15 .

Staphylococcus aureus
T cu khun
Nhm thc hin :
1) Hunh Phng Anh 2) L Th Khnh An 3) Ng Th Thy An 4) L Minh Ch 5) Nguyn Th Kim Cng 6) L Minh Duy 7) Trng Th Thanh Hin
8) Dng Minh Hong

0918005 0918019 0918022 0918052 0918056 0918074 0918149 0918161 0918200 0918201 0918225 0918355 0918596

9) Trn Phm Tu Hng 10) Th Xun Hng 11)Trn c Kin 12)Bi Th Kim Phng 13)V nh Uy Vin I) GII THIU

Chi Staphylococus l cu khun Gram dng, c gii thiu trong cun S tay H thng vi khun hc ca Bergey. c chia lm 2 nhm: Hiu kh (Micrococcus, planococcus, and Deinococcus) v k kh (Staphylococcus, Stomatococcus, Streptococcus, Leuconostoc, Pediococcus, Aerococcus, Coprococus, v Sacina). H Micrococcaceae gm 4 loi: Micrococcus, Stomatococus, Planococus, v Staphylococus. Thuc tnh khc bit ca cu khun Gram dng gm s sp xp ca t bo, hiu kh bt buc, hiu kh ty hoc vi hiu kh, k kh bt buc, phn ng catalase, s hin din ca cytochrome, sn phm ln men k kh ch yu t carbohydrates, peptidoglycan, acid teichois trn thnh t bo, menaquinones. S phn loi ca Staphylococus aures nh sau: Gii: Procaryotae. Ngnh: Firmicutes. Lp: Firmibacteria. H: Micrococaceae Chi: Staphylococcus. Loi: Aureus. Hin ti c 32 loi Staphylococcus c xc nhn. Mt vi loi khc ang c nghin cu. Bng 1 l danh sch 332 loi c tm thy trong h ny. Tn Staphylococcus c ngun gc t ting Hi Lp, Staphylo c ngha l chm nho v cossus c ngha l qu trong chm, do Staphylococcus c ngha l qu trong chm nho. Staphylococci l t bo hnh cu c ng knh t 0.5 n 1.5m, n c th tn ti nh cc t bo n l, thnh cp hoc thnh cm. Cm hnh thnh ch yu khi tng trng trn mi trng rn v l kt qu t s phn chia t bo theo nhiu mt phng, kt hp vi xu hng cc t bo con gn nhau. S hin th trong khng gian 3 chiu th hin r s gn kt bao ph ln nhau ca cc t bo, tuy nhin, cc t bo mu thng mang li s xut hin ca cc phin bt thng ca t bo. Staphylococci l vi khun Gram dng, khng di ng v khng sinh bo t, nang c th c hnh thnh trong nui cy, nhng ni chung l khng xut hin trong pha tnh t bo. Sc t khun lc trn mi trng khng chuyn
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bit nh tryptic soy agar c th nm trong khong t mu trng kem n m cam sng. Hu ht cc chng ca Staphylococci c xem l nhm cc tc nhn gy bnh mc II (potential hazard). Staphylococcicus hyicus c xp vo nhm cc tc nhn gy bnh mc IV (potential danger, cc k nguy him). Loi Staphylococal l cc vi khun hiu kh hay ym kh ty v c c h hp v ln men bin dng. Staphylococci thng qua chu trnh glycolysis, ni hexose monophosphate, v chu trnh tricarboxylic acid. Chng th dng tnh vi catalase va c th s dng mt lng ln carbonhydrates. Staphylococcicus aureus l loi ph bin nht trong chi Staphylococcicus. N cn s c mt ca amino acid v vitamin cho s tng trng hiu kh, v ngoi ra : uracil, ln men ca amino acid v vitamin cn cho s pht trin ca sinh vt hiu kh. Trong khi S. aureus c kh nng tng trng k kh v tng trng tt nht trong iu kin hiu kh. Nhit ti u cho s tng trng k 350C v gii hn chu ng l t 10 n 450C. Gii hn chu ng ca pH cn cho s pht trin l t 4.5 n 9.3, pH ti u l khong gia 7.0 n 7.5. Khi iu kin mi trng tr nn kht nghit hn, gii hn chu ng ca pH gim. Thnh phn ch yu ca thnh t bo S. aureus l peptidoglycan, v n chim 50% trng lng t bo v quy nh hnh dng bn vng. Peptidoglycan l mt polymer polysaccharide khng phn nhnh, gm cc lin kt (1-4), chui c cha xen k cc n v N-acetylglucosamine, vi khang 60% N-acetylmuramic acid residues o-acetylated. Lung ln O-acetylation gip cho t bo khng c s thy phn ca lysozyme. Pentapeptide lin kt bn vi lng d acid muramic, v lin kt ng ha tr ca pentaglycine vi L-lysine ca mt chui D-alanine khc. Phc hp peptidoglycan l mt nhm ca chui polymer phosphate vi acid teichois. Acid Teichoic (chim 40% trng lng vch t bo) c th lin kt cng ha tr vi peptidoglycan hoc mng t bo. Xng sng ca thnh t bo teichoic acid l mt chui xen k ca ribitolphosphate lin kt vi D-alanine bi s lin kt hoc -glycosidic xuyn qua N-acetylglucosamine.
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Cc thnh phn chnh ca v ca S.aureus

l acid N-acetylaminouromic v N-

acetylfucosamine. Mui mt kiu nhm huyt thanh c xc nh, vi cc gen sn xut v nang nm trong 1 operon. Kt nang vi khun c kh nng chng thc bo. Mt vi chng S.aureus c kt nang, v chng c nguy c gy nguy him hn cho ng vt. Bao nang thng c pht hin trong c th sng nhng li khng xut hin khi nui cy. Phm vi mu sc t mu trng xm, n m vng cam v mu cam. S tay ca Bergey ni rng cc sc t triterpenoid carotenoids hoc cc dn xut ca chng nm trong mng t bo. Sc t thng co sau 18-24 gi cua s tng trng 37 0C, nhng co mau ro rt hn khi me nui cy c ct gi nhit phong trong vong 24-48 gi. Sc t khng c tao thanh trong qua trinh tng trng ki khi hoc trong me nui cy long. Noi chung S. aureus tng trng va canh tranh an ap cac sinh vt hin din khac. Mc d gi tr D khc nhau vi cc loi v nhit ca dung mi, nhng mt gi tr D gim i sau 3 pht ti 60oC cho thy rng S.aureus th khng chu nhit. Kha nng chiu nhit chiu anh hng cua pH, Aw , va thi gian cua me nui cy. un nong cac san phm thit n nhit khoang 770C thng la u git ht S. aureus. S.aureus khang vi nhit thp. Nhng lai rt nhay cam vi mi trng co tinh acid va mt loat cac hp cht hoa hoc va thuc khang sinh. II. BNH NGOI NG RUT :

Staphylococci la mt phn binh thng cua h vi sinh cua c th ng vt, chung c tim thy trn b mt da va toc, trong mui, ming va c hong. Staphylococcus epidermidi, xm chim hu nh tt ca cac b mt biu bi, trong khi S. aureus xm chim chu yu mang nhy. Cac sinh vt nay xm chim vao tre s sinh trong 1 vai ngay sau khi sinh. Polledo et al a nghin cu x ly trn 300 mu thc phm. Ho phat hin rng co 83 (27.6%) la th truyn bnh co tu cu dng tinh vi coagulase (enzyme gy ng tu) va 118 (39.3%) dng tnh vi coagulase t cu m tnh hc mi. Thu vi hn la nam gii c phat hin co mang tu cu dng tinh coagulase nhiu hn so vi n gii. Ba mi sau ca th c phat hin co tu cu enterotoxigenic. Ty l nay trai ngc vi ty l 4050% theo bao cao cua Bergdoll. Th truyn bnh khng co triu chng cua tu cu co th
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c chia lam nhiu kiu: th truyn bnh man tinh, ngi mang bnh nui dng mt loai S. aureus trong thi gian keo dai, th truyn bnh/ ngi mang bnh cp tinh, ho khng thng xuyn mang Staphylococci gy bnh, lc c lc khng, ho mang mt loai cho 1 thi gian nht inh, va sau o mang 1 loai khac; ngi khng mang mm bnh, la nhng ngi him khi hoc khng bao gi mang mm bnh c hai S. aureus. Ngi mang bnh/ th truyn bnh co triu chng nhim S. aureus la nhng ngi mang bnh cng khai. Con ngi c coi la mi trng sng cua S. aureus trong t nhin. Staphylococci co th gy bnh hu ht cac m trong c th, S. aureus.la nguyn nhn ph bin nht gy bnh nhim trung suppurating ( nh mun nhot, ni mun) va la mt trong cac vi khun gy bnh lu nht. Mt trong cac bao cao u tin cng b v bnh do S. aureus la vao nm 1871 vi nha bac hoc ngi c - Van Reclinhausen, ngi a quan sat khun cu, ma ng a c lp t thn cua 1 bnh nhn a cht sau mt cn st. Cac tu cu c coi la tac nhn gy bnh c hi ma qua trinh xm nhim thng la qua da hoc qua mang nhay. Cac tn thng thng gp nht la 1 ap xe trong da hoc vt bong, ma vung trong tm cua nhim trung thng co mu mt phn hoc hoan toan, co vach bao quanh xut phat t cac m xung quanh. Cac vung cua s hinh thanh ap xe thng la nang long va tuyn m hi. Nhot la mt loat cac ap xe kt ni vi nhau. S ly cua nhim trung trong c th, cac c quan chinh va cac m co th xay ra bng cach ly lan qua cac m tip giap hoc ly lan qua h bach huyt va mau. Tn thng di cn c tao ra trong mt loat cac m. Nhim trung nghim trong hn co th xay ra nhng ngi nhay cam nh: tre em, ngi bi bong hoc chn thng, va ngi ri suy nhc man tinh (ung th, x nang vv). Bnh tu cu nghim trong thng c gy ra bi tu cu khun m tinh coagulase. Chung xm nhp vao c th qua cac vt thng h cac m biu bi nh: tim chich, ng thng, hoc cac dung cu y t khac. Mt h thng bach cu binh thng giup c th co th chng lai s xm nhp cua cac vi khun nay, s bt thng cua cac min dich dich th co th gop phn lam cho c th d mc bnh. Bnh tu cu co th c ly lan bng hai cach la thng qua h hp va do tip xuc vi da va qun ao cua ngi bnh. Bnh tu cu thng
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co bnh vin vi 90% cc bin th c c tnh khng peniciline. Cc bin th xut hin nhanh chng trong mt vi bnh nhn sau khi c iu tr bng liu php khng sinh. Mt s khng sinh khc ti ni mt vi bin th khng c erythomycin, neomycin, streptomycin, gentamicin, tetracycline, v chloramphenicol. C plasmics v nhim sc th staphylococus c tm thy m ha cho kh nng khng khng sinh. Mt s bnh nghim trng gy ra bi chng bao gm: nhim trng mu, vim mng tim, vim ty xng, vim phi. Mt s bnh khc nh: triu chng shock do c, vim rut kt, v ng c thc phm.

