia trn th gii

Quc gia T l pht hin % S lng mu nhim

Ty Ban Nha 64 28

Norway 61 71

Anh 60 61

M 23 6

Belgium v Php 10 15 48

Ireland 59 91

2.2.9.2. Tnh hnh Vit Nam

Nm 1996, trong s 215 mu thc phm cc loi gm sa chua, hi sn ng

lnh, tht ti sng, rau xanh v thc phm ch bin dng ngay, Vin Pasteur TP HCM

phn lp c 2 dng L . monocytogenes, chim t l 0.93%. n nm 1999, vin65

V Sinh Y T Cng Cng cng phn lp c 2 dng L .monocytogenes t 20 mu

thc phm, chim t l 10%.

Nm 2006, theo tng kt ca Phng Kim Nghim Ha L Vi Sinh thc phm

vin Pastuer TPHCM, cc mu hi sn ng lnh c t l nhim L. monocytogenes

23/138 mu, chim 23.9%. Nh vy t l xut hin ca chng vi khun ny cng c

khuynh hng gia tng theo thi gian nc ta v thng c tm thy cc mu thc

phm ng lnh.

Theo Quyt nh s 2670 ca B Nng Nghip v Pht Trin nng thn cp ngy

29/8/2008 v vic Cng b danh mc cc ch tiu ch nh kim tra i vi l6 hng


SVTH: V Thnh Hng 41

thy sn, ch tiu L. monocytogenes quy nh gii hn pht hin l 0cfu/25g i vi

hu ht cc loi sn phm thy sn.

Bng 2.9: Gii hn pht hin cho php i vi L. monocytogenes trong sn phm

thy hi sn.

Loi sn phm Gii hn cho php i vi L. monocytogenes

Thy sn n lin 0 cfu/25g

Thy sn ng lnh (bao gm

nhuyn th), khng cn nu trc

khi n

0 cfu/25g

C ng lnh (nguyn con, fillet,

ct khc...) 0 cfu/25g

2.3. Cc phng php pht hin Listeria monocytogenes

2.3.1. Phng php truyn thng

Thng quy k thut nh lng listeria monocytogenes trong thc phm.

2.3.1.1. Nguyn l phng php

S dng phng php cy m Listeria monocytogenes trn mi trng nui cy

chuyn bit sau khi m nhit 37 0C trong 48 gi. Chn cc khun lc nghi ng

xc nh Listeria monocytogenes bng cc th nghim sinh vt ho hc.

2.3.1.2. Phm vi p dng

Pht hin s nhim do Listeria monocytogenes trong thc phm v sn phm

sa.

2.3.1.3. Dng c, mi trng v thuc th

Dng c, thit b:

1. 0,1g mu

2. Giy bc dng c thy tinh

3. Bnh tam gic 500 ml

4. ng ln men (Durham)


SVTH: V Thnh Hng 42

5. Cc loi bt nh du

6. T m 30 - 350C

7. Du ngm

8. Que cy vng

9. Que cy thng

10. Knh hin vi

11. Mu ngm du (quan st th knh 100x)

12. a Petri

13. Pipettes, 25, 10, v 1 ml

14. ng nghim c np y

15. My xay hoc ti nghin mu

16. Cc dng c, thit b c v trng hoc s dng mt ln

Mi trng, thuc th:

1. Acid acetic, 5 N

2. Acriflavine monohydrochloride

3. Agar

4. N-(1-naphthyl) ethylene diamine

5. -Naphthol tinh khit

6. Thch mu (Unipath)

7. Cycloheximide

8. Natamycin (pimaricin)

9. Mu cu

10. Ethanol tuyt i

11. Khng th hunh quang (FA) m (Difco)

12. Glycine anhydride

13. B nhum gram

14. Hydrogen peroxide 3% cho th nghim catalase

15. KOH 40% (R65)


SVTH: V Thnh Hng 43

16. Huyt thanh cha Listeria (Difco)

17. Lithium chloride-phenylethanol-moxalactam (LPM) agar (M81) cng

esculin v st (M82)

18. Nalidixic acid (mui sodium)

19. Nitrate trung bnh (M108) v thuc th pht hin nitrat (R48)

20. Mi trng dinh dng (M114)

21. Nc mui sinh l 0,85% (R63)

22. Dch ng ln men (M130), cho cc th nghim 0,5% dextrose,

esculin, maltose, rhamnose, mannitol, v xylose

23. SIM trung bnh (Becton Dickinson, h thng Vi sinh vt, M137) hoc

nhu ng th nghim trung bnh (MTM, Difco) (M103)

24. Sulfanilic axit tinh khit (R48)

25. Trypticase u nnh agar vi chit xut nm men 0,6% (TSAye)

(M153)

26. Trypticase canh u nnh vi chit xut nm men 0,6% (TSBye)

(M157)

27. Oxford trung bnh (OXA) (M118)

28. Canh m tng sinh Listeria (M52)

29. PALCAM agar (M118a)

30. Carageenan (Sigma loi II)

31. BCM agar (M17a)

32. MOX agar (M103a)

33. ALOA agar (M10a)

34. Thch to mu cho Listeria (M40b)

35. Mi trng di ng ca Listeria (M131a)

36. mu Listeria (M40a)

37. canh v thch Tryptose (Difco) (M167)

Lu : cc mi trng thay th c th c s dng khi cc sn phm l tng ng.


