t i y i INtjI-ll l \ P l U / \ n y i ^ L - U I M U INOni? o u i n n v " - l*-l^»l^r •-.-»«i;
NGHIEN CO-U sy BA DANG VA DAC TINH K H A N G K H A N G SINH C O A C A C
D 6 N G £. COU G A Y B E N H P H A N LAP TO- BENH P H A M V A THU-C P H A M * Phgm Vu Vi^t Dung, L^i Trjnh Anh Khoa, V6 Van Gliu, Nguyln Tiin Dung
Tmng lim Chit luqng Nong lim thuy san vung 4
T 6 M TAT
Sv bien dii ciia cic dong E. coli gay b^nh cho ngircri ngay cang da d^ng. Ben canh sy biin dii ve kicu gen, dan den su: bifti d5i v^
d?c difim gay b^nh thi svr khang khing sinh ciia cfic chiing vi sinh v^t niy ngay cing gia tang. Nghien cdu tinh da d ^ g v^ kiau gen giy benh va d?c di6m khang khing sinh ciia cic d6ng E. coh dirge th\rc hi|n frfin 215 chiing phin lip tir thvc pham va 89 chung ti b?nh phim. S\r da d^ng cua E. coli gay benh dugc xac dinh dua tren k^ &mi PCR v6i cic moi duijc thiit l|p trfn cic gen stxl, six2, eae. bJpA, eaf. Dac dinn khing khing sinh b E coh dugfc khio sit v6i cic lo?i khang sinh Ampicillin, Ciprofloxacin, Gentamycin, Ccfotaxim, Ceflazidim, Amikacin, Imipenem. Ket qua nghifin cmi tinh da d?ng cua cic d6ng E. coli cho thay trong si 215 chiing E.
coli CO nguon gic tu th^c phim thi c6 7 chiing thu^c nhom AEEC; trong d6 c6 5 chiing thuoc EPEC, 2 chung thupc EHEC. Trong 89 chung E. coli tu benh phim thi c6 4 chiing thuoc nhom AEEC; trong do c6 2 chiing thu^c EPEC, 2 chun^ thugc EHEC. Kit qui khao sit cung cho thiy c6 s\r hen h^ gjiia dong gay b?nh AEEC vAi trifu chiing tieu chay is ngu&l 16n. V6 ket qua nghiSn cihi tinh khing khing sinh cho thiy ty 1? cic chung khing v6i Anq)icillin la 78,6% 6 nh6m thvrc pham va 91,0% 6 nh6m benh pham; khing vih Ciprofloxacin li 27,9% 6 nhom Ihuc pham va 46,1 % a nh6m b6nh phim; khing vdi Gentamycin 25,6% 6 nh6m thirc phim vi 32,6%
6 nh6m b?nh phim. C6 xuit h i ^ E coli khing vdi Ccfotaxim, Ceflazidim, vi Amikacin nhung c6 ti 1§ thip. Chua ghi nhgn duiirc truing hqp E. coli khing tmipenem. Nghien dJru cQng cho thiy chiing E. coli phan l|p tii thjt gi luon c6 ty 1? khing cao nhit vdi tat ci cic khing sinh. E. coli trong mlu thiiy san c6 ty 1? khing Ciprofloxacin d^n 20,3%, cao gap 2 lan so vdi Gentamycin. Nghien cihi Cling phit h i ^ dugc 5 chiing thupc nh6m ESBL; 2/5 chiing nay c6 ngufin goc tir thjt gi. Cic E. coli ESBL ihi khing voi Anmicillin, Ciprofloxacin, Gentamycin. Diy la nghien ciiu diu tien b ViSt Nam chi ra vi khuin E. co/i ESBL c6 hifn di6n trong tiiuc phim. Cic nghien cuu trudc dSy chi phit h i ^ E. coli ESBL trong moi truing l)?nh v i ^ vi tit bfnh p h ^
Tii khoa. AEEC, khing khing sinh, b ^ phim, EHEC, EPEC, Escherichia coli, Ihuc phim.
mCroku
NgO die thyc phim da trd fhSnh mil quan tUm cua toSn xS hOi. Hi^n nay c6 r i t nhiiu nguySn nhan gSy ra c^c vy ngO d^c fhi,rc phim nhu-: i nhiSm vi sinh vgt, 6 nhiem h6a chit c6ng nghiip, t i n du- thuic bio v§ thyc vgt, 6 nhiim die tij sinh h p c . T r o n g d i i n h l S m vi sinh v§f gay hgi la nguyen nhSn Ion nhit trong s i cic vy n g i die thyc phim hi§n nay.