III.

C TNH

S.areus l mt loi vi khun c quan tm trong cng nghip thc phm. Ng c thc phm do Staphylcocus l nguyn nhn hng u trong cc v ng c thc phm khng ch ring Hoa K, m cn rt nhiu quc gia khc. N gn nh c th so snh vi Samonella nh l mt trong nhng tc nhn gy ng c thc phm ph bin nht v c cho rng l dn u cc nguyn nhn gy ng c thc phm. Ng c do Staphylcocus din ra l kt qu ca vic tiu th c t enterotoxin chu nhit c sn xut bi cc vi khun trong sut qu trnh sinh trng. A. Cc hot ng trong nc Mt l do m S.aureus c quan tm nghin cu l do kh nng pht trin 0.83 a w tc l rt thp so vi s pht trin ca cc loi vi khun. Phn ln cc bin th S.aureus th c kh nng chu ng cao trong mi trng mui hoc ng c th pht trin trong iu kin vt qu 0.83 n 0.99 aw. S.areus pht trin mnh nht trong iu kin khong ln hn 0.99 v s sinh trng iu kin aw thp ph thuc vo cc iu kin tng trng khc mt cch thch hp. Ch s aw thp nht cho s pht trin ca chng ln hn iu kin k kh (0.90). S.aureus c th pht trin tt trong iu kin nc mui 16% (0.88aw) v ng 50% (0.97 aw).

B. Enzymes ngoi bo S.aureus sn xut mt lng ln enzyme ngoi bo. Iandolo (8) cho rng S.areus c kh nng sn xut t nht 34 loi protein ngoi bo khc nhau, mc d khng c dng n no sn xut tt c cc loi protein. C kin cho rng sn xut protein ngoi bo gip cho cc vi khun c th xm nhp vo cc m khe mnh v trnh khi b my min dch ca c th. Mt vi dng enzyme ny c gi tr nhn din vi khun. Bjorklind v Arvidson pht hin ra phn ln cc protein ngoi bo c sn xut vo cui qu trnh tng trng. 1. Coagulase Bng 2: Protein v c t ngoi bo ca Staphylococcus aureus Coagulase Endopeptidase Fibronolysin Enterotoxins A-E Elastase Protein A Lipase Leukocidin Collagenase Phospholipase Hemolysins Colagen Binding protein Nhn t Clumping Metalloprotease Penicillinase Serine Protease Nuclease Pyrogenic exotoxin Staphylokinase Acid phosphatase Alkaline phosphataseotein Fibronectin binding B Nhn t Matrix Adhesin c t gy hi chng shock c t trc da A v B Staphylococcal Lysostaphin Thiol protease Fibrinogen biding protein lysozyme Nhn t Sucinic oxidase

Fibronectin binding proetin a Elastin binding protein

Coagulase l mt trong nhng enzyme quan trng c sn xut bi S.aureus. Hai loi ca enzyme coagulase gm mt dng t do v mt dng nm trong t bo. Hai loi khc nhau v c tnh min dch v mt s hot ng nh. Coalgulase ngoi bo c th tn ti trong t nhin v c nhiu kiu c nhn dng. C 93-100% tng hp gia sn phm ca Coagulase v enterotoxin. S.aureus dng tnh vi coagulase c kh nng hnh thnh cc cc mu ng. Enzyme ny c sn xut bi cc bin th c c bit n
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c kh nng gy bnh do hnh thnh cc cc mu ng... Tuy nhin, tm quan trng ca hng ro chng li s ly nhim th khng ton vn, bi v nhng dng S.aureus m tnh vi coagulase cng c th l nguyn nhn gy bnh. S ng cc ca mu trong c th lin quan n s lng ca cc thnh phn. Mt cch vn tt, mt cc mu ng c nh hnh bi cc yu t ng mu c trong huyt thanh v c chuyn ha thnh mt mng li c tn l fibirn, Staphylococus aureus hot ng bng vic chuyn i fibrinogen thnh fibrin. Mt trong nhng l do cho vic s dng rng ri coagulase xc nhn dng tnh vi S.aureus l kim tra thc hin kh d dng v d nhn bit. T bo ca loi sinh vt nghi ng c trn vi huyt tng ca ngi hoc ca th (c thm vo citrate, oxalate, EDTA kh ion Ca, yu cu ng mu invivo), hoc s dng phng php xt nghim ng, hoc s dng phng php xt nghim da trn bn mu trn knh hin vi. Slide hay ng xt nghim c 370C trong 1 3 gi. Tuy nhin, cc phng thc hin ti c m t trong Bacteriological Analytical Manual (10), yu cu mt khi kt ng bn chc hin din trong ng xt nghim trt ngc th c xem l dng tnh. Kt qu ca phng php bn mu, ni no t bo kt thnh khi th c xem l dng tnh. Weer-Pothoff al.(11) so snh by coagulase test ca phng php nhn dng staphylococus. Nhn chung, cc th nghim thng mi cho kt qu nhy hn cc ng xt nghim thng thng hay xt nghim bn mu. 2. Nuclease chu nhit Nuclease chu nhit l mt trong nhng chn on quan trng ca S.aureus. Nhng Nuclease ny c sn xut trong qu trnh tng trng bnh thng c kh nng tn cng c ribonucleic v deoxyribonucleic. S.aureus cng c kh nng sn xut nuclease ngoi bo c kh nng chu nhit. S hin din ca hai loi nuclease (chu nhit v nhy nhit) c th l nguyn nhn to nn s khc bit trong cc chng loi. Gudding cho bit l S.aureus. S.intermedius and S.hylius Subsp. Tt c Hyicus u sn xut nuclease chu nhit. Nh tn gi ca n, neclease chu nhit v cng bn vng trong iu kin
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nhit cao v c th bn vng trong nc si trong vng 30 pht m khng c bt k mt bin i nghim trng no. Mt bi test din t cc hot ng ca nuclease chu nhit. Phng php dng Toluidine blue deoxyribonucleic acid agar c th dng nhn dng. Trn mt khi agar (trn c khun lc S.aureus c c lp pht trin) c ly mu c cy vo mt mi trng ng mu c un si trong 15pht. Tiu bn c lm bi vic ly mu Toluidine blue deoxyribonucleic acid agar cng vi mt cht agar tt cha khun lc. Mu c un si c th ch v c trong mi trng m t trong 4 gi 350C. Kt qu dng tnh cho thy vng mu hng sng ca DNA c thy phn. Ging nh coagulase, vic sn xut nuclease chu nhit v enteroxigenicity ging nhau n 95 ti 100%. Ratner v Strattion cho bit khng c s i lp gia mt th nghim gia thermonuclease v coagulase trn 250 mu mu ngi. Tuy nhin, Adekeye cho bit thermonuclese khng c hiu qu nhn bit S.aureus c c lp t ng vt (khng phi ngi). Ngc li vi Staphylcococal ngoi bo, nuclease chu nhit c sn xut trong sut qu trnh tng trng ca t bo. 3. Cc loi Enzyme khc. S.aureus sn xut nhiu loi enzyme ngoi bo khc nhau c gi tr chn on v gip th sinh bnh. Staphylokinase gy ra s tan khi khi mt vi lipase c kh nng to ra nhng vng tan trn thch trng c. Hyaluronidase c sn xut bi hu ht cc dng ca S.aureus, l mt loi enzyme thc y s lan ta trong m bi vic kh trng hp ca hyaluronic acid. Nhng loi enzyme khc c sn xut bi S.aureus bao gm c penicillinase, protease (collagenase, elastase, endopeptidase, metalloprotease, serine protease, thioprotease), lipase (phospholipase v phosphatase acid v alkaline), fibrinolysin (bin i fibin thnh cc sn phm ha tan), protein A (dng phc hp ca lp mng ngoi ca thnh t bo lm suy yu oposin ha bi huyt thanh, v lytic enzyme (staphylcococal lysozyme v lysostaphin). C. Toxins Hemolysins v cc loi khc

Hemolysin, cng tng t nh staphylolysin, c sn xut bi S.aureus v c t tn l alpha, beta, gamma v delta; mi dng n s sn xut mt hay nhiu emolysins. Nhng protein ngoi bo khc bit bi kh nng dung gii hng cu ca chng trn nhng loi ng vt khc nhau. Gamma hemolysin v Panton- Valentine leukocidin (phn gii leukocyte) c hp thnh t hai cu t, mt trong s c c hai. Tt c ngoi tr delta-hemolysins to ra vng tan bao quanh khun lc S.aureus khi pht trin quanh thch mu. Staphylococi sinh m phn gii hemoglobin u phn ng dng tnh vi coagulase. Mt s c t khc c sn xut bi S.aureus l exfoliatin, leukocidin, pyrogenic exotoxin (29,30). S tng phn vi enterotoxins vi khun gram m ( c sn xut bi protein chuyn ch c bit) l mt phn ca operon m ha c t. SEs c xem l exotoxins kt hp ca protein vi phn khng c phi protein (31). SEs hon chnh kt hp ca chui polypeptide n vi 230 239 amino acid, sp xp thnh phn t nng t 26,360 ti 28,494 v c tht nt (loop) disulfide gn gia phn t (Bng 4). Phn tch amino acid ca SEs cho thy s giu lysine, asparatate v glutamic acid(6) . (Bng 5). Ngoi ra, s tch in dng ca chng c th ng vai tr quan trng trong gy nn ma.

IV.

CC C T TRONG RUT
1) Cu trc.

