DOT BIEN GEN gyrh, parC
6 NHUfNG CHUNG PSEUDOMONA AERUGINOSA KHANG CIPROFLOXACIN VA IEVOFLOXACIN
Le Van DuyftS ITran Quang Himg', Tran Thi Giang Hirong^, Nguyen Vu Trung^^.
Pseudomonas aeruginosa khang fluoroquinolone do thay doi tren gen gyrAwaparC. Muc tieu: Xac dinh dot bi^n gen gyrA vaparC a nhiing chung P. aeruginosa khang ciprofloxacin va Ievofloxacin.
Ddi twang vd phirong phdp: 107 chimg P. aeruginosa (82 chung khang, 19 chung nhay cam va 6 chiing khang trung gian vai nhom fluoroquinolone) dugc phat hien cac dot bien tren gen gyrA va parC b^ng PCR va giai trinh tu gen. Kit qud: 57/107 chung co dot biln tren gen gyrA (codon 83 - Thr, 84 - Ala va 85 - Vai). 22/107 chung co dot bien tren gen/jarC (codon 76 - Vai, 87 - Ser, 103 - Arg va 123 - Ala). S6 lugng cac diem dot bien tren gen gyrA, parC ty le thuan voi gia tri MIC cua cac chiing P. aeruginosa. Ket luan: Cac dot bien phat hien chu yeu a chiing khang va s6 lugng dot bien ty le thuan voi miic do de khang. Dot bien tren gen gyrA dong vai tro quan trpng toi su de khang vol nhom fluoroquinolone cua P. aeruginosa, cac dot bien tren gen/ja/C co vai tro bo trg cho su de khang voi nhom fluoroquinolone cua P. aeruginosa.
Tir kh6a: P. aeruginosa, dot bien gen gyrA, parC, fluoroquinolone.
DAT VAN DE
Pseudomonas aeruginosa (tru'c khuan mu xanh) la can nguyen gay benh cd hpi quan trpng trong cgng dong va rat hay gSp trong nhiem khuan benh vien nhu" nhilm khuan vet thu'dng, vet mo, nhiem khuan vet bong, nhiem khuan huyet... Day la vi khuan Gram am, thupe chi Pseudomonas, hp Pseudomonadaceae. Loai vi khuan nay CO sire chju duhg tot d ngoai canh, gay ra tinh trang benh rat da dang va nang ne do co nhieu yeu to dpc lu'c cung tham gia vao qua trinh gay benh... Viec dieu tri eung rat kho khSn, thu'dng dan den nhieu hau qua nghiem trong nhd t3ng ty le tCf vong, keo dai thcfl gian nam vien, tang ehi phi dieu tri.
NhiJng nam gan day, viec su" dung khang sinh chu^
"•Hoc viVn Qudn \ '-'Benh vien Bfnh nhtet dai Trung uang "'Bp Y te.
"•Tniong Dgt hgc YHaNgi.
\^a\ n h i n b i i : 02/10/2015 NBa> pbiD bifR long: OS/IO/2015 NRSI duyft d i n g : 30/10/2015.
Ngiriri chju t r i c h nhifm ngi dung khoa hijc: Le Van Duyet, Benh vien Bfnh nhiet doi Tnmg uang
Diin thogi: 0936647979. E-mail duyetibK^gmad com
hpp ly trong dieu trj nhiem khuan benh vien do R aerug- inosa da lam gia tang tinh trang khang khang sinh cua loai vi khuan nay. Cac nghien culi trong nu'dc ve mu'c do nhay cam khang sinh deu cho thay mii'c dp de khang khang sinh cua P. aeruginosa ngay cang gia tSng d mii'c bao dpng va dien bien vo eiing phii'c tap. 100% cac chung R aeruginosa da eo bleu hien khang khang sinh. Oac biet, t / le phan lap cae chung R aeruginosa da khang khang sinh ngay cang tang, gay rat nhieu kho khan trong cong tac du" phong va i^eu tri.
Cac nghien eiiti tren the gicfi cho thay, R aeniginosa CO nhieu cd che de khang doi vdi nhom fluoroquinolone.
