Tim Hieu Ve Vi Khuan Salmonella CvEbb9ZTTe 20130427110121 11208

Kha lun: Tm hiu v vi khun Salmonella
Chng I: GII THIU

1.1 t vn
Trong nhng nm gn y, vn ng c thc phm ngy cng tr nn cp
thit, cc bo co cho thy phn ln cc v ng c thc phm l do vi sinh vt. c
nhiu cnh bo, nhng tnh trng ng c thc phm vn ang leo thang v ngy cng
nghim trng.
C rt nhiu vi sinh vt gy ng c thc phm, v d nh Clostridium
butolinum, Escherichia Coli, Listeria monocytogenes trong , Salmonella l loi vi
sinh vt gy ng c rt nguy him. Salmonella thuc h Enterobactriaceae, gy ra
bnh thng hn, nhim trng huyt v nhiu bnh nghim trng khc.
Xut pht t nhu cu tm hiu v c t, kh nng gy bnh v cch pht hin
cng nh cch phng phng chng bnh vi khun Salmonella,ti thc hin nghin cu
ti tm hiu v vi khun Salmonella c ci nhn tng quan hn v vi khun
Salmonella v mt s chng vi sinh vt khc.
1.2 Mc ch
- Tng quan v mt s loi vi sinh vt thng ly nhim vo thc phm
v tm hiu v Salmonella vi c t ca n.
1.3 Ni dung nghin cu
- Nghin cu v c im sinh ha, sinh l, khng nguyn, c t v c
ch gy c ca mt s chng vi sinh vt.
- Nghin cu v cch pht hin Salmonella v xut bin php phng
nga.
GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Hu Lim

Chng II: TNG QUAN
2.1 Tng quan v mt s vi sinh vt nhim trong thc phm
2.1.1 Escherichia Coli
2.1.1.1
Gii thiu
- Escherichia Coli do Theodore Escherich (1857 1911), mt bc s nhi
khoa ngi o pht hin nm 1885.
- E.Coli l mt trong nhng thnh vin chnh ca h vi khun bnh
thng ng rut, nhng cng l cn nguyn ca nhiu bnh nhim trng.
- E.Coli c s dng lm m hnh nghin cu v sinh hc phn t
trong lnh vc vi sinh hc ni ring v sinh hc ni chung. Trong , chng E.coli K12
l m hnh c nghin cu nhiu nht. Nhiu thnh tu v di truyn hc, ha sinh
hc thu c da trn c s nghin cu vi khun ny.
2.1.1.2 Phn loi
- V phn loi, Escherichia Coli c xp vo:
Gii: Bacteria
Ngnh: Proteobacteria
Lp: Gamma Proteobacteria
B: Enterobacteriales
H: Enterobacteriaceae
Chi: Escherichia
Loi: Escherichia Coli
2.1.1.3
c im
- E.Coli l trc khun, Gram m, kch thc trung bnh t 2 3 m 0,5
m. Mt s t chng E.Coli c v, nhng hu ht u c lng v c kh nng di ng.
- E.Coli c kh nng ln men nhiu loi ng v c kh nng sinh hi
E.Coli c kh nng sinh Indol, khng sinh H 2S, khng s dng c ngun carbon ca
citrat trong mi trng Simmons, c deoxycarboxylase nn c kh nng phn gii
carborxyl ca lysin, ornithin, arginin v acid glutamic. Th nghim VP (Voges
Proskauer) sau 24h m tnh, sau 48h c th dng tnh .

SVTH: Nguyn Hu Lim

2.1.1.4
Hnh 2.1 : Vi khun Escherichia Coli

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).
a) Nhm STEC
- Hin nay c kh nhiu thut ng khc nhau gi tn cho nhm E.Coli
ny. Tn gi Verotoxigenic E.Coli (VTEC) c Konowalchuk v cng tc vin
(1977) t cho nhm ny khi pht hin vic sn xut c t gy c cho dng t bo
Vero. Tn gi Enterohaemorrhagic E.Coli (EHEC) l do dng ny gy vim kt trng
xut huyt v hi chng huyt niu (Nataro v Kaper, 1989). V thut ng Shiga
toxin-producing E.Coli (STEC) (trc y gi l Shiga-like toxin producing E.Coli SLTEC) ch r kh nng sn sinh c t gy c t bo ging nh c t Shiga.
- 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.
- Hai c t Stx1 v Stx2 u c cu to t 5 tiu n v B c khi
lng phn t 7,7 kDa v 1 tiu n v A c khi lng phn t 32 kDa. Tiu n v A
gm peptide A1 28 kDa v peptide A2 4 kDa ni vi nhau bng cu ni disulfur.
Peptide A1 c hot tnh enzyme v peptide A2 c nhim v gn kt tiu n v A vo
nhng tiu n v B. Nhng tiu n v B gip c t kt hp vi receptor c hiu
Gb3 (globotriaosylceramide) hin din trn b mt ca nhng t bo eukaryote (Stx2e
c receptor l Gb4). Sau khi c chuyn vo bn trong t bo, tiu n v A n t
bo cht v tc ng ln tiu phn 60S ca ribosome. Peptide A1 c hot tnh enzyme
hot ng nh mt N-glycosidase ct mt gc adenin khi rRNA 28S ca ribosome,
do gy tr ngi cho s tng hp protein. Do khng tng hp c protein, nhng t
bo b Stx tc ng (t bo ni m ca thn, t bo biu m rut, t bo Vero, t bo
SVTH: Nguyn Hu Lim

Hela hay bt c t bo no c receptor l Gb3, receptor Gb4 i vi Stx2e) s cht.
Hu qu gy c cho t bo rut do Stx v cc yu t c lc khc ca STEC l gy s
h hi nhng t bo nhung mao rut, gy tiu chy v vim kt trng xut huyt
(Haemorrhagic colitis HC). S h hi nhng t bo thnh mch mu do Stx2e s gy
nn hin tng ph thng heo.
- Yu t bm dnh ca STEC/EHEC c chng minh l ng vai tr
quan trng trong s nh v vi khun rut. chnh l intimin, mt protein mng
ngoi c trng lng phn t 94 97 kDa. Intimin c m ha bi gen eae (E.Coli
attaching and effacing). Intimin gy tn thng dng bm dnh v ph hy (attachingand-effacing, A/E) rut gi do vi khun bm cht vo t bo biu m (Donnerberg v
ctv, 1993). Gen eae ny cng c tm thy nhm EPEC. Gen eae gy tn thng
kiu A/E l mt trong s cc gen nm trn vng gy bnh 35,5 kb (gi l vng gy h
hi t bo rut locus of enterocyte effacement, LEE). Vng LEE ca STEC/EHEC
cha nhng gen m ha cho intimin, m ha receptor ca intimin Tir (translocated
intimin receptor) v mt s gen khc. Vng LEE khng ch l iu kin cn m cn l
iu kin cho vic hnh thnh tn thng A/E. Tuy nhin khng phi tt c STEC
u c gen eae, nhng tt c EHEC u c gen eae.
- 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
(enteroaggregative heat stabe toxin 1). 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
mt vi trng hp tiu chy khng c mu m thng thy nhng ngi nhim
STEC.
b) Nhm EPEC

SVTH: Nguyn Hu Lim

- 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)
c) 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.
LT c 2 serogroup chnh l LT-I v LT-II. LT-I v LT-II
khng c phn ng cho v mt min dch. LT-I c tit bi nhng dng E. coli gy
bnh trn ngi v th. Cn LTII c tm thy ch yu E. coli trn th v him khi
ngi. LT-I l mt oligopeptide khong 86 kDa, c cu to bi 1 tiu n v A 28
kDa v 5 tiu n v B 11,5 kDa. Tiu n v A chu trch nhim trong hot tnh
enzyme ca c t gm peptide A1 v peptide A2 lin kt nhau bi cu ni disulfur.
Nhng tiu n v B sp xp thnh vng, lin kt chc chn vi ganglioside GM1 v
lin kt lng lo vi GD1b v vi glycoprotein rut chng l cc receptor ca LT.
Hai loi LT-I c lin h gn nhau v c th phn ng cho mt phn vi nhau l LTp
(LTp-I) u tin c phn lp t heo v LTh (LTh-I) c phn lp t ngi. Gen m
ha cho LT l elt hay lt-I nm trn plasmid m plasmid ny c th cha c gen m ha
SVTH: Nguyn Hu Lim

ST v/hoc gen m ha nhng khng nguyn ca yu t nh v (colonization factor
antigen - CFA).
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 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.
Nhm LT-II ging vi LT-I v CT khong 55 - 57%

tiu n v A, nhng khng ging vi LT-I v CT tiu n v B. LT-II lm gia tng
cAMP trong t bo qua c ch tng t nh LT-I, nhng LT-II s dng GD1 lm
receptor thay v GM1. Nh ni trn, LT-II khng c lin quan n bnh trn
ngi v th.
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
SVTH: Nguyn Hu Lim

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.
STa l mt peptide gm 18 -19 amino acid vi trng
lng phn t khong 2 kDa. STa c chia thnh 2 loi l STp (ST porcine hay STIa)
phn lp c u tin trn heo v STh (ST human hay STIb) phn lp trn ngi. C
2 loi c t c th c tm thy dng ETEC ngi. Receptor chnh ca STa l
enzyme xuyn mng GCC (guanylate cyclase C) thuc h nhng enzyme receptor
cyclase. S kt hp ca STa vo GC-C kch thch hot tnh GC, dn n vic gia tng
lng cGMP ni bo. Hot ng ny cui cng dn n s kch thch tit Cl - hoc
ngn cn s hp th NaCl, gy ra s tit cht lng trong rut. STb: STb ch yu c lin
quan n dng ETEC phn lp t heo mc d cng c bo co v vi chng ETEC
ngi cng sn sinh STb. Khng nh STa, STb gy ra nhng tn thng v mt m
hc trn lp biu m rut nh mt t bo nhung mao ca biu m rut v teo nhung
mao mt phn. Receptor ca STb cha c bit r mc d gn y ngi ta cho rng
c t c th kt hp khng c hiu vi mng t bo cht trc khi vo trong t bo.
Khng to ra s tit Cl- nh STa, STb kch thch t bo rut tit bicarbonat (HCO 3-)
STb khng lm tng cAMP hay cGMP ni bo mc d n kch thch tng lng calci
ni bo t ngoi bo. STb cng kch thch phng thch PGE2 v serotonin, t ngi
ta cho rng ENS c th cng c lin quan n p ng tit gy ra bi c t ny
(Hitotsubashi, 1992).
- Yu t nh v (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.
d) Nhm EAEC
SVTH: Nguyn Hu Lim

