Staphylococcus Aureus

Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm
Chng I: Gii thiu

1.1. t vn Trong x hi hin nay tnh trng ng c thc phm trn th gii v Vit Nam l rt cao. Tnh trng ny cha c du hiu dng li v cng ngy cng tng. Qua kim tra cho thy hu ht cc v ng c thc phm ny l do vi sinh vt gy ra. y l iu c cnh bo v c cch thc phng nga nhng vn xy ra cc v ng c tp th gy nguy him v c th dn n t vong. Hu ht cc v ng c thc phm thng l do Salmonella, E.coli, Staphylococcus aureus v mt s loi khc gy ra. c bit Staphylococcus aureus l 1 trong nhng vi sinh vt gy ng c cao nht. Ngoi ra Clostridium botulium v nm mc cng l nhng loi gy ng c thc phm cho con ngi. Chnh v vy chng ta cn phi tm hiu nhng yu t gy bnh ca cc vi sinh vt ny c nhng cch phng nga c hiu qu hn. Xut pht t nhng l do trn chng ti tin hnh thc hin bi kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm. Ni dung bi kha lun ny s p ng cho ta mt ci nhn tng quan v mt s c t vi sinh vt gy bnh trn thc phm, phng php xc nh v cc bin php phng chng ly nhim vi sinh vt trn thc phm. 1.2. Mc ch Nghin cu v nhng c t vi sinh vt gy bnh trong thc phm v i su tm hiu, tng quan v mt s loi thng xuyn gy nhim trong thc phm nh: Clostridium botulinum v c t botulin, nm mc v cc c t thng gp ca nm mc. Quan trong nht l tm hiu tng quan v Staphylococcus aureus v xut mt s bin php phng nga ly nhim vi sinh vt ny trn thc phm. 1.3. Ni dung nghin cu Nghin cu cc c im v hnh thi, cu to, di truyn, hot ng sinh l, ha hc ca 1 s nhm vi sinh vt. Nghin cu cc c ch gy c, c tnh v ca 1 s nhm vi sinh vt gy bnh cho ngi v ng vt. GVHD: KS. Hunh Vn Thnh
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SVTH: Nguyn Trn Hi Hong
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Nghin cu v cc phng php pht hin v xut bin php phng nga ly nhim trn thc phm.
GVHD: KS. Hunh Vn Thnh
Chng II: Tng quan