Trnh t nucleotide ca cc gen m ha cho SEA, SEB, SEC1, SEC3, SSD, SEE ( sea,seb,sec1, sec3 ,sed v see) (32-37) v trnh t amino acid ca SEA, SEB, SEC1 (38-40) c bo co. SEs thuc v mt h enterotoxins ln gi l pyrogenic toxins ( PTs), gm c pyrogenic toxins A v B, TSST-1 , v streptococal pyrogenic toxins A-C ( SPE A-C) (41-47). PTs tit l nhng gi tr v mt sinh hc, bao gm cc tc nhn ca s nguyn phn t bo lympho, s chn min dch, s st, v lm tng tnh nhy cm vi endotoxin. So snh gia cc PTs c th nhn ra 43- 85 % trnh t nucleotid c trng( 33). Nucleotid c bit v amino acid tng ng gia entC1, entB v SPE A c nhn ra, trong khi entC1 v entB ging nhau hn vi SPE A(36). Couch v Betley (33) bo co rng SEC3 c 98% trnh t nucleotide tng ng vi SEC1, trong khi SEC1 v SEB th hin 68% amino acid tng ng. C SEB v SEC 1 c tng ng cu trc cc k
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cao, c bit l vng u amino v carboxy, vi tnh cht quyt nh khng nguyn ca n, trng lng phn t tng ng, cu disulfile n v khng c nhm sulhydryl t do (41,51,55,56). Vng u amino SEB v SEC1 th hin tnh khng nguyn chu trch nhim cho phn ng cho vi khng th huyt thanh (57), trong khi mc cao ca s khng tng ng u amino ca SEA c th gii thch l do khng huyt thanh SEA khng c phn ng cho vi SEB hay SEC1 (38).

SEA Amino acid (c t hon ton) (Gen enterotoxin) Trng lng pt (c t hon ton) (Gen enterotoxin) im ng in T l Nitrogen Liu gy nn ( ED50 g/ kh Hp th ti a( nm) im gy cht (Em) H s lng( Sow), S
a

SEB

SEC1

SEC2

SEC3

SED

SEE

233 257

239 266

239 266

239 266

238 266

228 258

230 257

27078 29700 6.8 16.5

28494 31400 8.6 16.1

27500 30511 8.6 16.2

27531 30608 7.0 16.0

27438 _ 8.15 _

26360 _ 7.4 _

26425 29358 7.0 _

5 277 14.3 3.04

5 277 14.0 2.78

5 277 12.1 3.00

5-10 277 12.1 2.90

<10a _ _ _

20 278 10.8 _

10-20 277 12.5 2.60

mi os 0.05mg/kg bi IV phn (19)

Ngoi ra, Huang v cng s (38) c th chng minh s tng ng cu trc c bit SEA bi cystein bn phn ti v tr 106 vi SEB v SEC1. Huang at al (58) t ra gi thuyt rng cystein bn phn ny c th gy nn ma v chu trch nhim cho s vim d dy. SEA hon chnh c 82, 72, 74 v 34 amino acid ph bin ging vi SEB, SEC 1, SPEA v TSST-1 ( 37, 39, 59-61).
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Amino acid Alanine Arginine aspartic acid Cysteine glutamic acid Glycine Histidine isoleucine leucine lysine methionine

SEA 1.9 4.0 15.5 0.7 12.6 1.0 3.2 4.1 9.8 11.3 1.0

SEB 1.3 2.7 18.1 0.7 9.5 1.0 2.3 3.5 6. 9 14.9 3.5 6.2 2.1 4.1 4.5 11.5 1.0 5.7 1.7

SECs 1.7 1.7 18.2 0.7 8.6 3.1 2.9 3.8 6.4 14.0 3.5 5.4 2.2 5.0 5.7 10.1 1.0 6.0 1.5

SED 2.0 3.4 16.7 0.7 13.2 2.7 2.7 6.0 9.3 12.9 1.1 408 1.4 5.1 4.5 7.2 0.6 4.1 1.7

SEE 2.4 4.5 15.1 0.8 12.2 4.1 3.0 4.3 10.1 10.8 0.5 4.5 1.9 4.7 6.4 9.8 1.7 4.4 1.7

phenylalanine 4.3 proline serin threonine tyrosine trytophan valine amide NH2 1.4 3.0 6.0 10.6 1.5 4.9 1.8

Bng 5: Thnh phn amino acid ( g/100 g protein) ca Staphylococcal Enterotoxin

Betley v Mekalanos (34) c th lai u d DNA ly t dng gene entA vi cc type khc ca SE, iu ny gi rng t l cao ca cc trnh t khc bit c th c gia SEC 1
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v SEB vng u carboxyl. Trnh t nucleotid ca SPE A tit l s tng ng c bit vi entB v entC1 (55,56), trong khi Bloomster- Hautamara v cng s (59) bo co TSST-1 c s tng ng trong hot tnh sinh hc nhng khng c trnh t amino acid no tng ng vi SPE A , entB v entC1. Couch v Betley (33) cng bo co s so snh ging nhau gia cc gene entC3, entC1 v entB, iu ny gi rng gen ging entC1 c c hnh thnh bng s ti t hp gia gene entC3 v entB. Couch v cng s (35) bo co rng gene entE c 84 % trnh t nucleotid tng ng vi gene entA. H cng bo co rng SEE c quan h gn vi SEA hn SEB, SEC1 hay SPE A. Nhn chung, t l phn trm ca trnh t tng ng cc amino acid gia cc dng protein hon chnh ca eterotoxins khc nhau t 29% ti 82 % , vi SEA v SEE l c quan h gn nht (35) . Bohach v Schlievert (36) gi rng s tng ng gia cc PTs yu cu nhng chc nng sinh hc thng thng, trong khi ngc li nhng vng a dng khc nhau quy nh tnh c c bit. Ngoi ra, Bohach v Schlievert (62) nhn ra nhng s tng ng khi so snh trong cc nghin cu khc v trnh t amino acid ca PT th hin rng: mt vi u carboxyl v vng trung tm ca trnh t s cp c bo tn ng u trong h PT. V d, H c th khm ph thm bng chng v khu vc carboxyl ca t cu khun v lin cu c t gy st c dng chung hot tnh sinh hc v nhiu phn ng cho epitopes. Hu ht cng vic c thc hin vo vic xc nh thnh phn acid amin, gen ent, nhng thuc tnh ha sinh ging nhau, s ging nhau thng thng ,v cc hot ng ca thuc sinh ha, cng vic lin quan n cc phn t trung gian nh hng ca SEs vn cn tng i cha c bit n. K t khi nhng cuc trin lm ging nhau ca Sec ng ch , Singh v Betley ngh rng cc trnh t cn thit c th cu trc ln v d lng axit amin cn thit cho chc nng sinh hc thng c bo tn trong Sec. 2) Tnh cht Tnh chu nng Tnh chu nng ca SE l ph thuc vo s tinh khit ca s iu ch, cc loi c t, s lng ban u, pH, dung mi t nng, v phng php ca s d tm. c t rut
13

non B c th hin bng cch gi li hot ng trn 16 gi 60oC v pH 7.3. Lm m cho SEB ngm trong nc si 30 n 40 pht khng c ph hy hon ton kh nng to ra nn ma mo v kh. Borja v Bergdoll chun b t nng SEC trong 30 pht 60oC vi khng c s thay i trong huyt thanh, trong khi t nng SEA 80oC trong 30 pht hoc 100oC trong 1 pht lm mt hot tnh ca n phn ng serologically. Ni chung, tnh chu nng ca SEA, SEB v SEC l SEC>SEB>SEA. Tuy nhin, iu cn lu l nhit bt hot ca n nhanh hn b m thi gian trong cc h thng phc tp hn trong cc tn hiu truyn i ca thc phm.V d, Denny v cng s ca mnh pht hin tnh n nh nhit tt hn ca SEA trong nc dung tht b so vi m phosphate, Fung v cc cng s bo co rng ti 60oC, SEC trong m phosphate c bt hot trong 60 pht so vi 180 pht trong mi trng nui cy. D121 ca SEB c phm vi t 9.9 n 11.4 pht v mt gi tr ca Z l 25.8 ti 32 oC. Tibana v cc cng s, v Bennett v Berry cung cp bng chng cho thy rng SEA, SEB, SEC, v SED c th tn ti tr li trong iu kin ng v nhit v thi gian. Kh nng khng phng x Cc c t xut hin kh nng khng phng x gamma. Read v Brashaw chng minh nhiu hn so vi 2.7 v 9.7 l cn gim tc dng ph ca SEb trong sa v m tng ng. Gn y Modi v cc cng s kim tra nh hng ca chiu x trong SEA bng cch s dng h thng elisa nhy. Trong m galatin phosphate, SEA hon ton bt hot bi phng x 8.0 kGy. Tuy nhin, 15% (mince slurry) 27-37% ca SEA vn 8.0kGy. Tht th v, tc dng bo v ca (the mince slurry) c nhn thy tp trung ca 30%, trong khi tc dng bo v gim khi (the slurry) tng trn 30%.

Kh nng khng enzyme

SES c kh nng chng li hot ng ca enzyme thy phn protein nh trypsin, chymotrypsin, rennin, v papain (ph>2.0). Tuy nhin, SE d b nh hng vi pepsin gi tr pH nh hn 2.0. y l kh nng chu c s tn cng ca nhng s thy gii protein h tr kh nng ca chng vn hot ng sau khi ung hoc hot ng duy tr thc phm trong nhiu nm.
14