CP die chmh la bien doi dich tac dpng cua hai enzym md vong xoan DNA la enzym DNA gyrase (co dich tac dong chinh la gen gy/A nam b'en nhiem s3c the ciia vi Wiuan) va enzym topoisomerase IV (co dieh tae dpng la gen paiC nam tren nhiem sac the eua vi khuan). 6 nddc ta hien nay ehu^ CO cong trinh nghien cdu nao ve nhiJng gen nay d eac Chung R aemginosa. Trong nhuhg nam gan day, da co mpt so cong binh nghien a i l i ve aJ che de khang khang sinh cua R aeruginosa. Tuy nhien, van chu^ co cfing trinh
- T R U Y ^ N H i M V l f T N A M * SO 4 ( 1 2 ) - 2 0 1 5
nghien ciili ve cd che de khang khang sinh cua R aerug- inosa vcfi nhom fluoroquinolone, mpt trong nhutig nhom khang sinh dang du'dc sd dung rpng rai trong dieu b-i cac benh nhiem khuan do P. aeruginosa, dac biet la ciprofloxacin va levofloxadn'". Vi vay, de cung cap cP sd khoa hpc eho cac nghien cdu sau nay, gop phan lam ro them cd che de khang cua R aeruginosa vcfi nhom fluo- roquinolone, chung toi tien hanh nghien culi nham muc tieu: Phat hien dot bien gen gy/A, paiC 6 nhiJng ehung Pseudomonas aeruginosa khang ciprofloxacin va Iev- ofloxacin.
D O I TU'ONG VA PHl/ONG PHAP
Thiet ke nghien ciiu: Nghien ciili mo ta cat ngang. Thdi gian nghien ciili: tir 04/2012 - 07/2015.
D&i tu'dng nghien ciTu: 107 chung R aeruginosa (82 chiing R aeruginosa de khang, 19 ehung nhay cam va 6 chiing CO mtfc dp khang tmng gian vdi ciprofloxacin va Iev- ofloxacin) phan lap tu" 13 benh vien: Benh vien da khoa Trung u'dng Thai Nguyen, Benh vien da khoa Uong Bi (Quang Ninh), Benh vien Benh nhiet ddi Tmng u'dng, Benh vien Bach Mai, Benh vien Quan Y103, Benh vien da khoa Thai Binh, Benh vien da khoa TTianh Hoa, Benh vien da khoa Trung u'dng Hug, Benh vien da khoa Binh Djnh, Benh vien da khoa Bac t i c , Benh vien Chd Ray, Benh vien Benh nhiet ddi thanh pho Ho Chi Minh, Benh vien da khoa Can Thd tir 09/2012 - 09/2014. Cac chiing vi khuan sau khi phan lap du'dc tien hanh xac dmh mii'c dp khang Wiang sinh sir dung he thong Djnh danh va Khang sinh do td dpng vrrEK®2 Compact. Sau do tien hanh tach chiet ADN tong so bang bp kit thu'dng mai Stratec molecular RTP®Bacteria ADN Mini kit - 250 (Cong hoa lien bang Diit).
Vat lieu: (+)Vat lieu tach ADN ciia eac chiing R aemgi- nosa: kit thu'dng mai Stratec molecular - RTPCDBaeteria ADN Mini kit (Du'c). (+)Vat lieu de thu'c hien PCR: *PCR master mix (Qiagen - My) ; *Cac cap moi dae hieu cho gen gy/A va pa/C.
Bang 1 : Cac cap moi dac hieu cho gen gyrA va parC
Gen dich gyiA
paiC TSn moi gyrft-l gK/ft-Z pa/C-l parC-2
Trinh tu" nucleotide ( 5 ' - 3") S' - GTCTGGTTATGCCATGAG - 3' 5' • GGnrCCTTTTCCAGCTC - 3' 5' - CATCGTaACGCCATGAG - 3' 5' - AGCAGCACCTCGGAATAG - 3'
Kich t h u ^ c (bp)
287
267
Agarose cho dien di; Gel red dimg de nllupm gel; TAE (Tris Acetate EDTA) dung cho dien di.