2.1. Mt s c t vi sinh vt trong thc phm 2.1.1. c t botulin 2.1.1.1. Gii thiu v Clostridium botulinum Clostridium botulinum l trc khun, k kh bt buc. Clostridium botulinum tn ti trong t, nc thi, bn, m ly, h v cc vng nc ven bin, thc vt v trong h thng ng rut ca c. Tri cy v rau qu c th b nhim c t t, c b nhim c t nc. Ngoi ra, cc thc phm khc nhau c th b nhim c t nhiu ngun nht nh. Clostridium botulinum pht trin thun li 26-280C, chng tit ra c t toxin botulinum, sinh kh hydro sulfur (H2S) v sinh hi. V c im nui cy cc t bo ny nhy cm v khng pht trin vi nng pH thp (<4,6), nng mui cao trn 1% c th ngn cn s pht trin ca vi khun. Clostridium botulinum khng th s dng lactose nh l mt ngun carbon chnh. 2.1.1.2. Gii thiu v c t botulin a. Cu trc c t botulin c tng hp nh l mt chui polypeptide duy nht vi trng lng phn t 150000 dalton, cu trc ny phn c t c hot lc tng i thp. Khi b mt s enzyme ca vi khun v trypsin tch ra th c t ny to thnh 2 chui nh gm 1 u cha carboxyl (mnh A) c trng lng phn t l 50000 dalton v nng gm 1 u cha amino (mnh B) c trng lng phn t l 100000 dalton c ni vi nhau bng cu ni disulfur c gn vi 1 phn t Zn. Cc on im ct A ca c t trn mt trng lng phn t c bn tr thnh c t mnh nht trong t nhin.
Hnh 2.1: Cu trc ca c t botulin b. C ch c t botulin Tnh gy bnh ca Clostridium botulinum ph thuc hon ton vo vic sn xut c t thn kinh. Cc c t hot ng trn dy thn kinh ngoi vi tit ra acetylcholine ngn chn s phng thch acetylcholine on giao thn kinh c. iu ny ngn cn c hot ng bnh thng v l nguyn nhn gy ra bnh bi lit. Hot ng ca c t khi u bi nhng cch sau: Nhim c s cp: y l kt qu ca vic tiu th cc loi thc phm trong S truyn nhim s cp i theo sau bi nhim c: y l kt qu t vic ung b nhim cc bo t sn xut ra cc c t. thc phm c cha bo t Clostridium botulinum sn sinh, pht trin v sn xut c t trong rut. Sau khi n phi cht c c sn xut trong c th, n c hp th bi cc phn trn ca ng tiu ha. T ng tiu ha n i qua mu v h thng bch huyt n ch ni thn kinh c ngoi vi. Qu trnh nhim c thn kinh ny gm 4 bc: + c t rng buc: ti min rng buc ca chui nng cc cht c thn kinh lin kt vi cc th th protein v lipid gangliosides (mt nhm thuc cc cht glucolipid trong no, gan, l lch v hng cu) trn mng t bo thn kinh.
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm + Tip thu c t: c t thn kinh c tip thu bng nng lng ph thuc vo qu trnh thm nhp ni bo. c t c a vo bn trong c quan ni bo l trung gian gia cc min di di ca chui nng. + S di chuyn qua mng t bo: cht c thn kinh ca chui nh di chuyn theo mch mu c quan ni bo n t bo thng qua s tng gim pH. pH ca c quan ni bo c chui nh cho php di chuyn tng phn thng qua cc knh hnh thnh bi chui nng. Nhng knh ny iu chnh s chuyn ng ca chui nh vo trong t bo cht. Ngoi ra, chui nh s tch ra thng qua s gim bt ca mi ni disulphide, y l lin kt duy nht ca 2 chui. + Gy tt nghn do s gii phng acetylcholine: chui nh ca c t thn kinh l 1 endoprotease chng lin kt vi cc th th N-ethylmaleimide nhy cm vi cc yu t thy phn protein. Nhng protein ny gii phng cc cht truyn thn kinh c bit l acetylcholine. Enzyme thy phn cc protein c t ngn cn s hp thnh cc ti tit acetylcholine trn b mt bn trong ca mng t bo vi cc mng nron trc khp thn kinh (sinap). iu ny dn n vic c ch gii phng acetylcholine ti nhng khp thn kinh tit acetylcholine ngoi vi, cng chnh l mc ch hot ng ca c t botulin. u tin cc dy thn kinh ca no b nh hng gy t lit sau n cc dy thn kinh vn ng v cc c bp. 2.1.2. c t nm mc 2.1.2.1. Gii thiu chung v nm mc a. Hnh thi Nm mc l vi sinh vt khng c dip lc t nn khng c kh nng t tng hp cc cht dinh dng cho chnh bn thn. Do , chng ch pht trin trn ngun dinh dng c sn. Nm mc l loi vi sinh vt pht trin thnh hnh si phn nhnh. Nhng si phn nhnh ny pht trin thnh tng m, ngi ta gi l khun ty. Khun ty khi pht trin trn mi trng c thng phn ra 2 loi: khun ty k sinh v khun ty dinh dng.
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Hai loi khun ty ny ng vai tr v nhim v khc nhau, khun ty dinh dng c nhim v ht cht dinh dng, khun ty k sinh c vai tr l sinh sn. mi si nm th pht trin thnh nhng b phn khc nhau. Nm mc khng di chuyn c v khng c c quan vn chuyn, nm mc ch pht trin trong iu kin mi trng thong kh. So vi vi khun nm mc chu c nhit v acid thp hn, y l 1 trong nhng c im c bn cn thit trong qu trnh phn lp nm mc. V mu sc v hnh thi khi lng bo t cng c nhiu kiu khc nhau. b. Cu to Do cu to c bit, nm mc hon ton khc vi vi khun v nm men. Da vo cu to ngi ta chia nm mc ra lm 2 loi: Loi nm mc c vch ngn: y l trng hp khun ty to thnh do 1 chui t bo ni tip nhau. Ngn cch 2 t bo l mt mng ngn. T bo nm thng c c quan ca 1 t bo, trong quan trng l c nhn thng thy Aspergillus v Penicillium Loi nm mc khng c vch ngn: y l nhng nm mc nhiu hch, gia cc hch khng c mng ngn, hu ht cc t bo nm khng c lp v cellulose nh thc vt m c lp v kitin nh lp v cng ca su b. T bo nm rt giu cc hot tnh sinh hc v giu khng sinh nn c con ngi s dng nm mc sn xut nhng sn phm phc v cho i sng.
Hnh 2.2: Nm mc Aspergillus
Hnh 2.3: Penicillium chrysogenum c. Hnh thc sinh sn +, Sinh sn sinh dng Nm mc c th sinh sn pht trin bng khun ty, trong lng khun ty c s xut hin ca mt hay nhiu t bo hnh cu, c mng dy bao bc, bn trong c nhiu cht d tr. Gp iu kin thun li th cc t bo hnh cu ny s pht trin thnh mt si nm mi. Nm mc cn c th sinh sn bng hch nm, y l 1 khi hnh trn u, bn trong l mt t chc si xp v thng c mu trng. khi gp iu kin thun li chng s pht trin bnh thng. +, Sinh sn v tnh y l kiu sinh sn ch yu bng bo t, cc bo t c th c to thnh t nhng phng php sau: Bo t c to thnh do s ct on ca cc si nm Bo t c th c to thnh t t bo sinh bo t bng cch ny chi Bo t c to thnh bng cch ngn vch vi t bo ngay khi bo t mi Ngoi ra nm mc cn c th sinh sn bng hu tnh bng cch sinh sn bng bo t tip hp.
hnh thnh.
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 2.1.2.2. Gii thiu v mycotoxin C n 30-40% s nm mc c phn loi c th sn sinh ra c t vi liu lng v c tnh khc nhau, nhiu loi nm mc khc nhau c th sn sinh ra cng mt loi c t. Mt loi nm mc c th sn sinh ra cc loi c t khc nhau ty thuc vo iu kin mi trng v c cht. Cc loi c t ny c gi chung l mycotoxin. Mycotoxin l cc hp cht trao i bc 2 c c tnh v do mt s vi nm tng hp trong qu trnh trao i cht xy ra t bo trong cc iu kin xc nh Mycotoxin l c t c kh nng gy c cp v mn tnh trn ng vt v con ngi. Hi chng c do n phi mycotoxin c gi chung l mycotoxicoses. S sinh trng ca nm mc trn thc phm rt ph bin kh hu m v m. c hng trm loi mycotoxin c sn sinh t cc ging Aspergillus, Penicillium v Fusarium Mycotoxin c th c phn loi theo bn cht v cu trc ha hc, theo tc nhn tng hp mycotoxin hoc theo bnh l do mycotoxin gy nn Nhng mycotoxin thng gp trong chui thc phm l: Aflatoxin B1, B2, G1 v G2 Ochratoxin A Fumonisin B1, B2 v B3 Patulin C th tm thy aflatoxin trong cc loi thc phm khc nhau nh: ng, go, bnh m v cc loi hat cha du. Aflatoxin l mt sn phm trao i th cp bc hai trong qu trnh pht trin ca vi nm, n khng phi l cht d tr v cng khng phi l cht cn b. a. Ngun gc Cc chng nm mc tng hp aflatoxin ch yu thuc Aspergillus flavus, A.parasiticus, A.nomius. Loi Penicillium puberulum c th sn sinh ra cc aflatoxin nhng vi s lng t. Khng phi tt c cc chng Aspergillus flavus c kho st u sn sinh ra aflatoxin, ch c 71% cc chng l c kh nng sn sinh ra aflatoxin, trong 23% cc chng sn sinh aflatoxin mc cao nht. GVHD: KS. Hunh Vn Thnh
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2.1.2.3. Aflatoxin
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Loi Aspergillus flavus c khp mi ni: t, cc hp cht hu c, cc loi ht nhng ch yu l cc loi ht c du. A. flavus thng gp trn la m, trn cc ph phm bt sng v trong bnh m. Ngoi ra, A. flavus cn c tm thy trn ng, go, trn cc si bng v ht bng. A. flavus rt d nhn bit, A. flavus c cc bo t tng i ln mu vng nu n hi lc. Chng ny thch hp pht trin trong iu kin kh hu m v nng, nhit thch hp sn sinh A. flavus l t 250C-280C, nhit trn 450C A. flavus s b c ch. Trong nui cy c t aflatoxin B1 c to ra nhiu nht, sau l aflatoxin G1, tip sau l aflatoxin B2, c t aflatoxin G2 rt t xut hin v t nguy him hn. Aflatoxin B1 pht trin nhit 250C-280C cn aflatoxin G1 pht trin nhit 300C. b, Cu trc ca aflatoxin Cc aflatoxin B1, B2, G1, G2 c nghin cu v xc nh cu to ha hc. Cng thc ca aflatoxin B1 l C17H12O6, cng thc ca aflatoxin G1 l C17H12O7. Trong cu trc phn t ca 2 aflatoxin B1 v G1 c nhm lacton v metoxyl, khng c nhm hydroxyl t do. Aflatoxin B1 cha 1 vng lacton cn aflatoxin G1 cha 2 vng lacton. Sau , hai aflatoxin B2, G2 cng c pht hin. Chng c cng thc ha hc hon ton ging aflatoxin B1, G1 ch khc l ni i trong vng hydrofuran b kh. Cng thc ca aflatoxin B2 l C17H14O6, cn cng thc ca aflatoxin G2 l C17H14O7
Hnh 2.4: Cu trc phn t ca aflatoxin B1, B2, G1 v G2 Nm 1963 trong nghin cu cht c sa v tht b n phi thc phm c aflatoxin. Alicroft v Carnaghan nhn thy trong 2 loi thc phm ny c dn xut ca aflatoxin B1 v B2. c t ny c gi l c t sa, l cc cht hydroxyl ha ca aflatoxin B1 v B2 ti v tr 9a ln lt c gi l aflatoxin M1 v M2. Cng thc nguyn ca aflatoxin M1 l: C17H12O7, cng thc nguyn ca aflatoxin M2 l: C17H14O7.
Hnh 2.5: Cu trc phn t ca aflatoxin M1
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Hnh 2.6: Cu trc phn t ca aflatoxin M2 c. C ch gy c ca aflatoxin Aflatoxin c kh nng lin kt vi DNA trong nhn t bo, s lin kt ny gy c ch enzyme polymerase ca RNA lm hn ch s tng hp RNA v gy c ch polymerase t-RNA. y l nguyn nhn lm gim s tng hp protein trong t bo. Ngoi ra, vng lacton, -lacton khng bo ha c trong phn t aflatoxin lm cho cht ny c hot tnh gy ung th, ng thi vng lacton ny gy c ch tng hp DNA trong nhn t bo v lm ri lon s tng trng bnh thng ca t bo. Cc qu trnh gy c ca aflatoxin ln t bo qua 5 giai on: Tc ng qua li vi DNA c ch cc polymerase chu trch nhim tng hp Ngng tng hp DNA Gim tng hp DNA v c ch tng hp RNA truyn tin Bin i hnh thi nhn t bo Gim tng hp protein. c tnh ca aflatoxin c hai loi l c tnh cp v c tnh mn c tnh cp l s ng c cp tnh th hin bng ci cht ca cc ng vt th nghim trong nhng khong thi gian thay i ty theo kh nng chu ng ca tng loi. Gii phu bnh cho thy hoi t v chy mu nhu m gan, vim tiu cu thn cp, t mu phi. gan dn mt mu cn th tch th tng ln. Khi khng c ni i vng furan u th c tnh gim i 4,5 ln. Nh vy B 1 c hn B2 v G1 c hn G2. DNA v RNA
d. c tnh ca aflatoxin
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm V c tnh cng gim khi c hai vng lacto (G1 v G2) do aflatoxin loi B c hn loi G. c tnh ca aflatoxin rt cao v lm tn thng n t bo. c tnh mn l nhng triu chng do nhim c mn tnh. Biu hin u tin l n km ngon v chm ln, thm ch xung cn, gan l ni chu nh hng nng nht ca cht c. nh hng v mt ha sinh ln t bo c nhiu nghin cu v s tc ng ca aflatoxin trn cc acid nucleic v s tng hp protein. 2.1.2.4. Ochratoxin C th tm thy ochratoxin trong la m, ng, la mch, bt m, go, ht c ph v cc thc n gia sc hn hp khc nhau. Cc mu thc phm cha ochratoxin A (OTA) nh nho, ng cc, c ph. S nhim OTA ph thuc rt nhiu vo xut x a l ca nguyn liu. a. Ngun gc Cc chng nm mc c kh nng tng hp ochratoxin cha c xc nh, nhng mt s nghin cu gn y li cho thy cc chng nm mc c th rt khc nhau trn cc i tng khc nhau v thuc vo ging nm mc ph bin Aspergillus v Penicillium. Cc nghin cu ch ra rng s pht trin ca nm mc sn sinh ra ochratoxin khc nhau v vic hnh thnh ochratoxin t chng ph thuc rt khc nhau v nhit , m, hot ng nc ca sn phm v bn cht sn phm. Cc chng sinh ochratoxin khc nhau theo khu vc a l, kh hu v bn cht ca sn phm bi nhim. Cc chng tng hp ochratoxin cng c th tng hp ng thi nhiu loi mycotoxin nh acid penicillic hoc citrinin. b. Cu trc ca ochratoxin Cu to ha hc th ochratoxin A l hp cht ca izocumarin lin kt vi 1 nhm L-phenylalamin. c tnh ca achratoxin khc nhau lin quan ti vic nhm hydroxyl phenol c tch ra kh hay d.
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Hnh 2.7: Cu trc phn t ca ochratoxin A, B v C c. C ch gy c ca ochratoxin Ochratoxin gy c ch s vn chuyn ca ribonucleic axit (tRNA) v cc axit amin. Ochratoxin cn c ch vi khun, nm men v phenylalanine-tRNA gan. S c ch cnh tranh phenylalanine lm o ln cc axit amin cn d, cc phenylalanine d cng tc ng lm c ch s tng hp protein trong t bo v c th. S c ch min dch ca ochratoxin c biu hin lm gim thc bo v c ch t bo lympho. Hp cht tng t ca OTA trong cc phenylalalanine c thay th bi cc axit amin khc c tc dng c ch tng t nh trn cc amino axit synlaza tRNA tng ng c th. 4P-hydroxyochratoxin A, 1 cht chuyn ha ca OTA cng c nhng hot dng tng t trong khi -ochratoxin v OTB khng c nhng hot ng ny. Ochratoxin c th hot ng trn cc enzyme khc nh 1 cht nn phenylalanine. OTA gy c ch hydroxylase phenylalanine, 1 na phenylalanine ca OTA l 1 phn hydroxyl ha tyrosin gy bnh cc t bo gan trong c th.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Ochratoxin c ch s tng hp RNA lm nh hng n cc protein trong vng tun hon, lm gim mc hot ng ca phosphoenol pyruvate carboxykinase, 1 enzyme quan trng trong s hnh thnh gluoza trong ng vt, c ch ny l do s phn hy ca mRNA. Ochratoxin cn tc ng n cc t bo mng ty th v gy ra cc hiu ng khc nhau trn ti th. Kch thch s hnh thnh DNA trong thn, gan v l lch. Cc DNA ny l cc si n b ph v. d. c tnh ca ochratoxin c tnh ca ochratoxin A (OTA) l cao nht. y l hp cht khng mi, kt tinh, ha tan trong dung mi phn cc v trong dung dch bicabonat, ha tan hn ch trong nc. OTA l c t nm mc lin quan ti bnh thn cp tnh ca ln, gy qui thai cho chut v phi g. OTA cn gy chng bnh suy thn ngi. Nhng trng hp gy ng c OTA cp tnh c th gy t vong. Ngoi ra OTA cn b nghi ng l cht c th gy nhim c thn kinh. Bn cnh ochratoxin cn gy ra bnh grout trn gia cm. 2.1.2.5. Fumonisin Trong s cc mycotoxin, mi quan tm v fumonisin ngy cng cao. Fumonisin l c t mi pht hin gn y do Fusarium moniliforme tng hp nn. y l nhm cc mycotoxin c c tnh cao vi ng vt v ngi. Vic nhim fumonisin trong thc n cho ngi v gia sc quy m trn ton th gii. a. Ngun gc Fumonisin ch yu do cc nm mc thuc ging Fusarium tng hp nn, loi in hnh nht trong nhm ny l Fusarium moniliforme. Cc chng nm mc ca loi ny kh ph bin trong mi trng v thng nhim vo lng thc c bit l ng. Cc chng khc thuc Fusarium cng tham gia tng hp nn c t ny bao gm: F. proliferatum, F. anthophilum, F. subglutinans, F. annulatum, F. succisae, F. beomiforme, F. diamini, F. napiforme v F. nygamai. F. moniliforme tng hp fumonisin B1 ti a nhit 200C. Hm lng c t gim mnh khi nhit tng ln 250C, 300C hoc gim xung 150C, 100C. Ti 350C F. moniliforme pht trin mnh nht trn mi trng nui cy phng th nghim v
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm trn lng thc, tuy nhin lng fumonisin c tng hp l khng nhiu. Trong iu kin ym kh, nm mc pht trin rt yu v khng c kh nng sinh c t. b. Cu trc phn t ca fumonisin Fumonisin l nhm hp cht dieste ca acid tricacboxylic vi cc ru bc cao khc nhau, fumonisin cha nhm amin bc nht, tan trong nc v bn vng vi nhit . Trong s cc fumonisin, ch s fumonisin B1, B2 v B3 c pht hin vi mt hm lng ng k trong t nhin. F. moniliforme khng ch tng hp fumonisin B1 m cn tng hp cc dn xut khc ca c t ny. Cc dn xut ny ch khc nhau nhm hydroxyl nh vi nguyn t carbon v tr th 10 ca mch chnh fumonisin B. Trong , cc dn xut B1, B2 v B3 ca fumonisin l cc c t ph bin nht trong t nhin.
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Hnh 2.8: Cu trc cc c t ca fumonisin B1, B2, B3 c. C ch gy c ca fumonisin Cu trc ca fumonisin gn ging vi cu trc ca sphingosin, iu ny cho php gi nh l fumonisin c th nh hng ti trao i cht ca sphingosin trong c th. Sphingosin l cc tin cht ca mi sphingolipid, bao gm sphingomyelin, ceramid v gangliosid. c tnh ca fumonisin B1 lin quan mt thit ti cc hiu ng ln s trao i cht cc sphingolipid, bao gm cc qu trnh sinh tng hp mi, tch ly cc sphingolipid t do, qu trnh thi loi cc sphingolipid phc tp, tng cng phn gii cc sphingoid t do, tng hm lng cc lipid v sphingosin. Cc hiu ng ny dn n hng lot cc phn ng sinh ha gy ra cc s nhim c khc nhau. Cc fumonisin c tnh c hiu ti s tng hp cc sphingosin, th hin s c ch serin. Khng pht hin cc hiu ng tng t ca fumonisin n s tng hp cc phosphatidylserin, phosphatidylcholin v cc acid bo. V tr hot ng ca fumonisin l sphingosin v sphingosin N. transacetylase trong phn ng kt hp ca thiolase vi sphingosin v sphingosin to thnh dihydroceramid v ceramid. Do s c ch ca fumonisin, s lng t bo gan gim xung 25% sau 24 gi v 50% sau 4 ngy, cc hot ng ca fumonisin nhy cm vi t bo gan hn so vi t bo thn. S nhim fumonisin lu di nng cao c th gy ra cc nh hng mc t bo hon ton khc vi s nh hng ca sphingolipid. Do cc t bo no rt giu sphingolipid nn cc tn thng thn kinh c th do s nhim c fumonisin B1 gy nn. Hot tnh gy ung th ca fumonisin B1 cng c GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm th do c ch c ch tng hp sphingolipid gy nn v cc c t ny km hm cc hot ng ca sphingosin th hin vai tr ca tc nhn chng khi u ni bo. d. c tnh ca fumonisin Fumonisin B1 l c t c c tnh mnh nht, fumonisin B1 c th gy ra cc triu chng nhn no, suy gan, m, gy cc triu chng bt bnh thng cho ti t vong nga, ung th gan chut, bnh gan g v suy tim cp kh. Fumonisin B1 c xp vo nhm 2B, nhm cc hp cht gy ung th cho ngi. Nghin cu nh hng fumonisin ngi, ngi ta thy c s lin quan n bnh ung th thc qun v vic s dng lng thc nhim fumonisin ca cc bnh nhn. Ngoi ra fumonisin cn c nhng tc ng n c tim, c t ny ngn cn dng ion canxi v gy ngng tr hot ng ca tim. Fumonisin thng c tng hp cng vi aflatoxin, do vy c tnh ca c t ny cn c th cao hn na. Cho n nay tng tc ca cc c t ny vn cha c bit r. 2.1.2.6. Patulin a. Ngun gc Patulin l hp cht trao i bc hai do nm mc Penicillium, Aspergillus v Byssochlamys to nn. Cc chng tng hp patulin ch yu l cc loi thuc Aspergillus nh A. clavatus v A. giganteus. Trong s cc Penicillium th loi tng hp patulin nhiu nht l P. expansum, P. urticae, P.griseofulvum, cc loi nm mc ny thng gp trong t, trn b mt ca mt s loi hoa qu nh to Ty thuc vo kh hu cc h vi sinh vt ny c th b thay i. c bit l A. clavatus thng a thch mi trng c hm lng m cao, trn cc cht ang thi ra. V vy rt hay gp A. clavatus cc tri chn nui, trn phn gia sc, gia cm. b. Cu trc phn t ca patulin Patulin hay cn gi l clavaxin l sn phm trao i cht bc 2 ca nm mc c bit ti trc tin nh l mt loi thuc c th cha bnh cm lnh. Trong qu trnh s dng ngi ta mi nhn bit c tnh ca n. N l hp cht vng lacton khng no hot ng vi cng thc ha hc 4-hydroxy-4-furo [3,2] pyran-2(6H)-1. Patulin l hp cht khng mu, kt tinh c, tan trong nc v cc dung mi phn cc. Patulin c th c tng hp trn rt nhiu nng sn thc phm cng nh GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm trn thc n gia sc. Ngi ta phn lp c patulin trn ng cc, trn cc sn phm dng ht, trn hoa qu. Thc phm c kh nng nhim patulin cao nht l to v cc sn phm t to.
Hnh 2.9: Cu trc phn t ca patulin c. C ch gy c ca patulin Patulin l mt c t gy tn hi cho DNA hoc cc NST trong 1 thi gian ngn. Ngoi ra, patulin cn ngn cn s h hp hiu kh, lm gim s hot ng ca triphosphatase adenosine. Patulin kch thch cc si DNA gy v cc t bo Hela v lm NST b sai lch dn n DNA, protein v s tng hp RNA u b nh hng. Nghin cu c ch ca cc t bo lin kt vi c tnh ng rut ca patulin, ngi ta nhn thy 2 t bo biu m rut ngi (HT-29-D4 v CaCO-2-14) tip xc vi mycotoxin, dn n cc chu trng vim rut do patulin gy ra. d. c tnh ca patulin Patulin nh hng n hot ng ca mt s enzym nh ATPase, alkaline phosphatase, aldolase, hexokinase, ng thi kch hot enzym glycogen phosphorylase lm cho nng glucose trong mu tng 60%. Patulin c ch s tng hp ca cc protein, c coi l cht c c kh nng gy ung th cho ngi. Hot tnh suy gim min dch ca patulin cng c pht hin. Patulin c lin quan ti cc chng xung huyt, gy lot nim mc, c bit l nim mc rut.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 2.2. Tng quan v Staphylococcus aureus 2.2.1. Lch s pht hin Staphylococcus aureus do Robert Koch pht hin nm 1878 sau khi thc hin phn lp t m ung nht Nm 1880 Louis Paster cng thc hin tin hnh phn lp v nghin cu v Staphylococcus aureus Ngy 09/04/1880 bc s ngi Scotland Alexander Ogston trnh by ti hi ngh ln th 9 hi phu thut c mt bo co khoa hc, trong ng s dng khi nim t cu khun (staphylococcus) v trnh by tng i y vai tr ca vi khun ny trong cc bnh l sinh m lm sng n nm 1881 Ogston thnh cng trong vic gy bnh thc nghim, y l tin cho nhng nghin cu v S.aureus sau ny n nm 1884 Rosenbach thc hin mt lot cc nghin cu t m hn v vi khun ny. V ng t tn cho vi khun ny l Staphylococcus aureus Nm 1926 Julius von Daranyi l ngi u tin pht hin mi tng quan gia s hin din hot ng men coagulase huyt tng ca vi khun vi kh nng gy bnh ca n. Tuy nhin mi n nm 1948 pht hin ny mi c chp nhn rng ri. 2.2.2. Phn loi 2.2.2.1. Phn loi khoa hc V phn loi khoa hc Staphylococcus aureus c xp vo: Gii: Eubacteria Ngnh: Firmicutes Lp: Bacilli B: Bacillales H: Staphylococcaceae Ging: Staphylococcus Loi: Staphylococcus aureus Tn khoa hc l: Staphylococcus aureus Rosenbach 1884
2.2.2.2. Phn loi theo khng nguyn GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Cc t cu c nhiu loi khng nguyn: protein, polysaccharide, acid teichoic ca vch. Nhng da vo khng nguyn th vic nh loi rt kh khn. Di y l mt s khng nguyn trn b mt t bo c quan tm nghin cu: Acid teichoic: l khng nguyn ngng kt ch yu ca t cu v lm tng tc dng hot ha b th. y cn l cht bm dnh ca t cu vo nim mc mi. Acid ny gn vo polysaccharide vch t cu vng. y l thnh phn c hiu ca khng nguyn O. Protein A: l nhng protein bao quanh b mt vch t cu vng v l mt tiu chun xc nh t cu vng. Tt c cc chng t cu vng c protein ny. S d khng nguyn ny mang tn protein A, v protein ny gn c phn Fc ca IgG. iu ny dn ti lm mt tc dng ca IgG, ch yu l mt i s opsonin ha (opsonisation), nn lm gim thc bo. V v biofilm: v cu to bi polysaccharide c t nht 11 serotyre. Trong cc serotyre 1, 2, 5, 8 c nghin cu v cu trc phn t. Gy bnh cho ngi thng l nhng chng t cu vng c v mng v thng l serotyre 5 hoc 8. Ch mt s t chng S. aureus c v v c th quan st c bng phng php nhun v. Lp v ny bao gm nhiu tnh c hiu khng nguyn v c th chng minh c bng phng php huyt thanh hc. Biofilm l nhng lp mng, sn st v nhn do S. aureus tit ra v bao bn ngoi t bo vi khun. N c tc dung cho S. aureus bm v xm nhp vo nim mc. Khng nguyn adherin (yu t bm): ging nh nhiu vi khun khc, t cu c protein b mt c hiu, c tc dng bm vo receptor c hiu t bo. Adherin c th l cc protein: laminin, fibronectin, collagen. 2.2.2.3. Phn loi bng phage (phage typing) Cc phng php phn loi da trn khng nguyn ca t cu l rt kh khn, v vy vic phn loi t cu vng ch yu da trn phage. S k sinh ca phage trn vi khun mang tnh c hiu rt cao. Do vy phng php ny rt c ngha trong phn loi vi khun Cn c vo s nhy cm ca phag, ngi ta chia t cu thnh typ phag. Nhng b phage cho php xp loi phn ln cc chng t cu thnh bn nhm phag chnh. nh typ phage t cu xc nh cc nhm t cu khc nhau. GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 2.2.3. Hnh thi Staphylococcus aureus (cn c gi l t cu vng) c dng hnh cu, gram (+), ng knh 0,8 1m v ng thnh hnh chm nho, hnh thc tp hp ny do vi khun phn bo theo nhiu chiu trong khng gian Trong bnh phm th vi khun thng thng hp li tng i mt hay to thnh nhng m nh. Vi khun ny khng di ng, khng c lng, khng sinh nha bo v thng khng c v.
Hnh 2.10: Hnh thi c trng ca Staphylococcus aureus 2.2.4. c im 2.2.4.1. Tnh cht nui cy Staphylococcus aureus pht trin d dng mi trng thng thng, l vi khun k kh ty nghi. Pht trin c nhit 10 - 450C, mc tt 370C nhng to sc t tt 200C mi trng canh thang th sau 5 - 6 gi lm c mi trng, sau 24 gi th lm c r, lu c th lng cn mi trng thch, khun lc trn li, bng lng, ng nh, ng knh khong 1 - 2 mm, c th c mu vng m, mu vng cam hoc mu trng, tng i ln sau 24 gi mi trng thch mu, t cu vng pht trin nhanh, to tan mu hon ton. T cu vng tit ra nm loi dung huyt t (hemolysin): , , , , . 2.2.4.2. Tnh cht sinh ha
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm T cu c h thng enzyme phong ph, nhng enzyme c dng trong chun on l: Coagulase c kh nng lm ng huyt tng ngi v ng vt khi c chng ng. y l tiu chun quan trng nht phn bit t cu vng vi cc t cu khc. Coagulase c tt c cc chng t cu vng. Coagulase c 2 loi: mt loi tit ra mi trng (coagulase t do), mt loi bm Catalase dng tnh. Enzyme ny thy phn H2O2, catalase c tt c cc t Ln men ng mannitol. Desoxyribonuclease l enzyme phn gii DNA. T cu vng c kh nng khng vi nhit v ha cht cao hn cc vi khun khng c nha bo khc. N b dit 800C trong mt gi. C th sng mi trng c nng NaCl cao (9%) Kh nng khng vi nhit thng ph thuc kh nng thch ng nhit ti a (450C) m vi khun c th pht trin. t cu vng cng c th gy bnh sau mt thi gian di tn ti mi trng Nhy cm thay i vi khng sinh, nhiu chng khng sinh vi penicillin v cc khng sinh khc. 2.2.5. S phn b T cu vng c ri rc trong t nhin nh trong t, nc, khng kh, c bit ngi l ngun cha chnh ca t cu vng, ch yu l vng mi hng (30%), nch, m o, mn nc trn da, cc vng da try xc v tng sinh mn T l mang vi khun cao hn cc nhn vin y t, bnh nhn lc mu, c bnh tiu ng, nghin ht, nhim HIV, mc bnh da mn tnh. Khong sau hai tun nm vin t l ny ln n 30%-50% v thng nhim chng khng thuc. 2.2.6. Cc yu t c lc 2.2.6.1. Cc protein b mt v cc protein tit ra mi trng vo vch t bo (coagulase c nh). cu m khng c lin cu.
2.2.4.3. Kh nng khng
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Protein A (SPA): tt c cc chng t bo t cu vng u c lp protein A bao xung quanh. Lp protein ny c tc dng gn phn Fc ca IgG v do v hiu ha tc dng ca khng th ny. IgG l loi khng th c t l cao nht (70%) trong cc loi khng th v ng vai tr quan trng nht trong chng nhim trng. Protein gn fibronecctin A v B (fibronecctin binding Proteins A and B, FnBPA, FnBPB) bm vo th th fibronecctin trn b mt t bo biu m, gen m ha c xc dnh l FnBPA v FnBPB . Yu t kt t A v B ( Clumping factor A and B, CIfA, CIfB): adherin gn vo fibronecctin to cc yu t kt t CIfA v CIfB hot ha gy ngng t tiu cu. Protein gn collagen (collagen binding Proteins, Can): adherin y mnh s gn kt ca protein vi collagen, s tng tc vi collagen l bc quan trng trong vic thc y s gn kt ca vi khun gy tn hi m. S gn kt ny gy ra bnh vim xng ty v nhim trng khp. Protein gn sialoprotein xng (Bone sialoprotein binding Proteins, Bbp): adherin cho sialoprotein xng gy c ch cc Bbp vi cc t bo t cu, lm gim kh nng khng ca xng gy ra cc bnh v vim khp. Protein nhy cm plasmin (Plasmin sensitive Protein, PIs): l protein c b mt ln, c th tng tc vi vi khun v t bo c th nh l fibronectin, khng th. Lm cho vi khun khng th bm dnh c. Protein lin quan ti Biofilm (Biofilm associated Proteins , Bap): cu trc ca biofilm l cc vi khun v lp v glycocalyx bn cht l polysaccharide c th tng tc vi vi khun ang xm nhp t chc, gip cc vi khun ny bm vo thnh t bo, khng bi o thi ra bn ngoi, trnh c cc tc ng ca thc bo, khng th v khng sinh. Protein gn elastin (elastin binding Proteins, EbpS): adherin y mnh s gn kt ca cc protein vi elastin gy nh hng n ng mch v lm cho mu ngng lu thng trong c th. Protein gn ngoi t bo (Extracellular matrix binding Proteins, Ehb): protein cng hp rt ln vch t cu vng, thc y s kt dnh cc protein vi vt ch nh laminin v fibronectin ngi, to thnh cc cht gian bo trn b mt ca biu m v trn b mt ni m. GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Cc protein b mt hoc protein tit ca t cu vng tham gia vo cc tc dng sinh hc khc nhau ca chng. 2.2.6.2. Cc yu t xm ln a. Hemolysin C 4 loi Hemolysin c xc nh l , , v . Mt chng t cu c th to thnh nhiu hn mt loi hemolysin. l nhng phm vt bn cht protein gy tan mu nhng tc ng khc nhau trn nhng hng cu khc nhau. Chng c tnh sinh khng. Mt vi loi hemolysin gy hoi t da ti ch v git cht sinh vt th nghim. Bng 2.1: Tnh cht ca cc loi hemolysin ca t cu vng Type Loi hng cu nhy cm Th, cu Loi bch cu nhy cm Th, ngi Ngun gc vi khun Ngi Tc ng trn ng vt th nghim Gy hoi t da th, gy cht chut v th, gy c t bo Cu, b v ngi Th, ngi, cu, chut, b, nga Ngi, th, nga, cu v chut b. Leucocidin Khng Cha xc nh c Th, chut v ngi Ngi ng vt Ngi nui cy Liu cao gy cht th, hoi t tng m t bo nui cy Gy hoi t nh da th v da chut, gy cht th Lm x cng da th v da chut, gy hoi t t bo nui cy
Mc d mt s staphylolysin cha c t bch cu, nhng ch mt c t t cu tht s c vi bch cu v c gi l leucocidin. T cu gy bnh c th b thc bo nh t cu khng gy bnh nhng li c kh nng pht trin bn trong bch cu c t ny c bn cht l protein, chng to ra cc protein nhiu thnh phn v gy tn hi mng, khng chu nhit v gy c cho bch cu ngi v th, khng gy c cho bch cu cc loi ng vt khc. N cng c tc dng hoi t da th
