Nghien
CIFU- Ky thuat
Phat Men mot so gen de khang khang sinh
tren vi khuan Acinetobacter baumannii phan lap tu*
Benh vien Da khoa tinh Binh Duong bang ky thuat multiplex PCR
Ngo Thi Bich Thuy, Nguyen Tii Anh*
Khoa Duac - Dgi hoc YDuac TP. Ho Chi Minh
*E-mail: [email protected] Summary
A multiplex PCR detection procedure of the three antibiotic resistant genes of A. baumannii, including the
^^^oxAsuke d^tie, the AmpC gene, the aph (3)-Vla gene, was set up. The positive PCR resulted genes are: (1) the bla^^^, - 825 bp, (2) AmpC ~ 663 bp, (3) aph (3')-Vla ~ 234 bp. The results of the multiplex PCR are the same as those of the In vitro antibiotic sensitivity testing of A. baumannii by antimicrobial susceptibility technique, except for amikacin. However, the traditional microbiological methods such as the antimicrobial susceptibility technique showed time-consuming, especially in combination with the bacterial identification test Meanwhile, application of the multiplex PCR directly from the bacteria colony enabled to proceed the bacterial identification and the antibiotic resistant gene detection at the same time, and therefore, much saving time, labor and cost.
Therapeutically, the striking advantage is the time-savingness of the test, which may allow the clinician indicate the proper antibiotics in time to reduce the treatment time and cost
Keywords: Hospital Binh Duang - acguire infection, Acinetobacter baumannii, antibiotic resistant gene, multiplex PCR.
Dat van de
Hien nay, tren t h i giai cung nhu tai Viet Nam, Acinetobacter baumannii la mpt trong nhirng nguyen nhan chinh gay nhiim khuin benh vien''''.
Ciing vol su gia tang ti le nhilm A. baumannii la hien tugng xuit hien eae ehung khOng nhay cam vol khang sinh, k l ea khang sinh t h i he mai earbapenem. Theo bao eao cua Trung tam Phong ehing va Kiem seat djeh benh IVIy (CDC) nam 2013, eo kheang 63% truang hgp nhilm A. baumannii da khang thule'^'. Theo tong kit (2009) cua ca quan Hgp tac toan c i u v l khang khang sinh cua Viet Nam thi tren 60% cac chiing A. baumannii phan lap tai mpt s l benh vien nhu Benh vien Bach Mai, Benh vien Chg Riy va Benh vien Benh Nhiet doi Trung uong la cae chiing da khang thulc'^'.
De CO ehl djnh khang sinh som, hgp ly, an toan va hieu qua cho nhirng benh nhan nhilm A.
baumannii d l khang khang sinh thi viec xae djnh sam dae tinh d l khang eua nhOng chiing nay la rat can thilt. Vi vay, nghien ciru phat hien cae gen d l khang khang sinh tren vi khuan A. baumannii phan lap tif Benh vien Da khoa tinh Binh Duong bang ky thuat multiplex PCR da duge thuc hien.
Trong thue nghiem nay, ba gen khang khang sinh dugc chpn, g i m co gen Wao^.j,.,,^^ - d l khang earbapenem, gen ampC - d l ktiang cephalosporin va gen aph(3)-Vla-dh khang amikacin. Gen bla^.^
jj, 1,^^ ma hoa che enzym earbapenemase thuy phan earbapenem'"'. Gen AmpC ma hoa ehe enzym AmpC cephalosporinase thupc lap C p-lactamase, enzym nay thiiy phan nhom 3-lactam, nhom cephalosporin phi hep va rpng'^i. Gen aph(3)-Vla ma hoa enzym aminoglycosid phosphotransferase phan buy amineglyeosid'^'.
Vat lieu va p h u a n g phap nghien CLPU W khuin: A. baumannii dugc phan lap tif 48 miu benh phim ciia benh nhan diiu trj tai cac khoa lam sang ciia BVDK tinh Binh Duang trong thai gian tir 15/08/2014 d i n 31/10/2014.