SVTH: V Thnh Hng 44

2.3.1.4. Chun b mi trng v mu th

Chun b mi trng

Mi trng nui cy, canh thang, nc pha long v mi trng sinh vt ho hc

c iu ch theo cng thc. Cc mi trng c ng sn vo bnh cu, bnh nn,

ng nghim v c hp tit trng (110 0C/30 pht hoc 121 0C/15 pht ).

Chun b mu v dung dch mu th:

Chun b mu

Mu thc phm c ct nh hoc xay nhuyn bng my trong iu kin v

trng cho ti khi c th ng nht.

Chun b dung dch mu th 10-1:

Cn chnh xc 25g thc phm c chun b (hoc ht 25 ml thc phm lng),

cho vo erlen cha sn 225 ml nc pepton. Lc u 2-3 pht, thu c dung dch mu

th 10-1 .

Chun b dung dch mu th 10-2 , 10-3 , 10-4 ...

Ht chnh xc1ml dung dch mu th 101 cho sang ng nghim cha sn 9ml

nc pepton. Lc u trong 2-3 pht thu c dung dch 10-2 . Tip tc lm tng t

nh vy ta thu c cc dung dch mu th tng ng 10-3 , 10-4 ,10-5 .

2.3.1.5. Phng php tin hnh

Thu mu v tng sinh

1. Bo qun mu: Mu c bo qun 40C, trong qu trnh bo qun v vn

chuyn mu th L. monocytogenes vn pht trin c, mc d rt chm nhit ny.

Tuy nhin, nu mu c ng lnh, th khng nn x cho n khi phn tch.

2. Mu phc hp: Thng thng, phc hp mu c chun b sao cho mu i

din c b mt v bn trong ca thc phm. Cc mu ly i din trn mt thc

phm c trn li v cho vo 250 ml dung dch m tng sinh Listeria c cha sodium

pyruvate, khng c cha mi trng chn lc. Cc mu khc cng thc hin tng t.

Thng thng 50g ca hn hp pha trn c pha vi 200 ml dung dch m trc khi

tng sinh. Mu c bo qun trong khong nhit t 00C 50C.


SVTH: V Thnh Hng 45

3. Mu khng phc hp: Nu mu khng yu cu phc hp, n gin chn 25g

mu thc phm pha vo 200 ml dung dch m v thc hin cc bc tng sinh tip

theo.

4. Tin tng sinh v tng sinh: mu trong 4 gi 300C, thm cc mi trng

chn lc v tip tc thm 48 gi 300C. Nu cycloheximide khng c sn, th c th

thay th bng pimaricin (natamycin) nng 25 mg/l. Natamycin an ton v d s

dng hn cycloheximide.

5. Lit k cc mu dng tnh vi L. monocytogenes: Dng mt s phng php

xc nh nhanh lit k cc mu dng tnh vi L. monocytogenes, sau cc mu

tip tc c tng sinh v s lng L. monocytogenes c trong thc phm l rt thp

(khong 1 cfu/25g mu).

Cc bc xc nh

1. Chn mu nghi ng c cha L. monocytogenes. C th quan st vi nh sng

ca cc thit b chiu sng.

2. mu 300C, ngm du, quan st di knh hin vi kim mc pht

trin. Chn mt mu vi tc tng trng t yu cu xc nh.

3. Kim tra sinh ha ca cc loi Listeria l catalase dng tnh.

4. Tin hnh nhum Gram. L. monocytogenes l vi khun Gram dng.

5. Th nghim ln men ng. Cc mu dng tnh c gi li xc nh cc

bc tip theo.

6. Th ngim vi thch mu cu: mt s loi L. monocytogenes lm tan thch

mu. L. monocytogenes c cy vo thch mu v trong 24 48 gi 350C. C gng

cy L. monocytogenes vo lp thch sao cho gn n lp di cng ca thch mu.

7. Xc nh cc loi Listeria thng qua hin tng tan huyt: L.monocytogenes

to vng lm, L.seeligeri to hi, L.innocua cho thy khng c tn huyt, trong khi L.

ivanovii to ra mt vng cng c xc nh r rng xung quanh. Tn huyt c xc

nh d dng hn khi lp thch mu mng hn 5mm.

8.Th nghim Nitrate. 350C trong 5 ngy. Cho vo 0,2 ml thuc th A, tip

theo l 0,2 ml thuc th B (R48) Mt mu sc -tm cho thy s hin din ca nitrit.


SVTH: V Thnh Hng 46

9. Xc nh kh nng di ng vi thch SIM: L. monocytogenes dng tnh vi

hnh anh di ng hnh d.