3) Sn xut Cc t c t t cu khun c sn xut trong ngun giu dinh dng v cc ni truyn ngun dinh dng kh khn. Theo Smith v cc cng s cc yu t sau nh hng n c s pht trin v sn xut c t: kch c ca chng, nng oxy, nhit , pH, nng natriclorua, Aw, cc ion khong, v thnh phn truyn tn hiu. S sn xut ca SEs l khc nhau thnh hai nhm da trn c im sn xut. u tin cc nhm bao gm SEB v SECs, khi chng c sn xut vi s lng ln v ph thuc vo iu kin p trng hoc iu kin nui. iu kin th hai bao gm cc nhm ca SEA, SED, v SEE, khi chng c sn xut vi s lng nh hn, qu trnh sn xut lin quan cht ch n s tng trng ca s cng thng ch khng phi iu kin bnh. Ni chung, mt s yu t hin din trong thc phm xc nh c hay khng SE c th c sn xut. Bt k thc phm no c mi trng thun li v b nhim vi mt loi enterotoxigenic ca S.aureus l c kh nng gy ng c thc phm do t cu khun. Cc yu t nh hng n s tng trng v sn xut ca t cu v c t rut c xem xt v s c cp vn tt y. Ngi c mun tm hiu xa hn na v s pht trin ca S.aureus v sn xut ca SEs iu vt ra ngoi phm vi ca chng ny. Mc nhim ca vic sn xut SE l ng k.Mc cy cao hn s cho kt qu sn xut SE cao hn. Ngoi ra mc cy cao hn s gim thiu c p lc vi mi trng. SEs c chng minh c sn xut gii hn nhit 10 oC-45oC m ti S.aureus c th pht trin. Ni chung nhit h tr kch thch s pht trin c t trong thc phm. Phm vi pH sn xut SE l hi hn ch so vi s tng trng v c bo co pH gia 5.2-9.0. trong gii hn pH m ti S.aureus khng pht trin tt, t c kh nng sn xut c t rut non. Phm vi ti u cho vic sn xut c t rut non chu nh hng bi Aw. SEA (0.90) c sn xut gi tr Aw thp hn so vi SEB(>0.90). S sn xut ca SEs c th chu nh hng bi iu kin tng trng k kh hay hiu kh. Staphylocci c th pht trin di c hai iu kin. Tuy nhin pht trin di iu kin k kh kh s chm hn, v t bo thng khng t c nhng ci m di iu kin hiu kh. Mc d c t rut non thnh thong c pht hin c sn xut di iu
15

kin hiu kh, nhng s quan st ny c th thay i. Hin nay iu khng th chng minh cho gi thuyt rng c t rut non c th c sn xut trong thc phm ng gi. S xut hin ca SEs trong thc phm c thng bo(94). Xp x 25% dng S.aureus c phn lp ngu nhin trong thc phm cho thy s sn xut cc chng khc nhau ca SEs(95). Gourama et al.(96) cho bit rng s pht trin ca S.aureus t mc ti u trong s Chowder vi s lng t bo m c ln hn 108 cfu/g sau 12 gi nhit 42oC. S sn sinh ca SEs v SED bt u sau 3 ting. Thm vo , s pht trin trong nc luc nghu sn sinh ra lng SEA v SED vi nng ln lt l 21.9 v 36.3ng/mL, sau 24 gi 37oC. Tuy nhin, Daoud v Debevere tm ra s sn xut ca c t enteretoxin ch mc thp mc cho s tng trng tt ca S.aureus trong cc loi rau qu c un nng. c t enteretoxin l cc protein ngoi bo c biu hin mc cao v c th c sn xut mc vt ngng 100ug/mL, trong lc nhng c t khc c sn xut ngng thp (thng vo khong mt vi ug/mL)(2). Bt chp iu , phn ln cc v bng pht nhim c thc phm xy ra do cc kiu huyt thanh A v D. Mt nhn t chnh trong nhim c thc phm bi c t enteretoxin bi tt c cc type ca khun cu l do kh nng chu nhit ni bo cao ca chng, th m cho php cc c t tn ti c qua cc phng php x l nhit c th git cht sinh vt trong quy trnh sn xut. Vy nn, khng him thc phm c hi c tm thy trong m khng c staphylococci c th c phc hi. V mt di truyn, cc ni c t kh a dng. Gene (sea) ca ni c t A(SEA) c hnh thnh t mt nhm cc thc khun th n ha, ti t hp vi nhim sc th trong cc gen beta-c t (hlb) bt hot n v chuyn i th th thnh mt kiu hnh Hlb - SEA+. Mt s b phn ca nhm thc khun th ny cng mang gene cho staphylokinase (sak) v ng thi chuyn i th th thnh kiu hnh Hlb- Sak+ SEA+ (98-100). Gene (seb) ca ni c t B (SEB), ngc li, l gene nhim sc th, l mt phn ca yu t ring bit lc u c cho l thc khun th khim khuyt cao(101) nhng nhiu nhng bng chng gn y(102; R.P.Novick, personal communication) lin kt cc gene thnh nhm gy bnh di ng. Hn na, cc yu t ca tst v sab nm trn cng mt locus v loi tr
16

ln nhau. Cc gen ca ni c t C(SEC), E(SEE), G(SEG) v H(SEH) u c cho l thuc v nhim sc th(26), v d c kh nng, ngi ta khng bit n liu c phi l mt phn ca cc nhn t di truyn ln hn nh l cc gen ni c t A v B. Ni c t D (SED) c m ha bi mt gene nm trn mt staphycococal plasmid ln (pIB485 l plasmid nguyn mu), th m ng thi cng mang gene (sej) ca ni c t J(SEJ) (103), th nh v trc tip ngay trn mch trn ca sed v c sao chp theo hng ngc li. Khng c gene ni c t no l gene thit yu c i hi cho s tng trng ca S.aureus. V ngun gc ca chng c th bt ngun t cc yu t di ng, chng thng c xem l vt liu di truyn ph. Ngoi tr SEA, biu hin ca cc gene ni c t c iu ha dng bi protein ngoi bo tng th t cu kim sot mch agr(gene iu ha ph) (102). S biu hin ca b iu bin ny c kch hot trong pha lgarit mun, v chc nng ca n th c bit cn thit cho s phin m ca cc gene ca protein ngoi bo. Chnh v vy, s hot ha ca agr trong pha lgarit mun gii thch v sao cc ni c t (ngoi tr SEA) c sn xut trong pha cn bng ca s tng trng. SEA c sn xut trong pha lgarit ca s tng trng, s phin m ca gene khng nm di s nh hng ca mch iu khin agr(8). 4) Du hiu nhn bit Vic pht hin cc ni c t trong thc phm vn lun l mt vn . Ch mt vi ng vt c th thng qua phn ng nn loi b cc cht c c dng, v ng tin cy nht trong s ny l loi kh(20). M hnh nhim c ni c t ny th tn km, phc tp v i hi vic xy dng cc trung tm chm sc ng vt. Tuy nhin, phng php chun ca vic d tm l c tnh min dch v da vo cc phn ng kt ta c quan st trong nhiu nhng th nghim khuch tn gel khc nhau. Mc d nhiu cc c t c phn ng min dch cho, nhng phng php xt nghim Ouchterlony nh lng v nh cht cht c tinh khit chun thng c s dng nhn bit huyt thanh c th hin din. Nc chit t nhng mn thc phm c nghi vn c s dng trong xt nghim ny, nhng trong thc t him khi c nguyn liu cn st li sau mt t bng

17

pht c th thc s tin hnh c xt nghim ny, v mt chn on v nhim c thc phm thng c thc hin mt cch truy li. Khi mt thc phm b ng rng gy nhim c thc phm bi t cu phi c xt nghim tm kim s hin din ca S.aureus cn hot tnh v s hin din ca SE. iu ny l v kh nng chu nhit ca SE tt hn nu so snh vi S.aureus, n c th l thc phm ng vai tr l ngun gc ca mt dch ng c do thc phm nhim staphycococal nhng khng c s hin din ca sinh vt hu hiu. xc nhn mt t bng pht nhim c thc phm do t cu, iu cn thit y l ch ra s hin din ca SE trong mn thc phm lin quan. Cc quy trnh truyn thng pht hin SE th tn thi gian, vt v v thiu nhy. Th nghim thnh cng lc trc cho s hin din ca SE s dng mu nn ca kh vn cn c s dng n ngy nay. Mt mu thc phm ng cht vi nghi vn (thng l 50 mL) c a vo d dy ca nhng con kh nu tr (Macaca mulatta). Nhng con vt ny s c theo di lin tc trong nm gi tip . Nhng kt qu dng l s i ma ca cc con vt. Nhng con mo ( Mt mu kt qu tch cc trong nn ma ca con vt. Mo con (a vo mng bng hoc tim tnh mch) c dng thay th cho nhng ch kh trong d tm SE. Nhng xt nghim trn cc ng vt chu nhiu nhng rc ri nh kinh ph v nhn cng tham gia trong vic duy tr mt by ng vt, s tht thng, nhy vi nhng ng vt khc nhau, v s hin din ca cc cht gy nhiu trong mu. Cc phng php hin c dng d tm SE th kh nhy nhng b nh hng bi vic thu hi c t khng hiu qu sau khi tch chit t thc phm, c bit l khi nng c t trong thc phm thp. Ni chung, hu ht cc loi thc phm lin quan n vic bng pht nhim c thc phm do staphycocal u cha lng c t ngng thp (<1ug)(19). Lng c t ti thiu cn thit cho cc triu chng lm sng c cho l vo khong 0.1ug/100g thc phm(64). V vy, xc nh an ton ca thc phm c cung cp, cc phng php d tm phi pht hin c t nht 1ngSE /g thc phm (104). Cc phng php s dng kim nghim huyt thanh trong ng nghim c pht trin p ng cc tiu ch v nhy vi SE. Cc xt nghim c lin quan n nhng
18