- 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. 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.
e) Nhm EIEC
- 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.
2.1.1.5 Kh nng gy bnh
- E.Coli l nguyn nhn gy ra cc bnh nh tiu chy, vim ng tit
niu, vim ng mt. L mt trong nhng nguyn nhn chnh gy ra bnh nhim
khun huyt, l cn nguyn thng gp trong bnh vim mng no, vim phi tr
mi sinh. E.Coli cn gp trong nhim trng ngoi khoa, nhim trng trong bng.
- Trong cc loi c t ca E.Coli, c t Shiga l nguy him nht c
bit n trn ngi, lm hy hoi cc vi nhung mao hp thu ca t bo biu m rut.
N xm nhp vo t bo biu m i trng, c ch qu trnh tng hp protein lm cht
t bo. Hu qu l gy vim i trng xut huyt, gy tiu chy phn nh mu. Nhng
trng hp hai t nng c th gy thng rut.
2.1.1.6
Cc thc phm lin quan

SVTH: Nguyn Hu Lim

- Cc trung tm kim sot dch bnh (CDC) tnh rng c 85% ngun gc
E.Coli c ngun gc l thc phm. Trn thc t, bt k s tiu th thc phm nhim
phn (c bit l phn gia sc) u c th dn n vic nhim bnh. Cc thc phm
c xc nh l ngun gy bnh bao gm tht b xay, tht nai, xc xch sy kh
(khng nu chn), sa cha tit trng, ph mai, nc tri cy cha tit trng, c linh
lng, mm c ci, rau v nc.
2.1.1.7 Bin php phng nga v kim sot
- Hin nay cha c bin php phng nga c hiu. phng nhim
khn ng tiu ha do E.Coli, thc hin cc bin php phng nga khng c hiu
ging cc loi vi khun ng rut khc.
- phng nhim khun ng tit niu do E.Coli : thc hin v sinh
cng hu mn v b phn sinh dc ngoi. iu tr loi tr cc yu t nguy c khc v
E.Coli l vi khun gy bnh c hi quan trng.
2.1.2 Listeria monocytogenes
2.1.2.1
Gii thiu
- Nm 1924, Muray v cng s trong qu trnh nghin cu mt dch
bnh trn cc ng vt c th nghim, phn lp c mt chng vi sinh vt t cc
hch lympho ca cc ng vt b bnh. Sau ng ly chng vi khun ny tim cho
cc con vt khe mnh th thy c hin tng tng bch cu n nhn (monocytosis),
v vy ng tn cho n l Bacillus monocytogenes. Ba nm sau, ti Nam Phi, Pirie,
khi ang nghin cu s mang Yersinina pestis trn chut, phn lp c t gan
chut mt chng vi khun v t tn cho n l Listeria hepatolitica. Loi vi khun ny
hon ton khng th phn bit c vi chng vi khun ca Muray. Chnh v vy, Pirie
xut mt tn mi cho cc chng vi khun ny, l Listeria monocytogenes.
2.1.2.2
Phn loi
Gii: Bacteria
Ngnh : Firmicutes
Lp: Bacilli
B: Bacillales
H: Listeriaceae
Chi: Listeria
Loi : Listeria monocytogenes
2.1.2.3
c im
- Cc loi Listeria l trc khun Gram dng, c kch thc ngn (0,4
0,5 0,5 2,0 m), chng mc trn cc mi trng nui cy khng acid, khng sinh
SVTH: Nguyn Hu Lim

nha bo. 20oC chng di chuyn bng lng mc xung quanh thn (peritrichous
flagella), nhng s di ng khng quan st c 37 oC. Chng l vi khun k kh
khng bt buc v c th sinh trng trong mt khong nhit dao ng rng t 3
45oC (ti u l 30 36oC), mc d tc mc nhit thp l rt chm. Chng cng
mc trn mt khong pH dao ng rt rng, mt s chng c th mc pH 9,6, nhng
t iu kin ti u pH hi kim hoc mi trng trung tnh.
Hnh 2.2:
Vi
khun Listeria
monocytomonogenes
2.1.2.4
Yu t c lc
- Listeriolysin O (LLO) l yu t c lc chnh ca Listeria
monocytigenes cho php n thot khi s thc bo. LLO c khi lng phn t 58,6
kDa, hnh thnh bi mt chui polypeptide n gm 529 amino acids. Trnh t u N
i din cho c im ca trnh t du hiu ca vi khun Gram dng in hnh: phn
u tin 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
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 bi hu ht cc vi khun Gram dng. Hot tnh ly gii ca cc c t ny bi
gim bt mt s tc nhn 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 trong nhng mng
cha cholesterol. C ch hot ng ca chng da vo s tng tc vi cholesterol c
SVTH: Nguyn Hu Lim
10

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.
- 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 gc 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 LM. Sau 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. Trong 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. LM 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.
2.1.2.5
Kh nng gy bnh
- Ngoi nhng trng hp rt him gp nhim trc tip qua ng da
hoc ng kt mc, hu ht ngi trng thnh vi khun xm nhp qua ng tiu
ha. Vi thai nhi, nhim Listeria hu ht xy ra qua ng mu v mt s t trng
hp xy ra qua ng sinh . Cc trng hp khi nhim Listeria mun tr mi
sinh l do qu trnh ly nhim trc tip hoc gin tip t ngi sang ngi.
2.1.2.6
Cc thc phm lin quan
- Ngun ly nhim Listeria ch yu l t gia cm, nhng kt qu theo
di gn y cho thy, hn 62% mu tht g sng c kh nng b nhim khun. T l
nhim L.monocytogenes tht chn t hn tht sng nhng cng c kh nng gy ra
bnh Listeriosis trn ngi.