10. Th nghim ln men tao acid vi: dextrose, esculin, maltose, rhamnose,

mannitol, v xylose. 7 ngy 350C. Listeria spp thng dng tnh cho sn phm

acid khng c gas. Tt c cc loi Listeria u dng tnh vi dextrose, esculin, v

maltose. cc th nghim esculin c th b qua.

Th nghim Methyl Red v Voges Proskauer (MR-VP):

T canh thang TSB-YE cy vo canh thang Clark-Lubs .

m : 35 oC/48h.

Ly 1ml vo ng v trng v nh thuc th: 0,2ml KOH 40% v 0,6 ml a

Napton.

nhit thng trong 1h. c kt qu. Nu dng tnh c mu .

m canh thang cn li thm 2 ngy. Nh Metyl 0,5% trong cn 600 .

c kt qu: Nu dng tnh c mu .

Th nghim CAMP Test

Ria chng Staphylococcus aureus NCTC 1803 v Rhodococcus equi

NCTC 1621 to thnh nhng ng ngang qua a thch mu cu 7%.

Ria khun lc kim tra gia, vung gc vi Staphylococcus aureus v

Rhodococcus equi.

m 37 oC/24h.

c kt qu: Nu thy vng tan mu tin v pha ng cy chng

S.aureus l Listeria monocytogenes.

Ch : ng cy chng kim tra khng c chm vo chng Staphylococcus aureus

v Rhodococcus equi v cch chng khong :1-2mm.

B mt thch mu phi kh.

Tiu chun xc nh Listeria monocytogenes trong thc phm:

Trc khun, Gram ( + ) Catalaza ( + ) MR ( + ) VP ( + )

Xylose ( - ), Rhamnose (+), Manitol (-), Di ng (+) 20 0C-25 0C


SVTH: V Thnh Hng 47

Nu cn : Ngng kt vi cc khng huyt thanh : H v O

CAM Test:

+ Staphylococcus aureus (+)

+ Rhodococcus equi ( -)

S nh lng L. monocytogenes.

m: p ngc a thch, m 37 OC 48 gi.

Chn a thch: Chn a c t hn 300 khun lc m.

Tng sinh thun nht.

Xc nh tnh cht sinh vt, ho hc nh bng sau:

Bng 2.10: Tnh cht sinh vt ha hc ca L. monocytogenes.

STT Phn ng sinh ha Biu hin ca L. monocytogenes

1 Nhum gram + (Gram dng)

2 Catalase +

3 Kh nng di ng (200-250C) + (Hnh d)

4 Ln men Xylose -

5 Ln men Rhamnose +

6 Phn ng KOH -

7 Phn ng CAMP +

8 Kh nng tan huyt +

9 Manitol -

10 MR +

11 VP +

12 Sinh hi -

2.3.2. Cc phng php hin i

Pht hin vi sinh vt bng phng php nui cy c xy dng v pht trin

trong thi gian di, c nhiu nc cng nhn v chng tr thnh nhng phng

php tiu chun. Tuy vy nhc im ln nht ca phng php nui cy l tn nhiu


SVTH: V Thnh Hng 48

thi gian cho kt qu chm, mt nhiu cng sc, cng knh v ngy cng t ra khng

p ng c cc yu cu phn tch phc v nhu cu thc t hin nay. khc phc

nhng nhc im trn ca phng php nui cy, nhiu phng php nhanh v t

ng c hnh thnh v pht trin da trn nhiu nguyn tc sinh hc v vi sinh vt

hc khc nhau. Cc phng php mi ny nhm mc ch rt ngn thi gian phn tch,

gim thiu s phc tp trong thao tc, d dng thc hin v c nhy cao v chnh

xc cao.

Cc phng php nhanh v t ng ha trong phn tch vi sinh vt thc phm

u da trn nguyn tc ca vi sinh vt hc, sinh ha hc, ha l, min dch hc, sinh

hc phn t pht hin v nh lng vi sinh vt mc tiu hay sn phm c hi ca

chng trong thc phm, mi trng. Tt c cc yu cu k thut lin quan n vic thu

v chun b mu phn tch u c nghin cu, ci tin theo hng t ng ha, n

gin ha cho kt qu nhanh v chnh xc.

2.3.2.1. Phng php Elisa (Enzyme Linked ImmunoSorbent Assay)

Nhng tin b khoa hc trong nhng nm gn y thc y s pht trin ca

cc k thut chn on bng huyt thanh. Cc phng php ny rt hiu qu trong vic

chn on nhanh v chnh xc cc vi sinh vt gy bnh. Ngy nay phng php ny cn

c pht trin xc nh cc cht c hi trong mi trng nh c t, d lng

khng sinh Nguyn tc ca phng php Elisa da trn phn ng kt hp gia khng

nguyn vi mt khng th c hiu. Phn ng min dch xy ra c pht hin bng

cch s dng nhng khng th c nh du (bng cch nhum pht hunh quang,

ng v phng x, hay enzyme).

