JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol 10 - N°3 l2tM
Moi tircng dong kieu gen cua cac chung Acinetobacter baumannii phan lap tren benh nhan nhiim khuan huyet b^ng ky thuat dien di xung trucrng (PFGE)
Typing Acinetobacter ibaumannii \so\ated from sepsis patients by pulsed field gel electrophoresis (PFGE)
Nguyin Thi Thanh Ha*, Phan Quoc Hoan* *Benh vien Nhi Dong / - TP. Hd Chi Minh
**Benh vien Trung uang Qudn doi 108
Muc tieu: Tim hieu mdi taong dong ve kieu gen cua cac ehCmg Acinetobacter baumannii gay nhiem khuan huyet phan lap dupc tai mot sd benh vien Idn cCia Viet Nam. Boi tdgng va phuong phap: 73 chung A. baumannii dupc phan lap tren benh nhan nhiem khuan huyet tai 6 benh vien Idn ca 3 mien Bac, Trung Nam. Thdi gian nghien cdu t d thang 6/2011 den thang 5 nam 2013./fef qua-Sddung kythuat PFGE phan tich 73 chung A. baumannii phan iap t d 6 benh vien da eho thay cd 16 chum (Cluster) vi khuan ddpc ky hieu t d I den XVI vdi mdc do tdOng dong ve kieu gen cua cac chung A. baumannii nay t d 89,5% tdi 100%.
Cac chung A. baumannii khang vdi nhom carbapenem phan lap dope co kieu gen tdong ddng tren 90%
nam rai rac d ca 6 benh vien. Cac chung A. baumannii phan lap dUpc tai khoa Hoi sdc tich cdc cua Benh vien TLf Hue cd sU tUOng dong kieu gen rat cao.
Tdkhda: Kieu gen, Acinetobacter baumannii, PFGE.
Summary
Objective: To investigate the relationship of genotype Acinetobacter baumannii isolated from sepsis patients in Vietnam. Subject and method: 73 strains of A. baumannii isolates in patients with sepsis in 6 major hospitals in Northern, Central and Southern Vietnam. Study period from 6/2011 to June 2013.
Result: Using PFGE analysis on 73 A. baumannii strains isolated from six hospitals, 16 clusters A.
baumannii have been found and symbols from I to XVI. The level of similarity of genotypes of A.
baumannii was isolated from 89.5% to 100%. A. baumannii strains resistant to carbapenem w/ith similar genotypes are scattered over 90% in the six hospitals. The strains isolated in the ICU at Hospital Central Hue resemble very high genotype.
Keywords. Genotype, aclnetobacterbaumannii, PFGE.
Phan bien khoa hgc, PGS. TS. LE HUU SONG 132
^»P CHl Y DU^OC L A M SANG 108
Tap 1 0 - 3 6 3/2015
1. Dat van de
-Ac/nefotacfer baumannii {A. baumannii] la m o t trong nhiJng vi khuan Gram am hien nay ddoc quan tam nhieu vi kha nang gay benh nang, khang lai nhieu khang sinh t h d d n g d u n g , va t J le t d v o n g cao hdn so vdi cae t r u d n g hpp nhiem khuan do eac tac nhan khac [2], [3]. A baumannii ed dae tfnh sinh hpc dac biet, do la tao ra cac mang sinh hpc (biofllm) giup vi khuan gan chat vao be mat d u n g cu, m o i trudng va tao dieu kien cho vi khuan dg dang t d n tai lau dai, t h u nhan, tich luy gen khang sinh va t r d thanh tac nhan gay kho khan t r o n g dieu trj va kiem soat lay nhiem [4].
Hien nay cd nhieu p h u d n g phap sinh hoc phan tCf giup xac d i n h n g u o n goc va each lay truyen cua cac chung A. baumannii phan lap dddc. Tuy vay, ky thuat dien di x u n g t r u d n g (PFGE) van dupe xem la
"tieu chuan vang-gold standard" t r o n g phan loai, xac dmh n g u o n goc vi khuan khong ehl rieng cho Acinetobacter ma cdn cho nhieu loai mam benh vi khuan khac [2, 4]. Chung toi sCr d u n g ky thuat PFGE, vdi muc dich t i m hieu moi t d o n g d o n g ve kieu gen cua cac chung Acinetobacter baumannii gay nhiem khuan huyet phan lap dupe tai m o t so benh vien ldn cua Viet Nam.