Vi khuin dugc phan lap tren moi truang MacConkey (Merck) va nuoi c i y tren Tryptic Soy Broth (Merck).
Phan U'ng multiplex PCR phat hien gen de khang khang sinh. Ba cap mli dae hieu dugc thilt k l , diing d l khuleh dai eae gen Wa„,.., ,„
' ^ OXA-51-like
(~ 825 bp), gen ampC (~ 663 bp) va gen aph(3')- Vla (~ 234 bp) (bang 1)'«'.
TAP CHI DirpC HOC - 4/2016 (SO 480 NAM 56)
• Nghien CLFU
Moi OXA-51-like F OXA-51-likeR
ACIS ACI6 aph (3')-Vla F aph (3')-Vla R
- Ky thuat
B a n g i : Trinh tu mdi Trinh tirnucleotid (5'-3'j ATGAACATTAAAGCACTC CTATAAAATACCTAAnGnC AClTACnCAACTCGCGACG TAAACACCACATATGrrCCG ATACAGAGACCACCATACAGT
GGACAATCAATAATAGCAAT
phan ung multiplex PCR Kich (hiroc gen
825 bp
663 bp
234 bp
1
Muc dich Khuich dai gen Wa„^^„,,^
Khuech 3a\ gen AmpC
Khuech dai gen aph (SJ-Via
Sau khi tiin hanh til uu hoa, thanh phin phan irng multiplex PCR da xac dinh gim co dung djch dem PCR (1 X), dNTPs (0,4 pM), MgSO, (4,0 pM), mil OXA-51-like F - R va ACIS - ACI6 (0,4 pM), moi aph (3')-Vla F - R (1,0 pM), Tag ADN polymerase (2,5 Ul), vi khuan (khuich tan mpt khOm vi khuan trpng 20 pi moi truong TSB va diing 1,0 pi cho phan irng PCR), nuac cit vira dii 25 pi.
Chuang trinh phan irng multiplex PCR gim eae giai doan: biin tinh (94 "C/l phut), bat cap mli (47
°C/1 phiit), keo dai (72 "C/l phut). Phat hien cac dean gen khuich dai bang phuong phap dien di tren thach agarose 1%.
Xac djnh tinh nhay cam v&i khang sinh cua A. baumannii bang ky thuat khang sinh do. Liy mpt khuin lae A. baumannii, khuleh tan vap nuac mull 0,85% sao cho dat dp due ~ 0,5 MaeFariand (1-3.10= vi khuan/ ml). Pha bang 100 lin de thu dugc huyIn djch vi khuin eo ning dp 10^ vi khuin/ ml va sir dung ngay (trpng vpng 15 phiit). Diing que bong vo khuin trai diu vi khuin tren mat thach Mueller Hinton Agar (Merck)'". De kho mat thach va dat 4 leal dTa giay khang sinh imipenem, cefepim, cefetaxim, amikacin (tuang irng vol khang sinh khae sat gen d l khang va co trong danh mue thulc cua benh vien). De duang kinh vong ifc ehe va se sanh vol bang bien giai eua CLSI(2015).
Ket qua va ban luan
Phan ipng multiplex PCR phat hien 3 gen de khang l<hang sinh
Phan img PCR xac djnh tirng gen rieng le da dugc thuc hien trong cac cong b l truac day Tuy nhien, multiplex PCR de phat hien ding thoi 3 gen 6/3^^,.,,.^^, gen ampC va gen aph(3')-Vla chua duge nghien ciru. Vi vay nhim tii uu hoa dieu kien multiplex PCR, cac ylu t l anh huang da duge khao sat nhu ning do thanh phin PCR, nhiet dp bit cap mli,...(sl lieu khong trinh bay)' Phan irng sir dung ADN khuon miu la ADN nhilm sae t h i eua .4. baumannii mku 5199 chilttaeh theo qui trinh cua nhom nghien ciru (hinh 1)"='.