Phng php ELISA (Enzyme Linked ImmunoSorbent Assay) l phng php

hp ph min dch lin kt enzyme Nguyn tc k thut Elisa l s dng khng th n

dng (khng th s cp) ph bn ngoi nhng ging nh nhm mc ch thu gi nhng

khng nguyn mc tiu. Nhng khng nguyn thu gi c pht hin bng cch s

dng khng th th hai c gn vi enzyme pht tn hiu (thng l horseradish

peroxidase hay alkaline phosphatase). Khi cho vo hn hp phn ng mt c cht c


SVTH: V Thnh Hng 49

hiu ca enzyme, phn ng xy ra v to cc sn phm lm i mu phn ng. V vy

chng ta c th pht hin c s hin din ca khng nguyn.

Hnh 2.14: C ch phn ng ELISA

Quy trnh thc hin phn tch ELISA c cc bc chnh nh sau:

t mu vo trong cc ging c cha khng th s cp, cho khng th th cp

lin kt vi enzyme to mu vo to sandwich, ra loi b cc khng th mang

enzyme khng tham gia phn ng cho c cht to mu vi enzyme lin kt trong hn

hp, o cng mu to thnh xc nh lng khng nguyn trong mu.

Hin nay Elisa c s dng rng ri phn tch Salmonella, E.coli gy

bnh, Listeria, c t Staphylococcus, thuc tr su, d lng khng sinh i vi vi

sinh vt, phng php Elisa c th s dng pht hin v nh lng vi sinh vt trong

thc phm sau vi gi tng sinh nhm tng nhy ca phng php.

2.3.2.2. Phng php PCR (Polymerase Chain Reaction)

Tt c cc mc tiu khuch i c pht hin bng mt s c im c bn. S

dng quy trnh cng ngh enzyme trong mt enzyme n hoc enzyme tng hp

nhiu bn sao ca cc mc tiu axit nucleic. Tuy nhin, phn ng chui polymerase

(PCR) l phn ng u tin v vn l k thut c p dng rng ri nht. PCR l mt

phng php n gin nhanh chng khuch i chui DNA mc tiu. mt DNA

polymerase nhit c kh nng tng hp DNA c th, v si kp DNA chc nng nh

mt mu khun thc hin phn ng DNA polymerase. Qu trnh PCR bt u

nhit cao ( 940C) bin tnh v tho xon DNA si kp thnh DNA si n, tip


SVTH: V Thnh Hng 50

theo l nhit tng i thp (540C) kch hot qu trnh bt cp ca cc nucleotide

v mch khun, v sau l 720C cho php sao chp DNA polymerase ca mu.

Ton b qu trnh c lp i lp li 25 - 30 chu k mt bn sao ca mu DNA c

th bin thnh hng t bn sao trong vng 3 - 4 gi.

Gel in di thng c s dng pht hin cc sn phm khuch i. Khi ht

mi xunh quanh DNA khun, khi lng v kch thc ca gel ch c th c d

on v pht hin vi mt vt DNA (Wang et al., 1993; Manzano et al., 2000; Volokhov

et al, 2002.). Bng cch xc nh s khc bit trong phn t gen 16S v 23S rRNA,

vng gel m, hly, inlA, inlB, IAP v gen khc ca L. monocytogenes c nhanh

chng v phn bit chnh xc gi Listeria khc loi v vi khun thng thng. PCR

kho nghim a thnh phn c s dng khuych i mt cch chn lc gene

IAP v to thun li cho vic xc nh s khc bit ca tt c su loi Listeria trong mt

th nghim n (Bubert et al., 1999). Tuy nhin, nn cn thn trng xc nh kch

thc ca sn phm khuch i, c th thy s khc bit kch thc gia cc loi

Listeria. Nh vy, xc nh gen mc tiu cho mt loi Listeria duy nht c th c tt

hn, v n cung cp mt biu hin c lp xc nhn ca loi (Gilot v ni dung, nm

2002; Liu et al., 2003b, 2004a, 2004b, 2004c, 2005b). Nu ng hin din ca mt s

loi vi khun Listeria lm phc tp vic xc nh cc L. monocytogenes, th kho

nghim a thnh phn l rt hu ch nhn dng v c tnh ca vi khun phn lp.

Vic s dng cc on mi PCR nhm c th, cung cp cng c b sung xc nh

L. monocytogenes (Jinneman v Hill, 2001; Borucki v Call, 2003; Doumith et al. ,

2004a).