2.DciitUdng, phUcTng phap
2.1. Chung vi khuan
73 chung A. baumannii ddgc phan lap tren benh nhan nhiem khuan huyet tai 6 benh vien IPn ca 3 mien Bac, Trung Nam. Thdi gian nghien cdu t d thang 6/2011 den t h a n g 6 nam 2013.
2.2. Kythuat nudi cay, djnh danh vi khuan Thdc hien tai Khoa Vi sinh vat, Benh vien TLTOO 108, theo t h d d n g qui cua To chdc Y te The gidi.
2.3. Ky thuat dien di xung trUdng PFGE ThUc hien tai phong xet nghiem Vi khuan hpc, Vien Ve sinh dich te Trung dOng, Ha Npi, cu the nhd sau:
Pha loang va do dp due vi khuan: Chung A.
baumannii da dddc d i n h danh se ddpc nuoi cay tren
moi t r d d n g thach d m h d u d n g , sau do ddpc lay ra hda vao thach long roi do vao nhdng khuon nho.
Gan vi khuan vao thach: Mieng thach sau khi do dupc cat va tao cac nut thach nhd chda toan bo te bao vi khuan.
Ly giai va cat ADN cCia vi khuan bang Protein K va Xbal: Vi khuan ddpc cd d m h t r o n g thach se chiu tac d u n g cua Protein K se ly giai te bao vi khuan giai p h d n g toan bp ADN, tiep theo ADN cCia vi khuan se bi giang hoa dudi tac d u n g phan cat tai nhdng vi t r i dac hieu bdi enzym gidi han Xbal.
Oien di thuc hien bang may dien t r d d n g xung (PFGE): Cac n u t thach chda ADN A. baumannii sau khi ddpc phan cat, dat vao cac gieng tUOng Ung tren khuon thach agarose va tien hanh dien di. Oua trinh dien di dupe thdc hien vdi d d n g dien thay doi d cac khoang xac djnh trade sao cho dien t r d d n g cho phep cae doan ADN co kich thUdc Idn khoang 10 - 800 kb phan tach rdi ddoc vdi nhau.
N h u p m gel va chup anh: Mau thach sau dien di se ddoc n h u p m vdi thuoc n h u d m huynh quang n h d e t h i d i u m bromide va quan sat dudi den cdc tim (UV light). Hinh anh bp gen cua VK dUOc chup bang may chup GelDoe (BIO-RAD).
2.4. XU ly sd lieu
So lieu ddpc quan ly bang phan mem excel. Phan tich chum VK bang phan m e m Fingerprinting ll (BIO- RAD). Cac chum VK ddoc the hien qua bieu do cay (dendrogram). Cac chung A bgumannii cd mdc do t d o n g d d n g ve kieu gen ADN>90% duac xem cd quan he di truyen chat che.
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol10-N°3/2g15
3. Ket qua
3.1. TUdng dong kieu gen cUa 73 chung A.baumannii
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Hinh 1 . So do hinh cay kieu gen cae chung A.Baumanii d 6 benh vien Hinh 1 cho thay do 16 chum gen ddpc ky h i e u t d
I den XVI vc(i mdc dp tUOng d o n g ve kieu gen ADN thay doi t d 89,5%-l 00%, dac biet cd chum IV gom 13 chung A.baumannii cd do t d o n g dong la 96,8%;
t r o n g do co 8/13 chCing phan lap d Benh vien TL/
Hue va 05 chijng cdn lai dddc phan lap d cac benh vien khac. Tddng t u n h u vay, Chum XVI cd dp tUdng d o n g 92% va cung co den 4/5 chung phan lap ddoe tal Benh vien Trung d o n g Hue.