T h i a bj: Tu lanh lu\J giif cluing - 80°C; May dieu nhiet tlT dgng GenAmp PCR system 9700 AB (ABI - My); May ly tam lanh; Bq dien di Horizon 58 (Gibco - BRL); May soi gel Wealtee Corp Model MD-20 (My), may chup anh gel Gel- doc (Biorad, My); May giai trinh tif sequencer ABI PRISM 3130 (My).
Ky thuat
Ky thuat tach chiet /l/VD; Theo qui trinh cua bp kit Stratec molecular - RTP®Bacteria ADN Mini kit (BCrc).
Ky thuat PCR: TIILTC hien theo nghien cuU cua Neda Gor- gani va cong su™i, hon hpp phan utig PCR gom 200iJl moi loai dNTP (dATP, dCTP, dGTP, dTTP), lOOpM moi cap moi, 1 U Taq ADN polymerase, lOmM Tris - HCi, 50mM KCI, l,5mM MgCI2, lOpI ADN khuon. Phan ung du'dc thiTc hien vfli 32 chu ky. Moi chu ky gom 3 budc: Bien tinh (95°C/30 giay), gan moi (51»C/30 giay), tong hpp chuoi (72°C/lphut). San pham PCR du'dc dien di va chup anh theo thu'dng qui. Cac san pham PCR dUdc giai trinh tir gen trifc tiep sir dung cap moi ciia PCR. Trinh tir cac san pham PCR du'dc phan tich tren phan mem ATCG 7.2 (Nhat) va so sanh vdi trinh tir goc tren ngan hang gen (GenBank) de tim cac dot bien diem tren gen gyrA va paiC.
Thong ke: Sir khac biet giCfe hai ty le dupc danh gia bang test x2, p < 0,05 la sir khac biet co y nghia thong ke.
KET QUA
200bp lOObp
Hinh 1. Ket qua dien di san pham PCR Mlu 04 mang gen gyiA (+), paiC (+) M: Mariier
TRUrfelNHliM V i r r I M A M * S 6 4(12) - 2015 - 9
Ket qua giai trinh W gen
umtn Tyr His j n l b Gl; A^ XM. Ah Vd T)T A^ Tb He Vil Arg TAC CAC CCC CAC GGC GAd AYC GCG OTC TAC OAC ACC ATC GTC CCC
230 MO I 550 2 M ™ l-SrA^tf^*'TACCACCCGCACGGCGACJACCCCOGrCTACGACACCATCGTCCGC l - g r A ' " ' TACCACCCGCACGGCGACATCGCGGrCTACGACACCATCGTCCGC
Hinh 3.2. Dot bien d codon 83 tren gen gyrA cua Chung P. aeruginosa
Ciiu thicii: (*) - Trinf] ti/doan gen gyrA cua diung goc.
(**) - Tr'mt) tl/ doan gen gyrA ciia chiing so 01.
Bang 1 . Ket qua phan tich trinh tu* gen gyiA, paiC cua cac chung P. aeruginosa
So chung (n = 107)
8 1
MIC ( l i g / m l ) CIP
5 5 5 5 5 5 5 5 5 5 S 5 5 5 5 5 5 5 S 5 I
LEV 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9
gyrA 83 Thr lie
He He
He He He He He He He He He He He He
He 84 Ala
Giu
Giu Giu
85 Vai
Asp
Asn
Asp He
pare 76 Vai
Met 87 Ser
Leu
Leu
Leu
Leu Leu
Leu Leu
103 Arg
Leu 123 Ala
Ser
( n = 107)
6 2
MIC ( e g / m l ) CIP
2 2 0,24 0,24 0,24 0,24 0,24 0,24 0,24
LEV 9 9 0,5 1 1 0,25
0,5 1 2 Tgng
8 3 Thr
58 gyrA
8 4 Ala
8 5 Vai
pare 76 1 8 7 Vai . Ser
1 103 1 123 Arg
3 i 4
•
Ala
—-
' • —
1 1 20 1 1 1 1
Ctiu thidi: 6 CO dau "-"la giong vdi chiing diuan.
Bang 2. Phan bo chung P. aeruginosa dot bien tren gen gyrA
MIC ( u g / m l ) CIP
< 1 2
> 4 LEV
< 2 4
> 8 Tong
Tong so chung 19 6 82 107
So lu'dng dpt b i f n n 0 1 57 58
%
0 1,8 98,2 100,0 58 Chung c6 dot bien (57 chung Ichang va 1 chung de khang trung gian). Cac chung niiay cam khong c6 i3iem dot bien tren gen gyr/^. Nhin chung, nong dp ii'c che toi thieu (MIC) tang thi so lu'dng dot bien 6 moi chiing tren gen gyrA cung tang.