Tt c cc DNA polymerase u cn nhng mi chuyn bit tng hp mch

DNA mi t mch khun. Mch khun thng l nhng trnh t DNA ca gen nh c

trng cho loi vi sinh vt mc tiu hoc cc gen quy nh vic tng hp mt loi c t

chuyn bit. Mi l nhng on DNA ngn c kh nng bt cp b sung vi mt u

ca mch khun v nh hot ng ca DNA polymerase on mi ny c ni di

hnh thnh mch mi. Khi cung cp hai ngun mi chuyn bit bt cp b sung vi hai

u ca trnh t DNA, vi iu kin DNA polymerase hot ng trong phn ng PCR,


SVTH: V Thnh Hng 51

mt on DNA nm gia hai u mi s c to nn. Nh vy khuch i trnh t

DNA xc nh, ta c nhng thng tin ti thiu v trnh t to cac mi b sung

chuyn nghip. Cc mi ny gm mt mi xui (sens primer) v mt mi ngc

(antisens primer) so vi chiu phin m ca gen.

Phn ng PCR l mt chui nhiu chu k ni tip nhau. Mi chu k gm 3 bc:

bin tnh (denaturation), lai (anealation), tng hp (elongation). Phn tch sn phm

khuch i bng s in di trn gel agarose. c trng ca phn ng khuch i i vi

tng vi sinh vt mc tiu thng qua kch thc ca sn phm khuch i.


SVTH: V Thnh Hng 52

S phn ng PCR:


SVTH: V Thnh Hng 53

K thut PCR (Polymerase Chain Reaction) vi cp mi LM-F LM-R:

Chng vi sinh vt: L. monocytogenes

Chun b th nghim:

Trnh t gen hlyA ca cc chng L. monocytogenes c cng b trn

ngn hng gen.

Phn mm BLAST dung xc nh trnh t gen ch hlyA ca cc L.

monocytogenes. (Kanamyxin, Xgal, Eco RI, b kit tch dng Topo TA Cloning Kit, b

kit tch v tinh sch plasmid QIA prep Spin Miniprep Test Kit),kit xc nh trnh t

BigDye Terminator v3.1

Cp mi LM-F v LM-R c thit k bao gm 20 bp/mi cho sn

phm PCR 468 bp.

Ho cht s dng cho tch chit DNA tng s, ho cht cho in di trn

gel agarose, thang DNA chun 100 bp v 1000bp .Cc ho cht khc tinh khit

phn tch.

Phng php tin hnh th nghim : Mi cho phn ng PCR

Da trn cc trnh t gen hlyA ca cc chng L. monocytogenes c cng b

trn ngn hng gen, cp mi LM-F v LM-R c thit k bao gm 20 bp/mi cho sn

phm PCR 468 bp .

Xc nh tnh c hiu ca mi

Kh nng bt cp chnh xc ca mi c khng nh bng cch phn tch trnh

t sn phm PCR theo phng php ca Sanger v cng s v so snh vi trnh t cc

gen ch hlyA ca cc L. monocytogenes theo phn mm BLAST. Tnh c hiu ca mi

c kho st bng cch kim tra kh nng bt cp ca mi thit k vi cc khun DNA

ca vi khun L. monocytogenes v khng phi L. monocytogenes trong phn ng PCR.

Phn ng PCR

DNA khun t L. monocytogenes c thu nhn cho phn ng PCR bng cch

tch v lm sch DNA vi chloroform hoc x l nhit t bo 100 0C trong 10 pht.

Ti u ho iu kin PCR v nng Mg2+ (1,5 - 4,0 mM), nng mi (0,1 - 0,5

pmol/mi) v nng dNTPs (100 - 250 mM/mi loi). Phn ng PCR c thc hin


SVTH: V Thnh Hng 54

vi th tch hn hp 25 ml vi chu trnh nhit gm 94 0C - 3 pht, (94 0C 1 pht, 60 0 C

1pht, 72 0C 1 pht) x 30 trnh, 72 0C- 5 pht v 4 0C - 5 pht. nh gi kt qu phn

ng PCR da trn phn tch sn phm PCR trn gel agarose 1% vi 3 ml sn phm.

Xc nh nhy ca phng php

Mi trng nui qua m ca L. monocytogenes trn mi trng LEB c nh

lng, pha long ti cc mt thch hp. Ly tm thu sinh khi ca 1 ml canh trng

ha tan trong 100 ml TE, x l nhit thu DNA cho phn ng PCR. 3ml dch t bo

qua x l nhit c s dng lm khun cho phn ng PCR. nh gi kt qu phn ng

PCR nh phn tch sn phm bng in di trn agarose 1%.

DNA b gen

DNA b gen t L. monocytogenes c tch v lm sch dng lm khun cho

phn ng PCR khuych i on gen mc tiu hlyA vi cp mi LM-F v LM-R, Kt

qu phn ng PCR vi khun DNA tinh sch cho thy mt bng DNA c kch thc

nm trong khong kch thc sn phm PCR thit k.Vi mc ch thu nhn DNA bn

mu cho phn ng PCR mt cch n gin hn, huyn ph L.monocytogenes c x l

nhit 100 0C trong 10 pht ph v t bo, gii phng DNA, ly tm 10000 vng/pht

x 10 pht, thu dch ly tm cha DNA lm khun cho phn ng PCR.