3.2. TUdng dong kieu gen vdi khdng carbapenem
T r o n g 73 chCing A.baumannii cd 20 chCing k h a n g c a r b a p e n e m , p h a n t i c h b a n g PFGE cho thay cd 3 c h u m vi khuan khac nhau (hinh 2) trong do: Chum (A) g o m cd 11 chung cd kieu gen tddng d o n g den 96%, cd 6/11 chCing A. dot/mann/Vtd Benh vien TLf Hue. Chum B g o m co 4 c h u n g co kieu gen
TAP CHl Y DL^O-C U M S A N G 108 Tgp 10-So 3/2015
tddng d o n g 90,5%, t r o n g do 2 t d Benh vien TLT Hue; Chiim C g o m cd 5 chung, ed 4 t d Benh vien TL/ Hue.
1 t d BV T r u n g V d o n g va 1 t d Benh vien Nhi O o n g 1.
19 Hue 43 H U B 52 BM 56 Hue 67 ND
75 Hue 224 H L E 36 Hue 195 TV 55 ND 146 Hue
Hinh 2 . Phan bo cac chum co kieu gen khang vdi carbapenem
3.3. TUdng dong kieu gen cdc chung A.
Baumannii tgi Khoa Hoi sUe
18 chiling A.baumannii phan lap dupe tU cac khoa HSTC ciia 6 benh vien chia lam 3 chum khac nhau (hinh 3) bang ky t h u a t PFGE t r o n g do chum D CO moi t d o n g d d n g kieu gen tdi 96,5% va cd 7 chung
phan lap ddpc tai Khoa Hoi sdc tich cdc cua benh vien TL/ Hue, chum E cd 5 chdng kieu gen tUOng d o n g den 92% va 4 chung thude Benh vien TL/ Hue, chum F ed 4 chung kieu gen t d o n g d o n g den 9 1 % va 3 chung thuoc ve Benh vien TL/ Hue.
52 BM ICU
Hinh 3. Phan bo churn A.baubamannii tai khoa Hoi sdc tich cue
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol10-N°3/2Q15
4. Ban luan
Ap d u n g ky t h u a t PFGE phan tich 73 chung A.
baumannii phan lap t d 6 benh vien da cho thay co 16 chiim (cluster) dddc ky hieu t d I den XVI vdi mdc dp t d d n g d o n g ve kieu gen cua cac chung A.
baumannii t d 89,5% tdi 100%. Dac biet chum IV, g o m CO 13 chung A. baumannii cd do t d o n g d o n g tdi 96,4% phan lap ddpc d 4 benh vien ca 3 mien Bac (1 BM), Trung {8 t d TWH) va Nam (2 t d BVCR va 2 t d BVND), dieu nay eho thay co sU Idu hanh cua cae chdng cd cung kieu gen t u o n g d o n g cao d nhieu benh vien khae nhau. Phai chang eac chCing A.
baumannii nay co cung nguon goc va ddpc phat tan t d benh vien nay sang cac benh vien khac hoac ngdoc lai. Van de nay tiep tuc dat len vai cac nha kiem soat nhiem khuan va vi sinh, can phai lam gi tiep theo de co the biet ro eon d d d n g lUu hanh cua cae chung nay cung nhU xac dinh rd nguon lay nhiem, d d d n g lay nhiem de kip thdi ngan chan sU tiep tuc phat tan cua cac chung nay. PFGE la mdt p h u o n g phap hdu hieu tiep can djch t§ hpc phan t d ma nhieu quoc gia tren the gidi da ap d u n g de phat hien nguon nhiem va kha nang lay lan cCia cac chung nay d trong cung m o t khoa hay nhieu khoa p h d n g , benh vien, vung, quoc gia tren the gidi.