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- Listeria monocytogenes cng n t cc ngun khc nh pat, rau (k
c loi salad ng sn), hi sn c bit l tht cua v c hun khi, trong cc sn phm
ng lnh.
2.1.2.7
Bin php ngn nga v kim sot
- Lm sch b mt tip xc vi thc phm, c th dng cn hoc kt hp
vi amoni 4 tng hiu sut kh trng. Thc phm lu tr cn c gi lnh di
4oC m bo an ton.
- Thc hin cc bin php v sinh an ton v sinh thc phm cho cng
ng. Kim tra cc c s git m cung cp tht cho th trng. i vi c nhn, thc
hin v sinh n ung, n chn, ung si, ra tay trc khi n, dit khun, dit rui
- Pht hin ngi mang vi khun, c bit l nhng ngi c lin quan
trc tip n n ung ca tp th nh cp dng ca cc c quan, ngi ch bin thc
n, ngi phc v cc nh hng n ung.
- Chun on sm v cch ly bnh nhn kp thi, x l cht thi ca
bnh nhn. i vi sc vt b bnh cn cha trit hoc tiu hy.
2.2 Tng quan v Salmonella sp.
2.2.1 Lch s pht hin
- Nm 1885 Slamon v Smith (M) tm c Salmonella t ln mc
bnh dch t v gi tn l Bacilus cholerasuis, hin nay gi l Salmonella. Nhng sau
Schweinittz v Dorset 1903 chng minh bnh dch t l do mt loi vi rt gy
nn v xc nh S.choleraesuis l vi khun gy bnh ph thng hn.
- Nm 1888 A.Gartner phn lp c mm bnh t tht b v lch ngi
bnh, ng gi vi khun ny l Bacillus enteritidis v ngy nay vi khun ny c gi
l S.enteritidis. Vi khun ny cng c gi bng nhiu tn khc nh: Bacterium
enteritidis, Bacillus gartner
- Nm 1889 Klein phn lp c S.gallinarum v Rettger cng phn lp
c S.pullorum nm 1909. Trc y ngi ta cho rng y l hai loi vi khun gy ra
hai bnh khc nhau ln gi chung l bnh ph thng hn g (Typhus avium) v cn
bnh c tn chung l S.gallinarum pullorum.
- Nm1896 C.Archard v Rbensauded phn lp c vi khun
S.paratyphi equi v S.paratyphi bacilus. Ngy nay vi khun ny c gi tn l
S.paratyphi B v n nm 1898, S.paratyphi A tm c do N.Guyn v H Keyser.
2.2.2 Phn loi
- V phn loi khoa hc Salmonella c xp vo:
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Gii : Bacteria
Ngnh: Proteobacteria
Lp: Gramma Proteobacteria
B: Enterobacteriales
H: Enterobacteriaceae
Ging: Salmonella lignieres 1900
- Lc u, cc loi Salmonella c t tn theo hi chng lm sng ca
chng nh S.typhi hay S.paratyphi A, B, C (typhoid = bnh thng hn, para = ph),
hoc theo vt ch nh S.typhimurium gy bnh chut (murine = chut), v sau ngi
ta thy rng 1 loi Salmonella c th gy ra nhiu hi chng v c th phn lp c
nhiu loi khc nhau. V nhng l do m cc chng Salmonella mi pht hin
c t tn theo ni m n c phn lp nh S.teheran, S.congo, S.london.
- Salmonella tng c chia thnh cc chi ph v nhiu loi, mi loi
li c kh nng c chi ph. V d nh loi Salmonella enterica c chia thnh 6 loi
ph gm S.enterica, S.salamae, S.arizonae, S.diarizonae, S.houtenae v S.indica.
- Bng cc k thut sinh hc phn t hin i, nhng nghin cu sau ny
cho php xp tt c cc loi Salmonella vo 1 loi duy nht. Mc d kin ny
c nu ra nhng cch truyn thng c s dng qu quen v c ngha ring
nn n khng c chp nhn.
- Da vo cu trc khng nguyn, ch yu l khng nguyn thn O v
khng nguyn lng H, Salmonella c chia thnh cc nhm v cc type huyt thanh.
Hin nay c xc nh gm trn 2500 type huyt thanh Salmonella.
2.2.3 c im
2.2.3.1 c im chung v c im nui cy
- Salmonella l trc khun Gram m, kch thc trung bnh t 2 3 x 0,5
1 m, di chuyn bng tin mao tr S. gallimarum v S. pullorum, khng to bo t,
chng pht trin tt nhi t 60C 420C, thch hp nht 350C 370C, pH t 6 9
v thch hp nht pH = 7,2. nhi t t 180C 400C vi khun c th sng n 15
ngy.
- Salmonella l vi khun k kh ty nghi. pht trin c trn cc mi
trng nui cy thng thng. Trn mi trng thch hp, vi khun s pht trin sau
24 gi. C th mc trn nhng mi trng c cht c ch chn lc nh DCA
(deoxycholate citrate agar) v XLD (xylose lysine deoxycholate), trong mi trng
XLD t cht c ch hn nn thng c dng phn lp Salmonella. Khn lc c
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trng ca Salmonella trn mi trng ny l trn, li, trong sut, c tm en, i khi
tm en ln bao trm khn lc,mi trng xung quanh chuyn sang mu .
Hnh 2.3 : Vi
khn
2.2.3.2
Salmonella
Tnh cht
ha
sinh
Salmonella khng
ln men lactose, ln
men
ng
glucose v sinh hi. Thng khng ln men sucrose, salicin v inositol, s dng c
citrate mn trng Simmons.
- Tuy nhin khng phi loi Salmonella no cng c nhng tnh cht
trn, cc ngoi l c xc nh l S.typhi ln men ng glucose khng sinh hi,
khng s dng citrate trong mi trng Simmon, hu ht cc chng S.paratyphi v
S.Cholerasuis khng sinh H2S, khong 5% cc chng Salmonella sinh c t sinh
bacteriocin chng li E.Coli, Shigella v ngay c 1 s chng Salmonella khc.
2.2.4 Cu trc ca Salmonella
- Salmonella c ba loi khng nguyn, l nhng cht khi xut hin
trong c th th to ra kch thch p ng min dch v kt hp c hiu vi nhng sn
phm ca s kch thch , gm: khng nguyn thn O, khng nguyn lng H v
khng nguyn v K. Vi khun thng hn (S.typhi) c khng nguyn V (Virulence) l
yu t chng thc bo gip cho vi khun thng hn pht trin bn trong t bo bch
cu.
- Khng nguyn vch t bo (khng nguyn thn O)
+ Thnh phn c bn l vch t bo c cu trc phc t p gm 2
lp. Trong cng l mt lp peptidoglycan mng, cch mt lp khng gian chu cht v
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ti lp mng ngoi (outer membrane) l phc hp lipidpolysaccharide gm lipoprotein
v lipopolysaccharide.
+ Bao bn ngoi lp peptidoglycan l lp phospholipid A v B
(quyt nh c t ca Ni c t), sau l hai lp polysaccharide khng mang tnh
c hiu. Khng nguyn ca ni c t c bn cht ha hc l lypopolysaccharide
(LPS). Tnh c hiu ca khng nguyn O v LPS l mt, nhng tnh min dch th
khc nhau : khng nguyn O ngoi LPS cn bao gm c lp peptidoglycan nn tnh
sinh min dch ca n mnh hn LPS. Mng ngoi c cu trc gn ging t bo cht
nhng phospholipid hu nh ch gp lp trong, cn lp ngoi l
lipopolysaccharide dy khong 8- 10 nm gm 3 thnh phn : Lipid A, polysaccharide
li, khng nguyn O. Mng ngoi cn c thm cc protein: Protein c cht: porin vi
khun cn gi l protein l xuyn mng vi chc nng cho php mt s loi phn t i
qua chng nh dipeptide, disaccharide, cc ion v c. Protein mng ngoi: chc nng
vn chuyn mt s phn t ring bit v a qua mng ngoi. Lipoprotein: ng vai
tr lin kt lp peptidoglycan bn trong vi lp mng ngoi
- Khng nguyn lng (khng nguyn H)
+Khng nguyn H: Ch c cc Salmonella c lng. Hu ht
Salmonella u c lng ch tr S.galilarum, S.pulorum gy bnh cho gia cm. Khng
nguyn H l mt loi khng nguyn c bn cht l protit, km bn hn khng nguyn
O. Khng nguyn H rt d b ph hu nhit cao hoc x l bng cn, axit yu.
+ Khng nguyn H chia lm 2 phase :
Phase 1: C tnh cht c hiu gm c 28 loi khng
nguyn lng c biu th bng cha s La tinh thng: a, b, c
Phase 2: Khng c tnh cht c hiu, loi ny c th
ngng kt vi cc loi khc i khi thnh phn ny c th gp E.coli. Pha 2 gm c
6 loi c biu th bng cha s Rp 1-6 hay cha s La Tinh e, n, x
- Khng nguyn v K
+ Khng nguyn K: Khng phc tp, c mt khng nguyn v l
khng nguyn Vi v cng c 2 type huyt thanh S.typhi v S.paratyphi. Khng
nguyn Vi angtigen c Felix v cc cng s pht hin nm 1935. Khng nguyn
Vi gy hin tng ngng kt chm v xut hin cc ht v, khng nguyn Vi l khng
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nguyn v bao bc bn ngoi khng nguyn O, khnh nguyn Vi khng tham gia vo
qu trnh gy bnh.
2.2.5 Yu t c lc
- Vi khun Salmonella c th tit ra 2 loi c t: Ngoi c t v ni
c t.
+ Ni c t ca Salmonella rt mnh gm 2 loi: Gy xung
huyt v mn lot, c t rut gy c thn kinh, hn m, co git.
+ Ngoi c t ch pht hin khi ly vi khun c c tnh cao cho
vo ti colodion ri t vo bng chut lang nui, sau 4 ngy ly ra, ri li cy
truyn nh vy t 5 n 10 ln, sau cng em lc, nc lc c kh nng gy bnh cho
ng vt th nghim. Ngoi c t ch hnh thnh trong iu kin invivo v nui cy k
kh. Ngoi c t tc ng vo thn kinh v rut.
2.2.5.1
Ni c t - Endotoxin
- Mng ngoi t bo vi khun gram m ni chung v vi khun

Salmonella ni ring, c cu to bi thnh phn c bn l lipopolysaccharide (LPS).
LPS c cu to phn t ln, gm 3 vng ring bit vi c tnh v chc nng ring
bit: Vng a nc, vng li v vng lipit A.
- Vng a nc bao gm mt chui polysaccharide cha cc n v cu
trc khng nguyn O. Vng li c bn cht l acid heterooligosaccharide, trung tm
ni khng nguyn O vi vng lipit A. Vng lipit A m nhn chc nng ni c t ca
vi khun. Cu trc ni c t gn ging cu trc ca khng nguyn O. Cu trc ni
c t bin i s dn n s thay i c lc ca Salmonella.
- Ni c t thng l lipopolysaccharide (LPS) c phng ra t vch
t bo vi khun khi b dung gii. Trc khi th hin c tnh ca mnh, LPS cn phi
lin kt vi cc yu t lin kt t bo hoc cc receptor b mt cc t bo nh: T bo
lm ba cu B, lm ba cu T, t bo i thc bo, tiu thc bo, t bo gan, lch.Rt
nhiu cc c quan trong c th chu s tc ng ca ni c t LPS: Gan, thn, c, h
tim mch, h tiu ho, h thng min dch; vi cc biu hin bnh l: Tc mch mu,
gim trng lc c thiu oxy m bo, toan huyt, ri lon tiu ho, mt tnh thm n
- Ni c t tc ng trc tip ln h thng min dch ca c th vt ch,
kch thch hnh thnh khng th.
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- LPS tc ng ln cc t bo tiu cu, gy st ni c t, theo c ch :
+ Gii phng cc cht hot ng mnh nh: Histamin.
+ Ngng kt cc tiu cu ng mch.
+ ng vn, tc mch qun.
- LPS tc ng ln qu trnh trao i gluxit: LPS lm tng cng hot
lc ca cc men phn gii glucoz, cc men phn gii glycogen, lm gim hot lc cc
men tham gia qu trnh tng hp glycogen
2.2.5.2
c t ng rut
- V c ch min dch v di truyn cc Enterotoxin ca Salmonella c

quan h gn gi vi Choleratoxin, nn c gi l Choleratoxin like enterotoxin (CT).
Cn v c tnh sinh hc Enterotoxin ca Salmonella khng ch vi ging CT m cn
ging vi Enterotoxin ca E.Coli.
- c t ng rut ca vi khun Salmonella c hai thnh phn chnh:
c t thm xut nhanh Rapid permeability facto (RPF) v c t thm xut chm
Delayed permeability facto (DPF).
- RPF gip Salmonella xm nhp vo t bo biu m ca rut, n thc
hin kh nng thm xut sau 1-2 gi v ko di 48 gi v lm trng cc t bo CHO
(Chinese Hamster Ovary cell). c t thm xut nhanh c cu trc, thnh phn ging
vi c t chu nhit ca E.Coli, c gi l c t chu nhit ca Salmonella. ST c
kh nng chu c nhit 1000C trong 4 gi, bn vng nhit thp, c th bo
qun nhit -20oC. Cu trc phn t gm mt chui polysaccharide v mt chui
polypeptide.
- RPF kch thch co bp nhu ng rut, lm tng s thm thu thnh
mch, ph hu t chc t bo biu m rut, gip vi khun Salmonella xm nhp vo t
bo v pht trin tng nhanh v s lng. Vi khun tch cc tng cng sn sinh c t
lm ri lon cn bng trao i mui, nc v cht in gii. Qu trnh bnh l ng
rut v hi chng tiu chy cng thm phc tp v nghim trng.
- DPF ca Salmonella c cu trc, thnh phn ging c t khng chu
nhit ca vi khun E.Coli, nn c gi l c t khng chu nhit ca Salmonella. N
thc hin chc nng phn ng chm t 18-24 gi. LT b ph hu 70 0C trong vng 30
pht v 560C trong vng 4 gi. LT c cu trc gm 3 chui polypeptid.
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- DPF lm thay i qu trnh trao i nc v cht in gii, dn n
tng cng bi xut nc v cht in gii t m bo vo lng rut, cn tr s hp thu,
gy thoi ho lp t bo villi ca thnh rut, gy tiu chy.
2.2.5.3
c t t bo
- Khi c th ngi v ng vt b tiu chy th km theo hin tng mt