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Mt s chng S.aureus tit ra 1 loi c t gi l Panton-Valentine leucocidin (PVL), c t ny c mt trong c th ngi khe mnh (khong 0,6%) gy triu chng bnh vim khp, vim phi ngi. PVL l 1 synergohymenotropic exotoxin. y l 1 c t thuc h c t gm 2 thnh phn v hot ng thng qua s h tr ca 2 protein. c t ny gy c ch cc t bo bch cu ht, i thc bo kch thch bch cu ngi to ra cc enzyme (glucuronidaza v lysozyme), cc thnh phn chemotactic (Leucotriene-B4 v interleukin-8) v cht chuyn ha oxy gy hoi t cc t bo Cc PVL hot ng mnh gy v mng v phn gii t bo, sau tc ng ln cc t bo ch nh bch cu trung tnh. Ngoi ra, PVL cn lm tn thng cc m, to cc t bo mu ngoi vi trong qu trnh ly nhim sinh bnh vim phi. Leucocidin bao gm 2 mnh F v S v c th tch ri bng sc k ion, trng lng phn t l 32000 v 38000 Dalton. Nu tch ri hai mnh ny th mt tc dng gy c c. Hyaluronidase Enzyme ny phn gii cc acid hyaluronic ca m lin kt, y l 1 thnh phn chnh ca c cht ngoi bo ca cc m trong c th. Enzyme ny nhim vo m v to ra 1 ngun carbon v nng lng gip vi khun lan trn vo m. Cc nghin cu cho thy cc protein t Staphylococcus aureus UAMS-1th hin dng t bin do 2 protein sarA v sarA agar gy ra, v s tham gia ca protein sarA l iu quan trng ca c tnh trong qu trnh hot ng ca hyaluronidase. Vai tr chnh ca S.aureus hyaluronidase vn cha c tm hiu r rng, ch bit s tham gia ca sarA l 1 yu t quan trng i vi 1 s c tnh c th hin bi S.aureus hyaluronidase. Cc coagulase c th c lp c hyaluronidase trong 1 s trng hp, cc phn ng DNAse l 1 trong nhng yu t gip cho hyaluronidase hot ng. d. Coagulase Coagulase l mt protein ngoi bo lin kt vi prothrombin trong vt ch hnh thnh 1 phc h gi l staphylothrombin. Prothrombin b bin i thnh enzyme thrombin nh enzyme prothrombinase. Cc protease hot ng c trng ca thrombin GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm c cc qu trnh hot ha trong phc h dn n vic chuyn i fibrin (dng khng ha tan) thnh fibrinogen (dng ha tan). Cn theo cc nghin cu ca Soulier, Tager v Zajden th coagulase v prothrombin khng c hot tnh enzyme, s tham gia ca chng to nn cc phc hp bn vi cc hot ng ly gii c hiu gi l Staphylothrombin, staphylocoagulase khng c hot tnh ly gii, chng phn ng mt cch chuyn bit vi cc prothrombin v hot ha cc hp cht ny a n s kt hp cc fibrinogen thnh khi fibrin. S hot ng nh sau: Staphylocoagulase Staphylothrombin Prothrombin Fibrinogen Fibrin Coagulase l du hiu nhn bit S.aureus trong phng th nghim. Tuy nhin cha c bng chng no cho thy coagulase l mt yu t gy c. Mt s kin cho rng cc vi khun t bo v mnh khi thc bo v min dch bng cch gy ng mu C mt vi nhm ln cho rng coagulase chnh l yu t ng kt (clumping). nhng mt s nghin cu ch ra rng khi thiu coagulase th vn duy tr s hot ng ca yu t ng kt, trong khi yu t ng kt vn th hin coagulase 1 cch bnh thng. e. Staphylokinase Staphylokinase (Sak) l 1 protein to ra bi 136 amino acid v c sn xut bi cc chng S.aureus Sak kch hot plasminogen, tin thn ca protease plasmin phn gii fibrin. V mt cu trc, Sak c cu trc tng ng vi cc cht kch hot plasminogen khc, cha mt v tr gn plasminogen v 1 vng serine protease. Tuy nhin, Sak khng phi l 1 enzyme. N to thnh 1 phc hp vi plasminogen c th chuyn i cc phn t plasminogen khc thnh plasmin, 1 enzyme mnh phn hy protein ngoi bo. i lc cao ca phc hp Sak plasminogen vi fibrin hnh thnh nn mt tc nhn gy tan huyt.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Sak c th to iu kin cho S.aureus plasminogen gn vi th th trn b mt t bo vi khun v qua y mnh qu trnh xm nhp vo cc m ch y l mt enzyme c trng cho cc chng gy bnh ngi, gip t cu pht trin trong cc cc mu v gy v cc cc mu ny, to nn tt mch. f. -lactamase S khng li khng sinh ca t cu vng l mt c im rt ng lu . a s t cu vng khng li penicillin G do vi khun ny sn xut c men penicillinase nh gen trn R-plasmid S khng penicillin ca t cu vng l do a s t cu vng sn xut c enzym -lactamase Mt s cn khng li c methicillin gi l methicillin resistance S. aureus (vit tt l MRSA), do to ra c cc protein gn vo vi tr tc ng ca khng sinh Hin nay, mt s rt t t cu cn khng c vi cephalosporin, cc th h. khng sinh c dng trong cc trng hp ny l vancomycin. g. Mt s enzyme khc S.aureus cn c th c s hin din ca protease, lipase, deoxyribonuclease v cc acid bo. y l nhng yu t cung cp cc cht dinh dng cho cc vi khun v c th n cn gn vai tr trong qu trnh gy bnh. Cc enzyme ny gip ko di s sng ca cc vi khun. 2.2.6.3, Cc yu t chng li s t v ca t bo ch a. Capsular polysacharide Phn ln cc chng lm sng ca Staphylococcus aureus u hin din mt polysacharide b mt ca mt trong hai serotype (kiu huyt thanh) 5 hoc 8. N c gi l microcapsule bi v n ch c th xc nh c bng knh hin vi in t khng ging nh mt s cc vi khun khc c nhn thy d dng bng knh hin vi nh sng Capsular polysaccharide (CP) chng li cc c ch phng v ca c th cng nh khng khng sinh. Cc CP bo v vi khun chng li s thc bo bng cch khng cho cc khng th to hin tng opsonin ha trn vch vi khun
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Do khng c hin tng opsonin ha nn cc i thc bo v bch cu trung tnh tip cn km hoc khng th tip cn c vi khun Cc thc bo khng tiu dit c vi khun th cng c gng tit nhiu cytokine hn na nhm lm sch vi khun xm nhp, nhng chnh iu ny li thu ht cc bch cu a nhn v i thc bo khc n vim. Phn ln cc CP chng li c thc bo l do ngn cn cc t bo thc bo bm, c ch sinh C3 convertase v C3b ca b th, hoc che ph C3b lm cho thc bo khng nhn ra. Cc chng S.aureus phn lp t bnh nhim trng th hin mt mc cao polysaccharide nhng nhanh chng b mt kh nng khi nui cy trong phng th nghim. Chc nng ca cc capsular polysacharide khng phi hon ton l c tnh. b. Protein A Protein A l mt protein b mt ca S.aureus m IgG gn kt cc phn t theo vng Fc. Cc mnh Fc ny l ca globulin min dch. Chnh nh hin tng gn kt ny m s lng mnh Fc gim xung. mnh Fc ca globulin min dch c vai tr quan trng trong hin tng opsonin ha .Trong huyt thanh vi khun lm cho IgG ph v opsonization v phagocytosis Cc mnh Fc chnh l cc receptor cho cc i thc bo, qu trnh gn kt trn gip cho t cu vng trnh khng b thc bo bi cc i thc bo t bin ca S.aureus thiu protein A c hiu qu hn phagocytosed trong ng nghim, cc t bin trong cc trng hp b ly nhim th nghim c hin tng gim c tnh. c. Exofoliative exotoxins y l mt ngoi c t, gy nn hi chng phng rp v chc l da (scaded skin syndrome) tr em, gm 2 loi l ETA v ETB. C ch gy bnh: ET gy ra s phn ly bn trong lp biu b gia cc lp t bo sng v cht lm da phng phng ln, lm mt dn i nhng lp biu b da mt nc v c th tip tc nhim trng
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Nhng c t ny c kh nng esterase v proterase v n tn cng nhng protein c chc nng duy tr s nguyn vn ca cc t bo biu b Bnh thng bt u vi s nhim trng da ti nhng v tr xc nh nhng sau vi khun bt u sn sinh c t nh hng n da trn ton b c th Tr pht st, pht ban v phng da. Pht ban bt u t ming lan rng n bng, tay, chn. Khi vt phng b b ra th pht ban kt thc. Lp da ngoi cng b choc ra v b mt tr nn . au ging nh mt vt bng. 2.2.6.4. Cc siu khng nguyn a. Toxic shock syndrome toxin-1 (TSST-1 ) c t shock nhim c thng gp nhng ngi ph n c kinh dng bng bng dy bn hoc nhng ngi b nhim trng vt thng, hay cn gi l nhim trng huyt. c t ny kh phn bit vi enterotoxin F ca t cu vng Trong mt s trng hp nhim khun gram m, cc ni c t bn cht l polysaccharide kch thch hot ha i thc bo gii phng TNF (Tumor necrosis factor, yu t hoi t u) v cc interleukin 1, 2. Cc cytokine ny tham gia vo c ch sc do nhim khun huyt, cch thc gy c y nh ni c t LPS c ch nh sau: - Cu trc ca LPS gm 3 phn: phn li, chui bn c hiu O mang tnh khng nguyn c hiu v lipid A chu trch nhim v c tnh. Phn t LPS nm trong mng ngoi v phn lipid A ca phn t c tc dng LPS c gii phng t mng ngoi ca vch vi khun gram m, vo mu gn gn LPS vo vch vi khun. vi protein (LPS binding protein) to thnh phc hp. LPS bm vo th th (phn t CD14) hoc trc tip vi TLR-4 (toll like receptors-4) trn monocyt v i thc bo. Chng kch thch t bo tit ra TNF- (tumor necrosis factor alpha), IL-1 (interleukin-1), IL-6, IL-8 v PAF (platelet activating factor) dn n st, gin mch v tng tnh thm huyt tng gy gim huyt p, gim mu n c quan, gim chc nng gan, thn v h hp. Trong cc trng hp nhim c thc n do S.aureus v hi chng sc do nhim c lng cytokine cao cng gy ra nhng triu chng nht nh. Trong hi chng sc nhim c cng xut hin cc triu chng nh ca nhim c do c t rut nh: GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Cc c t rut hot ng nh nhng cht kch thch phn bo hot ha tt c cc t bo T biu hin 1 h gene V (th th ca t bo T). Cc cytokine do cc t bo T gii phng ra khi c hot ha bi cc siu khng nguyn SE s gy nn nhiu triu chng st, a chy, sc trong nhim c thc n do S.aureus. Trong trng hp ny ngoi c t ca t cu c gi l c t 1 gy hi chng sc, hot ng nh 1 siu khng nguyn kch thch cc t bo T hot ha i thc bo tit ra nhiu TNF. Cc vi khun sng bn trong t bo thc bo c kh nng hot ha t bo T dn ti ph hy cc m. Cc cytokine do cc t bo T hot ha tit ra s tp trung cc i thc bo v hot ha chng hnh thnh cc khi u. Cc enzyme lysosom c gii phng t cc khi u ny s gy ra hoi t ng k cc m. b. Enterotoxin Cu trc: L nhng chui protein n tng i chu nhit, mi chui c v tr khng nguyn ring bit. Khng b hy bi s un nu, trng lng phn t t 28000-30000 dalton, bao gm 6 type k hiu t A-F. c im chnh l c vng cystein gia gip n nh cu trc phn t v khng s phn gii protein. C cc chui amino acid, trong nhiu nht l aspartic, glutamic, lysin, tyrosine.
Phn loi: S loi SE khc nhau nhiu ti liu khc nhau ty thuc vo nm pht hin v vai tr ca cc SE trong cc v ng c thc phm do t cu. Do s lng SE kh ln nn rt cn thit phi phn loi v sp xp chng.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Nm 1962, ngi ta a ra h thng sp xp cc c t theo bng ch ci. u tin 5 loi SE c tm thy v phn loi da vo tnh cht khng nguyn ca chng, l c t A (SEA), c t B (SEB), c t C (SEC), c t D (SED) v c t E (SEE). Trong SEC c chia thnh SEC1, SEC2, SEC3. Sau , cc SE mi cng vi cc gen tng ng c tm thy v nh du t SEG n SER v SEU. Khng c c t SEF, v F l k t ch TSST-1. Tuy nhin s lin quan gia cc SE mi ny n cc v ng c th cha r, hin nay hu ht cc b test thng mi ch thch hp xc nh cc c t t SEA n SEE l cc c t thng gp nht trong cc v ng c, khong 5% cc v ng c do cc c t enterotoxin m ta cha bit gy ra. Trong cc loi c t trn th SEA thng gp nht trong cc v ng c do t cu. Cc dng S.aureus to c t SEA c tn s cao nht trong cc mu thc phm (61.5%) v trn nhng ngi khe mnh (53,6%). SEA l nguyn nhn ca 75% cc v ng c do t cu, tip n l SED, SEC v SEB, cc v dch do SEE thng rt t gp. Tnh cht: SE l nhng protein n gin, ht m, d tan trong nc v nc mui, l nhng protein c bn, ng in pI l 7-8,6, tr SEG v SEH c ng in pI tun t l 5,6 v 5,7. m cao nht l 277 nm, cao hn so vi nhng protein thng thng. D c 1 mc tng ng gia cc SE, nhng vn c s khc nhau gia cc trnh t amino acid lm cho cc c t c cc v tr khng nguyn khc nhau. SE giu lysine, acid aspartic, acid glutamid v tyrosine. Hu ht c vng cystein to cu trc thch hp c th lin quan n hot tnh gy nn. Chng c tnh n nh cao, khng vi hu ht cc enzyme phn hy protein v v th chng gi c hot tnh trong ng tiu ha sau khi c n vo bng. Chng cn khng vi chymotrypsine, rennin v papain. c bit, tnh bn nhit l mt trong nhng tnh cht quan trng nht ca cc SE trong lnh vc an ton thc phm. Chng khng b phn hy 1000C trong 30 pht, thm ch 1210C trong 28 pht th nhng SE vn gi c hot tnh sinh hc (khi th nghim trn mo). Tnh khng nhit ca SE trong thc phm cao hn so vi mi trng nui cy. GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm C ch c lc: Hot tnh siu khng nguyn tc ng trc tip ca SE vi th th khng nguyn t bo T v phc hp ca t bo ha mng ca t bo nhn din khng nguyn S nhn din khng nguyn l bc u tin trong p ng min dch t bo v cng l vn then cht quyt nh mc chuyn bit ca p ng min dch. Mt khng nguyn thng thng nhn din c th th t bo T bng cch hnh thnh nhng peptide gn kt vi phc hp ha mng MHC lp I hoc II. Ch 1 vi t bo T c th nhn bit c mt khng nguyn chuyn bit trn phc hp ha mng ca t bo nhn din khng nguyn Trong khi , cc c t siu khng nguyn tc ng trc tip ln nhiu t bo T bng cch nhn din cc chui V chuyn bit ca th th khng nguyn t bo T. Cc c t ny c th lien kt cho vi th th khng nguyn t bo T v phc hp tng ng lp 2 ca t bo nhn din khng nguyn Chnh s lin kt cho ny dn n vic hot ha khng chuyn bit lm tng nhanh lng t bo T v lng interleukin khng l l nhng yu t c t lin quan n c ch gy c ca SE. Do , SE c th hot ha 10% t bo T ca chut, trong khi nhng khng nguyn thng thng kch hot t hn 1% t bo T.
Hnh 2.11: Hot tnh siu khng nguyn SE Theo hnh 2.11 th cc siu khng nguyn kch thch t bo T. Siu khng nguyn rng buc trc tip lp II ca t bo nhn din (MHC II) v cc rnh bn ngoi khng nguyn, sinh ra 1 lng ln cytokines gy ra cc triu chng sc c hi. GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Ngoi ra SE cn c hot tnh gy nn khi i vo c th. SE tc ng trc tip ln biu m rut v kch thch trung khu gy nn dn n nhng triu chng ca ng c thc phm. c im chung nht ca cc SE l vng cystine, y l yu t quan trng nht nh hng n hot tnh gy nn. 2.2.7. Tnh hnh nhim Staphylococcus aueus trn th gii v ti Vit Nam hin nay 2.2.7.1. Tnh hnh nhim S.aureus trn th gii Trn th gii cc v ng c thc phm do vi sinh vt chim khong 70% tng s ca ng c thc phm. Ti cc nc chu S.aureus l nguyn nhn hng u gy ra cc v ng c. chu M in hnh l Hoa K nhng v ng c thc phm ch yu u do S.aureus gy ra, theo thng k cho thy t 1972-1976 ng c S.aureus chim 21,4% trong tng s cc v ng c. T nm 1983-1987 con s ny thp hn vi ch 5,2%. Theo mt thng k mi nht th n thng 9 nm 2009 Hoa K c 32 v ng c thc phm lin quan n S.aureus chim 10,3% trong tng s cc v ng c. Nhng phn tch gn y cho thy ti Hoa K hng nm c khong 48000 ngi t vong v MRSA (Methicillin resistant Staphylococcus aureus). c tnh c khong 19000 ngi M t vong v MRSA trong nm 2005. chu cc v nhim S.aureus ch yu cc nc Nht Bn, Trung Quc v trong khu vc ng Nam . Trung Quc trong nm 2008 xy ra 1 v ng c S.aureus tr em v ung sa b nhim S.aureus. Cn Nht cng c 2 v ng c S.aureus ln vo thng 8 nm 1955 lm ng c hn 1936 em hc sinh ti 5 trng tiu hc Tokyo v thng 6 nm 2006 lm 14780 ngi b ng c vng Kansai. Nguyn nhn ca 2 v ng c ny u do h ung sa c nhim S.aureus ca tp on Snow. Trong khu vc ng Nam 2 quc gia c t l ng c S.aureus cao l Indonesia v Philippines. Vit Nam cng l mt trong nhng nc c t l nhim S.aureus cao trong khu vc chu . Cn chu u thng nhim S.aureus t cc bnh vin, t l nhim S.aureus chim 7% trong cc v nhim khun huyt ti bnh vin. cc v nhim khun huyt Anh th nhim khun do MRSA chim n 96%. GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 2.2.7.2. Tnh hnh nhim S.aureus ti Vit Nam Ti Vit Nam tnh hnh nhim S.aureus l rt ng bo ng. Nm 1974 t l nhim S.aureus l 2% trong tng s cc v ng c thc phm th n nm 1995 con s ny tng ln 22%, nm 2004 ln n 63%. Theo bo co ca b y t nm 2006 xy ra 35 v ng c thc n trong c nc. trong 25 v ng c tp th th c 11 v xy ra trong cc trng hc, trong c n 9 v l do nhim S.aureus. c bit cng trong nm 2006 xy ra 1 v nhim S.aureus tr em, iu tra cho thy 2/6 tr b sc vacxin c nhim S.aureus, khi xt nghim 5 ngi trong i tim chng th c 3 ngi b nhim S.aureus. Trong nm 2007 nc ta c 2 v ng c thc phm tp th do nhim S.aureus. Vo thng 9 nm 2007 tnh Ph Th xy ra 1 v ng c tp th, gn 100 hc sinh ti trng mm non Vnh Li b ng c thc phm do nhim S.aureus. Vo thng 12 nm 2007 ti thnh ph H Ch Minh xy ra 2 v ng c thc phm tp th, ng ch l c 2 v ng c ny u xy ra trong trng hc do nhim S.aureus. Trong , c 31 em ti trng tiu hc u C v 44 em ti trng mm non Vn Hng. Trong nm 2009 c nc c 116 v ng c thc phm trong c 6 v do nhim S.aureus. ng ch vo thng 7 nm 2009 ti tnh Hi Dng xy ra 1 v ng c tp th do nhim S.aureus, s ngi b ng c trong v ny ln ti 258 ngi. T u nm 2010 n nay c nc c 67 v ng c thc phm, trong c 5 v l do S.aureus gy nn.
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Chng III: Cc phng php pht hin v xut bin php phng nga ly nhim trn thc phm
3.1. Tng quan Staphylococcus aureus l vi khun hiu kh hay k kh ty , hnh cu, gram dng, c th nghim coagulase, phn ng DNAse, phosphatease (+), c kh nng ln men v sinh acid t mannitol, trehalose, sucrose. Mt s dng c kh nng lm tan mu trn mi trng thch mu, ng knh vng tan mu ph thuc vo tng chng nhng u nh hn ng knh ca khun lc. S hin din vi mt cao ca S.aureus trong thc phm ch th iu kin v sinh v kim sot nhit km ca qu trnh ch bin. S.aureus c xc nh trn c s cc c im tng trng v phn ng ng huyt tng ca cc dng thun t cc khun lc c trng trn mi trng phn lp. 3.2. Cc phng php truyn thng 3.2.1. Mi trng v ha cht s dng Mi trng MSB: mi trng ny dng phn tch nh tnh S.aureus, hoc Mi trng thch BP: b sung egg yolk vo mi trng sau khi hp kh nh lng bng phng php MPN. trng v lm ngui n khong 50-600 C. GVHD: KS. Hunh Vn Thnh
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Mi trng thch TSA Mi trng thch nghing BHI Huyt tng th: c c nh bng 0,1% EDTA hay sodium oxalate v c
phn phi 0,3ml vo cc ng nghim nh. 3.2.2. Phn tch nh tnh Staphylococcus aureus 3.2.2.1. Phng php Cy 2ml dung dch mu vo ng nghim cha 8ml mi trng MSB. Trn u Dng que cy vng cy ria dch mu t ng (+) (mi trng t mu sang Tm khun lc c trng ca S.aureus trn mi trng phn lp c mu en v 370 C trong 24h. mu vng) ln mi trng phn lp l thch TGA hay BP, tip tc 370 C trong 24h. nhnh, sng, trn, li, ng knh 1-1,5mm c vng sng chung quanh khun lc. chn khun lc c trng cy vo mi trng BHI, tip tc 370C trong 24h. vt. 3.2.2.2. Kt qu Mu c S.aureus khi th nghim coagulase (+).Mu c s xut hin khi ng trong khi ng i chng khng c. 3.2.3. nh lng Staphylococcus aureus bng phng php m khun lc 3.2.3.1. ng nht mu Cn 10g mu cho vo ti PE v trng, thm 90ml dung dch pha long, ng Chun b dy pha long mu thch hp ty theo mc nhim ca tng loi mu, nht mu bng my dp mu khong 30 giy. sau khi cy bng mt th tch xc nh trn mi trng BP s xut hin 20-200 khun lc/a. 3.2.3.2. Phn lp trn mi trng chn lc Cy 0,1ml mu pha long vo mi trng BP. Dng que cy tam gic tri u mu trn b mt mi trng cho n khi kh (khong 10 pht). Gi nguyn a trong khong 1 gi sau lt p li. Mi pha long lm 3 a mi trng thch BP. em 350C- 370 C trong 45- 48 gi. Chn a cha 20-200 khun lc, hoc cc a GVHD: KS. Hunh Vn Thnh
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Cy vo cc ng nghim nh cha khong 0,3ml huyt tng v 370C trong
24 gi th phn ng ng kt, lm 1 ng i chng khng c cy dch vi sinh
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm c pha long thp hn nhng c s xut hin in hnh cc khun lc c trng ca S. aureus. Khun lc c trng ca S.aureus c hnh dng trn, li, m t, ng knh khong t 2-3mm, c vng sng c bao quanh. i vi mi trng thch mu lm tng t nh cc bc trn mi trng Sau 24 gi khun lc trn mi trng thch BP c ng knh khong 0,5thch BP. Nhng ch 370 C trong 24 gi. 1mm, li, en bng c vng sng rng khong 1-2mm bao quanh. Sau 48 gi th khun lc S.aureus c ng knh khong 1-1,5mm, mu en bng, li, c vng trng c hp v vng sng rng khong 2-4mm quanh khun lc. Khun lc ca mt s chng S.aureus c th khng to vng sng quanh khun Trn mi trng thch mu sau 24 gi th S.aureus cho khun lc bng long, lc nh trn. Cn m v nh du c hai dng khun lc. c, li c mu xm hay vng nht, ng knh khong 1-2mm. Hu ht S.aureus c vng tan mu, tuy nhin mt s dng khng to vng tan mu ny. Cc chng phn lp t thc phm ng lnh hoc kh c lu tr trong thi gian di th pht xut hin cc khun lc c trng t hn.
Hnh 3.1: Khun lc c trng ca S.aureus trn mi trng BP 3.2.3.3. Khng nh Dng que cy vng cy 5 khun lc c trng v khng c trng t mi trng thch BP sang mi trng thch TSA, em 37 0C trong 24 gi.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Cy sinh khi vi khun t mi trng TSA vo cc mu ng nghim cha huyt tng th v 370C. theo di kt qu phn ng ng huyt tng sau cc khong thi gian 2, 4, 6, 8 v 24 gi. T l khng nh da trn s khun lc c trng hoc khng c trng. Thc Kt qu th nghim l (+) khi khi ng huyt tng hnh thnh, kt qu l (-) Ngoi ra ta c th ly sinh khi vi khun cho vo nhng ng nghim nh cha hin tng t vi cc khun l c trng trn mi trng thch mu. khi khng c hnh thnh khi ng, hn dch vn ng nht nh ng khng cy. 0,2 - 0,3ml mi trng BHI. nghing mi trng thch BHI v em i 18-24 0C trong 35 gi. Thm 0,5ml huyt tng cho vo mi trng thch nghing BHI, trn k v em 350C, kim tra nh k qua 6 giai on hnh thnh khi ng huyt tng. Kt qu dng tnh khi khi ng vn nm trn mt thch nghing ngay c khi o ngc ng nghim.
Hinh 3.2: Kt qu th nghim coagulase 3.2.3.4. Kt qu Mt S.aureus trong mu c tnh nh sau:
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Mt (CFU/g hay CFU/ml) =
Trong : F: pha long Nt: tng s khun lc c trng Na: tng s khun lc khng c trng Ht: t s gia s khun lc c trng cho th nghim khng nh (+) so vi s khun lc c trng Ha: t s gia khun lc khng c trng cho th nghim khng nh (+) so vi s khun lc khng c trng. 3.2.4. nh lng S.aureus bng phng php MPN Phng php ny c dng nh lng S.aureus trong mu c mt S.aureus thp nhng mt vi sinh vt cnh tranh cao, kh c th xc nh bng phng php m khun lc. 3.2.4.1. Phng php Dung dch mu c pha long vi 3 pha long 10-1, 10-2, 10-3. Ty theo yu cu v chnh xc ca kt qu phn tch m c th s dng phng php MPN vi 9 hay 15 ng nghim ( 3 pha long lp lai 3 hoc 5 ln ). gi. Chn cc khun lc c trng trn mi trng BP thc hin th nghim Cc a cho kt qu khng nh S.aureus (+) c i chiu vi cc ng khng nh S.aureus tng t nh phng php m khun lc. nghim trong h thng cc dy ng nghim ban u v ghi li s ng nghim (+) tng ng vi mi pha long. 3.2.4.2. Kt qu Mi trng c s dng l canh MSB. Cy 1ml dch mu c pha long Cc ng nghim c vi sinh vt pht trin s lm c mi trng v cho kt qu khc nhau vo ng nghim ch 10ml mi trng, 370C trong 48 gi. (+). Tip tc phn lp khun lc n trn mi trng thch BP, em 370C trong 48
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Tra bng MPN suy ra mt vi khun S.aureus trong mu (s MPN/g hay MPN/ml) 3.3. Cc phng php hin i 3.3.1. Xc nh S. aureus khng methicillin (ch to b th nghim multiplex PCR) 3.3.1.1. Vt liu Cc chng vi khun S. aureus ATCC 25923 (MSSA), chng S. aureus ATCC 43300 (MRSA) v mt s chng khc. Cc on mi dng trong nghin cu ny tham kho theo Mehrotra v do Sigma Aldrich cung cp. B kit PCR v mt s ha cht sinh hc phn t khc.
Bng 3.1: Cc mi dng trong nghin cu (a kch thc sn phm khuch i) Gen v tr Tn FEMA-1 FEMA-2 MECA-1 MECA-2 3.3.1.2. Phng php Chun b DNA vi khun c thc hin theo Feriera. un cch thy nhit si huyn ph vi khun (5 khm/500l TE 1X) trong 10 pht. Ly tm 14000 vng trong 5 pht. B cn, thu phn dch ni. Pha long 10 ln bng TE 1X. Multiplex PCR dng 2l DNA chit nhanh trc tip huyn ph vi khun m t trn, thm vo 48l hn hp PCR 1X (16mM (NH4)2SO4, 67mM Tris HCl (pH 8,8)), 0,2mM cho mi th trong 4 loi dNTP, 1,5mM n 2,5mM MgCl2, 5pM n 20pM ca mi femA, 25 pM ca mi mecA v 1,25 U ca Taq DNA polymerase. GVHD: KS. Hunh Vn Thnh
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Kch thca (pp) 132 132 163 163
Trnh t AAAAAAGCACAT AACAAGCG GATAAAGAAGAA ACCAGCAG ACTGCTATCCAC CCTCAAAC CTGGTGAAGTT GTAATCTGG
Nucleotic femA 14441463 femA 15751556 mecA 1182-1201 mecA 1344-1325
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Chu k lun nhit l 950C trong 10 pht, cc bc (940C trong 30 giy, 50-650C trong 30 giy, 720C trong 1 pht) lp li 35 ln, 720C trong 10 pht (iCycler, BioRad). Sau khi xc nh iu kin ti u ca multilex PCR, mt phn ng c tin hnh vi mi femA nhn nh cc chng S. aureus, vi mi mecA pht hin du hiu khng, vi c hai mi cho mi chng vi khun. Sau khi khuch i bng PCR, ly 5l sn phm phn tch in di gel agarose 2% c tnh kch thc ca sn phm bng cch so snh vi thang chun 100bp. Gel c nhun vi ethidium bromide v quan st di tia UV (GelDoc, BioRad). Xy dng chng ni ti dng chung mi ca mecA nhng cho sn phm khuch i c chiu di ln hn (khong 240bp) kim sot s c ch phn ng PCR vi mi mecA. Tin hnh qua cc bc: PCR khuch i mecA, tinh ch v ct vi enzyme ct gii hn thch hp. Ct on DNA tng hp ha hc, c mang trnh t 2 u nhn bit Lai v bin np vo E.coli. Chn lc dng ti t hp, nui v chit plasmid Kho st pha long thch hp ca chng ni ti cho vo PCR mix. Thit k b th nghim gm cc thnh phn: L dung dch lu mu cha mi trng tng sinh chn lc cho MRSA. Que bng qut mi. ng dung dch m ly trch mu: cha cc cht ph mng t bo vi khun Tube cha hn hp thc hin phn ng PCR, dung dch m PCR, MgCl2, ng cha chng ni ti. Gen mecA dng xc nh cc chng MRSA v gen femA c trng cho S.aureus v khng phn ng cho vi cc vi khun khc. Tham kho cc nghin cu trc y, nng 25pM mi mecA trong mt phn ng c chn c nh v kho st tm nng ti u cho mi femA. cc nng ca femA 5pM v 10pM (hnh 3.3), ch cho mt bng tng ng mecA ti cc ging khun MRSA, trong khi MRSA khng cho bng. nng 15pM GVHD: KS. Hunh Vn Thnh
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dng lm DNA chng ni ti.
(Triton-X-100, Tween 20, Tris HCl pH 8, EDTA). dNTP, cc mi Taq DNA polymerase. 3.3.1.3. Kt qu v tho lun
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm ca femA, MRSA trong ging 12 cho mt bng mecA nhng trong ging 13 v 14 cho hai bng femA v mecA. Ging 15 khng xut hin bng no (hnh 3.3). nng 20pM ca femA, cc khun MRSA (cc ging 16-19, hnh 3.3) cho 2 bng femA v mecA, khun MRSA ch cho bng femA c trng ca S.aureus (ging 20, hnh 3.3). Chn cc nng ca cc cp mi mecA v femA trong M-PCR ln lt l 25pM v 20pM cho cc th nghim tip theo.
Hnh 3.3: Gel agarose phn tch M-PCR vi cp mi femA cc nng khc nhau Nng cp mi mecA gi c nh 25pM v ln lt bn mc nng femA: 5pM (ging 1-5), 10pM (ging 6-10), 15pM (ging 11-15) v 20pM (ging 16-20) trn DNA cc chng vi khun, thang chun DNA l 100bp. Chng S. aureus ATCC 25923 (MSSA) l ging 5, 10, 15 v 20. Chng S. aureus ATCC 43300 (MRSA) l ging 1, 6, 11 v 16. Cc ging cn li l ca cc chng khc. Thc hin kho st nm mc MgCl2 v tm mc nhit lai (hnh 3.4) cho thy mc 1,75mM MgCl2 v nhit lai 55,70C cho hai bng r v p (hnh 3.4B, ging 5). loi tr s hin din ca cc cht c ch phn ng PCR trong mu chng ni ti dng mi mecA c thit k v a vo hn hp phn ng.
Hnh 3.4: Gel agarose phn tch M-PCR gradient nng MgCl2 v nhit lai trn chng S. aureus ATCC 43300 (MRSA) GVHD: KS. Hunh Vn Thnh
42
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Nng MgCl2 ln lt l 1,5mM (A), 1,75mM (B), 2mM (C), 2,25mM (D) v 2,5mM (E). Nhit l 500C, 50,10C, 530C, 55,70C, 59,50C, 62,30C, 640C v 650C tng ng vi cc ging t 1-8. Kt qu xc nh pha long ti a ca chng ni ti c trnh by trong hnh 3.5, nng pha long nht c cho bng chng ni ti l 10-6 (hnh 3.5A, ging 5), cn trong multiplex PCR, ba bng 132bp (femA), 163bp (mecA) v 243bp (chng ni ti) xut hin 3 pha long ca chng ni ti (hnh 3.5B), ch c bng s 6 hin th r rng nht. do , nng pha long 10-6 c chn cho vo multiplex PCR.
Hnh 3.5: PCR kim tra pha long chng ni ti Hnh A l pha long chng ni ti ln lt t ging 1-9 l 10-2, 10-3, 10-4, 105
, 10-6, 10-7, 10-8, 10-9 v 10-10. Hnh B l pha long ca chng ni ti trong multiplex
PCR trn khun MRSA ln lt t cc ging 2 n ging 10 l t 10-2-10-10, ging 1 khng cho chng ni ti.
Hnh 3.6: B kt M-PCR pht hin MRSA 3.3.2. Phng php hp ph min dch dung enzyme-ELISA (Enzyne Linked Immunosorbent Assay)
43
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Phng php ELISA c ng dng pht hin S.aureus trong thc phm kh ph bin, y l phng php n gin, nhanh, nhy, c th pht hin cng nh nh lng c t, c nhiu b kt pht hin c t ang c bn trn th trng. Kiu ELISA thng gp nht l phng php sandwish. Trong phng php ny khng th c gn vi mu cha c bit, sau phc hp khng th - c t c gn vi cng hp enzyme khng th. Qui trnh ny c s dng nhiu v lng enzyme v mu c to ra t phn ng enzyme c cht t l thun vi lng c t c trong mu cha bit. Hai enzyme alkaline photphatase v horseradish peroxidase u c dng trong phng php ny, nhng alkaline photphatase d gn vi khng th hn. Tuy nhin horseradish peroxidase cho phn ng nhy hn, khi kt hp vi c cht cho mu xanh l, xanh dng hay cam, cn mu c to ra vi c cht khi s dng vi alkaline photphatase, -nitrophenyl photphatase (pNPP) l mu vng. Hu ht cc phng php ELISA nhy t nht l 0,5 ng c t/ml. Nhiu nh khoa hc s dng phng php ELISA pht hin c t S. aureus trong phng th nghim vi nhiu qui trnh khc nhau. Phng php ELISA ch yu s dng a ging (microtitre plate hay strip) khng th gn vo. Mu c cho vo ging, nu nh trong mu c khng nguyn mc tiu, khng nguyn s gn vi khng th. Sau , ly mu ra v ra a. Cng hp enzyme khng th (khng th c gn vi enzyme horseradish peroxidase hoc alkaline photphatase) c thm vo v cho php phn ng vi phc hp c t - khng th. Ra a, thm c cht c hiu ca enzyme vo. Enzyme xc tc phn ng thy phn c cht to ra sn phm c mu. i chng dng v i chng m cng c a vo th nghim. Phn ng dng tnh c ghi nhn nu nh mu c c m hn mu i chng m. Bng cch theo di s i mu, c th pht hin s hin din v nh lng lng khng nguyn (c t). Ngoi ra ngi ta con s dng ht polystyrene c gn khng th. Phng php ny nhy hn v ch dng 20 ml dch trch thc phm. Nhng ht m mu c ly ra khi dch trch v em ra, mi ht c cho vo ng tube mu mt m. Cho cng hp enzyme - khng th vo mi tube v c th phn ng vi phc hp c tGVHD: KS. Hunh Vn Thnh
44
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm khng th. Ra ht trong tube nhiu ln, cho vo 1 ml c cht. Mu c to ra c th c bng mt thng v lng c t khng cn xc nh trong vic xc nh phn ng dng tnh. Nu s dng my c mu th phn ng to mu s c dng li sau 30 pht bng acid sulfuric. Hin nay trn th trng c nhiu b kit pht hin SE. Trong phi k n TECRA, b kit pht hin v nh danh c t rut enterotoxin (loi A n E). Qui trnh ny c thi gian ngn (4 gi), nhy (1ng/ml hay mg) v c th pht hin ng thi s hin din nhiu loi c t ca t cu, nhng khng th phn bit cc loi c t. B kit ny dng microtitre plate vi cc ging c ph hn hp cc khng th chuyn bit ca cc c t t A n E; enzyme horseradish peroxidise, EDTA v NaH2PO4 l c cht. Phn ng ELISA c thc hin da trn k thut sandwich, v c tn thng mi l TECRATM. y l sn phm u tin c chng nhn bi AOAC v c s dng kh rng ri pht hin cc c t SE.
Hnh 3.7: Mt s b kt ELISA 3.3.3. Phng php RPLA (Reversed Passive Latex Aggulutination)
45
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm K thut RPLA c ng dng pht hin c t trong thc phm cng nh xc nh Staphylococcus c c t dng tnh. Trong k thut ny, ht nha c ph khng th ca c t trc khi cho vo ging. Sau , cho mu vo to phn ng. Nu c c t trong mu th phn ng gia c t v khng th s to ra s ngng kt, ty mc d ngng kt m xc nh c lng c t. Phng php ny nhy pht hin c t trong hu ht thc phm ca cc v ng c, cng nh trong vic pht hin nhng dng Staphylococcus to ra lng c t thp m cc phng php khc khng pht hin c.
Hinh 3.8: B kt RPLA Ngoi ra cn c 1 s phng php khc pht hin S.aureus c s dng rng ri nh phng php min dch phng x RIA (Radio Immunoassay) v phng php khuch tn trn gel (gel diffusion) 3.4. xut bin php phng nga ly nhim trn thc phm Mun phng ng c thc n do t cu, cn phi khng ch s pht trin ca vi khun v s hnh thnh c t rut. ng vt S.aureus ly truyn ch yu l do ung sa b, nguyn nhn l do b b vim v nn trong sa c t cu sinh c t rut. V th, khi b b vim v th phi vt ht sa v khng c dng n. Qu trnh vt sa phi tun theo yu cu v sinh 1 cch nghim ngt, trnh tnh trng b nhim t cu lan rng.
46
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Gim nguy c ly nhim t cc loi thc phm ti l vn rt quan trng, phi m bo cc quy trnh ch bin, d tr, phn phi, ng gi thc n, theo di tnh hnh sc khe ca ngi lm thc n, v sinh ni ch bin v d tr thc n. Trc khi ch bin thc n phi ra tay tht sch. Cc thc n t khu ch bin n khi tiu th phi c bo qun lnh hoc ch mt m bo v sinh. Cc thc n c nu chin nn dung ngay khi cn nng v khng nn qu 2 gi. Cc thc n tha phi ct gi trong t lnh, khi dng li phi nu li trc khi n v vi khun c th xm nhp vo thc n trong lc thc n ngui v trong la chng ta ang n. i vi nhng ngi n hng, qun, nh n tp th phi quan tm n an ton ca thc phm, ch nhiu khu ch bin v d tr phi m bo v sinh. C th thay th nhng thc n kh bo qun thnh nhng thc n pha ch sn hp v sinh cho ngi tiu dng. Trong qu trnh d tr thc n cn c bit quan tm n nhit , nu khng s gy n cc trng hp ng c vi c t t cu. Nhng ngi lnh mang trng c khuyn co khng nn lm vic phng sinh, phng tr s sinh, phng m v cc cng ty ch bin thc phm. Khi pht hin ngi b nhim c t t cu cn cch ly ngay trnh tnh trng ly nhim sang nhng ngi khc. Tm li vn phng nga lun c t ra, tuy nhin phi trn c s theo di tng trng hp c th, tng v dch b nhim c t do t cu, phn tch cc loi thc phm c lin quan, cc yu t ly nhim, cc tc ng n s pht trin dch bnh ri mi c nhng bin php can thip thch hp.
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Chng IV: Ni dung v phng php nghin cu