Vi khuan Escherichia coli \i mOt trong s i nhu-ng tac nhSn vi khuin gay ngg d$c thyc phim fhifdng ggp,
£ coli AEEC (Attaching and ef^clng E. coli) giy thuong t i n d niSm mgc ruit cda ngu^i va dOng v^t, diy cOng 1^ tSc nhin hing d i u gSy b§nh ti6u chay * ngu-W. Thuiong ton ni§m mgc nj^t c6 Ii6n quan fo-i sy biiu hi§n cCia intimin, mOt protein ngoii m^ng 3isac ma hoa b6i gen eae, nam tr§n locus enterocyte effacement (LEE). Vung LEE cung ma h6a cho cic He nhin g i y bgnh it hai ding EPEC (Enteropathogenic £ coli) va EI-IEC (Enfenshaemorrhagic E. coli) (Nataro at al., 1998). EPEC dupe chia lam 2 nhom: nhom dlin hinh c6 mang ptasmld ma hba cho protein pilln (bfpA); nhdm khing dlin hinh cung c6 mang plasmid giy bgnh nhung khing ma hia cho protein niy. EHEC cin gpi l i VTEC l i nhirng £ coli mang dOc t i Sbcl v i Sbt2 giy nen cic trigu chCrng viim dg diy, viem dgi tring xuit huyet (hemontiagic colitis - HC) v i hOi c^Cmg t i n huyit ting ur§ (Hemolytic-uremic syndname - HUS), d i n t6i suy than cap ir tre em. Trong chSn nuii, AEEC giy ra nhOng thiit hgi rit to 16n v i kinh t i . Mgt khio sat trin 258 con heo 6" Cin The vio nim 2002 cho thay khoing 9% heo bj nhiem £ coli giy bf nh, phan Idn l i AEEC va EHEC (Kobayashi et al., 2002). Khi gia sOc b|
nhiim cic ddng vi khuan g i y bgnh niy, ngu'dl chin nuii thudng diiu tri bing cic logi thuic khing sinh. Nhi/ng cic nghi§n ci>u gan diy cho thiy cic ding vi khuin niy khing Igi khing sinh ngiy cing gia ting. Giy l i mil quan nggi aDa nhiiu bic sT thii y. Cic vi sinh vgt giy b^nh c6 khi nang khing khing sinh tr§n d^ng vgt nuii cd t h i xSm nhiim vio thyc phim. Mdt nghiin ci>u tgi Iran cho thiy da phin Igp dug'c nhOng chung £ coli 0157 tCr pho mat, sifa chua c i M\i ning khing Igi khing sinh. MCrc khing khing sinh cua cic chiing nay nhif sau: ty Ig khing Ampicillin v i Gentamycin id 44%, khing Erythromycin l i 33,3%, khang Amoxicillin, Tetracyclin. add Nalidixic l i 11,1%. Nghien CCPU cQng da cht ra ring cac chOng niy con nhgy cam vdi Chloramphenicol, Cefunsxlm, v i Streptomycin (Rahimi et al., 2011).
Cic nghiin ci>u cQng cho thiy da c6 sy xuit hien nhim E. coli cd enzyme p-Lacfamase p h i rgng (Extended Spectrum beta-lactamase - ESBL) giiip chung khang Igi hiu hit cic khing sinh tri> Carbapenem v i Cephamycin. Nghiin ciJu ciia Manjor v i ding tic gia (2011) cung da chl ra rang viic si> dgng cic logi khing sinh khing hgp 1;^ thi c i the tgo nhi^ng phin ijng die bi§t trong £ cdi 0157, khong chi giijp dong vl sinh v^t nay khing Igi khing sinh m i cin thOc diy nhanh vi§c sinh die to shiga-foxin lim cho biiu hi§n b§nh cing trim trpng v i kh6 diiu tri ho-n (Manjor ef al.. 2011).
Kit qua nghiin ci>u tren cho thiy rang vl^c chpn lya khang sinh trong diiu trj cac b^nh do vi khuin E. coli giy ra d ngydi v i ding vit la phii cin nhac. TInh khang khing sinh d i ding lan tnjyin gii/a eic dong vi khuin giy binh duxrng ru$t. Vi§c Igm dyng khang sinh c6 the la nguyen nhin gay ra tinh trgng khang khing sinh cao d cic chung £ coli trin ngirtri, h-in dgng vit v i txing ty nhlin. Vige nghi6n ci>u tinh nhgy cam khing sinh d cic vi Wiuin gay b0nh se cung cap thing «n c6 gii trj cho cac bie sT trong viic chpn lya phat do trj ligu thich hop.
VAT LlfU VA PHl/aNG PHAP V|t ii^u nghien cthj
Chung chuin vi khuan E. coli: ChCing vi khuan £ coli ATCC 25922 du-yc cung cip tir Tmng tam Chit lyg-ng Ning Lim Thuy sin vCing 4 (NAFI 4). Cic chung vi khuin £ coH 0157, £ coli 0104 va E coli 0111 d u ^ cung cip tir Viin
V i sinh Djch t i Tmng LTang.
II Miu nghiin cOv: Nguin miu thire pham gim thjt heo, thjt b6, thit g i v i fhiiy san, cic miu niy auqrc thu th$p ti> cic Cher tr§n khu vyc thinh p h i H i Chi Minh trong nim 2012.
II Nguin mSu bSnh phim duwc thu thip fir binh vi«n Nhi £)6ng 2 va binh vifn Chg Riy, thinh phi H i Chf Minh. Cic ITA ^ " " i P ^ f " ^ " ^ * i " ^^P trong quy 3, 4 nam 2012, gim c i 2 kiai: mau tiiu chiy ciia t r i em tO 6 thing tuii din 3 tuoi v i miu tigu chay cOa ngudi frirfrng thinh h> 12 tuii trd ten. Mau diroc bio quin d 6 - B^C cho tdi l^i phin tich, thdi gian bio quin trong khoing 2 - 6 gid.