Kim tra tnh c hiu ca cp mi LM-F, LM-R.

khng nh kh nng bt cp chnh xc ca cp mi LM-F, LM-R vi gen

hlyA ca L. monocytogenes, trnh t sn phm PCR ca cp mi ny c xc nh v

so snh vi kt qu x l plasmid trnh t gen ch hlyA ca cc L. monocytogenes. Sn

phm PCR sau khi c khuch i bng cp mi LM-F,LM-R c gn trc tip vo

vect pCR4 TOPO. DNA plasmid cha sn phm PCR c tch dng trong E.coli

DH5a. Tin hnh tch chit v tinh sch plasmid ti t hp (s dng b kit QIA prep

Spin Miniprep Test Kit), kim tra sn phm trn agarose 1%. Kt qu in di cho thy

cc plasmid ca khun lc trng c kch thc ln hn so vi i chng .

Sn phm PCR gn trong plasmid c kim tra bng cch ct vi EcoRI. Ti t

hp vi EcoRI cho thy on DNA chn vo plasmid c kch thc tng ng vi kch

thc sn phm PCR. Trnh t sn phm PCR c xc nh bng thit b xc nh trnh


SVTH: V Thnh Hng 55

t t ng ABI 3100 Avant. Trnh t ny c so snh vi trnh t hlyA ca ccL.

monocytogenes c cng b. Kt qu so snh cho thy sn phm PCR khuych i

bng cp mi LM-F, LM-R c kch thc 468 bp, t tng ng trnh t >98 % vi

trnh t hl yA ca 53 chng L. Monocytogenes cng b. Nh vy, cp mi LM-F, LM-R

thit k cho php nhn chnh xc on gen hlyA ca L. monocytogenes.

Xc nh iu kin ti u cho PCR.

Nng Mg 2+ :

Phn ng PCR c thc hin cc nng cui cng ca Mg 2+ t 1,5 mM n

4,0mM.

Nng dNTPs:

Phn ng PCR c thc hin vi nng dNTPs t 100 mM n 250 mM.

Nng mi

PCR c thc hin ti cc nng mi khc nhau t 0,1 n 0,5 pmol.

Dch t bo c xc nh mt bng phng php m khun lc. Pha long

dch vi khun ti cc nng khc nhau , x l nhit cc dch huyn ph vi khun. 3ml

dch sinh khi vi khun sau khi x l nhit dng lm khun cho phn ng PCR cho thy

phn ng PCR cho kt qu dng tnh vi cc mu c mt t bo 6x102 /phn ng

tng ng vi mt t bo t 2.104 CFU/ml.

Nh vy, phng php PCR pht hin L. Monocytogenes c nhy tng

ng vi kt thng mi BAXTM ca Qualicon. Inc., (104 CFU/ ml) nhng nh hn ca

Listeria monocytogenes LightCycler ca Roche (103 CFU/ ml). Cp mi LM-F, LM-R

thit k c hiu vi L. monocytogenes, cho php pht hin vi khun gy bnh L.

monocytogenes mt 6.102 CFU/phn ng (2. 104 CFU/ml).

B kit PCR:Vi nhy rt cao vi cc vi khun, c trong 25g mu thc

phm c mt con vi khun cn tm th PCR s pht hin ra chng. iu c bit l cc

b kt khng ch gi tn mt loi vi sinh vt gy ng c thc phm m chng c th

pht hin ra 12 loi vi khun khc nhau nh: e.coli, e. coli 0157:h7, salmonella spp,

shigella spp, nm mc... Ty thuc vo loi thc phm v s loi ch tiu vi khun gy

bnh cn kim sot i vi mi loi thc phm, cc quy trnh v b Kit PCR cho php


SVTH: V Thnh Hng 56

xt nghim v gi tn tt c cc vi khun nu trn trong vng 24 gi (tr Clostridium

botulinum, cn thi gian nui cy tng sinh di). Chng c thit k cho 50 phn ng

PCR vi 50 ng phn ng, cha y thnh phn dung dch m PCR, mi, nc...

gip kim tra chnh xc mc nhim vi sinh vt trm trng hay khng. So vi phng

php nui cy truyn thng, phng php ny cho hiu qu chnh xc v hay hn ch,

n pht hin nhiu vi khun gy bnh hn v tn rt t thi gian.

u im ca phng php PCR:

Thi gian cho kt qu nhanh.

C th nhn din nhng vi sinh vt kh nui cy. vic nui cy tng sinh

l n gin hn v c khi khng cn thit.

Ha cht cn cho phn ng PCR sn c hn v d tn tr hn so vi

trng hp huyt thanh hc. Khng cn dng c v mi trng chn on phc tp, c

th thc hin hin trng.

t tn km v mt nhn s. C th c t ng ha lm gim chi ph

pht hin cc vi sinh vt gy bnh trong thc phm.

Mc d vy phng php vn cn mt s khuyt im cn khc phc:

S c ch hot tnh ca Tag DNA polymerase bi thnh phn ca mu

vt. mu thc phm thng c nhng thnh phn phc tp. vic chit tch v tinh ch

DNA t thc phm hai mi trng trc khi thc hin phng php PCR cho php loi

b nhng hp cht c ch. Tuy nhn mt vi quy trnh pht hin vi sinh vt gy bnh

thc phm bng PCR khng cn tch chit, tinh ch DNA.