4.1. a im v thi gian Thi gian: T ngy 31/05/2010 n ngy 12/06/2010. 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. 4.2. i tng v phm vi nghin cu Nghin cu v pht hin Staphylococcus aureus trn mt s mu tht g ti sng ti mt s ch nh trn a bn hai qun Ph Nhun v Bnh Thnh thnh ph H Ch Minh. 4.3. Vt liu tin hnh th nghim 4.3.1. Cc dng c v ha cht tin hnh th nghim Mu: 15 mu tht, mi mu ly 25g. Ko ct mu. Pipet 10ml. a petri. Que cy trang thy tinh. Micropipet 10l-200l, u tp dung cho micropipet Ti pe v trng. Cn in t. ng nghim rng. ng ong 100ml. T sy 370C, ni hp kh trng 500C n cn. Qu bp. Chai chu nhit dung tch 250ml. Ga ng nghim inox. T cy. Bng khng thm. Que cy.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Cn 960. Dung dch egg yolk (Ty Ban Nha) Huyt tng th, nc mui sinh l Mi trng SPW (n ) Mi trng BP (n ) Mi trng TSA (n )
4.3.2. Cc mi trng s dng
4.4. B tr th nghim Mu tht G
Thu mu ti cc a im kho st
nh gi cc ch tiu
Cm quan
Staphylococcus aureus
Nhn xt, Kt lun Hinh 4.1: B tr th nghim 4.5. Phng php nghin cu
49
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 4.5.1. Quy trnh phn tch 25g mu + 225ml SPW ng nht trong 10 pht
pha long l 10-1
Cho 0,1 ml mu pha long 10-1 cho vo a mi trng BP c b sung egg yolk cy trang
370C trong 48 gi Khun lc c trng ca S.aureus trn BP: trn, li, tm en, c qung sang bao quanh Chn 5 khun lc c trng cy chuyn trn mi trng TSA 370C, trong 24 gi Cy chuyn vo ng nghim cha 0,2 ml huyt tng th 370C v theo di sau 2, 4, 6, 8 gi. Nu khng c biu hin ngng kt s tip tc n 24 gi Tng s S.aureus (cfu/g):
S=
Hnh 4.2: Quy trnh phn tch Staphylococcus aureus 4.5.2. Thuyt minh quy trnh 4.5.2.1. Phng php tng sinh vi khun: Phng php ny nhm tng sinh vi khun kt hp vi pha long mu Cn 25g mu cho vo ti PE v trng, thm 225ml mi trng SPW (pepton water), buc kn v bt u ng nht mu bng tay trong khong 10 pht, pha long s dng l 10-1. GVHD: KS. Hunh Vn Thnh SVTH: Nguyn Trn Hi Hong
50
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 4.5.2.2. Phng php phn lp mu Sau khi tin hnh ng nht mu v tng sinh vi khun trn mi trng SPW ta tip tc giai on phn lp vi sinh vt trn mi trng BP. Chun b 10 a mi trng thch BP trc mt ngy v bo qun trong t lnh. S dng micropipet 10l-200l ht 100l mi mu pha long 10-1 v dng que cy tam gic tri u mu trn b mt mi trng BP. Thc hin cy trang mi mu t 5-10 pht cho n khi kh hn mi lt p a li. Mi mu thc hin lp li 2 ln trn 2 a mi trng BP, sau em i 370C trong 48 gi. 4.5.2.3. Phng php cy chuyn khun lc c trng: Sau khi 370C trong 48 gi ta tin hnh m v xc nh cc khun lc c trng xut hin trn mi trng thch BP. Chun b 10 a mi trng thch TSA trc mt ngy v bo qun trong t lnh. Khun lc c trng l khun lc c dng trn, li, m t, c vng sng bao quanh, nh du cc khun lc c trng chun b cy chuyn sang mi trng thch TSA. Dng que cy vng cy cc khun lc c trng t mi trng thch BP sang mi trng thch TSA. Tip tc em 370C trong 24 gi. 4.5.2.4. Phng php thc hin ng t huyt tng Dng micropipet 10l-200l ht 200l huyt tng th vo mi ng nghim rng c hp kh trng. Dng que cy vng tin hnh cy sinh khi vi khun t mi trng thch TSA vo cc mu ng nghim cha huyt tng th v em 370C. Theo di kt qu phn ng ng huyt tng trong cc khong thi gian 2, 4, 6, 8 v 24 gi. 4.5.2.5. Phng php tnh ton kt qu Kt qu th nghim l (+) khi khi ng huyt tng hnh thnh, kt qu l (-) khi khng c hnh thnh khi ng.
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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm Mt S.aureus trong mu c tnh nh sau: S(cfu/g)= Trong : N: tng s khun lc c trng n: s a thc hin V: th tch f: pha long R: h s ngng t 4.6. Kt qu 4.6.1. Kt qu cm quan
Hnh 4.3: Cm quan tht g V mu sc, tht g phi c mu c trng ca tht, phn tht c mu hng, phn m v da c mu vng ti hoc trng. V mi th tht g phi c mi c trng ca tht, khng c mi l. V tnh cht, th b mt ca tht c nc, tht c n hi tt
52
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm V cm quan v tht nhn chung l tt, tht nhn cn ti, tnh cht, mu sc v mi v ca tht khng c g l. Nhng nh gi cm quan khng xem xt mc sch ca tht. 4.6.2. Kt qu nh gi mc nhim Staphylococcus aureus Theo TCVN 4830-89 (ISO 6888:1983) vi quy nh mt nhim Staphylococcus aureus l 102 CFU/1g
Hnh 4.4: T l nhim S.aureus trn cc mu thc nghim Theo TCVN 4830-89 (ISO 6888:1983) mu 1, 2, 3 v 5 nhim Staphylococcus aureus vi quy nh 1.102 CFU/1g. (chim 4/5 mu thc nghim). 2 mu s 1v s 5 nhim vi t l 33,33% (chim 2/5 mu thc nghim). Cn 2 mu s 2 v s 3 nhim vi t l 66,67% (chim 2/5 mu thc nghim). 2 mu s 1 v s 5 trong 3 ln kim tra th ch c 1 ln nhim iu ny c th l do b nhim t ngi bn hoc do khch mua hng chn la tht. Mc d b nhim nhng t l nhim ca 2 mu ny khng cao hn nhiu so vi tiu chun. Kh nng nhim cng c th l do ngi bn khng eo bao tay khi phn phi tht hoc do ch bn khng hp v sinh. 2 mu s 2 v s 3 th c n 2 ln nhim thm ch t l nhim l rt cao, ring mu s 2 th ch c 1 ln kim nghim l va ng tiu chun. iu ny chng t 2 c s bn mu ny khng m bo v sinh hoc do thao tc ca ngi bn khng hp v sinh. V sinh 2 a im ny l khng tt iu ny chng t b nh hng ca mi trng xung quanh nh: tp nhim trong khng kh, do ni bn hng khng GVHD: KS. Hunh Vn Thnh
53
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm m bo v sinh hoc c th do ni bn hng nm gn khu vc cha rc, cng nc thi. Tm li, mu s 4 l mu v sinh v m bo an ton nht, 2 mu s 1 v s 5 th c th s dng c nhng phi m bo cc quy trnh v sinh thc phm, cn 2 mu s 2 v s 3 th nu m bo v sinh tt hn na th mi c th bo m an ton cho ngi s dng.
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Chng V: Kt lun v kin ngh