PhiFo^g phap nghiin cuu Phan lap vi khuin E. coli
Mpt luwng mlu xic djnh du^c ding nhlt trong mffl frifdng Saline peptone water. Pha loing mlu thinh cac ning d i
^A^^^^^t' "^ ^1 ""' ''''^^ ^'^^ '°^"9 *^^' ^ ^ " '^" "'^i t r ^ n g MacConkey agar, nuoi 0 cac dTa cay d 37°C trang 24 gid Cic khuin igc dlin hinh ciia £ coll tren m i i tnrdng MAC se dinirc ciy phin Igp Igi tren moi fnjdnq Eosin Methylene Blue, sau dd xic djnh E coli bing cic phan dng sinh h6a theo khda phin logi vi khuin cua Bergey (9" edition, 2010).
Phin tich su- da dang ve kiiu gen giy binh ciia vi khuin E coli bing phuvng phip PCR
Ly trich DNA: Vi sinh v$t dinyc nuii ciy trong canh thang Brain Heart Infustion qua dim d 37°C. HOt 1 ml canh khuin vio ing eppendorf, ly t i m 10000 v6ng/philt trong 5 phut d i ttiu nhin sinh khii. Sinh khii vi khuin sau ly tim se di^rc huyin phCi Igi trong 500 pi dim TE, dun cich thOy 10 philf d 100°C, lim lanh nhanh v i ly tim 10000 vdng/phiit trong 1 phOt. Dich noi thu du^c diing lim khuin DNA trong cic (rfiin dng PCR.
PCR khuich dgi gen: phan dng PCR duyc ttiyc hign trong ing eppendorf 0.2 ml vdi dung tich phin dng l i 25 pi bao gim: 12,5 pi dung djch Fast start PCR Master Mix 2X, 1 pi khu&i DNA. vdi mSi gen myc tiiu khic nhau b i sung 0,5 pi mil moi vdi ning dp 10 pM, them nydc c i t de d i 25 pi. Chirong trinh phan dng PCR vdi nhigt d i bat c$p duvc thyc hiin theo nhu (Bing 11. San phim khuich dgi duyc phit hi$n bang c i d i di$n di frin gel agarose 2%. Quan sit v i ghi nhin kit qua bing thiit bj Universal Hood II (Biorad) v i Video graphic Printer (Sony). Xic djnh kich thudc sin phim bang thang DNA d i u l n cOa hing Invitrogen.
Phin nhdm c i c dong E. coll gay b^nh: tiiu chl d i nhin diin cic nhdm E. coli giy b$nh bing phin dng PCR thing qua sy hi$n dign cua cic gen giy b^nh: gen eas dgi di$n cho nhdm AEEC, gen eae v i stx1/stx2 dgi dlin c^o nhom EHEC, gen eae v i eaf/bfpA dgi di§n cho nhim EPEC diin hinh, gen eae v i r\on-stx1/stx2 dgi di$n cho nh6m EPEC khOng cnln hinh.
B i n g 1 : Trinh t y m i i sir d ^ n g trong c i c p h i n d n g PCR M i l
Sbc1 S t x 2 Eae BfpA EAF
Trinh np (5" - 3") GAG CGA AAT AAT TTA TAT GTG TTG ATG ATG GCA ATT CAG TAT ATC CTA TTC W G GGA GTT TAC G GOG TCA TCG TAT A c CAG GAG C TCA ATG CAG TTC CGT TTA TCA GTT GTA AAG I C C GTT ACC CCA ACC TG TTC TTG GTG GTT GCG TGT GTT TT TTT TGT TTG TTG TAT GTT TGT AA CAG GOT AAA AGA AAG ATG ATA A TAT 6 G G GAC CAT GTA TTA TCA
N h i ^ d 9 b i t c^p 54° C 52° C 58°C 53° 0 56° C
Kich t h u d c dogn khufich d f i (bp) 520 587 482 367 397
Xic djnh kha nang khing khing sinh
Khi ning khang khing sinh cua £ co// dup-c dinh g i i bing phuong phip khuich tin trgn thgch. Sy nhgy cam khing sinh dup-c xic i^nh bang cich do ving khing khuin trfin dTa thgch. Phucng phip thyc hi§n v i biin luin kit qui khang khing sinh va xic djnh sy hi§n dign ciia nhdm vi khuin E. coli sinh enzyme p-Lactamase phi md ring theo tiiu cfiuln cua Vign Tiiu chuin Lim sing va Xit nghiim Hoa Ky (CLSI), (2011). NghiSn cdu niy da khio sit tren 4 nhom gim 7 Iogi khing sinh nhu sau; /^picillin. Ciprofloxacin, Gentamycin, Cefotaxim, Ceftazldim, Amikacin. Imipenem. Tinh nhay cim vdi tdng logi khing sinh ciia vi khuin duyc xic ^nh theo nhu Bang 2.
B i n g 2: TiAu chl d l i n g l i l tinh nh^y cam c i c lo^l k h i n g sinh I h u nghiem (CLSI Nh6m k h i n g sinh Loai k h i n g sinh
AmpKillm Penfelllln (p-lactaf") , .