10
Sau khi xac djnh dugc thanh phin va diiu kien phan irng, multiplex PCR dugc thuc hien true tilp tren 48 khuan lac cua 48 mau vi khuin A.
baumannii. Do djnh huang tiep theo la nghien ciru c h i tao bp kit phat hien A. baumannii va gen de khang khang sinh cua chung; vi vay, tren mli miu vi khuin, da tiin hanh ding tho'i (1) multiplex PCR de dinh danh A. baumanniP va (2) multiplex PCR d l phat hien gen d l khang khang sinh. Khuan lac ciia E. coli ATCC 29522 dugc su' dung lam chirng am. Kit qua chay dien di gel agarose cua 10 miu dai dien the hien a hinh 2 (miu 5370, 2123, 2009, 2074, 2147, 5199, 5308, 5249, 5270 va 5377).
100 bp (-) PCR
Hinh 1: Gel agarose chay dien di san pham multiplex PCR (v&i ADN khudn miu la ADN nhiim sic thd cua A. baumannii mau 5199) khuich dai 03 9^" ^'aox4.5<-/*e ^825 bp), AmpC (663 bp), aph (3-)-Via
(234 bp). Chung (-): E. coll ATCC 29522 Ve kit qua djnh danh, tif 48 m i u vi khuan diu khuich dai dugc 02 bang ADN co kieh thuoc ~ 425 bp (gen recA) va ~ 208 bp ( dean ITS 168 - 23S rADN), day la nhirng trinh tu nucleotid dac hieu ciia loai A. baumannif-^\ V l kit qua phat hien gen d l khang khang sinh, co 10 mau (5199, 5308, 5249, 5270, 5377, 5251, 1571, 1525,1672, va 2146) khuich dai dugc ea 03 gen: gen bla^^, 5i-i,ke ~ 825 bp, gen AmpC ~ 663 bp, va gen aph (3')-Vla ~ 234 bp. Mpt m i u (5370) ehi khuleh dai 01 dpan gen AmpC, 37 m i u khuich dai 02 dean gen bla^^^^^^^ va gen AmpC. Kit qua nay
TAP CHI DirOC HOC - 4/2016 (SO 480 NAM 56)
Nghien
CIFU- Ky thuat
cho thay tat ca 48 mau A. baumannii khao sat d i u d l khang khang sinh nhom cephalosporin, treng do 37 m i u d l khang them khang sinh
nhom earbapenem va 10 mau da d l khang voi ca 3 khang sinh cephalosporin, earbapenem, amikacin.
1 kbplus (-Xa) (-)(b)5370(a)5370(b)2123(al2l23(b) 2009(a) 2009(b)2074(al2074(b)2147(a)2147(b)
lkbplus(-Xa)(-Xb) 5199(a) 5199(b) 5308(al 5308(b)5249(a)5249(b)5270(a)5270(b)5377(a)5377(b)
Hinh 2: Gel agarose chay dien di san phim (b) Multiplex PCR phat hien gen di khang Xac djnh tinh nhay cam vai khang sinh cua A. baumannii bang ky thuat khang sinh do
Song song viec phat hien eae gen d l khang earbapenem, cephalosporin, amikacin bing ky thuat multiplex PCR, thi> nghiem in vitro tinh nhay cam khang sinh ciia 48 miu A. baumannii bing ky thuat khang sinh do cung dugc thue hien. Vol thiP nghiem khang sinh do, chgn 4 khang sinh la imipenem, eefepim, cefetaxim, amikacin. Duang kinh vong ire ehl eua 10 miu dai dien (5370, 2123, 2009, 2074, 2147, 5199, 5308, 5249, 5270 va 5377) duge trinh bay a bang 2.