Bang 3. So iUOng diem dot bien tren gen gyiA theo mu'c do khang CIP
MIC ( j i g / m l )
5 2 0,24 Tcing
So chung ( n )
82 6 19 107
Tfing s 6 trSn gyrA
57 1 0 5 8
SS lirgng d i l m d $ t biSn tren gyiA
0 Z5 5 19 49
1 53
1 0 5 4
2 1 0 0 1
3 3 0 0 ,3 Ket qua tir bang 3 cho thay, so lu'dng cac diem dot bien tren gen gyd< tang theo mufc dp de khang vdi CIP va chii yeu xuat hien d chiing R aemginosa co MIC > S^jg/ml.
10 - T R U Y ^ NHlilUI VIETNAM * S 6 4 { 1 2 ) - 2015
Bang 4. So lu'dng diem dot bien tr€n gen gyrA theo mu'c do khang LEV
Bang 7. So IUdng diem dot bien tren gen parC theo mu'c do de khang v6i LEV
MIC (Mg/ml)
9 2 1 0,5 0,25 TSng
So Chung (n)
88 2 13 3 1 107
T6ngs5 irengyrA 58 0 0 0 0 58
So lu'dng diim dpt bien tr£n gytA
0 30
2 13 3 i 49
1 54 0 0 0 0 54
2 1 0 0 0 0 1
3 3 0 0 0 0 3 Theo bang tren, so lu'dng cac diem dot bien tren gen gydK tang theo mirc dp de khang vdi LEV va chii yeu xuat hien d chiing R aemginosa co MIC > 9Mg/ml.
Bang 5. Phan bo chung P. aeruginosa dot bien tren gen parC
MIC (Mg/ml) a p
< i 2
> 4 LEV
< 2 4
> 8 Tong
Tong so chiing 19
6 82 107
So lu'dng dgt bien n 0 0 22 22
%
0 0 100 100,0 So lieu d bang 5 cho thay, trong 107 chiing TKMX co mang gen parC, sau khi giai trinh tir thay co 22 chiing xuat hien dot bien. Trong do, 22 chiing co xuat hien dot bien nay deu ia cac chiing khang, cac chiing nhay cam va de khang trung gian khong co diem dot bien tren gen pa/C.
Bang 6. So lu'dng diem dot bien tren gen paiC tiieo mu'c do khang CIP
MIC (lig/ml)
5 2 0,24 T6ng
So chung (n)
82 6 19 107
Tong SO tren parC
22 0 0 22
SoluVng diem dot bien t r i n parC 0
60 6 19 85
1 21 0 0 2 1
2 1 0 0 1
3 0 0 0 0
4 0 0 0 0
Theo bang 6, so lu'dng cac diem dot bien tren gen paiC tang theo mu'c dp de khang vfli CIP va chu yeu xuat hien tren chung P. aeruginosa co MIC > 5|jg/ml.
MIC (|ig/ml)
9 2 1 0,5 0,25 Tong
So chung (n)
88 2 13 3 1 107
Tdng 50 tren pare 22
0 0 0 0 22
S5 lu'dng diem dot bi@n t r « i pa/C 0
66 2 13 3 1 85
1 21 0 0 0 0 2 1
2 1 0 0 0 0 1
3 0 0 0 0 0 0
4 0 0 0 0 0 0 Ket qua tLr bang 7 cho thay, so lu'dng cac diem dot bien tren gen paiC tang theo mirc dp de khang vdi LEV va chu yeu xuat hien tren chiing R aeruginosa co MIC >
9Mg/ml.