Imipenem Cefotaxim
Cephalosporins . ,
"^ Ceftazidim Gentamycin
Aminoglycosides ^ . ^ ^ . ^ Quinolones Ciprofloxacin
Ndng do (pg)
10 10 10 30 5 30 30
-2011)
GIdi hgn d u d n g kinh viing i><
Nhgy a 17 2 16 2 1 5 2 17 3 21 2 26 2 21
Trung gian 14-16 14-15 13-14 15-16 16-20 23-25 lft-20
i: ch& (mm) k h i n g
S 1 3 S 1 3 S 1 2 S 1 4 S 1 5
£ 2 2 S 1 7
KfeT QUA VABigN LUAN Phan l^p vl ithiiln E coli
Qui trinh nghiin cuu da thu duoc 304 chOng £ coli tir nhiiu n g u ^ Idiic nhau. S6 lu^mg chiing £ coli phin l i p duyc t h i hien fr^n Bing 4. Trong so do co 215 chiing td thyc phim, gim 93 chOng td thjt g i , chiim 43,3 %, 53 chiing td thft heo chiim 24.7%, 69 chGng Kr thuy sin, chiem 32.0%. C6 89 chOng £ coli thu thip h> cic nguin m l u binh phim, trong d i c i 40 chung tu b#nh phim tre em, chiim 44,9%, 49 chung tu binh phim ngudi Idn, chiim 55,1 %. Sau khi suu tip, cic chung niy duyc bio quin d -SO^C trong glycerol 20 % cho qui trlnh nghiin cdu.
Bing 3: S4 lu^ng va kf hifu ching vi khuin E. coll phSn l|p td b^nh phim va thyc pham N g u i n p h i n l i p
Thyc p h i m
B$nh phim
Thjt heo Thjtga Thiiy s i n B$nh p h i m t o em B$nh F^am ngudi li>n
S6 l u y n g 53 93 69 40 49
K i i r i f u c h i n g ^ T l 753
G I G93 C 1 „ , . , C 6 9 PT1....,PT40
P I P49 Sy da dgng ciia cac dong E. coll g i y bfnh
Vige Khio sat d i xic djnh cic ding E. coli mang cic gen giy bgnh duyc thyc hign tren 304 chdng £ coli phin l$p ti>
binh phim v i thyc phim. Trong 215 chung £ coil phin lip tu thyc phim c i 7 chung duomg tinh vdi gen eae. chiim 3,3%. Cic chung niy duqrc xip v i o nhdm AEEC. Trong s i cic chOng niy c6 5 chdng td mlu thit heo, 2 chiing td th|t g i . Cic nghiin a>u v i AEEC trong chan nuii cung da duyc thyc hiin tgi Vigt Nam, cic cing trinh niy duyc thyc hiin trin mlu phin. mlu nude thii hay mSu dat cua cic trang trgi nuii gia siic, Nhim tic gii Kobayashi (2002) cho thiy khoing 9% phin heo td trang trgi heo d Can The, Vi^t Nam cd nhiem £ coli, phin idn l i AEEC va £ coli sinh Shiga toxin. Cling vdi d i , nghiin cdu cua Trin Thanh Phong et al. (2007) cOng da phit hign STEC tir miu b§nh phim tiiu chiy ciia heo, khing phit hi^n nhim niy d cic trang trai nuii bd. Kit qui nghiin cdu niy ciing vdi cic kit qui tham khio nhu trin da budc diu cho thiy c i m i l Ilin h§ giua nguin gic liy nhiim AEEC tu trang trgi chin nuii v i o cic logi thjt thyc phim luu hinh trin thj truing.
Trong s i 69 mlu thuy san da khio sit, khing ghi nhin c i AEEC. Ngoii cong trinh nghien cdu ciia Kumar v i ding tic gli (2001) phit hi$n cd mit ty 1$ thap AEEC td cic chy c i d Mangaiore - An DO, cic^nghiin cdu khic gin diy vSn chua dua ra mit s6 liiu cu the nio ghi nhgn cd sy xim nhiim cua nhim AEEC d cic mau thiy sin. Cic tic g i i chl luu y ring nguin thOy sin, bao gim tim, c i , nhuyin fhl 2 minh vo... v i mii tnidng nude rat c6 t h i l i nguin lay nhiem vi khuin giy b$nh AEEC quan tn?ng (Kumar et a/., 2001),
Viic xic djnh ti l i EHEC v i EPEC trong so cic diung AEEC cOng da duyc thyc hiin. Kit qui khio sit d i phin nhim trin tdng chiing AEEC duyc Wnh bay frin Bing 5. Kit qui niy cho thiy c6 sy khic biit v i ty 1$ phit hign EHEC vS EPEC trong cic logi mlu thyc phim khic nhau. 0 thjt g i trong s6 2 tofdng hgp phit hi§n AEEC thi c6 1 trudng hyp phit hiin EPEC v i 1 tardng hyp phat hi§n EHEC. G thit heo, trong s i 5 chdng AEEC thi c6 4 chCing EPEC v i 1 chCng EHEC. Tit c i cic chdng EPEC td miu thit heo deu thuoc nhdm EPEC khing diin hinh. Mit s i nghiin cdu cda Martina (2008) cho ring nhim EPEC khing a i n hinh c6 thi co mii liin quan vdi nhim EHEC nhung bj dpt blin mit gen stx (EHEC lost Shiga toxin - EHEC_LST) (Bielaszewska ef at., 2008). Sy vyyt trii v i s i luyng EPEC khing diin hinh so vdi EHEC trong miu ^ j t heo d n duyc flip tgc nghiin cdu d l lam ro.