phng php vi nhy cao bao gm cc xt nghim th ng v vn cc th ng d tr (PHA v RPHA), xt nghim ngng kt th ng d tr (ELISA), v xt nghim min dch pht quang (LIA). S pht hin SE trong cc thc phm c truyn thng s dng cc khng th hnh thnh trong c th chng li s tim nhp ca cc khng nguyn ha tan, v d nh ni c t. Nhng khng th ny sau c th phn ng in vitro vi khng nguyn to thnh kt ta. H thng da trn xt nghim. H thng da trn th nghim lin quan n huyt thanh hc c th c phn thnh ba loi: (a) xt nghim gel khuch tn min dch, (b) xt nghim ngng kt , (c) xt nghim nh du min dch. TIU BN HIN VI Phng php truyn thng nh kho nghim microslide, kho nghim immunodiffusion gel c s dng cho pht hin ca SE. Kho nghim microslide l da trn s hnh thnh kt ta trn mt ma trn thch v cc th tc ang c ngh cho pht hin ca SE. Th nghim khuych tn microslide gel ny c s dng hnh dung kt ta v c m t chi tit trong Ti liu Phn tch Vi khun hc (10).. Nh th hin trong hnh. 1, microslide c chun b vi s tr gip ca mt mu, chng hn c nm ging c hnh thnh trong agar Noble tinh khit cao Khng huyt thanh c thm vo trung tm, v c t tham chiu c t trong ging 3 v 5 (Hnh 2). Mu chun b c thm vo cc ging 2 v 4. Cc slide sau c trong mt cn phng m nhit phng trong 3 ngy. Khng nguyn (enterotoxin) v khng huyt thanh khuch tn t cc ging, cc hnh thc kt ta ni tp trung ca khng nguyn v khng th c t l ti u. Slide sau c c bng cch gi n trn mt ngun nh sng v chng li mt nn ti. Nhng s kt ta c xc nh xuyn qua s kt t ca n vi tuyn c s (ca) s kt ta. (Hnh 2). t nht l 0,1 mg / ml hoc t hn ca SE c th c pht hin bng cch s dng phng php ny. Tuy nhin, mc nhy cm l kh t c (21), trong khi nhng ngi khc bo co mt s bt cp trong k thut ny. V d, Pereira et al. (105) pht hin ra rng s sinh ra mc thp ca SED (ng / mL) chng S. aureus c th trn
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cc phng tin truyn thng phng th nghim, khng pht hin c bng phng php khuch tn gel, c th sn xut SED (ng/g) trong thc phm gy ra ng c thc phm. Ewald v chris-tensen (106) bo co rng 51% ca 47 chng S.aureus th nghim sn xut SE bng k thut ELISA, trong khi ch 35% dc pht hin bng k thut khuch tn min dch microslide. MU XANH Vic s dng ca PHA v RPHA c xut khong 30 nm trc (107.108). PHA s dng cc khng th c th nng khng i trong khi mu c kim tra khc nhau. Sau bnh, x l khng nguyn t t bo mu ca cu (SRBC) c thm vo. Hemagglu tination (HA) xy ra khi khng th khng b rng buc vi khng nguyn. L mt thay th, RPHA utiizes khng th c th c gn trc tip vo SRBC Hemagglutinstion thng xy ra trong thi gian bnh 2 gi. nhy ca nhng k thut ny nm trong mt gii hn pht hin 1 ng / mL. Tuy nhin, do nhng kh khn lin quan n ngng kt SRBC v lp ph khng c hiu gy ra bi cc thnh phn thc phm, cc xt nghim ny khng c ph bin. khc phc nhng vn ny, Igarashi et al. (109) pht trin RPLA. Kho nghim ny s dng cc ht cao su ph cc khng th c th. Tuy nhin, khi mt b RPLA thng mi c so snh vi mt kit ELISA thng mi, RPLA khng th pht hin cc yu cu 0,1 mg SE/100g phn li thc phm (110). Fujikawa v Igarashi (111) sau ci thin RPLA bng cch s dng ht cao su mt cao, t c mc pht hin yu cu. Cc xt nghim huyt thanh hc cho thy nhiu ha hn nh du c hiu immunoassays, trong bao gm RIA, ELISA v LIA. Nhng xt nghim nh du c hiu c th c nh ngha l mt loi xt nghim rng buc s dng phn ng khng nguyn khng th c s v mt ng v phng x, phn ng enzyme hoc phn ng pht quang nh im nh du. RIA, cung cp nhy cao v kh nng xc nh mc ca SE, l mt trong nhng nh du c hiu u tin xt nghim min dch ultized SE pht hin. Tuy nhin, RIA khng c s dng rng ri v nhng ri ro v chi ph sn xut v x l ng v phng x.
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Mt gii php thay th cho RIA cho pht hin ca SE, trong cho thy trin vng rt ln, l phng php ELISA. ELISA c phn loi l mt trong hai ng nht, ni m cc xt nghim min dch b rng buc vo mt ma trn rn. Kho nghim heterogeneou l tip tc chia nh thnh cc bnh sandwich ELISA (hnh 3 v 4) v ELISA trc tip (Hnh 5 v 6). Cc th nghim ELISA da trn hai gi nh hp l: (a) cc khng th hoc khng nguyn c th c gn lin vi mt s h tr vng chc pha v vn cn gi li hot ng, v (b) hoc cc khng nguyn hoc khng th c th c gn lin vi mt enzyme v phc tp gi li c hai immuological v cc hot ng enzyme ca n. Cc khng nguyn, cng nh cc khng th c th c gn lin vi mt lot cc vt th rn, chng hn nh a giy, polyvinyl hoc polystyrene, duy tr hot ng v c hiu. Tng t mt s cc enzyme (perox-idase, -galactosidase, vv) thnh cng trong gn lin vi c hai khng nguyn v khng th. Cc xt nghim cnh tranh sau c th c thc hin cho c hai nh lng v xc nh s lng khng nguyn hin din. (hnh v v k hiu) K hiu s 3 Khng th thun (k thut ELISA). Khng nguyn c b sung vo mng lai vi khng th chuyn bit; ra sch cc khng nguyn khng bt cp; phc hp khng th th cp vi mt enzyme c thm vo; ra ln hai v b sung c cht khng mu, nu tip xc vi enzyme to sn phm c mu Mt th nghim xut cho SE pht hin lin quan n mt khng th, ELISA bnh sandwich gp i. Trong k thut ny, mt khay polystyrene c bao ph vi cc khng th chng li SE v khay ra loi b nhng khng th khng bt cp. Mt vn c t ra t thc phm nghi ng c cha SE, v sau c vi mng lai khng th bng vt liu tng hp. SE cn nh li c khng th gi c nh trn mng lai, trong khi ra cc cht khng bt cp. Mt phc hp (khng th chng li SE phc hp vi enzyme peroidase) c hnh thnh trn mng. Ra nhng phc hp tha. Cui cng, H 2O2 (c cht peroxidase) c b sung. T l H2O2 oxi ho ph thuc vo s ca enzyme c gn trn khng th, v th cng tu thuc vo s lng ca khng nguyn trn mu kim tra. S phn ng enzyme c xc nhn thng qua vch ph theo iu kin ca mu sc bin i do thuc th 5-amino-salicylic acid.
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(hnh v v k hiu) K hiu s 4 Khng th gin tip (k thut ELISA). Khng nguyn c b sung vo mng lai vi khng th chuyn bit; sau khi ra, khng th th cp c thm vo; ra ln 2 c trnh din v phc hp ca khng th c hnh thi i ngc v enzyme c thm vo; sau ln ra cui th 1 c cht khng mu c b sung; nu tip xc enzyme to sn phm c mu. (hnh v v k hiu) K hiu s 5 Khng th cnh tranh (ELISA trc tip). Khng nguyn c kim tra v khng nguyn enzyme chun c thm vo mng lai cng mt lc vi khng th chuyn bit; mng c ra v b sung c cht khng mu; nu tip xc enzyme to sn phm c mu. Phng php chn on bng khng th thun c s dng u tin bi Saunders and Barlett (115) pht hin ra SEA trong thc n. Ti nay, phng php c chnh sa v nh gi mt cch chnh xc cho tt c SE (116). Freed et al (116) hn nh 0.1ng ca SE/mL trong mu thc phm s dng git polystyrene nh cht h tr. Ocasio v Martin (104) thu c hn nh l 0.5 ng SEB/mL trong 1 mu thc phm bt k. Thm vo , Gomez-Lucia et al. (117) tm ra hn nh l 0.625 ng SEA/mL sa v 1 ng SEB/mL sa, trong khi Fey et al. (118) so snh s ging nhau ca cc phng php chun on ELISA v kt lun rng phng php chun on bng khng th thun l cch tt nht. (hnh v v k hiu) K hiu s 6 Khng th lin tip (ELISA trc tip). Khng nguyn c b sung vo mng lai vi khng th chuyn bit; sau khi s bt cp hon thnh, mng c ra v b sung c cht khng mu, s to mu tng xng vi s c mt ca khng nguyn ti . Mt cch thc mi c lin quan v c khc bit tip cn ti vit nh lng s hin din ca SE trong thc phm l LIA. K thut LIA s dng gn chemiluminescent cng nh l diazoluminol (119), isoluminol v cc dn xut mang tnh kim ca n, v acridium esters (120-122). Tuy nhin, hm lng thp (1%) luminescent c nh hng (t l photon pht ra cho s lng phn t c hnh thnh) n gii hn vic s dng LIA
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nhn din SE trong thc phm (123). T khi k thut LIA c gii thiu, nhiu s ci tin c ng dng. V d nh 1 dn xut ca isoluminol [N-(4-aminobutyl)-Nethylisoluminol hemisuccinamid; ABEI-H3], cht ny sn xut ra mt s lng lng t cao khi to phc hp vi proteins (124,125), c s dng bi Lohneis et al. (126) kim tra SE trong thc phm. Th nghim immunoluminometric ca h (ILMA), c s dng vo tip hp ABEIH-IgG, ch c th pht hin 0.40 ng SEB/mL, trong khi ELISA c th pht hin thy 0.15 ng/ML. Tuy nhin, S dng ILMA cho 30 pht mu bnh tng ng vi 90 pht s dng ELISA. Mt v d khc l s la chn hp cht hiu ng pht quang cho cc phn ng. Cch chn la ny s dng enzyme horseradish peroxidase (HRPO), hot ng nh cht xc tc hoc mt cofactor trong phn ng oxi ha ca luminal H2O2. S tip hp enzyme horseradish peroxidase vi khng th th n nh v c s dng trong cc xt nghim LIA cng nh ELISA. Cng nh cc phn ng pht quang, phn ng luminal-H2O2 cn l phn ng lng t thp (1%). Vn ny c gii quyt bng cc s dng cc enhancer ca phn ng luminal-HPRO-H 2O2. Cc enhancer nh cc cht phenol thay th (vd: p-iodophenol) c cung cp nhiu gp 2500 ln trong nh sng pht quang hn phn ng khng c enhancer. Ocasio v Martin (129) tng s dng phn ng ny ci tin k thut xt nghip h min dch bng hiu ng pht quang enhancer (CLIA) cho SEB trong thc phm. V. CC TN THNG GY T VONG Ngnh cng nghip thc phm thng tn dng mt s cc dng qu trnh hoc tch ly nhit thp nhm ko di thi gian bo qun cht lng ca cc thc phm d hng (130). Quy trnh bo qun thc phm, bao gm cc cht bo qun ha hc, sy kh, ng lnh, gi nhit, phng x hoc kt hp cc phng php trn, c thit k gim thiu hoc loi tr cc t bo thc vt gy bnh v cc loi sinh vt gy thi ra trong cc sn phm thc n. Mc d hu ht cc sinh vt c kch thc micro b tiu dit, nhng cc t bo b tn thng hoc b stress c th cn sng st v phc hi sau . Cc t bo ny thng d gy hi ngc li cho mi trng hn hoc cc khng th khng t bo trn. Cc khng th ny cng c th b tc ng bi cc tc nhn chn lc c k trn.
23