Mt vi sinh vt gy bnh hin din trong mu thc phm thng thp,

n trong a s trng hp cn c bc nui cy lm giu c c mt pht

hin bng PCR.

Phng php ny khng phn bit c t bo sng hay t bo cht. do

vy c th dn n trng hp dng tnh gi do DNA t t bo cht. Ngc li phng

php ny cho php pht hin bo t, dng tim sinh, hay t bo cht ca vi sinh vt

gy bnh hoc ng c.

2.3.2.3. Phng php lai phn t


SVTH: V Thnh Hng 57

Hin nay nhiu h thng c thit lp da trn DNA nh lng vi sinh

vt v c t. Tuy nhin, ch c phng php lai phn t (hay cn gi l phng php

mu d, probes) v phng php PCR l c thng mi ha di dng cc b kit pht

hin vi sinh vt gy bnh trong thc phm. Phng php s dng mu d pht hin vi

sinh vt trong thc phm c da trn s pht hin mt on gen c trng ca vi sinh

vt. C s ca vic s dng mu d l phng php lai phn t. Qu trnh ny bao gm

s tch ri hai mch i ca chui xon kp DNA khi nhit vt qu nhit nng

chy (Tm) ca phn t DNA v s ti bt cp cc trnh t nucleotide b sung khi nhit

tr li bnh thng. S lai phn t xy ra khi on mi c trnh t nucleotide b sung

vi mt vng trnh t trn DNA mc tiu gp nhau do chuyn ng nhit v khi nhit

mi trng thp hn Tm t nht vi . Qu trnh lai phn t chiu nh hng bi rt

nhiu yu t: nng DNA trong mi trng, nhit v thi gian phn ng, kch

thc cc trnh t lai v lc ion ca mi trng.

V d: h thng d Gen trak (Framingham, USA), h thng ny s dng que

th vi mu d pht hin Listeria trong mu b sa v mu mi trng. mu d l

nhng on oligomer DNA nh du bng ha cht pht quang. Quy trnh phn tch c

th chia lm 6 bc:

1. Ph v t bo thu nhn rRNA

2. Mu d pht hin cha fluorescein isothiocyanate u 5` v 3` ca phn t

c t vo phn ng.

3. Que th c bao bc bi polydeoxythymidine (dT) gn c vi oligodA

ca mu d.

4. Que th c t trong ng o cha mu d pht hin c nh du bng

enzyme

5. Sau khi ra loi phn enzyme tha, que th c t vo ng o cha c cht

to mu.

6. Sau khi hin mu, mu c pht hin bc sng 450nm.


SVTH: V Thnh Hng 58

CHNG III

KT LUN V KIN NGH


SVTH: V Thnh Hng 59

3.1. Kt lun Mc d bnh do Listeria monocytogenes gy ra l tn s thp, 2-5 trng hp

trn mt triu ngi mt nm, nhng t l cht do vi khun ny l rt cao, 25-30%

trng hp t vong trong cc ca nhim bnh.

Phng php truyn thng: Thng quy k thut nh lng listeria

monocytogenes trong thc phm.

-u im :phng php n gin d thc hin.

-Khuyt im: tn nhiu thi gian (vi ba ngy n vi ba tun). chnh xc khng

cao do ngi thc hin c th c sai xt trong qu trnh th nghim.

Png php hin i:

K thut PCR (Polymerase Chain Reaction) vi cp mi LM-F LM-R:

u im :c th pht hin nhanh thc phm nhim trc khn Listeria

monocytogenes vi nng rt thp ch khong 102 CFU/g i vi ngi trng thnh

v 10 CFU/g i vi tr em v ph n mang thai.

Khuyt im: Ga thnh kinh t dng thc hin cao v hu ht cc thit

b s dng trong th nghim u phi nhp t nc ngoi.Bn cnh th nghim cn

i nhiu k thut phc tp nn kh nng ng dng khng trong thc t khng cao.

B kit PCR:

u im:

+ Cho php xt nghim v gi tn hu ht tt c cc vi khun trong vng 24 gi.

+ Qa trnh thc hin nhanh gn,cho chnh xc cao.

Khuyt im: Khng s dng c vi Clostridium botulinum v vi khun

ny cn thi gian nui cy tng sinh di.

K thut ELISA : y l phng php hin i cho php xc nh

Listeria monocytogenes mt cch chnh xc v nhanh chng .


SVTH: V Thnh Hng 60

3.2. Kin ngh

Cc phng php xc nh L. monocytogenes trong thc phm vn cn

nhiu hn ch v mt thi gian cng nh chnh xc khi khng nh s c mt

ca loi ny trong thc phm. v vy nn s dng cc phng php xc nh hin

i nh PCR, ELISA trong phn lp v xc nh vi khun ny nhm m bo

chnh xc cao.

ti ny ch kho st trn mt s sn phm thy sn. v vy ngh tip

tc tin hnh trn nhiu sn phm khc c th p dng phng php ny rng

hn.