B i n g 4 STT
1 2 3 4 5 6 7 6 9 10 11
P h i n nhdm gSy b^nh Ki hl^u chiing
T9 T20 T38 T40 T52 G80 G89 P25 P31 P35 P45
ciia cac chiing E. coll Gen g i y b^nh eae+. stxl-, stx2; bfpA-,eaf- eae+, s(x^, sb(2; bfpA-,eaf- eae+. stxl-, sbc2-, bfpA-,eaf- 636+. stxl-, sbe2; bfpA-,eaf- eae+, stx1+, stx2-, bfpA-.eaf- eae*, sbtl-, stx2+, b^-,eaf- eae*, sbcl-, stx2-, bfpA-.eaf- eae+, sbtl*. sbc2; bfpA;eaf- eae+. slx1+, stx2*, bfpA-.eaf- eae+, sbd-, sbc2; bfpA-.eaf- eae+, stxl-, sbc2-, bfpA*,eaf+
P h i n nhdm gay benh EPEC khflng dien hinh EPEC khfing d i i n hinh EPEC khflng d i i n hinh EPEC khfing d i i n hinh
EHEC ( 0 26) E H E C ( O I I I ) EPEC khSng diSn hinh
EHEC EHEC EPEC khflng d i i n hinh
EPEC d i i n hinh
N g u i n gdc M i u thjt heo M§u thjt heo MSu thjt heo MSu thjt heo M i u th|f bo Mau thjt gd M i u thjt g i M i u b$nh p h i m M l u b$nh p h i m M i u b^nh p h i m M i u b^nh pham Trong 89 chdng £ coli co nguin gic bgnh phim ttiu chiy, c6 4 chdng duyc xic djnh thuic nhim AEEC, chiim t j l§
8,1% Tit c i cic chdng niy deu duo« phan lip td miu binh phim tiiu chay ngucri \(tT\. Trong s i niy c i 2 chdng du^c xic djnh l i EHEC v i 1 chdng l i EPEC dien hinh v i 1 chdng EPEC khing dlin hinh. Ket qui niy trai vdi mit s i nhSn djnh cho rang EPEC dien hinh l i nguyin nhin hang dau giy tieu chay d cac nude dang phat triin v i mit ^ li^nhi d cic nude cing nghiip, EPEC khing dien hinh mdi Igi la nguyen nhan chu yeu g i y tiiu chiy a cic nude phit triln. Ket qua khao sit niy cOng khong ghi nhin duyc chdng AEEC nio td cic mlu binh phim tiiu chiy a t r i em, trong khi nghiin edu cda Bui Thu Hien va ding tic gii (2007) tgi cic benh viin d H i Nil lai thiy ring t j AEEC trong cic ca tiiu chiy d tre em Igi la 9,2%.
- l U U U U N U N L j M t tollNM M U O I U A N U U U C 2 0 1 3
Kk dua x4c ainh kliu huy^ thanh cung da phat hien 1 chung EHEC tir mau ga (G80) la E coS 0111 va 1 chiing EHEC tu mau th[t bit (T52) la E. coff 026. Cdc nghien cuu trgn thS gidi deu cho ling cac chung EHEC c6 llgn quan ch§t che vol nhlmg catiSu chSy, cic kiiu huylt thanh nhif 026, 091, O103. O104, 0111. 0113. 0121 va 0145 l^l cd liSn quan m5i i h » " ? j ' " ' ' ' ' * ""^ ''"^ " " ® - " " " " ' » • da cd 81/ ai4l hi«n cua nhdm EHEC (026 vS 0111) trong m i u th|t ga v i hA kTl '?° • * ddc ch^ cua khu vvrc BiSnh phd Ho Chi Minh. Vi$c xSy dung mOt quy trinh dd kiem soSt nhdm E. coff g§y nenn la can thiet.
K h i nang khing lihang sinh
ih^i° h? " " ! ! *''"*' ' * ° r ^ ' * 5 " ^ ^'"'' "^ "^ * " " 9 ^ '=°" ^ W c Ihire hiin trSn 304 chung E coll phin l$p t(r cic k>?l nhS /Is "" ' * ° ' ''"^ ' * * ' ^ ^ " 5 " ' ^ " ' ' ^ ''^'' ^ * " 9 5 ' * " "^y ' ^ ^- ° * ** " ^ " " " P''*'" "3 I * * pnam ™ i co ty 115 khang cao vdi Ampicillin, gim 78.6% chung tir Ihvc pham v i 91.0% chung tu bdnh phim. Ty 1^ khing mang sinh Ciprofloxacin l i 27,9% chdng tu «nro pham v i 46,1 % chdng ti> b«nh phim. ty 1$ niy di Gentamycin l i 25.6%
cnung ur thuc phim v i 32,6%chung tir benh pham. Ty l« khang niy Blip hon dii vdi cic khing sinh Celbtaxim.
o e i i ^ d i m . Amikacin. Ty le E co* d i khing cao vdi cic lo^i khing sinh Ampicillin v i Gentamycin cd le do diy l i nhimg 1091 knang sinh d i mua v i de si> dung trong chin nudi, nuoi trAng thuy sin (Binh Thi^n Thuit el al, 2002).