Trong nghien cdu nay 20 chung A. baumannii khang vdi nhom carbapenem phan lap ddpc tao ra 3 chum VI khuan khac nhau (A,B,C) co tUong d o n g kieu gen tren 90% nam rai rac d ca 6 benh vien, dac biet chum A vdi 11 chCing A. baumannii khang vdi carbapenem eo kieu gen t d d n g d o n g den 96%, t r o n g do cd 5/11 chung t d Benh vien TL/ Hue, chum C g d m 5 chOng vdi kieu gen tUOng dong den 92%, trong do co den 4 chung A. baumannii la t d benh vien TU Hue. Theo nhdng nghien cdu ve dich te hpc phan t d , nhieu tac gia da chdng minh A. baumannii khang vdi khang sinh nhom carbapenem cd the khang vdi nhieu khang khae nhau t r o n g n h d m (5- lactam va chung eo the phat tan sd lay lan qua con d u d n g plasmid va hoac nhiem sac the. Oieu do giup giai thfch tai sao d Benh vien TU Hue t o n tai dai dang cac chung A. baumanniico kieu gen tUOng d o n g cao. Day rat cd the la nguy eo tiem tang phat tan cac chung vi khuan khang thuoc ra nhieu cac
khoa p h o n g hoac benh vien khac. Nghien edu cCia Pilar va CS (2010), tai Tay Ban Nha da cho thay co sd tai lap cua n h d n g chung co t d d n g d o n g ve kieu gen tai cac benh vien t r o n g n h d n g nam khac nhau va su Idu hanh x§y ra khi n h d n g n g u o n nhiem khong ddpc kiem soat [6]. Tuong t u , A.Nemec va es, khi phan tich 150 chCing phan lap t d 56 khoa Hoi sdc tich cue, t h u o c 20 benh vien va 15 thanh p h d khac nhau cua nude Cong hoa Czech, da phat hien cd 108 chung A baumannii co kieu gen t d d n g d o n g vdi kieu gen EU Clone I, va 16 chdng co kieu gen tUong d o n g vdi EU Clone II [5].
Trong nghien cdu nay, phat hien 18 chdng A baumannii phan lap dUdc t d cac khoa Hdi sdc tich cue cua 6 benh vien vdi 3 kieu gen khac nhau qua 3 chum D, E, F, t r o n g do dang chu y la chum D co 9 chung A. baumannii cd kieu gen tUOng dong den gan 97%, 6 chung tUong d o n g den 100%, 5 trong so 6 chung d d la cua khoa Hoi sdc Benh vien TU Hue.
Tdong t d d chum E cd kieu gen tUOng dong den 92%, t r o n g do ed den 4 chung t h u o c Benh vien TL/
Hue. Nghien cdu cua Trdong Thi A n h ThU va CS (2012), thdc hien tren nhiJng benh nhan viem phoi do A. baumannii t h d c hien tai khoa Hoi sdc tich cdc Benh vien Bach Mai da cho thay co sU t d d n g dong ve kieu gen tren 80% cua eac chung A. baumannii t r o n g dich phe quan t o n tai t r o n g sudt 8 thang nghien edu [1].
5. Ket luan
Cac chung A. baumannii phan lap dddc tai cac benh vien Idn cua Viet Nam c6 m o i tUdhg dong cao ve kieu gen. Cd m o i lien quan kieu gen giCJa cac chdng phan lap dupe tai khoa Hoi sdc tich cdc va gen khang sinh cda c h u n g .
Tai lieu t h a m khao
TrdOng Anh Thu (2012) Dac diem dich ti hoc nhiim khuan phoi benh vien tai khoa Hoi sdc tich cdc Benh vien Bach Mai 2008-2009 Vien Ve sinh Dich te Trung UOng http://www.nihe org.vn, Truy cap ngay 16 thang 2 nam 2013.
TAP CHl Y Dl^OC LAM S A N G 1 Tap 10-So 3/2015
Nguyen Vu Trung (2009) Acinetobacter, Vi khuan hoc. Nha xuat ban giao due, Oai hpc Y khoa Ha Noi, tr.319.
Callie Camp, Owatha L. Tatum (2010) A Review of Acinetobacter baumannii as a Highly Successful Pathogen in Times of War. Labmedicine 41(11):
650-657.
Villalon P, Valdezate S, Medina-Pascual MJ, et al (2011) Clonal diversity of nosocomial epidemic Acinetobacter baumannii strains isolated in spain.
Journal of clinical microbiology 49(3). 875 -882,
Alexandr Nemec, Lenka Krizova, Martina Maixnerova, et al (2009) Emergence of carbapenem resistance in Acinetobacter baumannii in the Czech Republic is associated with the spread of multidrugresistant strains of European clone II. J Antimicrob Chemother 62.484-489.
Pilar V, et al (2011) Clonal diversity of nosocomial epidemic Acinetobacter baumannii strains isolated in Spain. Journal of clinical microbiology 49(3):
875-882.