nc v mt cht in gii l hin tng hng lot cc t bo biu m rut b ph hu
hoc b tn thng cc mc khc nhau. S ph hu hay tn thng l do c t
t bo ca Salmonella gy nn, theo c ch chung l: c ch tng hp protein ca t
bo Eukaryotic v lm trng t bo CHO.
- t nht 3 dng c t ca t bo:
+ Dng th nht: Khng bn vng vi nhit v mn cm vi
trypsin. Dng ny c pht hin rt nhiu serovar Salmonella; c trng lng phn
t trong khong t 56 n 78 kDa; khng b trung ho bi khng th khng c t
Shigella toxin hoc Shigella - like. c t dng ny tc ng theo c ch l c ch
tng hp protein ca t bo Hela v lm teo t bo.
+ Dng th hai: C ngun gc t protein mng ngoi t bo vi
khun c cu trc v chc nng gn ging cc dng c t t bo do Shigella v cc
chng (ETEC) sn sinh ra. Dng c t ny cng ph bin hu ht cc serovar
Salmonella gy bnh.
+ Dng th ba: C trng lng phn t khong 62 kDa; c lin h
vi c t Hemolysin. Hemolysin lin h vi cc c t t bo c s khc bit vi cc
Hemolysin khc v trng lng phn t v phng thc tc ng ln t bo theo c
ch dung gii cc khng bo ni bo.
2.2.6 C ch gy bnh
- Tt c cc kiu huyt thanh Salmonella u mang cm gen inv
(invasion) gip cho qu trnh xm nhim vo trong thnh rut ca ngi v ng vt,
m u ca tin trnh gy bnh. Cm gen ny nm trong h thng gen SPI - 1
(Salmonella pathogenicity island) c mt trong tt c cc Salmonella, t nhm tin ho
thp nht l S.bongori n nhm tin ho cao nht l S.enterica.
2.2.6.1
C ch gy bnh thng hn
- Bnh thng hn do S.typhi v S.paratyphi A, B, C gy ra. Cc yu t
c lc chnh ca vi khun thng hn l kh nng bm v xm nhp vo t bo ch,
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kh nng nhn ln trong i thc bo v ni c t. Khng nguyn Vi c mt S.typhi
v S.pararatyphi C l yu t c lc quan trng, nhng chng vi khun gy bnh
thng hn khng c khng nguyn Vi th s lng cn thit gy bnh cao hn rt
nhiu so vi nhng chng c khng nguyn ny.
- Vi khun xm nhp vo c th qua ng tiu ha do thc n hay nc
ung b nhim bn, s lng cn thit gy bnh vo khong 105 n 107
- u tin, vi khun thng hn phi vt qua mi trng axit ca d
dy, mc d chng c kh nng khng vi axit nh c gen art (acid response
tolerance), nhng ngi bnh thng, vi khun thng hn khng th tn ti lu,
nui cy dch d dy m tnh sau 30 pht. Sau khi vt qua c ro cn d dy, vi
khun di chuyn xung rut non ri nhn ln , nhng trong tun u s c 1 t vi
khun o thi theo phn, cy phn dng tnh trong 5 ngy khng c ngha l bnh
thng hn s xy ra. T rut non, vi khun thng hn i vo hch mc treo rut nh
t bo M, mt i thc bo ca mng Peyer. Sau theo ng bch huyt v mu gy
nhim trng ton thn. Sau khong mt tun, nhim khun huyt th pht xut hin. Vi
khun theo gan qua ng mt li tip tc xm nhp vo rut non, tip tc nhn ln
cc mng Peyer. T mu, vi khun ti lch v cc c quan khc. Trong rut non, vi
khun cht v gii phng ra ni c t. Ni c t kch thch dy thn knh giao cm
rut gy ra hoi t chy mu v c th gy thng rut, v tr gy tn thng thng
nm cc mng Peyer. y l bin chng hay gp c bnh nhn n sm khi cha
bnh phc, nht l n cc thc n cng. Ni c t theo mu ln h thn kinh v kch
thch trung tm thn kinh thc vt no tht ba. Giai on ton pht bnh, bnh nhn
st cao, biu thn nhit tng ln theo thi gian. Thn nhit tng nhng nhit
khng tng gy ra hin tng mch v nhit phn ly.
- Thi k cha c iu tr bng khng sinh, khon mt tun bnh, din
bin in hnh ca bnh thng hn tri qua 4 giai on, mi giai on khong mt
tun, Tun th nht thn nhit tng cao, tun th hai th au bng, gan v lch to dng
thi c s xut hin ca cc m hng trn da, tun th 3 c th xut hin thm cc
bin chng nh xut huyt, thng rut, tun th 4 s xut hin thm cc bin chng
nguy him dn n t vong, nu khng bnh nhn s bnh phc.
- Bnh nhn thng au u, bun nn hoc nn, chng bng, tiu
chy, khon 50% bnh nhn s thy gan v l lch di sn. Nhng trng hp nng
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thng c du hiu ly b, hn m, try tim mch, khng cha tr kp thi c th dn
n t vong. Nhng bin chng khc nh vim phi cp,, vim mng no, vim gan,
vim ty xng cng c th gp.
- Nhng bnh nhn qua khi c khong 5 10% vn tip tc thi vi
khun qua phn trong qu trnh hi phc v 1- 4% tr thnh ngi mang vi khun lu
di do vi khun vn tn ti trong ti mt. Tnh trng ny c th ko di n nhiu nm
v h tr thnh ngun mang bnh rt nguy him.
2.2.6.2
C ch gy nhim khun v nhim c thc n
- Bnh thng xy ra do n phi thc n b nhim Salmonella, nhng
cng c th ly truyn trc tip. Cn nguyn thng do vi khun S.enteritidis v
S.typhimurium.
- Vi khun xm nhp qua rut non nh cc t bo M ca mng Peyer.
Trn b gen ca Salmonella c cc tc nhn gy c ch cc cht khng khun c trong
lysosome, bin i t bo ch m bo cho s tn ti ca vi khun, lm cn tr hot
ng ni bo nh gim lng NADH oxidase rt cn thit cho vic sn xut cc hp
cht khng khun.
- S hy hoi ca i thc bo v cc t bo biu m ln cn, s cht ca
vi khun gii phng ni c t gy nn tn thng cho c th vt ch v gy ra cc
triu chng.
- Thi gian bnh trung bnh t 10 48 gi. Sau thi gian bnh, ngi
nhim thng c biu hin nh st, nn, au bng v tiu chy. Mc st khc nhau
ty vo th trng tng ngi, tiu chy phn thng khng c mu. ngi ln
thng ch dn n tnh trng ri lon ng tiu ha, nhng tr s sinh thng gy
ra nhim trng rt nng, c th dn n tnh trng nhim khun huyt, vim mng no
v vim xng. nhng ngi khe mnh, nhim trng do nhim c thc n thng
t khi sau 2- 5 ngy ri t khi.
2.2.7 Ngun gc ly nhim
- Cc sn phm tht ni chung, nht l tht gia cm v tht ln. Tt c cc
thc n ti sng c ngun gc ng vt u c th l ngun vi khun Salmonella. Vi
khun ny sng t do trong rut ng vt v c trn lng. Gia cm c nhiu
Salmonella nht, tip theo l cc ng vt nui trong nh v ng vt hoang (vt, ra,
ch, ch, chim mng bin, loi gm nhm, rn). Vi khun ny c th c trong thnh
phn dn xut cc cht t ng vt nh gelatin hoc nc bt ng vt, bi cn trng,
loi gm nhm, chim hoc cc sn phm tht nhim khun gy nhim vo thc phm.
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Ngoi ra c th b nhim t ngi khe mnh c mang vi khun ny. Thc phm c
ngun gc thc v t t c nguy c nhim khun. V l do b c ch bi pH < 4 v c
mt vi khun lactic nn cc sn phm ln men t b nhim. Trng v cc sn phm
trng v d nh bt nho, nc st mayonnaise, protit ng t tch t sa, gia cm l
ngun mang nhiu Salmonella, nn trng ca n cng b nhim v vi khun ny c th
xuyn qua v trng v sinh sn trong lng trng.
- Cc sn phm sa nh sa khng thanh trng, phomt t sa ti, kem
cht bo sa, v cc sn phm t sa ni chung c ch bin t cc nng tri, cc
thit b u c th gy nhim vo nguyn liu, to mi trng thun li
choSalmonella, v t gy nhim c cho sn phm sa. Nu tin hnh axit ha
chm th vi khun d dng sinh sn trong phomt nhng n b ph hy vi pH < 4,5.
Nhng sn phm c sa phi c gim st cht ch bi chng khng c thanh
trng na, v vy nu cSalmonella trong sa bt th chng vn c th sinh sn c
bi chng c kh nng tn ti iu kin kh hn v ly nhim sang cc sn phm
khc
2.2.8 Tnh hnh nhim Salmonella trong nc v trn th gii
2.2.8.1 Trn th gii
- T l nhim Salmonella chu u gim u n t nhng nm 1990
tr li y, trong nm 2007 c khong 152.000 ca nhim Salmonella trn ngi c
pht hin, s sai lch ca bo co ny l rt ln, s lng thc t rt c th gp 10 ln
nh th. M, tnh trng c kh hn, n nh mc 15 ca trn 100.000 ngi t nm
2001 do kim sot tt Salmonella trong thc phm t nm 1990, bao gm cc thc
phm sa, trng, nc tri cy, sn phm ti sng, rau, bnh ko, v c bit l tht.
Mt t dch gn y M gy ra bi S.typhimurium nhim trong b u phng
gy nh hng n hn 700 ngi trn khp nc M
- T thng 7/2009 ti nay, s ca nhim khun Salmonella c pht hin
M tng ln 184 ngi, thuc 38 bang khc nhau. C quan y t ca nc ny vn
cha xc nh nguyn nhn chnh xc khin s ca nhim khun Salmonella tng nhanh
nh vy. Tuy nhin, mi y cc chuyn gia y t ca bang Oregon cho rng ngun ly
lan vi khun Salmonella t cc sn phm xc xch. Va qua, cc c quan iu tra M
thu hi hn 560 tn xc xch do nghi b nhim vi khun Salmonella, tt c s lng
ny u do Cng ty Daniele International sn xut.Tuy nhin, ng Jason Maloni, pht
ngn vin ca cng ty ny khng nh: Cha c bng chng no chng minh cc sn
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phm xc xch ca chng ti b nhim vi khun Salmonella. S bng n ca s ca
nhim khun Salmonella khu vc ty bc Thi Bnh Dng khin cc c quan
iu tra nghi ng rng ngun gy bnh cho dch ny l t cc sn phm xc xch sau
khi h pht hin rt nhiu ngi n xc xch mua ti cc ca hng khu vc ny b
nhim khun Salmonella. Cc nhn vin iu tra ti bang Washington cng cho bit 14
bnh nhn b nhim khun Salmonella bang ny, tng n xc xch ca hng
Daniele. Ngoi ra, cc chuyn gia y t khng nh h kim tra v pht hin khun
Salmonella c trong cc mu xc xch ca cng ty ny. Hin ti, cc nhn vin iu tra
lin bang M vn ang lm vic tm ra nguyn nhn chnh xc gy ra dch nhim
khun Salmonella nc ny. V th, h vn cha th kt lun sn phm xc xch ca
Cng ty Daniele c phi l ngun ly bnh chnh hay khng. V th, nhng sn phm
xc xch ca cng ty ny b thu hi l do kim tra b nhim khun Salmonella.
2.2.8.2 Trong nc
- Theo ng Nguyn Cng Khn, Cc trng Cc ATVSTP cho bit, hin
nay, mng li kim nghim ATVSTP c hnh thnh rng khp trong c nc
nhng thc t nng lc kim nghim nhiu ch tiu v an ton thc phm ti cc a
phng vn rt hn ch. Trong 330 mu hoa qu c ti 2,7% s mu c t l tn d
thuc bo v thc vt cao, nht l to , qut v l. c bit, trong s 1.416 mu
tht v sn phm t tht pht hin ti 40,9% s mu nhim khun Salmonella gy ra
cc bnh v ng tiu ha. Hn na, khu vc TP H Ch Minh v ng Nai l nhng
a phng c t l mu thc phm nhim Salmonella cao nht, chim t 84- 95%
mu c gim st.
2.3 Cc phng php pht hin Salmonella
2.3.1 Phng php truyn thng
2.3.1.1 Nguyn tc
- Salmonella c th c pht hin ( phn tch nh tnh ) bng mt quy
trnh bao gm 4 bc l tng sinh, tng sinh chn lc, phn lp v khng nh.
Salmonella thng c mt trong mu vi mt s lng nh, b tn thng v cng
hin din chung vi mt s lng ln vi sinh vt thuc h Enterobacteriaceae c tnh
cnh tranh mnh v c ch s tng trng ca Salmonella.
+ Bc tng sinh : ty theo c tnh thnh phn ha hc ca mu
cn chn quy trnh tng sinh ph hp. Thng thng t l gia mu v mi trng
tng sinh l 1 : 9, tuy nhin ty trng hp m t l ny c th thay i
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+ Bc tng sinh chn lc : Cc mi trng tng sinh chn lc
thng dng pht hin Salmonella trong cc mu thc phm l Rappaport
Vassiliadis (RV), Selenite Cystein Broth,Tetrathionate Mueler Kauffmanm Broth (TT )
Cc nghin cu gn y cho thy mi trng RV c th thay th cho cc mi
trng khc phn tch nhiu mu khc nhau. Tuy vy, mi trng TT thng c
dng phn tch cc mu tht ti sng, cc mu c mt nhim cao, cc mu thc
n gia sc Hin nay ngi ta khuyn khch l nn dng t nht hai loi mi trng
tng sinh chn lc pht hin tt c cc serotype Salmonella nu c hin din trong
mu.
+ Bc phn lp : nhm tch v nhn dng Salmonella khi cc
qun th vi sinh vt khc trong mu. Nhiu loi mi trng rn khc nhau c s
dng phn lp Salmonella, mi mi trng gip nhn dng cc ging ca nhm ny
da trn mt vi c tnh. Hin nay, cc tiu chun v sinh an ton thc phm khuyn
khch s dng t nht 2 loi mi trng phn lp khc nhau tng cng kh nng
pht hin tt c cc dng Salmonella, c bit l mi trng XLD c khuyn khch
s dng
+ Bc khng nh : nhm xc nh li cc khun lc c trng
ca Salmonella xut hin trn mi trng phn lp. Bc ny da trn cc th nghim
sinh ha v cc th nghim huyt thanh c trng cho Salmonella. Cc th nghim
sinh ha c khuyn khch s dng l KIA/TSI, indol, LDC (Lysine decarboxylase),
ODC ( Ornithine decarboxylase ), urea, manitol, sorbitol, cc th nghim huyt thanh
O v H a gi.
- Mi trng v ha cht
+ Buffered Pepton Water (BPW)
+ Rappaport Vassiliadis Soya Pepton (RV)
+ Malachite Green Magnesium Chloride
+ Tetrethionate Muller Kauffmanm
+ Xylose Lysine Deoxycholate (XLD)
+ Hektoen Entric Agar (HE)
+ Bismuth Sulphite Agar (BS)
+ Brillian Green Phenol Red Lactose Sucrose (BPLS)
+ Triple Sugar Iron (TSI)
+ Urea
+ Lysine Decarboxylase
+ Ornithine Decarboxylase
+ Manitol
+ Sucrose
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+ Sorbitol
+ Tryptone
+ Thuc th Kovacs
+ Khng huyt thanh Salmonella a gi
2.3.1.2 Phng php thc hin
a) Tng sinh
- i vi cc loi mu thng thng, tin hnh cn 25g mu vo ti PE
v trng, b sung 225ml dung dch BPW v ng nht mu trong 15 hoc 30 giy.
370C trong 18 24 gi. i vi mt s loi mu c cha cc cht gy c hoc c ch
s pht trin ca Salmonella cn tin hnh quy trnh c bit
+ i vi tht gia cm ti sng : t gia cm vo mt ti PE ln,
cho them 1 lt BPW, lc bng my lc 30 giy mi trng thm vo ton b mu
+ i vi mu sa kh : Cn 25g mu vo ti PE v trng, cho
thm 225ml BPW, yn nhit phng khong 60 pht. Sau lc cho tan hon
ton
+ i vi cc mu gia v hay cc loi thc phm cha nhiu gia
v : ng nht mu vi t l 1/100 trong BPW ( thay v 1/10 nh bnh thng ) trc
khi tng sinh. Bin php ny nhm gim nng cc cht c ch s tng trng ca
Salmonella trong bc tng sinh
+ i vi cc loi mu nh casein, phomat, b v cc sn phm
tng t khc : thc hin dng nht mu trong mi trng tng sinh c lm m
n 400C
+ i vi cc sn phm cha coca: ng nht mu trong mi
trng Skim Milk Broth c lm m n 400C
+ i vi da, cc sn phm t da v cc mu c hm lng
cht bo cao: ng nht mu trong mi trng BPW, sau cho thm 2 3 git Triton
X 100 trc khi tng sinh
b) Tng sinh chn lc
+ Lc trn u dch tng sinh, sau chuyn 0,1ml sang 10ml
mi trng tng sinh chn lc RV c m n 42 0C. 420C trong 18 24 gi,
khi cn thit c th ko di thm 24 gi
+ Mt s mi trng tng sinh chn lc khc cng c s dng,
mi loi mi trng ch c tc dng chn lc da trn mt c im pht trin ca
Salmonella, mt s dng ch c th tng trng trong mi trng ny m khng th
tng trng trong mi trng khc. Do vy, tng kh nng pht hin tt c cc dng
Salmonella hin din trong thc phm cn phi dng t nht hai loi tng sinh chn lc
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khc nhau cho cng mt mu. Ngoi ra mi mi trng cn c cc nhit
khc nhau
c) Phn lp v nhn din
+ Dng que cy vng thc hin k thut cy phn lp khun lc
n vi ging t dch tng sinh chn lc trn a mi trng phn lp c trng cho
Salmonella nh XLD, HE, BS Cng chn lc v mc phn bit th hin
hnh thi ca khun lc Salmonella trn tng mi trng cng khc nhau. chn lc
v nhn dng tt c cc dng Salmonella cn s dng t nht hai loi mi trng chn
lc phn bit khc nhau cho cng mt mu. Cc biu hin ca Salmonella trn tng
mi trng khc nhau nh sau
+ Mi trng XLD: khun lc trn, li, trong sut, c hay khng
c tm en, i khi tm en qu ln bao trm khun lc, mi trng xung quanh
chuyn sang mu hng
Hnh 2.4: Khn lc
c trng ca
Salmonella trn mi
trng XLD
+
Mi trng
HE: khun lc c
mu thay i t
xanh dng n xanh lc, c hay khng c tm en, i khi tm n qu ln bao trm
khun lc