5.1. Kt lun Qua qu trnh lm bi kha lun ny chng ti nhn ra rng Staphylococcus aureus l 1 loi vi khun nguy hin cho c con ngi v ng vt. Vi khun ny rt nguy him c bit l cc c t ng rut ca n. N gy ra cc v nhim khun ng rut cn c coi l 1 siu khng nguyn rt nguy him v l s bt u ca cc triu chng sc c hi. Ngoi ra vi khun ny cn ly nhim trong cc bnh vin, y l 1 iu ng bo ng v hu ht nhng v nhim trng ny nh hng n cc bnh nhn mi m xong v c bit l tr s sinh. S.aureus cn gy cc triu chng sc c hi rt nguy him i km vi nhim trng huyt. Ngi khe mnh hu ht u c S.aureus nhng ch khi c iu kin thch hp th vi khun ny mi sn sinh ra cc c t. Tm li, trn th gii hin nay c rt nhiu loi vi khun nguy him khng ch l S.aureus m cn l Clostridium botulinum, E.coli, Salmonella. y u l nhng vi khun nguy him n sc khe ca con ngi. Vic tm hiu v nghin cu v chng l rt cn thit ra cc bin php phng nga thch hp nhng c l thc ca mi con ngi l bin php phng nga hiu qu nht. 5.2. Kin ngh Trong thc nghim trn, c mt s phn do thi gian qu gp m ti cha th thc hin c l: 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
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TI LIU THAM KHO