Nhng hin tng ca s ti phc hi cc t bo tn thng hay b stress c tm thy trong mt s hng ha, vt phm. Cc iu kin gy tn thng t bo khun t cu bao gm sc nhit (132,133), ng lnh v ng lnh kh (134), bc x (135), gim ht nc(136) v tip sc vi mt s ha cht khc nhau nh axit v mui (137,138). Mt vi v tr b tn thng c tin hnh trong cc t bo ny. Cc bo co ph bin nht l mng t bo cht vi s tht thot tng hp ca cc cht cu thnh t bo cht (132,136,139,140) v s thoi ha ca cc ribosome v RNA (141,142). Cc v tr tn thng khc gm s bin cht ca protein v s bt hot enzyme. 1. Nucleic Acids S nh hng ca RNA ln u tin c tm thy khi cc nh xt nghim tm thy cc mt t huyn ph nhit ca S.aureus b mt 260 nm vt liu ht nc. Iandolo v Ordal (132) cho rng 260 nm vt liu ht nc trn thot ra t S.aureus b sc nhit l RNA hoc cc Rnu. Cc nghin cu sau cho thy rng cc vt liu trn do s thoi ha ca RNA ribosome v cc thnh phn trong t bo (141,133). S thoi ha ribosome v RNA ribosome trong invivo l kt qu ca s sc nhit trn S.aureus (141,144,145). Phn tch gradient nng ca sucrose trong ribosome t t bo sc nhit cho thy rng tiu n v 30S b tiu dit c chn lc. Hin tng chuyn in t trong gel polyacryamide ca RNA ribosome chit xut t t bo sc nhit cho thy RNA 16S b thoi ha trong khi RNA 26S th bnh thng. Rosenthal v Iandolo (141) cho rng sut thi gian sc nhit, RNA 16S hu nh b thoi ha hon ton v RNA 23S th bin i. S sc nhit trong t bo vi khun vi s c mt ca cc ion phosphate quy nh th thc y s thoi ha RNA bi s kch thch ca hot ng ribonulease v bi s ph v ca s n nh ribosome do chelation ca Mg 2+ ni bo. V vy, s thoi ha ny ca ribosome vi khun l do cc cu ni hidro b ph v. S phc hi cc tn thng tng ng vi giai on ti phc hi ca t bo. V tn thng ribosome v s thoi ha RNA ribsome l thng tn ch yu trong cc sinh vt stress nhit, c th thy s tng hp RNA ribosome v n v ribosome l ch yu trong qu trnh phc hi (130). Iandolo v Ordal (144) co rng s tng hp RNA l cn thit cho s phc hi. H cn
24

khng nh s tng hp RNA xy ra trc c s tng hp protein v din ra ngay c khi s tng hp protein b c ch. th nghim tip theo, Rosenthal v Iandolo (141) tm thy ch tiu n v 30S v 16S ca RNA b thoi ha trong qu trnh sc nhit. Tuy nhin, tiu n v 50S, cng nh 30S, c ti sinh sut qu trnh phc hi trong s tng hp protein. Flowers v Martin (148) xc nh qu trnh n v ribosome sut s phc hi ca t bo sc nhit S.aureus. Cc t bo phc hi c tm thy c cha 3 phn t ribonucleoprotein (49S, 26S v 30S). Hiu ng chuyn in t gel polyacrylamide cho thy phn t 49S cha RNA 23S, phn t 36S cha RNA 16S v tin 16S. Cc phn t vi c tnh lng ng tng t c tm thy trong cc t bo khng b sc nhit. 2. c t Oxygen Phn t oxy ch hon tan mt lng nh trong nc, vi ch 9mg/L c ha tan 20oC v 1atm (130,149). Ch 7mg O2/L ha tan trong cht dinh dng nc hm tht 30oC. Tuy nhin, cc phn t oxy hon tan gp t 7 n 8 ln trong dung dch hu c, v nh n hu nh tr nn c c trong mng t bo ht m (150). Phn t oxy l thun t v hai trong cc electron ha tr khng bt cp vi nhau. Dng phn t oxy ny cc cu hnh nng lng t nht vi cc electron khng bt cp song song v chuyn in t cho t hoc Ozon. Ozon c th cung cp nng lng cho cc Oxy n phn, m c hai e t do trong vng xoay khng i xng. Hai dng oxy n phn tn ti trong nng lng bt i xng. u tin, mt trong hai phn t c nng lng yu hn bin i khi cc e t do ca trng thi n nh tr nn bt cp trong cng mt qu o v trong vng xoay vt i xng. Phn t cn li bin i khi mt e b a vo vng xoay nghch chuyn, vi hai e t do cn li trong qu o khc. Oxy n phn l phn ng c bit v v th e doa cho ton b thnh phn ca t bo (150). Trong khi n c i sng ngn trong mi trng nc, n c th gy tn thng nht cho cc vng lipid/protein., v nh mng t bo, ni n tip xc vi cu ni C-C trong polyunsaturated fatty acids, v l ni oxy n phn khng b ngn chn v c th tn ti lu hn (149)

25

Ozon cha hai e t do ca vng xoau i xng. Cc vng xoay electron i xng ny ngn hng vo ca cc cp electron (149). V vy, qu trnh gim ha tr xy ra, mt e spin s b nghch chuyn trch s sp xp 2 spin i xng trong mt orbital. Kt qu l, khi c nng lng, con ng univalent ca phn ng oxy c a tin hn con ng divalent. Sn phm u tin trong phn ng kh univalent ca phn t oxy l cc gc t do hydroperoxyl trong cc dng proton v gc anion superoxide dng ion ha (bng 6). Gc t do superoxide anion (O2.-) c th hot ng nh mt cht kh mnh hoc tc nhn oxy ha v cng c th bt u chui cc phn ng gc t do. Gc superoxide v H2O2 c th tng tc hnh thnh cc gc hydroxyl OH, mt cht oxy ha mnh nht c bit n. Cui cng, nguyn t oxy c th c to ra bi cc phn ng ca O2.- vi OH. (150) hoc bi s tng tc ca hai phn t H2O2 (151). Hydrogen peroxide c hnh thnh bi s kh hai electron phn t oxy hoc do phn tch superoxide. N l mt cht oxy ha mnh v do c tc dng dit khun, nh l mt chc nng ca thi gian, phi sng, v tp trung, hnh thnh bo t v khng hnh thnh bo t vi khun (152). Hydrogen peroxide c sn xut bi hu ht cc t bo sinh trng hiu kh (vi khun, thc vt v ng vt) bi hot ng ca superoxide dismutase (SOD) v mt s enzymes flavin lin kt. Hydrogen peroxide l phn ng n nh hoc t nht l ca cc dng trung gian trong con ng univalent, nhng n vn l mt cht oxy ha mnh c kh nng gy tn thng khng th phc hi cc thnh phn khc nhau ca t bo. Hydrogen peroxide l mt cht dit khun c s dng nh mt ansiseptic cc b trong nhiu nm (153.154). Hiu qu ca H2O2 thay i ty theo nng , nhit , thi gian iu tr, loi v s lng sinh vt (152, 155). Cch thc hot ng ca H2O2 trn cc t bo vi khun l rt ln. V d, H2O2 c th bt hot enzym bng cch thay i cc acid amin (156,157). Cc tc ng thoi bin ca H2O2 trn DNA c chng minh. Hydrogen peroxide phn hy DNA bng cch thay i cu trc bc 1 v bc 2. ln tng i ca t l suy thoi (158-161) nh sau: ph hy base > ph v si n > ph v si i > lin kt ngang.

26

Sn phm th ba trong s kh univalent ca O2 l cc gc t do hydroxyl. Gc hydroxyl v mt l thuyt c th c to ra trong mt s cch khc nhau. Beauchamp v Fridovich (162) cho thy cc gc t do hydroxyl c th c to ra trong s lng ng ch bi mt phn ng gia O2.- v H2O2 trong mt h thng nc. Gc OH. l cht oxy ha mnh nht c bit n v c th gy ra nhiu loi tn thng. 3. Oxy v cc tn thng gy t vong Biu hin cc nh hng c hi ca oxy l kt qu ca nhng n lc ci thin mi trng c s dng trong s c lp ca S.aureuns. Nhng nghin cu ny kim tra tc ng ca tc nhn phn hy H2O2 (catalase, peroxidase, hoc pyruvate) trn cc mi trng rn v cht lng khc nhau (137.138.163.164). K thut m kp trn mi trng Tryptic Soy Agar (TSA) v TSA cng vi 7,0% NaCl (TSAS) o mc stress c pht trin bi Iandolo v Ordal (132). Trc khi t nng, vic m S. aureus MF31 tng t trn c hai mi trng. Sau khi lm nng 520C trong 20 pht trong m phosphate kali 100mm (pH 7.2), m trn TSAS gim xung cn 0,001% c tm thy trn TSA, ch ra rng 99% tng s t bo b tn thng(137). Tuy nhin, khi catalase hoc pyruvate c thm vo TSAS, vic m cc t bo b thng do nhit tng gp 14.000 ln, khong 76% c tm thy trn TSA. iu kin c catalase cho cc chng staphylococcal khc trn mi trng agar chn lc cng c tc ng lm tng s t bo m c. B sung ca catalase ng thi vi s hin din ca phosphatidylcholine v phn chit tht b trong thch Vogel v Johnson c hiu qu trong mt mi trng cho s lng m c bng hoc tt hn so vi thch Baird-Parker (164). Mi trng thch Baird-Parker hin ang khuyn co dng cho thc phm v thuc. S kim sot (10) s lng m c ca S. aureus. Brewer et al. (163) pht trin mt s phng php c kh nng b bin i trong iu kin dng catalase hoc pyruvate. Phng php ny thng xuyn pht hin s hin din vi s lng thp ca S. aureus b b st bi cc phng php hin nay.