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Hnh 2.5: Khun lc c trng ca Salmonella trn mi trng HE
+ Mi trng BS: khun lc c mu nu xm hay mu en, thnh
thong c nh kim tm, mi trng chuyn thnh mu nu v sau chuyn sang mu
en nu ko di thi gian
Hnh
ca
2.6:
Khn lc c trng
Salmonella
d)
trn mi trng BS
Khng nh
+ T mi mi
trng phn lp cy chuyn t nht 5 khun lc c trng qua mi trng khng chn
lc nh TSA. 370C trong 18 24 gi. Cc khun lc c trng trn mi trng
ny c s dng cho cc test sinh ha v huyt thanh
+ Cc th nghim sinh ha chnh cn thc hin cho Salmonella
l: ln men glucose, urea, manitol, indol, H2S, VP, ODC, LDC, saccharose, sorbitol.
Cc chng Salmonella cho kt qu th nghim nh sau
+ Th nghim H2S (KIA hay TSI): Salmonella ch ln men c
ng glucose trong mi trng, v vy phn nghing ca mi trng c mu ,
phn su c mu vng. a s cc chng Salmonella u c kh nng sinh H 2S nn c
cc vch mu en trong mi trng. C th quan st thy hin tng sinh hi qua hin
tng lm v thch hoc mi trng b y ln li mt khong h di y ng
nghim
+ Th nghim LDC (+): sau khi nui cy, mi trng b kim
ha, mu khng i ( mu tm hay xanh )
+ Th nghim urea (-): Salmonella khng phn gii urea nn
khng lm thay i pH ca mi trng, sau khi nui cy mi trng vn gi nguyn
mu
+ Ln men manitol, sorbitol (+): mi trng sau khi nui cy b
acid ha chuyn sang mu vng.
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+ Th nghim indol v VP (-)
Th nghim
H2S
LDC
Urea
Manitol,sorbitol
Indol
VP
Mi trng
TSI/KIA
Biu hin c trng