BAN LUAN
Trong nghien cuU nay, sau khi phan tich ket qua giai trinh ty" 107 chiing R aemginosa mang gen gyrA, co 58/107 chiing xuat hien dot bien vdi kieu dot bien pho bien ia dot bien d vj tri codon 83 tu* Thr -> lie. Co 3 chiing dot bien d vi tri codon 84 td Aia •'* Giu. 6 vj tri codon 85 CO 2 chung dot bien tiT Vai -• Asp, co 1 chiing dot bien tii Vai -* Asn va 1 chiing dot bien td Vai -* lie. Cac kieu dot bien d vj trf codon 84 va codon 85 luon phoi hdp vdi dot bien tai vj trf codon 83 tiTThr -. He. Trong nghien cu\j nay, CO 49 chiing R aeruginosa mang gen gyrA khong xuat hien diem dot bien. Tren 58 chiing mang gen gyiA, xuat hien dot bien chii yeu d chung R aeruginosa de khang vdi CIP va LEV vdi 57/58 chung mang gen py/A co dot bien.
Tat ca cac chiing R aeruginosa nhay cam vdi QP va LEV deu khong xuat hien diem dot bien. Chi duy nhat 1 chung R aeruginosa trung gian mang dot bien tren gen gyiA.
Tuy nhien, phan tich ket qua MIC tren chiing R aerugi- nosa nay thay vi khuan de khang trung gian vdi QP (MIC
= 2pg/ml) nhutig lai de khang vdi LEV (MIC > 8tjg/ml).
Nhu" vay, chiing nay van cd the xuat hien cac dot bien lien quan den de khang ciia nhom fluoroquinolone. Thong ke so lu'dng cac diem dot bien tren gen gyrA cho thay, co sir ty ie thuan vdi MIC ciia chiing R aemginosa. Khao sat vdi khang sinh CIP cho thay da so cac diem dot bien tren gen gyrA xuat hien d cac chiing R aeruginosa cd MIC >
5pg/ml. Trong so cac chiing R aeruginosa cd MIC = 2pg/ml chi cd 1 chiing xuat hien dot bien tren gen gyiA.
T R U > t N N H l i M V l 6 T N A M * s 6 4 ( 1 2 ) - 2 0 1 5 - 11
Vdi MIC < 0,24Mg/ml, khong cd chung R aemginosa nao xuat hien dot bien. Khao sat vdi khang sinh LEV, ket qua cho thay da so cac diem dot bien tren gen gyd^ xuat hien d cac chiing R aemginosa co MIC > 9jjg/ml. Cac chiing R aemginosa co nong dp MIC = 2\ig/m\, MIC = Ipg/ml, MIC
= 0,5pg/mi, MIC = 0,25^jg/ml deu khong cd diem dot bien. Nhu* vay, ta thay gia trj MIC cang Idn thi cang xuat hien nhieu diem dot bien.
Cac nghien cifu tren the gidi cung dio thay ket qua tu'dng t u vdi nghien cdu cua chung toi. Theo Minoru Yonezawa nghien ciilJ ve dot bien tren gen gyrA d cac chung R aemginosa phan lap du'dc b^n lam sang trong hai nam 1989 va 1993, cac chiing phan lap trong nam 1989 khong c6 chiing nao xuat hien dot bien, cac chiing phan lap trong nam 1993 co ba diem dot bien lien quan chSt che tdi tinh de khang ciia nhdm quinolone. 06 la dot bien d vj tri codon 83 tir Ser -• Leu, codon 87 Asp -* Asn va codon 87 Asp -• G l / ' ' . Hyam Mounrimne nghien ciitJ 30 chiing R aemginosa phan lap du'dc tren iam sang va xac djnh dot bien tren gen gyrA, gyrB va parC lien quan tdi tinh khang CIP. Ket qua cho thay, cd 28/30 chung xuat hien dot bien tren gen gy/A vdi cac vj tri dot bien la codon 83 va codon 87. Tren gen parC tac gia thay 10/30 chiing xuat hien cac dot bien mdi d vj tri codon 80 va codon 84'^'.