TI LIU TING VIT:
1. B mn vi sinh vt trng i hc y H Ni (2001), vi sinh vt y hc, Nxb y
hc, H Ni.
2. B mn vi sinh vt trng i hc y H Ni (2007), vi sinh vt y hc, Nxb y
hc, H Ni.
3. B mn vi sinh vt trng i hc y khoa Hu (2004), bi ging vi sinh y hc. 4. B y t (2008), vi sinh vt y hc, Nxb y hc, H Ni. 5. Trn Thu Hoa, Th Lan Hng, Nguyn Th Ngc Lan, Hong Hiu Ngc
v Phm Hng Vn (2009), nghin cu ch to b th nghin multiplex PCR pht hin Staphylococcus aureus khng methicilin, y hc TP. H Ch Minh, tp 13, ph bn s 2.
6. 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 TING ANH:

7. Anne-Laure Genestier, Marie-Ccile Michallet, Gilles Prvost, Gregory Bellot,
Lara Chalabreysse, Simone Peyrol, Franoise Thivolet, Jerome Etienne, Grard Lina, Franois M, Vallette Franois, Vandenesch and Laurent Genestier (2009), Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils, INSERM, Lyon, France.
8. Bibek Ray (2009), FUNDAMENTAL FOOD MICROBIOLOGY, ,Boca Raton
London New York Washington, D.C, USA.