Bang 2: Bien giai kit qua khang sinh dd ciia 10 miu A. baumanni
(a) Multiplex PCR phat hien A. baumannii va khang sinh. Chung (-): E. coli ATCC 29522
Miu
5370 2123 2009 2074 2147 5199
Im (mm) (ak1/dk2) S (27,7/22)
R (7,7/22) R (7,3/22) R (11/22) R(7/22) R(8/22)
Cm (mm) (i3k1/a<2) S (22/18) R (10/18) R (11,7/18) R (13,7/18) R (11/18)
R(8/18)
Ct(mm) (3iiim2) R (17,7/21)
R(7/21) R(7/21) R (7,3/21)
R(7/21) R(7/21)
Ak(mm) (dk1/dk2)
S (23/17) R (7/17) R (7/17) R(7/17) R(7/17) R(7/17)
5308 5249 5270 5197 5377
R (8/22) R(8,3/22)
R (10/22) R(9/22) R (7,3/22)
R (7,7/18) R(8/18) R (10,3/18)
R (10/18) R (8,7/18)
R (7,3/21) R(7/21) R(7/21) R(7/21) R(7/21)
R (7,3/17) R (14,3/17)
1 (15,3/17) R (7/17) R (7,3/17)
TAP CHI DLTOC H O C - 4/2016 (SO 480 NAM 56)
dk 1 - ducmg ifinh vong Crc ciie theo ket qua khang sinh do, dk 2 - duong kinh vong i>c chi cua khang sinh nhay vdi A. baumannii (theo CLSI). Imipenem (Im), Cefepim (Cm), Cefotaxim (Ct), Amikacin (Ak)
Nhom earbapenem (khang sinh Imipenem) ehi CO 01 miu 5370 con nhay, 47 miu con lai diu bj d l khang (ti le 97,9 %). Kit qua nay phii hgp vgi kit qua phan irng multiplex PCR - khong phat hien gen bla^^^^,^^.. a miu 5370 (hinh 2).
Nhom cephalosporin the he thir 4 (khang sinh cefepim) cung chi con 01 mau 5370 nhay 47 mau con lai deu bj d l khang (ti le 97,9 %). Tuy nhien, multiplex PCR lai phat hien ca 48 miu diu CO gen d l khang cephalosporin. Co tae gia cho ring mac dii vi khuin co gen AmpC - d l khang eae cephalosporin nhung ngoai tar cefepim v i n eon nhay'^'
Nhom cephalosporin the he thd" 3 (khang sinh cefotaxim) co ti le khang da len din 100%.
11
Nghien CIFU - Ky thuat
Oilu nay phii hgp khi t i t ea cac miu A. baumannii thir nghiem diu co gen AmpC d l khang cephalosporin.
Nhom aminoglycosid (khang sinh amikacin) CO 01 mau eon nhay ti le d l khang va d l khang trung gian cae (97,9%). Kit qua khang sinh d l kha khae biet so voi kit qua multiplex PCR. Hien tugng nay da dat ra vin d l : A. baumannii da thay dii eo ehl d l khang nhom aminoglycosid noi ehung va amikacin noi rieng chua? D l tra lo^i eau hoi tren, eo thi cin thilt phai giai trinh tu khong chi dean gen aph (3'j-Vla ma con toan bp he gen ciia cac chiing A. baumannii nay.
Ket luan
Qui trinh multiplex PCR phat hien 03 gen d l khang khang sinh eiia A. baumannii, gim gen '!''aoxA-5i-i*e' gen AmpC, gen aph (3')-Vla da dugc xay dung. Phan irng duong tinh khi phat hien dugc san phim PCR (1) dean gen WSoxA-swike ~ ^25 bp, (2) gen AmpC ~ 663 bp, (3) gen aph (3')-Vla ~ 234 bp. Kit qua nhan djnh v l tinh de khang khang sinh du/a tren multiplex PCR phii hgp vol thir nghiem in vitro dp nhay eiia A. baumannii bing ky thuat khang sinh d l , ngcai trir truong hgp cua arnikacin.