27

Quan st cho thy catalase hoc pyruvate lm tng ca s t bo m c ca S. aureus b nhit-stress c dng c 2 tc nhn thng qua s gim st ca H2O2. Bucker et al. (165) tha nhn cc t bo b tn thng do nhit phc hi gm c hiu kh v k kh. Cc t bo c tha nhn phc hi k kh cho thy khng c nhy vi NaCl, cho thy rng mc d mc catalase b gim, th c t hoc khng c peroxidase hydro c to ra. pH ti u i vi S. aureus MF-31 catalase l 5-6 (130.166). Cc enzyme n nh trong phm vi pH 4-9. Cc im ng in l 5,3 0,1 n v pH. Vi s ch cho s n nh nhit , S. aureus MF-31 catalase tinh khit n nh 520C sau 45 pht lm nng. Enzyme b bt hot 600C trong 10 pht. Tuy nhin, iu ny ph thuc vo nng ca cc enzyme v s hin din ca protectans. trng lng phn t cc n phn l 64.000 1.000 dalton, trong khi trng lng a phn l 235.000 5.000 dalton. Phn tch amino axit cho thy S. aureus MF-31 catalase tng t nh phn tch acid amin ca catalase t cc ngun khc. Hm lng st ca S. aureus MF-31 c tm thy l 0.098%. Enzyme b c ch bi nng milligram ca sodium cyanide, sodium azide, v hydroxylamine, cho thy s hin din ca cht xc tc kim loi nng. Tc dng ca mui, pH, nng mui cng nh hng ti tnh hot ng. Anion chloride c ch hot ng ca catalase pH thp, nhng c ch ny b nh hng bi cation. Natri clorua c ch pH thp hn so vi clorua, magi clorua. Hot ng superoxide dismutase c chng minh c h xung di nhit gy cht ca S. aureus MF-31 (520C, m phosphate) (167). Khi cc t bo c un nng 90 pht, nng SOD gim khong 20%, trong thi gian hn 99% ca cc t bo b cht. Tn tht quan st c trong hot ng l khng ln, nhng mc gim trong d tnh vi mc gim mc catalase c th ng k. T bo S. aureus nhit gn gy cht v c kho nghim mc SOD trong c qu trnh lm h hi v thi gian phc hi. C mt suy gim ng k (~ 85%) trong hot ng SOD lc bt u ca giai on phc hi. S suy gim ny xp x vi hot ng ca caralase trong qu trnh stress nhit. Mc

28

vn cn chm tr khong 2,5 gi cho n khi cc t bo bt u phn chia. Sau 2,5 gi, mc SOD tng ng thi vi s tng s lng t bo. VI. Pht hin v m C bn l do ti sao mt loi thc phm hoc bt k thnh phn no c kim tra xc nh s hin din ca S. aureus (130): 1. xc nhn s hin din ca S. aureus sau mt t bng pht ng c thc phm 2. xc nh c hay khng thc phm l mt ngun tim nng ca ng c thc phm do t cu. 3. chng minh nhim x l sau. 4. L mt phn ca mt chng trnh kim sot cht lng thng xuyn. N c quan st thy rng khi cc t bo t cu t nguy him c nhn mnh , nhiu ngi khng cn c th pht trin trn cc phng tin thng tin chn lc. Nhng l do cho vic ny tht bi, v cc phng php khc phc n, c nghin cu rng ri trong S.aureus. Vic la chn cc phng php pht hin ca S. aureus ph thuc vo mc ch thc hin kho nghim v sn xut c lin quan. Khi thc n b nghi ng l ngun gc ca s bng pht cc loi thc phm ng c t cu, s lng ln ca S, aureu thng xuyn hin ti v nhy cm phng php khng c th c yu cu. Phng php nhy cm hn l pht hin s lng nh ca S.aureus khng phi l sinh vt duy nht hoc thm ch chim u th trong mt mu. V l do ny, cc phng tin truyn thng c ch chn lc c s dng phn lp v m. Phng tin truyn thng la chn s dng mt s ha cht c hi khc nhau t c chn lc. Mt s nguyn liu s dng bao gm natri clorua, tellurite, lithium chloride, v cc khng sinh khc nhau. Mt s phng tin truyn thng c xut cho s c lp ca S. aureus t thc n khi c nhiu hn 100 gram c th hin nay. Mt s trong s ny bao gm Trung bnh Staphylococal 100, Agar Vogel-Jonhson, lng trng thch azide-natri, thch tellurite-polymyxin-trng lng , v Baird-Parker Agar (Bng

29

7). Thnh phn ca cc phng tin truyn thng tiu biu chn lc t cu c tm thy trong bng 8. Hu ht cc phng tin truyn thng chn lc ph hp vi iu tra ca S. aureus bnh thng hoc khng c chung m. Tuy nhin, do bo qun, ch bin, hoc tc dng ph khc iu kin, s cng thng it nguy him c th xy ra, kt qu l tng nhy cm ca S. aureus cho cc i l chn lc. Bi v cc t bo b thng tng nhy cm cho cc i l chn lc, S. aureus c th khng b pht hin trong cc th tc iu tra chn lc thng thng. Baird-Parker v Davenport (168) chng minh rng s phc hi ca cc t bo nng hoc kh ca S. aureus c th b mt hoc gim hot ng ca n bng cch nung nng hoc sy kh v mu, trong c catalase, b sung ca pyruvate, ng PED trong iu tra bng cch tiu dit H 2O2 sn xut bi cc t bo phc hi ca n thy rng thch Baird-Parker (BP) l t yu cu lit k cc t bo b thng khi so snh vi cc phng tin truyn thng chn lc t cu (169-171). Vic b sung ca catalase tryptic thch u nnh cng vi 7% NaCl (TSAS) (Bng 9) v cc phng tin truyn thng la chn khc c th lm tng cc lit k ca nhit nhn mnh cc t bo S. aureus, trong khi vic b sung nhit bt hot catalase c t nh hng trn iu tra (130, 137, 138). Vic b sung ca catalase khc la chn phng tin truyn thng c tm thy tng iu tra ca cc t bo c stress nhit v khng c chung m (138). Carlsson et al. (172) cho rng khi phosphat v glucose c hp vi nhau trong mt mi trng pH trung tnh hoc kim, cc sn phm kt qu nhanh chng t ng b oxy ha, to thnh nhng phn ng loi. Nhng n lc c thc hin pht trin mt phng tin chn lc t cu cung cp cho mt lit k bng thch BP trong khi khc phc kh khn trong vic s dng agar BP (v d vi cc sn phm sa) v chi ph ca qu trnh c m t bi Martin (130). Andrews, Martin (164) sa i Vogel v Johnson thch bng vic b sung cc chit xut t tht b 0,5%, 0,2% DNA, phosphatidycholine 0,2% (lecithin), v 780 n v ca catalase tri trn b mt thch trc khi tim chng. Vic tm thy ny phosphatidycholine-Vogel v Johnson.
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Bng 7 V d v thng tin chn lc ca Staphylococcus aureus Agar medium Staphylococus Medium 110 Vogel-Johnson Thnh phn chn lc Sodium chloride Lithium Potassium Glycine Lithium Egg yolk-sodium azide Potassium Ngun cp Mannitol Gelatin chloride Mannitol tellurite Tellurite Phenol red chloride tellurite Egg yolk

Baird-Parker

Tellurite Polymyxin B sulfate Lithium chloride Egg Potassium tellurite Tellurite

yolk

Bng 8 Phng tin truyn thng tiu biu chn lc t cu Thnh phn Nc mm Chit xut Chit xut tht b LiCl Glycine Sodium pyruvate Potassium phosphate dibasic Mannitol Phenol red Phosphatidyl choline DNA Agar Tellrite (1%) Egg yolk(50%) Catalase B-P(g/L) pH 7.0 10 1 5 5 12 10 _ _ _ _ _ 18 10 mL 53 mL _ PVCJ(g/L) pH 7.2 10 5 5 5 10 _ 5 10 0.025 2 2 16 10 mL _ 780 units VJ(g/L) pH7.2 10 5 _ 5 10 _ 5 10 0.025 _ _ 16 20 mL _ _

B-P = baird-parker agar; PCVJ = phosphatidyl choline-Vogel and Johnson agar; catalase v t Johnson agar Bng 9 enzyme VJ = Vogel and bo S.aureus stress nhit MF-31
31

Medium

Unstressed cells CFU/mL

Stress cells % enumerationb CFU/mL 100 90 108 87 100 79 90 77 87 56 64 108 110 2.4 x109 6.2 x108 2.9 x109 1.9 x109 2.5 x109 1.8 x107 1.2 x109 2.5x106 7.2x108 2.3x106 3.7 x108 8.0 x108 2.5 x109 % enumerationb 100 26 121 79 104 0.8 50 0.1 30 0.1 15 33 104

B-P VJ+ catc VJ+ catc TSA TSA +catc TSAS TSAS + catc MSA MSA+ catc S110 S110 + catc TPEY TPEY +catc

3.9x109 3.5 x109 4.2 x109 3.4 x109 3.9 x109 3.1 x109 3.5 x109 3.0 x109 3.4 x109 2.2 x109 2.5 x109 4.2 x109 4.3 x109

CFU = Colonyforming unit; B-P =Bard-Parker agar; VJ = Vogel and Johnson agar; TSA = Tryptic soy agar; TSAS = TAS +7%NaCl;MSA = Mannitol salt agar; S110= Staphylococcal 110 agar ;TPEY = telluritepolymysin egg-yolk agar. Nhng t bo b sc nhit dung dch m kali photphate 100 mM(pH= 7.2) 52 oC trong 15 pht. Phn trm ca s thng k c tnh bng cch chia CFU/mL trn nhng mi trng khc nhau, bi CFU/mL trn B-P v nhn vi 100. Catalase (cat) hot ha khong 780 n v trn 1 a. Agar (PCVJ) a n mt s lng lit k tng ng ca s stress t bo S.aureus v staphylococci t s nhim nhng mu thc phm mt cch t nhin m c tm thy
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cho s lit k B-P. S lit k vi cng c ny th d hn B-P v cho php xc nhn sn phm ca Dnase. Idziak v Mosse(173) bin i cng thc thch B-P bng cch thay i lng trng g bng huyt tng heo(B-PP) v nhn ra rng, da vo tnh chn lc, s m t triu chng v s gia tng tnh nhy cm, thch B-PP tt hn thch B-P. Lachia m t mt phng php n gin cho s thng k S.aureus t thc phm. ng y thay th lng trng trong thch B-P bng Tween 80(0.05%wt/vol) v MgCl2(0.1%). Khi nhng hn hp ny c thm vo thch B-P khng c lng trng g, s khi phc li s stress t bo c so snh phc hi li mi trng thch B-P hon thin. Metzer-Morgenstern v Katzenelson(175) pht trin mt staphylococcal mi trng c chn lc xc nh nh 4-S agar. Mi trng ny cho php c lp, xc nh staphylococci v t c trong bc u tin. Coagulase m tnh staphylococci dng nh, cm khun xm n xm m xung quanh dy c, trng c. Khng may l s stress t bo do nhit b c ch bi phng php ny,v trong 3 gi thi k tin bnh trong dch tim ph thuc vo thng k nhng t bo b thng. Phng php ny c bo cov tr nn rt nhy vi s.aureus. Khi s lng thp ca S.aureus c on trc trong 1mu thc phm, phng thc s lng c th ln nht(MPN) th thng c dng. Cng ngh MPN xem xt nhiu kt qu trong php thng k s lng thp sinh vt hoc khi s lng sinh vt hin din mc cao(176-178). Gi tr MPN l mt s c lng ca qun th v khng thng k chnh xc kh nng sng ca sinh vt. S m trong vi sinh vt hc cho bit s lng vi sinh vt trn khi lng mu bng phng php MPN. Th hin mt gia 7 v 89/g trong 95% ca tt c mu(179). Bi v tnh c chn lc ca n, NaCl(10%) c hp nht trong canh trypticsoy(TSBS) trong mt phng thc MPN. Sau 48h trong TSBS, nhng ng nghim phi c nh du trn B-P agar thm 48h cng c(180). S thng k nhng t bo S.aureus b tn thng trong TSBS cho thy s h xung(163,180). Brewer v cng s nhn ra rng thm 1% pyruvate hoc catalase gia tng ng k s thng k nhng t