Theo Joaquim Ruiz nghien cu\i ve dot bien gen lien quan tdi cd che de khang quinolone cung cho biet, co rat nhieu diem dot bien tren gyrA nhu'vj tri codon 51, codon 67, codon 81, codon 82, codon 83, codon 84, codon 87, codon 106. Hay tren gen parC, cd cac vj tri dot bien nhd codon 78, codon 80, codon 84. Day la nhutig vj trf dot bien xuat hien cd lien quan tdi tinh khang khang sinh vdi nhdm quinolone. Ket qua thong ke cho thay, chi cd cac chung R aeruginosa mang gen gyrA xuat hien dot bien d codon 83, codon 87 va chiing mang gen paiC xuat hien dot bien d codon 80, codon 84 co moi lien quan vdi tinh da khang thuoe tren lam sang. NhU vay, nghien cifU ciia chung toi cung cho ket qua tu'dng dong vdi tac gia khac khi xem xet dot bien tren gen gyrA d codon SB^^i, Mjnori Matsumoto va cong sd tien hanh nghien ciTu 114 chiing R aemginosa phan lap du'dc tren lam sang. Ket qua, co 22 chiing (19,3%) khang vdi LEV. Trong do, cd 6 dot bien axit amin da dude phat hien d vi trf codon 83 bJ'Thr - lie, 87 tir Asp
^ Tyr va 87 tir Asp -»Asn d gen gyrA va codon 87 td Ser - Leu, 87 tir Ser -* Trp va 91 tCr Giu ^ Arg d gen pafC^.
Mpt nghien culi ciia tac gia Katsumi Shigemura va cpng sd
cho thay, cac diem dot bien d codon 83 tii Ser -» Leu, codon 87 tir Asp -»Asn tren gen gyiA. Dot bien d codon 80 tir Ser -* He, codon 84 tir Giu -<• Vai tren gen patC co moi lien quan dang ke vdi MIC ciia nhdm khang sinh flu- oroquinoloneti.
Ket qua giai trinh tu" tren gen gyrA chung toi thay, o3 3 diem dot bien tai vj tri codon 83, codon 84 va codon 85.
Trong do, dot bien d codon 83 da dddc cac nghien cihj chi ra la diem dot bien lien quan tdi tfnh khang fluoro- quinolone. So sanh ket qua cua chiing toi vdi cac nghien cull tren the gidi thay co sd tddng dong. Doi vdi hai vi tri dot bien 6 codon 84 va codon 85 tren gen gyiA van chda cd tac gia nao chiTng minh dUdc dot bien dd c6 lien quan tdi cd che de khang ciia R aemginosa vdi nhom fluoro- quinolone.
Sau khi phan tach ket qua giai trinh td gen pa^C ciia 107 chiing R aeruginosa. Kieu dot bien pho bien la d w tri codon 87 tir Ser -» Leu vdi 20/107 chiing. Cac diem dot bien d codon 76 - Vai, codon 103 - Arg va codon 123 - Ala chi CO duy nhat 1 chiing. Trong nghien cuXi nay, cd 85 chiing khong xuat hien diem dot bien d gen parti. Ca 22 chiing xuat hien dot bien deu la chung R aeruginosa de khang vdi CIP va LEV. Tat ca cac chiing R aeruginosa mang gen paiC nhay cam va trung gian vdi QP va LEV deu khong xuat hien diem dot bien khi phan b'ch ket qua giai trinh tu" gen. Thong ke so lu'dng cac diem dot bien tren gen parC cho thay, co sir ty le thuan vdi MIC cua chung R aeruginosa. Khao sat vdi khang sinh CIP chung toi thay da so cac diem dot bien tren gen parti xuat hien d cac chiing R aeruginosa cd MIC > 5pg/ml (22 chung).
Trong dd, 21 chung xuat hien 1 diem dot bien va 1 chung xuat hien 2 diem dot bien. 6 cac chiing R aemginosa co MIC = 2Mg/ml va MIC < 0,24^Jg/ml, khong cd chung R aemginosa nao xuat hien dot bien. Vd^ khang sinh LEV, da so cac diem dot bien tren gen parC xuat hien d cac chiing R aeruginosa cd MIC > 9pg/ml. Cac chiing P.
aeruginosa cd nong dp MIC = 2pg/ml, MIC = Ipg/ml, MIC
= 0,5|jg/ml, MIC = 0,25pg/mi deu khong cd diem dot bien. NhU vay, gia tri MIC cang Idn thi cang xuat hien nhieu diem dot bien. Hay ndi each khac, khi cac chiing R aeruginosa tfch lijy cang nhieu dot bien, chiing co the trd thanh chiing khang vdi nhdm khang sinh fluoroquinolone va khang vdi MIC cang Idn.