9. Bradley G. Stiles, Teresa Krakauer, and Peter F. Bonventre (1994, 1995),
Biological Activity of Toxic Shock Syndrome Toxin 1 and a Site-Directed Mutant, H135A, in a Lipopolysaccharide- Potentiated Mouse Lethality Model, Divisions of Toxinology1 and Applied Research, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-5011, and Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267-0524, USA.
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10. K.A. Voss, G.W. Smith, W.M. Haschek (2001), Fumonisins: Toxicokinetics,
mechanism of action and toxicity, Toxicology and Mycotoxin Research Unit, USDA Agricultural Research Service, P.O. Box 5677, Athens, GA 30605-5677, USA. Department of Population Health and Pathobiology, College of Veterinary Medicine. North Carolina State University, Raleigh, NC 27695, USA. Department of Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.
11. K. M. Cunnion, J. C. Lee, and M. M. Frank (2001), Capsule Production and
Growth Phase Influence Binding of Complement to Staphylococcus aureus, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710,1 and Channing Laboratory, Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts 02115-58042
12. Mark E. Hart, Morgan J. Hart, and Anna J. Roop (1998), Genotypic and
Phenotypic Assessment of Hyaluronidase among Type Strains of a Select Group of Staphylococcal Species, Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Department of Biology, Ouachita Baptist University, Arkadelphia, AR 71998, USA.
13. Michael Palmer, Renate Jursch, Ulrich Weller, Angela Valeva, Karin Hilgert,
MiKcheaheole Q, and Sucharit Bhakdi (1992), Staphylococcus aureus &-Toxin, From the Institute of Medical Microbiology, University of Mainz, Augustwplutz, W-6500 Germany and the Department of Microbiology, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom.
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14. Nathalie Gaebler Vasconcelos and Maria de Lourdes Ribeiro de Souza da
Cunha (2010), Staphylococcal enterotoxins: Molecular aspects and detection methods, Biosciences Institute, UNESP Univ Estadual Paulista, Department of Microbiology and Immunology, Bacteriology Laboratory, Botucatu-SP, Brazil
15. Regina LaRocque, M.D. (2001), Staphylococcal Toxic Shock Syndrome: A
Superantigen-Mediated Disease, Joint Infectious Disease Conference, USA.