Phn nghing , su vng, nt thch v xut
LDC
Urea
Manitol, sorbitol
Trypton
hin vch en
Khng i mu
Khng mu
B acid ha,mi trng chuyn sang mu vng
Khng xut hin vng khi nh thuc th
VP
Kovacs
Khng i mu khi nh napthol 5% v
KOH 40%
Bng 2.1: Biu hin c trng ca Salmonella trong test sinh ha
- Cc th nghim khng nguyn cn c th nghim song song vi
mu i chng bng nc mui sinh l trnh hin tng ngng kt gi. Hai loi
huyt thanh a gi cn dng l Salmonella Polyvalent O v Salmonella Polyvalent H.
Phn ng (+) khi chng th nghim ngng kt khng huyt thanh nhng khng ngng
kt vi nc mui sinh l
2.3.2 Phng php hin i
- Phng php truyn thng mt kh nhiu thi gian (5 ngy) cho ra
kt qu, do ngi ta pht trin rt nhiu phng php th nhanh pht hin
Salmonella trong cc mu thc phm v mu nc trong thi gian ngn, t c bin
php x l kp thi i vi cc mu nhim. Xu hng s dng PCR v IMS thng
c s d ng. Phng php PCR gip pht hin nhanh; tuy nhin d xy ra hin
tng m tnh gi, i hi cc thao tc phi thnh tho. Multiplex PCR gip tit kim
thi gian, s lng mu v chi ph; tuy nhin li c nhy km hn. Ngi ta pht
trin thm phng php Real Time PCR t PCR truyn thng. Phng php ny c
nhy rt cao, cho kt qu sm hn, khng cn phi chy in di sau phn ng khuch
i. Phng php min dch t phn tch l mt la chn tt hn. N c nhy cao
(< 104 CFU/ml), thi gian ngn (th nghim c thc hin trong 4-5 gi).
- Bn cnh nhng k thut Sinh hc phn t c bn pht hin v nh
danh Salmonella, cn c nhng phng php khc, nh: phng php min dch
(huyt thanh hc, k thut ELISA, Western blot). V i khi tng hiu qu, tin
cy cho xt nghim ngi ta c n kt hp nhiu phng php vi nhau, v d: IMS-PCR

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assay (Immunomagnetic Separation and Polymerase Chain Reaction) pht hin
Salmonella trong thc phm.
2.3.2.1
Phng php PCR
- Nguyn tc : Phng php nh tnh ny da vo vic xc nh on
ADN ch c c khuch i hay khng. Qu trnh xc nh c thc hin bng
cch in di sn phm khuch i trn gel agarose, nhum mu ADN bng etyl
bromua v quan st bng n UV c bc sng l 302 mm. Nu trong mu c gen
ch, trn bng gel in di s xut hin sn phm khuch i c kch thc ph hp
vi di ca on ADN ch nh. Nu trong mu khng c gen ch, trn gel
din di khng xut hin sn phm khuch i hay sn phm khuch i c kch thc
khng ph hp vi on ADN ch.
- Thit b, dng c, ho cht v mi trng
+ Thit b, dng c
+ T m 37oC.
+ My lun nhit.
+ My ly tm dng cho ng eppendorf 1,5 ml.
+ My lc ng nghim.
+ Thit b in di ngang v b ngun in di c in th hot
ng t 80 150 volt.
+ Hp n soi UV c knh lc 302mm.
+ B chp nh trn n UV.
+ ng Eppendorf 0,2. 0,5 ml; 1,5 ml chuyn dng cho PCR.
+ Ho cht, mi trng
- Mi gm hai mi invA1 v invA2 c thit lp cch nhau 520 bp trong
gen invA c vai tr trong qu trnh xm nhim Salmonella vo thnh rut ng vt v
ngi. Trnh t ca hai mi nh sau :
+ invA1 : 5' - TTGTTACGGCTATTTTGACCA -3'
+ invA2 : 5' - CTGACTGCTACCTTGGCTGATG - 3'
Nng mi s dng trong phn tch c pha long thnh 6 pM trong m TE. m
TE c thnh phn nh sau : 10 mM Tris-HCl (pH = 8), 1 mM EDTA.
- Mi trng nui cy vi sinh vt v cc loi ho cht khc : Khuyn
khch s dng mi trng nui cy dng bt kh v cc vt liu dng cho phn ng
khuch i PCR c tng hp thnh kit ang c lu hnh trn th trng c thnh
phn ph hp nh. Qu trnh pha ch, bo qun, s dng phi tun th theo hng dn
ca nh sn xut. Trong trng hp s dng cc vt liu ho cht ring l, tinh
khit ca cc thnh phn v nc pha ch phi m bo cht lng dng cho vi sinh
v sinh hc phn t.
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- Thang DNA :Nn s dng thang o ph hp c th c lng c
on khuch i 520 bp. C th s dng sn phm khuch i c kch thc 520 bp
bit trc lm thang o trong phng php ny.
- Agarose : S dng trong k thut ny l loi dng in di ADN c
kch thc nh hn 1000 bp.
- Phng php tin hnh
+ Ly mu : Cn chnh xc 25 g mu (hoc mt khi lng chnh
xc tu theo yu cu) ri cho vo bnh tam gic hoc bao PE v trng.
+ Tng sinh : Nhm lm tng s lng Salmonella trong mu, cc
t bo b suy yu hay tn thng cng c phc hi v pht trin. Giai on ny
c tin hnh trong mi trng khng chn lc v trn nguyn tc c mt phn khi
lng mu s b sung 9 phn khi lng mi trng tng sinh. Nu ly 25 g mu, phi
b sung 225 g mi trng tng sinh dung dch pepton m. mu c mi trng tng
sinh nhit 37,0oC 1,0oC trong khong 18 gi 2 gi.
+ X l mu gii phng DNA : Giai on ny nhm thu nhn sinh
khi sau khi tng sinh, ra sch mi trng sau khi nui cy, ph v t bo gii
phng DNA. Cch x l nh sau:
Lc u canh khun tng sinh. Ht 0,5 ml vo trong ng
eppendorf c th tch 1,5 ml, ly tm vi tc 10 000 vng/pht trong 5 pht ri loi
b phn mi trng lng bn trn. Ra sinh khi bn di vi nc ct v trng ri
tip tc ly tm vi ch nh trn loi b phn nc.
Huyn ph sinh khi trong ng vi 0,5 ml nc ct v
trng. un si cch thu trong 10 pht. Ly tm huyn dch sau khi un vi tc 10
000 vng/pht trong 5 pht lng cc mnh v t bo xung y. Phn dch trong
bn trn c coi l khun DNA tin hnh phn ng khuch i.
- Khuch i
+ Giai on ny nhm lm tng s lng bn sao on DNA ch
trn my lun nhit (thermocycler) bng hai mi c trng. Qu trnh khuch i c
tin hnh trong khong 30 chu k.
+ Chun b ng khuch i
Ht 45 ml hn hp khuch i PCR 1,1 x cho vo trong
ng nghim PCR c th tch 0,2 hoc 0,5 ml, thm vo 1 ml mi mi invA1 v invA2
c nng 6 pM v 3 ml mu khun ADN. Tng th tch trong mt ng khuch i l
50 ml.
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i chng dng: thay dch khun DNA mu bng
dch ADN ca Salmonella chun bit.
i chng m: thay dch khun DNA mu bng nc
ct v trng.
+ Chng trnh khuch i
Cc ng khuch i c t vo trong my lun nhit.
Chng trnh khuch i nh sau: Duy tr nhit 95 oC trong 5 pht lm bin tnh
hon ton cc si DNA trong mu. Tip theo l 35 chu k, mi chu k c 3 bc nh
sau: 95oC/60 giy; 54oC/45 giy v 72oC/60 giy. Sau khi kt thc 35 chu k, mu
c gi nhit 72oC trong 10 pht, sau gi n nh nhit 20 oC cho n
khi in di.
- in di sn phm khuch i
+ Chun b gel in di agarose 1%
Gel agarose 1 % pha trong m TAE 1x c un chy
hon ton v vo khay in di c sn cc lc to ging. Gel in di phi c
dy khong 3 - 4 mm. Gel sau khi chun b c ngm chm hon ton trong m
TAE.
+ Chun b dch in di
Mu sau khi khuch i c nhum vi 10 ml m ti
mu 6x ri trn tht u.
+ in di
Mt ging trong gel in di c s dng cho thang
DNA chun hay mu i chng dng. Np 10 ml dch in di chun b vo trong
gel agarose. Tin hnh in di trong 60 pht hiu in th 100 vn.
- Nhum DNA, quan st sn phm khuch i
+ Chun b dung dch nhum mu
Pha dung dch nhum DNA nh sau: cho 0,2 ml etyl
bromua 10 mg/ml vo 0,5 lt nc, pha vo trong khay cha c ming rng hn bn
gel in di.
+ Nhum gel
Ngm bn gel in di vo dung dch nhum trong 10
pht. Ra gel bng nc trong khong 3 - 5 pht loi b phn etyl bromua d.
+ Quan st, chp hnh
Cho bn gel nhum ln hp n soi UV, ng knh
bo v, bt n ri quan st cc vch sng ca DNA xut hin trn bn gel. Sau ,
chp hnh lu tr kt qu.
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- c v gii thch kt qu
+ Kt qu phn tch ch c xem xt kt lun khi mu i chng
dng tnh c sn phm khuch i DNA vi kch tc 520 bp v mu i chng m
khng c sn phm ny.
+ Mu c kt lun l dng tnh Salmonella khi c sn phm
khuch i 520 bp trn bn gel.
+ Mu c kt lun l m tnh khi khng c sn phm khuch
i, hay sn phm khuch i c kch thc khc hn 520 bp.
2.3.2.2
Phng php ELISA
- ELISA c s dng rng ri di dng cc kit thng mi v c th
ci tin t ng ha. ELISA c th s dng pht hin v nh lng vi sinh thc
phm trong thi gian vi gi sau khi tng sinh. K thut ny c nhy khong
106CFU/ml. tuy nhin vn phi thc hin bc tng sinh v tng sinh chn lc trc
khi thc hin cc phn ng min dch
- Transia Salmonella (Diffchamb AB, Sweden) l 1 b kit pht hin
nhanh Salmonella spp. da trn nguyn tc E sanwich. Khi hn hp tng sinh chn lc
c cho vo ging, nu c s hin din ca vi sinh vt mc tiu, khng nguyn tim
mao ca vi sinh vt s to phc hp vi khng th c nh trn ging v khng th t
do c gn enzyme peroxydase to thnh 1 phc hp kp (sanwhich). Sau cc khng
th c gn enzyme dng t do khng to phc hp sanwhich s b ra khi phn
ng. Phc hp sanwihch s c pht hin nh s b sung c cht ca phn ng (ure
peroxide v teramethylbenzidine). Enzyme peroxide s thy phn c cht to phn ng
mu xanh dng. Phn ng kt thc bng cch bt hot enzyme bng dung dch kt
thc lm acid ha mi trng v lm mi trng chuyn t mu xanh sang mu vng.
S hnh thnh mu vng chng minh s hin din ca khng nguyn hay vi sinh vt
mc tiu v mt mu ca vi sinh vt c th xc nh c bng cch o cng
mu bng my so mu
2.4 Cc bin php kim sot Salmonella trong thc phm
- i vi gia sc v gia cm: Trong chn nui cn ch phng bnh
tt cho chng. Phi kim tra th y khi git sc vt, iu ny cng lm tt th cng t c
c hi bn hoc xut ra cc loi tht nhim Salmonella. Trong khi git tht phi m
bo tnh ring r, trnh s ly lan ca vi khun, ch ti cc loi dng c dng khi
git tht .
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- Trong bo qun thc phm, m bo thi gian ct gi thc phm
ch bin v cc nguyn liu. Ch tht bm, pat... tht nghin m khng p lnh
ngay sau s to iu kin cho ton b khi nguyn liu nhim trng mau chng.
- un si thc phm trc khi n l bin php tt nht. Tht p lnh
thi gian un nu phi ko di hn bnh thng, khi un phi m bo nhit si c
bn trong ming tht (t nht l 5 pht). Thc phm cn tha, thc phm d tr phi
un li trc khi n. Vi trng c th b nhim Salmonella rt sm ngay khi mi
(trng vt, ngan, ngng...), v vy phi ch bin chn hon ton, tuyt i khng n
sng hoc hng o.Bo m v sinh ni n, trnh rui, nhng, chut.
- Thc hin nghim ngt ch khm tuyn trc khi vo v khm nh
k (1 nm/1 ln) i vi ngi tip xc trc tip vi thc phm nht l thc phm
nu chn.
Chng III : NI DUNG V PHNG PHP NGHIN CU