BSngSMrtl qui khio sit gnh khing khing sinh cua cac ching E «>« c4 nguin g6c Ihvc phim v i b#nh pham M i u
Heo bo
X°^
Thuy s i n B«nh p h i m
T r ^ e m N g i r d l l d n
Ciprofloxacin 8/53 (15,1%) 38/93 (40,9%) 14/69 (20.3%)
14/40 (35%) 27/49(55,1%)
Ampicillin Gentamycin Amikacin 33/53 (62,3%) W53 (17%) 0 83/93(89.2%) 39/93(41,9%) 1/93(1,1%) 53/69 (76.8%) 7/69 (10.1%) 0
36/40 (90%) 9/40 (22,5%) 0 45/49(91,8%) 20/49(40,8%)
Cefotaxim 2/53 (3,8%) 4/93 (4,3%) 4/69 (58%)
1/93(1,1%) 1/69 (1,4%)
11/49(14,29%) 7/49 (22,45%)
• niifc ottim m Mnhi pliAm
Xit ning v i tinh khing khing sinh d cie chung E. coli phin lip td thyc phim thi t j 1$ Idling khing sinh d cic chdng phin l§p tu thjt g i luon cao hon so vdi cie logi thyc phim khic. Cic nghiin cuu trudc diy diu cho kit qui Krang ty (Ti Liin Thu et al, 2004). Ty 1$ d i khing vdi Cipnafloxacln tren cic chung h> mlu g i t i 40,9%, cao hon rit nhieu so vdi t* l i niy dii vdi cic diOng h> thjt l i 15,1% v i td mlu thdy sin l i 20,3%. TJ l i khing vdi Gentamycin tr^n mau g i t i 41,9% cung cao hern rit nhieu so vdi t j 1$ nay dii vdi cic chung tu mlu thjt v i thuy sin lan luyt l i 17% v i 10.1 %.
Xit ning vdi cac chdng £. coli phin lip tu thiy sin, t j Ig khing Ciprofbxacln cao gip dii (20,3%) so vdi ty Ig khing Gentamycin (10,1%). K i t qui niy phu hyp vdi thyc t i l i trong cic nim gin diy, du luyng khing sinh nhim qulnolone nhu Enrofloxaern v i Ciprofolxacln liin tyc duyc phit hi^n tgi eic ca sd nuii tring thuy san, diy c6 thi l i nguyen nhin dan din khi ning E. coli d i khing cao vdi Ciprofoxadn.
Trong qui trlnh nghiin cdu cung da phat hiin 1 Chung E. coli (G60) khang Amikacin duyc phan lip tu mlu thjt g i . Khing phit hiin chdng E. a^i khing vdi Imipenem. Sy de khing vdi Cefotaxim v i Ceflazidim cung duyc ghi nhin, nhung ty 1$ van cdn khi thip.
Nhung logi khang sinh niy co te vIn cdn tiim ning sd dgng. Tuy nhlin can phai phing ngua sy lay truyin tinh khang cie loai kf^ing sinh niy d cic ding vi khuin khic nhau trong mii trudng,
Ket qui khio sit cur>g da cho thiy cic chung AEEC phin l^p chu yeu khing Ampicillin vdi t^ l i khing la 71% d thyc pham v i 100 % d binh phim, mOt v i i chdng nay khing thim Gentamycin.
Bi^u d i 1: Ty 1% da khing cda cac chOng E. coli phin lap td b^nh pham va thyc pham
Kit qui nghiin cuu cQng ghi nhin nhung chung E. coli phan l^p co sy khing cCing luc nhieu logi khing sinh (bieu d i 1). Doi vdi cic chung E. co/i td thyc phim, c i 24/93 (25,8%) didng tu thjt g i , 4/69 (5,8%) chung tir thOy sin 1/53 (19%) chung h>
ttijt heo khing 3 iogi khing sinh frd len. Cac chung nay chu yeu khing cdng luc Ampicillin, Ciprofloxacin, Gentamycin. T^ 1$ cic chung vi sinh v i t nay khang cung luc 4 Iogi khang sinh cung duyc ghi nhgn vdi t^ 1$ 2/215 (0,9%). Sy da khang khing sinh d cac E. coli tu miu b#nh phim Iu6n cao hon so vdi cac chdng td thyc phim.
Ty l i khang 3 loai khing sinh Ampicillin, Ciprofloxacin, Gentamycin doi vdi cic chdng td binh phim l i 23/89 (25,8%), cao gap khoing 2 lan so vdi cie E. coli tir thyc phim l i 29/215 (13,5%). T^ 1$ cac chdng td b§nh phim khang cung luc 4 loai khing sinh l i 6/89 (6,7%), cao ho'n khoing 4 lan so vdi cic chung H> thyc phim.