SVTH: V Thnh Hng 61

TI LIU THAM KHO

[1]. PGS.TS Nguyn Phng Tin, GS.TS Bi Minh c, GS.TS Nguyn Vn Dp _ Vi

sinh vt trong thc phm k thut kim tra v ch tiu nh gi cht lng an ton

thc phm.

[2]. Trn Linh Thc _ Phng php phn tch vi sinh vt trong nc, thc phm v m

phm.

[3]. H Hunh Thy Dng, 1998 _ Sinh hc phn t, NXB Gio dc.

[4]. V Th Vit Hoa, T Minh Chu, V Th Lm An, Lm Thanh Hin, Nguyn Th

Ngc Dip, Nguyn Thy Hng, 2000 _ Vi sinh vt hc i cng, t sch trng H

Nng lm Thnh Ph H Ch Minh.

[5].http://www.biosyth.com/index.asp?topic_id=178&g=19&m=256

[6].http://www.cdc.gov/epo/dphsi/annsum/1998/98graphs/9844.htm

[7].http://www.en.wikipedia.org/wiki/Listeria

[8].http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnal

yticalManualBAM/UCM071400


SVTH: V Thnh Hng 62

PH LC

Mt s mi trng chnh trong phng php truyn thng nui cy v phn lp L.

monocytogenes

Thch OXFORD

Code: CM856.

Columbia blood agar base 39.0

Aesculin 1.0.

Ferric ammonium citrate 0.5

Lithium chloride 15.0

pH:7.0 0.2

Cn 27.75g thch Oxford trong 500ml nc ct. Ho tan thch trong nc nng.

Hp t 1210 trong15 pht.

Lm ngui xung cn 50 0C v cho thm 1 ng Listeria Selective Supplement

SR:140 ho vi 5ml cn/nc ct v trng(1:1). Trn u, a.

Thch mu cu

Tryptone 14.0

Peptone Neutralised 4.5

Yeast extract 4.5

Sodium chloride 5.0

Agar 12.5

pH:7.3 0.2

Cn 40g thch mu cu trong 1 lt nc ct. Ho tan thch trong nc nng. Hp

t 121 0C trong 5 pht.

Lm ngui xung 50 0C v cng thm 7% mu cu v trng.

Thch TRYPTONE SOYA (vi 0.6% yeast extract)

Tryptone 15.0


SVTH: V Thnh Hng 63

Soya Peptone 5.0

Sodium chloride 5.0

Yeast extract 6.0

Agar 15.0

pH:7.3 0.2

Cn 40g thch Tryptone Soya trong 1 lt nc ct. Ho tan thch trong nc

nng. Hp t 121 0C trong 5 pht.

Mi trng SIM:

Tryptone 20.0

Peptone 6.1

Ferrouss ammonium sulphate 0.2

Sodium thiosulphate 0.2

Agar 3.5

Cn 30g mi trng Sim trong 1 lt nc ct, un tan, ng ng, hp t 121 0C

trong15 pht. 5. Canh thang ng

Peptone 10.0

NaCl 5.0

Nc ct 1 lt

2% Alconolic B.C.P Solotion 2 ml

ng 50.0

(Sucrose/manitol. . .)

Ho tan cc cht chnh pH:7.2-7.5. San ra cc ng 16 x125mm mi ng 9ml c

cha ng Durhams. Hp t 121 0C trong 15 pht.

Ghi ch: cc loi ng trn dng phng php lc v trng, khng hp.

Mi trng Clark -Lubs

Peptone 5.0

Glucose 5.0

m


SVTH: V Thnh Hng 64

Phosphate buffer 5.0

pH: 7.5 0.2

Cn 15g mi trng Clark -Lubs trong 1 lt nc ct. Hp t 121 0C trong15

pht.

Canh thang TRYPTONE SOYA

Tryptone 15.0

Soya peptone 5.0

Sodium chloride 5.0

Yeast extract 6.0

pH:7.3 0.2

Cn 3g mi trng canh thang Trytone Soya trong 1 lt nc ct. Hp t 121 0C

trong15 pht.

Nc Pepton

Pepton 1.0g

Nc ct 1000ml v shy; pH=7.2

Cn 1g pepton trong 1 lt nc ct. Hp t 121 0C trong15 pht.

Listeria selective supplement (Oxford)

(B sung vo mi trng thch Oxford )

Cycloheximide 200 mg

Colistin sulfate 10 mg

Acriflavine 2.5 mg

Cefotetan 1.0 mg

Fosfomycin 5.0 mg

Ly 5 ml nc ct v trng v cn (pha theo t l 1:1), cho vo l lc u, sau ln

vo bnh cu cha 500 ml thch Oxford hp tit trng ngui 50 0C. Trn u v

vo a v trng.

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