3.1 a im v thi gian
- Thi gian: T ngy 31/05/2010 n ngy 12/06/2010.

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- a im thc hin: Phng th nghim Khoa Mi trng v Cng ngh
Sinh hc, Trng i hc K Thut Cng Ngh Thnh Ph H Ch Minh.
- a ch: 144/24 in Bin Ph, P25, Q. Bnh Thnh, TP. H Ch Minh.
3.2 i tng v phm vi nghin cu
- Nghin cu v pht hin Salmonella trn mt s mu tht g ti sng
trn a bn hai qun Ph Nhun v Bnh Thnh thnh ph H Ch Minh.
3.3 Vt liu th nghim
3.3.1 Cc dng c v ha cht tin hnh th nghim
- Ko ct mu.
- Pipet 1ml, 5ml, 10ml.
- a petri.
- Micropipet 10l-200l.
- Ti PE v trng.
- Que cy thng, que cy vng
- Cn in t.
- ng nghim kh trng.
- T m 37oC, 42oC.
- Ni hp kh trng 121oC.
- n cn.
- Qu bp cao su.
- Chai chu nhit dung tch 500ml, 1000ml.
- Ga ng nghim inox.
- T cy.
- Bng khng thm.
- Que cy.
- Cn 96o, 70o
- Nc ct kh trng
3.3.2 Cc mi trng s dng
- Mi trng BPW (n )
- Mi trng RV (n )
- Mi trng XLD (n )
- Mi trng dng test sinh ha :
+ LDC (n )
+ Manitol (n )
+ Urea (Merck)
+ VP (n )
+ TSI (n )
+ Trypton (n )
3.4 Ni dung thc hin
Mu tht g
Thu mu ti cc a im kho st
SVTH: Nguyn Hu Lim
33
nh gi cc ch tiu
Cm quan
Salmonella
25 g mu + 225 ml BPW,
ng
30 giy
Nhn
xt,nht
Ktmu
luntrong
-1
Hnh 3.1: Ni dungthc
hin
th
nghim
pha long 10
3.5 Phng php nghin cu
3.5.1 Quy trnh phn tch
em 37oC trong 24 gi
Ly 0.1ml canh trng cho vo mi trng RV
42oC 0,5 trong 24 gi
Cy chuyn trn mi trng XLD v nhit 37 oC trong 24

gi
KL c trng ca Salmonella trn mi trng XLD : trn, li,

trong sut, c tm en i khi tm en qu ln bao trm khun
lc, mi trng xung quanh chuyn sang mu
Cy chuyn vo mi trng test sinh ha : mi trng TSI,

Manitol phenol red, Urea, LDC, Trypton, VP
Hnh 3.2: Quy trnh phn tch Salmonella trong th nghim
3.5.2
37oC trong 24 gi
Thuyt minh quy trnh

34
SVTH: Nguyn HuBiu
Limhin c trng ca Salmonella : trn TSI:
/vng/H2S(+)/gas(+)/; Manitol(+); Urea(-); LDC(+); Indol(-);

VP(-)

a) Chun b mu.
- Dng ko v khun ct 25g mu (khng ly m, ly c cht lng nu
c), cho vo bao nilong v khun. Thm vo 225ml mi trng PBW hp kh trng
ngui. Tin hnh ng nht mu c pha long 10 -1. em nhit 370C
trong 24 gi.
b) Cy mu.
- Tng sinh: Cy 0,1ml dch mu c nng 10-1 vo ng nghim c
cha 10ml mi trng tng sinh chn lc RV, 42 0,5 oC trong 24 gi.
- Phn lp: Cy mu t mi trng RV sang mi trng thch a XLD
v nhit 370C trong 24 gi. Nhn dng khun lc c trng ca salmonella trn
mi trng XLD: trn, li, trong sut , c tm en i khi tm en qu ln bao trm c
khun lc, mi trng xung quanh chuyn sang mu (hng).
Hnh 3.3: Khun lc c trng ca Salmonella

- Chn 5 khun lc c trng cy chuyn sang mi trng thch a
TSA, tng sinh khng chn lc v 37 0,5oC trong 24 gi
c) Th nghim khng nh.
- Ly khun lc t mi trng TSA cy sang cc mi trng th nghim
sinh ha. Thc hin nh sau:
Th nghim
trn mi trng TSI:
- Hp kh trng mi trng TSI 1210C trong 15 pht v phn phi mi
trng vo cc ng v trng lm ng thch nghing.
SVTH: Nguyn Hu Lim
35

- Dng que cy vng cy sinh khi t khun lc ca ging thun su vo
y ca ng thch nghing.
- Sau khi cy xong th 370C trong vng 24h 48h.
- Salmonella ch ln men c ng glucose trong mi trng TSI
phn nghing ca mi trng TSI c mu , phn su c mu vng.
- Salmonella c kh nng H2S xut hi n cc v t mu en trong mi
trng TSI.
- C th thy hi n tng sinh hi qua hi n tng lm v thch
mi trng ho c mi trng b y ln trn to ra m t khong khng bn di ng
nghi m.
Negati
ve
Negati
ve
Positi
ve
Hnh 3.4: Th nghim trn mi trng thch TSI

Th nghim
Urea Broth:
- Cy VSV vo mi trng canh urea c cha cht ch th l
bromocresol purple
- 370C trong khong 12h 18h
- Th nghi m (+): mi trng chuyn sang mu vng.
- Th nghi m (-): mi trng khng i mu.