Tuy nhien, phuang phap truyin thing nhi^khang sinh do cin kha nhieu thoi gian, dae biet neu thue hien ca xet nghiem djnh danh vi khuin. Trong khi do, thuc hien ky thuat multiplex PCR true tilp tif khuin lae, co t h i tiin hanh ding thai vira djnh danh vi khuin vira phat hien met s l gen d l khang khang sinh. Nho vay, c6 t h i rut ngan dugc thai gian xet nghiem, bae sT lam sang se dua ra phae d l khang sinh diing ehi djnh trong thai gian sam nhit. Diiu nay se gop phan giiip benh nhan giam thoi gian eung nhu ehi phi diiu trj va tien lugng benh eung se tiin triln tit ban.
(Ngay nhan bai: 31/01/2016
Tai lieu tham khao
1. Worid Health Organization (2011), Report on the Burden of endemic health care-associated infection worldwide.
2. CDC-Centers for Disease Control ADN Prevention (2013), Antibiotic resistance threats in the United States.
3. GARP-Nhom nghien ciru quoc gia ciia Viet Nam (2011), Phan tich thuc trang sd dung khang sinh va khang khang sinh & Viet Nam, Ha Npi.
4. Coelho J., Woodford N., Afzal-Shah M., and Livermore D. (2006), "Occurrence of OXA-58-like carbapenemases in Acinetobacter spp. collected over 10 years in three continents", Antimicrob. Agents Chemother 50, pp. 756-758.
5. Stephane Corvee, Nathalie Garoff, Eric Espaze, Cecile Giraudeau, Henri Drugeon and Alain Reynaud (2003), "AmpC cephalosporinase hyperproduction in Acinetobacter baumannii clinical strains", Joumal of Antimicrobial Chemotherapy. 52, pp. 629-635.
6. Vila J, Ruiz J, Navia M, Becerril B, Garcia I, Perea S, Lopez-Hernandez I, Alamo I, Ballester F, Planes AM, Martlnez-Beltran J, de Anta TJ (1999), "Spread of amikacin resistance in Acinetobacter baumannii strains isolated in Spain due to an epidemic strain", Journal of Clinical Microbiolog. 37(30), pp. 758-761.
7. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement, IVI100S17, standard by Clinical and Laboratory Standards Institute, 01/2007.
8. Nguyin Tu Anh, Truong Thj Thu An, Ngo Thj Mong Thuy (2015), " Djnh danh va phat hien vi khuan Acinetobacter baumannii co trong miu benh pham cua benh nhan dieu trj tai Benh vien Da khoa tinh Binh Duong bang ky thuat sinh hoc phan tir". Tap chi Yhoc TP Hd Chi Minh. 19 (3), tr. 278-282.
9. Federico Perez, Andrea M. Hujer, Kristine M.
Hujer, Brooke K. Decker, Philip N. Rather and Robert A. Bonomo (2007), "Global challenge of multidmg- resistant Acinetobacter baumannii", Antimicob. Agents Chemother 51(10), pp. 3471-3484.
• Ngay duyet dang: 04/04/2016)
Nghien cmi bao che phirc hop lipid amphotericin B
/ TrSn Thj Hai Ygn'*, Dirong Thi Tiiuan'
\ / Tu-ffng Phi Vu•o^lg^ Pham Xuan Tha^g^ Pham Thi Minh Hue'
I 'Tru&ng Dgi hoc Duac Hd Noi ' Vien 69, Bo Tu lenh Bdo ve ldng Chit tich Ho Chi Minh ' E-mail: yentth@hup. edu. vn
Summary
In conventional formulations, large moleculed amphiphilic Amphotericin B (AmB) of board-spectnim antifungal activity, wide use for systemic fungal infection, is dispersed in micells, and as such, AmB Is unstable in the human blood and fanned toxic aggregates on release; in tendency of developing lipid fomiulations to reduce the dmg toxicity, and retain activity, fonvation of lipid complex of AmB by solvent displacement was 12 TAP CHI DirOC HQC - 4/2016 (SO 480 NAM 56)