Phit hi#n nhdm E. coil ESBL
Hinh 1: Kit qua thd nghiim E. coll ESBL 1: Cefatzidime (18,5 mm)
2- CeftazkJim/ Clavulanic acid (29.3 mm) Theo tiiu chuin cda Vign Tieu chuan Lim sing v i Xit ngiem Hoa Ky (CLSI),
(2011). cic chdng vi khuin nghi ngd thu^c nhdm E. c(^i ESBL co vong uc chl
cua Ceftazidim 5 22 mm ho^c Cefotaxim s 27 mm duyc khao sit cung luc vdi 2 ^- Cefotaxim (11,0mm) logi khing sinh Cefotaxim (30 pg) v i Cefatazidime {30pg) d dgng don le v i 4^ Cefotaxim/QavulacnK: aod (26.7mm) dgng kit hyp vdi chit de chi p-lactamase l i acid clavulanic (10 pg). Kit qui khao sit da phit hi§n c6 5 chung E. coli
H y i Niatii ^ n u A n y i , O U I N U iNtjMt; oiiNn M V U rOAl? >
ESBL, tat ca diu c i tinh khang vdi Cefotaxim, nhgy vdi Cefatazidime. Cic chung nay ed dudng kfnh vung uc c h i khi xd ly vdi dang kit hyp Cefotaxim/davulanic (30 pg/10 pg) deu a 5 mm so vdi Cefotaxim (30 pg) don le (Hinh 1). Trong s i 5 Chung E, cdi ESBL duyc phit hien, cd 2 chung c6 nguin gic tu thjt g i va 3 chdng ed nguin gic tir binh phim cSk ngudi Idn. Kit qui niy phin anh llnh trang vi khuin £ coil ESBL d i hi$n di^n trang thyc phim va m i i trudng. Cic nghiin edu v i tinh trgng vi khuin khing khing sinh tgi \flit Nam h> trudc din nay chf pftit hi§n sy hiin diin cua ding vi khuan niy trong mii trudng bgnh vi§n, chua phit hign duyc trong ty nhlin v i thyc phim. Trong diiu ki#n khi h^u nhigl ddi cua nude ta, nhom vi khuin niy rit d l ton tgi va phit triln ngoii mdi favdng, lim lan truyin cic gen siiu khing thuic fnang quin Vne vi khuin ty nhiin, tu d i lim gia ting tfnh siiu khing khing sinh d cic vi sinh vit giy b^nh cho ngud! v i dpng v$t
Cic E. coli ESBL duyc phit hign eh? d l khing chio vdi Ciprofloxacin v i Gentemydn. Kit qua niy phd hyp vdi nghiin cdu cua Ktianfar v i ding tac g i i (2009), cic E. coli ESBL td thyc phim c i t j l i da khang thip hern cic E. coli ESBL td binh phim [Khanfar et al., 2009]. T i t c i cic chung E. coli ESBL duyc phit hiin d i u cdn nhgy c i m vdi Cartiapenem, Diiu niy cho thiy diy vin cdn l i khing sinh hi^u qua de diiu trj cic nhiim khuin do E. coli ESBL.
K ^ LUAN
Qui trlnh nghien cdu duye tien hinh tren 304 chung E. coli phin l$p h> nhiiu nguin khic nhau bao gim 215 chung tj>
thyc phim gim mlu thjt g i , heo, thdy sin v i 89 chdng td nguin miu binh phim. Kit qua nghiin edu d i phit hi$n trong so 215 chdng E. co//h> thyc pham c6 7 chdng thuic nhim AEEC, trong d i cd 5 ching thuic EPEC, 2 chung thuic EHEC c6 kiiu huyit thanh E. coli 026 va E. c(di 0111. C i 89 chdng E. coli phin l i p td m i u binh phim tiiu chiy, trong s i niy c i 4 chung thuic nhim AEEC gim 2 chung l i EPEC, 2 chdng la EHEC. V i dgc diim khing khing sinh, c i 304 chung E. coli diu duye khio sit tinh nhgy cim vdi 7 logi khing sinh, bao gim: Ampicillin, Ciprofloxacin, Gentamycin, Cefotaxim, Ceftazidim, Amikadn v i Imipenem. Kit qua khio sit cho Ihly co 78,6% chung E. coli tir thyc phim vd 91,0% s i chdng td bgnh pham khing Ampicillin, 27,9% chdng td thyc phim v i 46,1% chung td b§nh phim khSng Ciprofloxadn; 25,6 % chdng tu thyc phim va 32,6 % chung td bgnh phim khing Gentamycin. CQng da phit hiin mpt tj i i thip eic chung E. coli td thyc phim va b^nh phim khing Ceiotaxim, Ceftazidim, Amikacin. Khong phit hi^n duyc E.
coli khing Imipenem. Cic chdng E. coli AEEC phin l i p chd y i u chi khing Ampidline. Ty 1$ da khing khing sinh d cic chung phin f$p td binh phim luon cao hon cic chdng tu thyc phim. Qui trlnh nghiin cdu cung da phit hiin duyc 5 chdng ESBL gim 2 chdng c i nguin gic td thjt g i v i 3 chdng co nguin goc tu binh phim ngudi Idn. Kit qui niy phin inh tinh trgng vi khuin E. coli ESBL d i xuit hi$n trong cdng ding.
TAI L I ^ U THAM K H A O
Bui Thi Thu Hien. Flemmmg Scheutz et al. (2007). Dlarrheaganic Esc/ien'c/iia co/r and 5hige//a Strains Isolated from Children in a Hospital Case-Conbol Study in Hanoi, Vietnam, Journal of Clinical Microbiology 46(3): 996-1004.