SVTH: Nguyn Hu Lim
36

Salmonella khng phn gii urea nn khng lm thay i pH mi
trng. Sau khi nui cy mi trng vn gi nguyn mu tm.
Negati
ve
Negativ
e
Negati
ve
Hnh 3.5: Th nghim Urea Broth

Ln men mannitol:
- Mi trng s dng l mi trng phenol red broth base c pH 7.4, ch
th mi trng ny l phenol red c mu s chuyn thnh mu vng khi pH < 6.8.
- Tin hnh cy vo cc ng mi trng cc chng VSV cn khng nh.
- 370C trong 18 24h.
- Kh nng ln men ca chng c nh gi da vo s sinh acid v
sinh hi
- Sinh acid (+): mi trng mu cam chuyn sang vng.
- Sinh hi (-): c bt kh trong ng Durnham.
Mi trng sau khi nui cy salmonella b acid ha v chuyn thnh
mu vng.

SVTH: Nguyn Hu Lim
37
Negativ
e
Positiv
e
Negativ
e
Hnh 3.6: Ln men Mannitol

Th nghim
LDC (Lysine Decarboxylase):
- S dng mi trng Falkow.
- Tin hnh cy vo cc ng mi trng cc chng VSV cn khng nh.
- Cht ch th mu trong mi trng l Bromocresol purple.
- Phn ng (+): mi trng gi nguyn mu tm ban u, canh khun
c.
- Phn ng (-): mi trng t tm chuyn sang vng.
Sau khi nui cy salmanella mi trng chuyn thnh kim, mi
trng gi nguyn mu tm ban u.

SVTH: Nguyn Hu Lim
38
Negati
ve
Hnh 3.7: Th nghim LDC
Th nghim
kh nng sinh Indol:
- Cy VSV th nghi m qua mi trng canh trypton khong 24 gi
37oC.
- Nh vi git ether ko indol ln b m t mi trng, thm vi git
thuc th Kovacs
- Quan st sau vi pht.
- Th nghi m (+): trn b m t mi trng xut hi n vng mu cnh
sen.
- Th nghi m (-): khng xut hi n vng .

SVTH: Nguyn Hu Lim
39
Positiv
e
Negativ Negativ
e
e
Hnh 3.8: Th nghim Indol

Th nghim
Voges Proskauer:
- Cy vi sinh vt vo trong mi trng glucose phosphate (MR-VP
Broth)
- nhi t 370C trong vng t 2-5 ngy
- Thm vo canh khun dung dch thuc th anpha- naphtol 5% trong
cn v dung dch KOH 40% hay NaOH 40 %. vi t l 3:1.
- Quan st phn ng xy ra trong 5 pht.
- Th nghi m (+):xut hi n mu hay hng sng trn m t mi trng.
- Th nghi m
(-): mi trng khng i mu.

SVTH: Nguyn Hu Lim
40
Negativ
e
Hnh 3.9: Th nghim Voges Proskauer
3.4.5.
Nhn nh tnh sinh ho c hiu:
Tnh cht sinh ho

Dng hoc m tnh
T l % vi vi khun Salmonella
TSI lactose
99,2
TSI sucrose
99,5
TSI glucose
+
100
TSI glucose sinh hi
+
91,2
TSI hydro-sulfua
+
91,6
Phn gii ur
100,0
Ln men mannitol
+
Phn ng Indol
98,2
Phn ng V.P
100,0
Bng 3.1: Pht hin hay khng pht hin Salmonella trong 25g mu
3.6 Kt qu
3.6.1 Kt qu cm quan
- V mu sc, tht g c mu c trng, phn tht hng, phn m mu
vng ti v da mu vng hoc trng
- V mi, tht c mi c trng, khng c mi l
- V tnh cht, b mt tht c nc, c n hi tt
- V cm quan nhn chung l tt, tht nhn cn ti, tnh cht, mu sc,
mi v khng c g l. Nhng nh gi cm quan khng xem xt mc sch
ca tht
3.6.2 Kt qu nh gi mc nhim Salmonella
- nh gi theo TCVN 4829:2001 (ISO 6579:1993) vi quy inh mc
nhim Salmonella l 0 CFU/25g
SVTH: Nguyn Hu Lim
41
Hnh
3.10 :
T l
nhim
Salmonella trn cc mu kho st

- Theo bng trn, mu 3 v mu 5 nhim Salmonella vi t l 33.33%.
Vic tp nhim Salmonella c th do thao tc ca ngi phn phi tht khng hp v
sinh, khng dng cc dng c bo h. Hoc cng c th tht b tp nhim do khng
kh, v v sinh khu bun bn cng c th l do nhim trung tm git m. Hai mu
nhim c th ni ln tnh trng v sinh an ton thc phm hai khu vc ny l rt
thp, v t l tp nhim trong quy nh ca TCVN l 0 CFU/25g
- Tm li, ba mu 1, 2 v 4 m tnh vi Salmonella c th s dng c,
nhng cng phi m bo v sinh an ton thc phm, v khng ch c Salmonella mi
gy ra nhng ca ng c thc phm nghim trng, khng nhim Salmonella khng c
ngha l khng nhim cc vi sinh vt khc.
- Hai mu 3 v 5 khng th s dng c, c s phn phi cn c bin
php v sinh tt hn, i khu vc ly thc phm khc trnh tnh trng ly nhim
Chng IV: KT LUN V KIN NGH
4.1 Kt lun
- Sau qu trnh nghin cu l thuyt v thc nghim, chng ti nhn thy
rng Salmonella l mt loi vi khun kh nguy him, chng gy ra bnh thng hn,
nhim trng huyt v mt s bnh khc. ng ni l loi ny thng tp nhim vo
thc phm, iu ny v cng nguy him v theo TCVN, mc nhim Salmonella l 0
CFU/25 g.
- Mng li kim tra an ton v sinh thc phm tuy tri rng khp c
nc, nhng nng lc kim dch cha c cao cc a phng , nht l cc thnh
ph ln nh Tp. H Ch Minh
SVTH: Nguyn Hu Lim
42

4.2 Kin ngh
- Trong thc nghim trn, c mt s phn do thi gian qu gp m ti
cha th thc hin c
+ Kho st tnh trng vi sinh ca khu bun bn
+ Kho st qua tnh hnh git m gia cm trn a bn thnh ph
H Ch Minh
- Lp cc trung tm kim dch trn a bn cc qun v yu cu tht trc
khi bn cn phi qua kim dch v s dng cc dng c phn phi hp v sinh
- Nn lp cc a im bn thc phm tp trung d dng kim tra v
sinh khu bun bn. Thng xuyn t chc cc t kim tra khu chn nui v git m
m bo v sinh an ton thc phm
TI LIU THAM KHO

TI LIU TING VIT
1. B mn vi sinh vt trng i hc y H Ni (2007), vi sinh vt y hc, Nxb y hc,
H Ni.
2. B mn vi sinh vt trng i hc y khoa Hu (2004), bi ging vi sinh y hc.
3. B y t (2008), vi sinh vt y hc, Nxb y hc, H Ni.
4. Trn Linh Phc (2009), phng php phn tch vi sinh vt trong nc, thc
phm v m phm, Nxb Gio Dc, H Ni
TI LIU NC NGOI
5. Bibek Ray (2009), FUNDAMENTAL FOOD MICROBIOLOGY, ,Boca Raton
London New York Washington, D.C, USA.
6. Camilla Giammarini and Mauro Magnani, Listeriolysin O from Listeria
monocytogenes, Diatheva, Centre for Biotechnology, University of
Urbino, Italy
7. Cynthia L.Sears and James B.Kaper (1996), Enteric Bacterial Toxins:
Mechanisms of Action and Linkage to Intestinal Secretion, American Society for
Microbiology
8. FAO and WHO (2009), Salmonella and Campylobacter in chicken meat
9. Jame M.Jay (2000), Modern Food Microbiology
SVTH: Nguyn Hu Lim
43

10. WHO, Risk assessments of Salmonella in eggs and broiler chickens

SVTH: Nguyn Hu Lim
44

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Kha lun: Tm hiu v vi khun Salmonella

Chng I: GII THIU

GVHD: KS. Hunh Vn Thnh

Kha lun: Tm hiu v vi khun Salmonella

GVHD: KS. Hunh Vn Thnh

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

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Kha lun: Tm hiu v vi khun Salmonella

Sau khi c t i vo ni bo, chng di chuyn trong t

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Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

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GVHD: KS. Hunh Vn Thnh

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Kha lun: Tm hiu v vi khun Salmonella

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Kha lun: Tm hiu v vi khun Salmonella

- Khi c th ngi v ng vt b tiu chy th km theo hin tng mt

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Hnh 2.4: Khn lc

GVHD: KS. Hunh Vn Thnh

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Biu hin c trng

GVHD: KS. Hunh Vn Thnh

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Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Chng III : NI DUNG V PHNG PHP NGHIN CU

GVHD: KS. Hunh Vn Thnh

Kha lun: Tm hiu v vi khun Salmonella

Kha lun: Tm hiu v vi khun Salmonella

Ly 0.1ml canh trng cho vo mi trng RV

42oC 0,5 trong 24 gi

Cy chuyn trn mi trng XLD v nhit 37 oC trong 24

KL c trng ca Salmonella trn mi trng XLD : trn, li,

Cy chuyn vo mi trng test sinh ha : mi trng TSI,

GVHD: KS. Hunh Vn Thnh

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Kha lun: Tm hiu v vi khun Salmonella

Hnh 3.3: Khun lc c trng ca Salmonella

Kha lun: Tm hiu v vi khun Salmonella