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bo stress. Bi v nhu cu thm vo catalase sau khi hp kh trng, pyruvate c cn nhc hn. Nhng khun t cu khc mi trng MPN c kim tra(181,182). A. Phng php d tm 1. S lng cao Khi mt mu c ngh cha ln hn hoc bng 1000 S.aureus t bo trn gram, mi trng ngh cho s thng k rng nht l B-P agar (183). Nhng mu cho trong nhng cht pha long v trng v 1.00 ml c tri a trong b ba mi trng B-P agar; a phi kh, lt ngc v trong 48h 35oC.Nhng vng S.aureus in hnh c mu en n xm m, mc vng quanh, trn, li, t, thng c mu sng ra ,c bao xung quanh bi mt min c ca s ta,v thng c mu sng hn vng ngoi. Nhng nhm ny nht(184). Khng may l nhng vng ny thng khng thy r gy ra bi mt s t bo S.aureus b hng. 2. S lng thp Phng php phn tch ha hc(AOAC) nhn ra s lng nh S.aureus l MPN m dng nhng ng MPN cha TSBS v 1% sodium pyruvate(185). Trong mt nghin cu, phng php ny c trnh by din t gia tng mc cao, trung bnh v thp ca s thng k ca S.aureus t s lm nhim sn phm mt cch t nhin th c chn lc cao. Nhng ng MPN c cy t nhng pha long thch hp v c 37oC trong 48h. Nhng ng c xc nhn m tnh bng s thnh sc trn B-P agar c 35oC- 37oC trong 48h. Lancette v cng s nhn ra rng khi thm 1% pyruvate vo TSBS th MPN cho thy s thng k tt hn c s lm nhim thc phm t nhin cng nh nhn to so vi TSBS mt mnh. H cng nhn ra rng khi thm pyruvate vo TSBS gia tng s khi phc stress nhit v staphylococci khng stress. Kt qu ca h th tng t ca Brewer v cng s(163), vi c catalase v pyruvate a n s tng cng khi phc. cng c, nhng ng MPN m tnh thng c dng cy truyn B-P agar, c theo di bng nhng th nghim sinh ha. B. Th nghim cng c
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Nhng th nghim cng c cho nhng S.aureus ng nht, m tnh bao gm cocci gram m, sn phm ca beta hemolysis trong thch mu, th nghim coagulase, th nghim catalase(sn phm ca O2 trong s phn hy H2O2) s dng glucose v mannitol k kh v sn phm ca nuclease chu nhit. Th nghim thm vo thng c trnh by l xc nh s nhy cm ca i tng nghi ng c lp vo enzyme lysostaphin. c bit lysostaphin dung gii nhng t bo ca ging Staphylococus. Hawcroft and Geary (186) tm ra hn ch v chiu di ca phn tch a hnh gen cho ribosomal RNA (ribotyping) l mt cng c hu ch cho vic nhn ra staphylococal. VII. NG C THC PHM

Mt vi thc n cung cp S.aureus cng ngun dinh dng v mi trng ph hp l tim nng cho ngun bnh t thc phm. M, nhng thc phm thng gm i nng, tht ln, rau sng (tht, khoai ty), bnh ngt vi kem hoc thm sa trng, sa bt, whey. Trong mt vi v d, ngun nhim S.aureu t nhng ngi cm thc n mang n nhng tn thng do sn xut ni c t S.aureus hoc ngi mang n khng c triu chng. Thit b x l thc phm b nhim v sa t b b vim v cha staphylococal l tim nng cho ngun sinh vt. Thng th, S.aureus c a vo thc phm l kt qu ca mt qu trnh nhim. Sau nhng thc phm ny khng c tr nhit _iu cho php S.aureus pht trin v sn xut c t. Mt ngoi l sau qu trnh x l nhim c th xy ra trong s sn xut xc xch ln men t nhin. Trong trng hp ny, S.aureus c th tng trng sn xut c t trc khi b c ch bi acid lactic, sn xut trong sut qu trnh ln men. Hu ht nhng triu chng thng c ca ng c thc phm staphylococal l bun nn, nn, bng co cng, tiu chy. Nhng triu chng ny c th khc nhau nh nn nhng khng tiu chay, tiu chy nhng khng nn. Khi xy ra trng hp nghim trng, c th thm vo cc triu chng trn nh au u, co tht c bp, kit sc. Thnh thong , nhng cn st nh c th c mt vi nn nhn. C ch ca phn ng SE khng c hiu hon ton, nhng n c i hi phi nhim c thng vo ni tng trong bng. S tn cng ca mt vi triu chng, tiu biu cho trng c thc phm, thng xy ra trong
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khong thi gian 1-6 gi sau khi tiu th th n nhm. Thi gian trung bnh cho s tn cng l 2-3 gi, nhng nhng triu chng c th pht trin t hn 1 hoc sau 6 gi. S pht trin ca nhng triu chng c xc nh bi c s nhy cm ca nn nhn v lng cht c tiu th. Phi mt 1-3 ngy phc hi hon ton ty sc khe tng c nhn; tuy nhin, vi triu chng nghim trng hn phi mt khong thi gian di hn phc hi. T l t vong do ng c thc phm bi staphylococal v cng thp mc d vn c, thng trong s ngi gi v tr em. VIII. PHN LOI TH THC KHUN

Trng thi cng ca S.aureus thng c nhn ra bi bacteriophage typing. Th thc khun c pht hin u tin bi Swort Anh vo nm 1915; th thc khun ny c kh nng dung gii cc t bo ca S.aureus. T thi gian b typing bacteriophage c a vo loi ca S..aureus. Nm nhm c chia vo host-range c trng, mi loi cha nhiu loi thc khun th, khc nhau bi nhng t bo ca S.aureus n b typing bacteriophage (bng 10). Mt git ca mi huyn ph chun ha phage (thng kim tra s pha long hoc RTD) th din ra trn b mt thch trc khi tim 1 chng nng ca staphylococal kim tra. Mu ca nhng vng ly gii c ghi li sau khi qua m 30 C. S dng phage typing l mt cng c quan trng trong dch t hc c th c s dng xc nh ngun c t. Nhng loi ca S.aureus c tm thy khi phn hy nhm III hu nh thng lin quan n s bng n nhng cn bnh do thc phm. Khng phi tt c mi trng nui cy c trng cho phng php ny, v nhng mu nhy cm lu thng thay i theo thi gian v v tr. Bng 10 Staphylococal Typing phage Group Phage group Group I Group II Phage within group 29, 52, 52A, 79, 80 3A, 3B, 3C, 55, 71
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Group III Group IV Miscellaneous IX. TM TT

6, 7, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 42D 81, 94, 95, 96

S.aureus l cu khun gam dng c th l nguyn nhn ca s nhim c nghim trng trong nhng ngi d b bnh v l mt trong nhng vi khun gy bnh dn u. Mt vi c tnh quan trng ca S.aureus gm kh nng tn ti pht trin khi gim nc v sn xut 1 khi lng ln nhiu c t v enzyme ngoi bo. Mt s quan trng hn gm coagulase, nuclease n nh nhit, penicillinase, hemolysins, hi chng sc c t, v c t t cu khun trong rut c sn sinh trong sut qu trnh pht trin ca S.aureus trong thc phm, v t hn 1 mg t cu khun c th l nguyn nhn gy ng c thc phm ngi. Ni c t ny bn nhit v c sn sinh di iu kin pht trin a dng. Chng c tm ra bng cch s dng m hnh ng vt hoc hoc bng phng php min dch. Nhng t bo ca S.aures c th b tn thng nh bng x l (nhit, ng lnh, v.v.) v nhng t bo ny tr nn nhy cm vi thay i ca mi trng cho thy nhng t bo ch khng c hiu lc. S gim nhng sn phm nh oxi v c t ca chng c bao gm trong s thiu chn lc mi trng lit k hoc tm ra nhng t bo b tn thng. Thch Baird-Parker v mt con s c th s dng cho phng thc l 10% NaCl v 1% pyruvate c ngh lit k cho nhng v d cha nhng con s cao v thp. Ng c thc phm do t cu khun l mt trong nhng nguyn nhn dn u s bng n dich bnh trn th gii. Cc triu chng bao gm bun nn, nn, tiu chy v kit sc. REFERENCES Sneath, P.A.,Mair, N.E., Sharpe, M.E., and Holt, J.G. (1986). Section 12 gram-positive cocci. Bergeys Manual of Systematic Bacteriology, Vol 2, williams and Wilkins, Baltimore, MD, pp. 999-1103.

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(1982). American Type Culture collection. Catalogue of Strains I 15th ed., Rockville, MD. Longree, K. (1972). Quality Food Sanitation, Wiley-Science, New York. Polledo, J.J.F., Garcia, M.L., Moreno B., and Menes, I. (1985). Importance of food handler as a source of enterotoxigenic staphylococi. Zbl. Bakt., Abt. Orig. B., 181:364. Bergdoll, M.S. (1989). Staphylococus aureus. In Foodborne Bacterial Pathogens (M.P.Doyle, ed.), Marcel Dekker, Inc., New York,p. 463.

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