Dinh Thi^n Thu^ vA c$ng sy (2002). Khio sat tinh hinh sd dgng khing sinh tn>ng chin nu^ vi du luyng khing sinh trong quSythjt heo, gi thucrng phim tren dja bin ttnh Blnh Duong, Chi cgc Thi) y t)nh Binh Ducng v i Khoa chSn nufii thu y DM Ndng Lim Tp.HCM.
Bielaszewska et al (2008). Shiga toxin-negative attaching and effacing Escherichia coli- distinct clinical associations with bacterial phylogeny and vimlence traits and infened In^iost pathogen evolution. Journal of Clinical Microbiology 35(8): 2184-2185.
IQianfar H., Bindayna KM, Senok AC and Botta GA (2009). Extended-spectmm beta-lactamases (ESBL) in Esc/isnc/ifa cdf and Klebsiella pneumoniae: trends in ttie hospital and community setBngs, The Joumal of Infection in Developing Countries 3(4),295 - 299, Kobayashi H, et al, (2002). Prevalence of Pathogenic Eschenchia coli in a swine Breeding Environment in the Canlho province of Vietnam, Japan (nfemafHWa/Researe/i CenterforAgncu/fura/Sciences (JIRCAS)(Tsukuba, Ibaraki 305-8686, Japan).
Kumar HS, Otta SK, Kantnasagar I (2001). Detection of Shiga-toxigenic Escherichia coli (STEC) in fresh seafood and meat marketed in Mangaiore, India by PCR. Letters in Applied Microbiology 33: 334-338
Manjor KS, Saikia D (2011). PCR detection of "stxl" and "stx2" toxigenic genes in multple antibiotic resistance Erchericbia coli population and phenotypic detection of ESBL producing Ercherichia coli isolates from local vanety of poutry. international Journal of Applied Biology and Pharmaceutical Techrv^ogy 2.
Nataro JP. Kaper JB (1998). Dianheagenic Eschenchia coli. Clinical Microbiology Review. 11:142-201.
STUDY ON DIVERSITY AND CHARACTERISTIC OF ANTIBIOTIC RESISTANCE OF PATHOGENIC E. COL/STRAINS ISOLATED FROM CLINICAL SPECIMEN AND FOOD
Pham Vu Viet Dung, Lai Trinh Anh Khoa, Vo Van Giau, Nguyen Tien Dung National Agm- Forestry- Fishenes Quality Assurance Department Branch 4 SUMMARY
The iranation of pathogenic E. coli which cause disease in human is increasingly diverse. Beside the genomic variation lead to the >«
variation of pathogenic characteristic, the antibiotic resistance in microbe also is more complicated. Study on diversity of pathogenic genotypes and characteristic of antibiotic resistance in E. coli was performed with 215 E. coli strains isolated from food samples and 89 strains isolated fiom clinical specimens. The diversity of pathogenic E. coli was studied by PCR method based on the pair of primers designed for detection of genes: stxl, stx2. eae, bfpA, eaf. The characteristic of antibiotic resistance in E. coli were studied with Ampicillin, Ciprofloxacin, Gentamycin, Cefotaxim, Ceftazidim, Amikacin, Imipenem, The study results in diversity of .„
pathogenic E coli showed that in 215 E coli strains isolated from food, there are 7 strains belonging to AEEC, in which there are 5
'_" r^yi Hianr i^HOA HQC C O N G N G H $ SINH HOC T O A N Q U O C 2013
whicT ?'*'* ^''EC and 2 strains were HIEC. Among 89 E. cob strains isolated from clinical ^jccimens, we found 4 AEEC strains, in a d u ^ A t J T * ^ * " ^^^ " ^ *^ '^^ belonged to EHEC. Our results ^uwed the rclatimishv between AEEC and diarrhea m fiom fomT^' * ^ antibiotic resistance characteristic, ihc study results showed that the rate of Anpicillin resistance were 78,6% stains strain.I°fr^ ^ l , ^ ' - ' ^ ' ' ' ^ ' " " ^ *»"> clinical specimens. The rate of Ciprofloxacin resistance were 27,9% strains from food and 46.1%
" ^ f f o m clmical specimens. The rate of Gentamycin resistance were 25,6% strains from food and 32,6% for strains from clinical
^ ^ e n s . The resistance for Ccfotaxim, Ceftazidim, and Amikacin were also recorded, but with low rates. We did not observe the
^ ^ c e of Imipenem. Study results also showed diat ttie resistance rates of aU antibiotics of E. cob strains from chicken were ays nigtiKt Separately fijr E. coli strains from seafood, (he resistance rate with Ciprofloxacin were very high, up to 20,3%, it Is imce as highcompared to die resistance rate with Gentamycin. This study also detected 5 strains E. cob ESBL, in which 2/5 of sna^ns were aom chickens. All E. cob ESBL strains were resiMant widi Anroicillin Ciprofloxacin and Gentamycin. It may be die first research in Viet Nam detected the E. cob ESBL strains in food and cm^omenL
Keywords: AEEC, antibiotic resistance, clinical specimen, EHEC. EPEC, Escherichia coli. food
•Author for correspondence: Tel: +84-918044558; Email: [email protected] / dungnt.nafi4@mard gov vn