16. Taishi Tanabe, Hisaaki Sato, Kengo udea, Hiroko Chihara, Takao Watanabe,
Katsushige Nakano, Hiroshi Saito, and Nobutoshi Maehara (1994), Possible Receptor for Exfoliative Toxins Produced by Staphylococus hyicus and Staphylococcus aureus, Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan.
17. Tao Jin, Maria Bokarewa, Timothy Foster, Jennifer Mitchell, Judy Higgins and
Andrej Tarkowski (2004), Staphylococcus aureus Resists Human Defensins by Production of Staphylokinase, a Novel Bacterial Evasion Mechanism, J. Immunol. 2004;172;1169-1176. TI LIU WEBSITE:
18. http://vi.wikipedia.org/wiki/T_cu_khun 19. http://vi.wikipedia.org/wiki/C_ch_c_lc_ca_vi_khun 20. www.bacsidakhoa.com 21. http://www.ncbi.nlm.nih.gov/pubmed/1820332 22. http://www.ncbi.nlm.nih.gov/sites/pubmed 23. http://www.benhhoc.com/index.php?do=viewarticle&artid=1427&title=dap-
ung-mien-dich-chong-nhiem-trung
24. http://tusach.thuvienkhoahoc.com/wiki/Min_dch_bnh_sinh_ca_nhim_trn
g_huyt
25. http://www.textbookofbacteriology.net/staph.html 26. http://textbookofbacteriology.net/themicrobialworld/Botulism.html
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PH LC
S liu khun lc c trng ca S.aureus trn mi trng BP t TN 1 t TN 2 t TN 3 Mu 1 3 2 2 Mu 2 3 80 9 Mu 3 21 9 1 Mu 4 10 2 2 Mu 5 4 3 1
S liu ng t huyt tng: S liu ng t huyt tng t th nghim 1 2 gi 4 gi 6 gi 8 gi 24 gi Mu 1 3 (-) 3 (-) 3 (-) 3 (-) 3 (+) Mu 2 3 (-) 3 (-) 3 (-) 3 (-) 2 (+) 1 (-) Mu 3 5 (-) 5 (-) 5 (-) 5 (-) 2 (+) 3 (-) Mu 4 5 (-) 5 (-) 5 (-) 5 (-) 1 (+) 4 (-) Mu 5 4 (-) 4 (-) 4 (-) 4 (-) 3 (+) 1 (-)
S liu ng t huyt tng trong t th nghim 2 2 gi 4 gi 6 gi 8 gi 24 gi Mu 1 2 (-) 2 (-) 2 (-) 2 (-) 1 (+) 1 (-) Mu 2 5 (-) 5 (-) 5 (-) 5 (-) 2 (+) 3 (-) Mu 3 5 (-) 5 (-) 5 (-) 5 (-) 2 (+) 3 (-) Mu 4 2 (-) 2 (-) 2 (-) 2 (-) 1 (+) 1 (-) Mu 5 3 (-) 3 (-) 3 (-) 3 (-) 2 (+) 1 (-)
S liu ng t huyt tng trong t th nghim 3 2 gi 4 gi 6 gi 8 gi Mu 1 2 (-) 2 (-) 2 (-) 2 (-) Mu 2 4 (-) 4 (-) 4 (-) 4 (-)
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Mu 3 1 (-) 1 (-) 1 (-) 1 (-)
Mu 4 2 (-) 2 (-) 2 (-) 2 (-)
Mu 5 1 (-) 1 (-) 1 (-) 1 (-)
Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm 24 gi 1 (+) 1 (-) 2 (+) 2 (-) 1 (+) 1 (+) 1 (-) 1 (+)
S liu mt S.aureus trong 5 mu tht t TN 1 t TN 2 t TN 3 Mu 1 1,5.102 50 50 Mu 2 102 16.102 2,25.102 Mu 3 4,2.102 1,8.102 50 Mu 4 102 50 50 Mu 5 1,5.102 102 50
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Staphylococcus Aureus

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Kha lun: Tng quan v Staphylococcus aureus v xut bin php phng nga ly nhim trn thc phm

Chng I: Gii thiu

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

Chng II: Tng quan

GVHD: KS. Hunh Vn Thnh

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GVHD: KS. Hunh Vn Thnh

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GVHD: KS. Hunh Vn Thnh

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Hnh 2.2: Nm mc Aspergillus

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

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SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

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Hnh 2.5: Cu trc phn t ca aflatoxin M1

GVHD: KS. Hunh Vn Thnh

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GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

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GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

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GVHD: KS. Hunh Vn Thnh

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2.2.4.3. Kh nng khng

GVHD: KS. Hunh Vn Thnh

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GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

GVHD: KS. Hunh Vn Thnh

SVTH: Nguyn Trn Hi Hong

SVTH: Nguyn Trn Hi Hong

Cy vo cc ng nghim nh cha khong 0,3ml huyt tng v 370C trong