Cac nghien cull tren the gidi cho thay sd tddng dong
12 - T R U Y ^ N H ^ M V I $ T M A M * SO 4 ( 1 2 ) - 2 0 1 5
vcfi nghien ciili ciia chiing toi. Takashi Deguchi nghien culi 22 chung R aemginosa phan lap dUdc tren lam sang. Tac gia da tim ra diem dot bien tren gen paiC d vi tri codon 87 - Ser va codon 84 - Giu. Day deu la nhOtig diem dot bien da dUdc cac bao cao trUdc day va cd lien quan tdi de khang khang sinh nhom fluoroquiolonet"'. Nhu vay, nghien cirtj ciia chiing toi cung thay cd diem dot bien tren gen gyiA va pa/C tUdng tU nhU nghien culi ciia tac gia tren'^'. Trong ddt biing phat cac chiing R aeruginosa da khang thuoe tai cac benh vien cpng dong d Nhat Ban, Jun- ichiro Sekiguchi tien hanh nghien culi 284 chiing R aerug- inosa phan lap dUdc tren lam sang. Tac gia sU dung cac gen nam trong vung quyet dinh tfnh khang thuoe eua R aeruginosa ddi vdi nhdm fluoroquinolone de tien hanh nghien cUu. Ket qua cho thay, tren cae chiing R aemgi- nosa ngoai nhiJng diem dot bien da dUdc nhieu nghien ciftj chuhg minh cd lien quan tdi tinh khang thuoe cua R aeruginosa doi vdi nhom fluoroquinolone con co mpt so diem dot bien mdi. 254/284 chiing (89,4%) mang gen gyrA cd dot bien hay gap nhat, bao gom dot bien d codon 83 tir Thr -* He va 87 ttr Asp ^ Asn. Trong dd, 251/284 chiing mang dot bien d vi trf codon 83 tU Thr -- He (88,4%), 9 chiing phan lap cd ca ddt bien d codon 83 - Thr va 87 - Asp. Vdi gen parC, cd 244/284 chiing (85,9%) cd dot bien, bao gom codon 87 tir Ser -* Leu va 91 tir Giu -> Lys. Trong do, 242/284 chiing mang dot bien d codon
87 tir Ser -> Leu, 3 chiing phan lap cd ca dot bien d codon 87 tir Ser -»Leu va codon 91 tir Giu -* Lys. Nghien culi ciia chung toi cung cd diem dot bien tren gen gyrA d vi trf codon 83 tir Thr -* He va paiC d vi tri codon 87 bJ' Ser -*
Leu tUdng tU nhU nghien cuU ciia tac gia tren[6]. Lomhott va cpng sU nghien cUu tren 106 benh nhan viem giac mac va phan lap dUdc P. aemginosa cung cho thay, gen gyrA ed dot bien chii yeu d vj tri codon 83 va gen patC dot bien d vi tri codon 87.06 la nhiihg vj tri dot bien lien quan tdi b'nh khang khang sinh ciia R aeruginosa doi vdi nhdm flu- oroquinolone''*'.
KET LUAN
Cac dot bien phat hien chii yeu d chiing R aemginosa khang va so IUdng dot bien ty le thuan vdi mu'c dp de khang.
Opt bien tren gen gyrA dong vai tro quan trong tdi sU de khang vdi nhdm fluoroquinolone cua R aemginosa, cac dot bien tren gen pafC cd vai tro bo trd cho sU de khang vdi nhdm fluoroquinolone ciia R aeruginosa.
Nghien cirU dUdc thUe hien nhd sU ho trd ciia de tai nghien ciili cap Nha nUdc, ma so KC 10.18/11 - 15
"Nghien cUu dng dung ky thuat sinh hpc phan tir xac dinh mUc do khang khang sinh eiia mpt so chiing vi khuan gay benh thUdng gap d Viet Nam".
T R U Y i N N H l i l V ! V l 6 T N A M * s 6 4 { 1 2 ) - 2 0 1 5 - 13
TAI UEU THAM KHAO
1. Le Oang Ha va CS. (2003), "Tinh hinh khang thudc ciia VI khuan gay benh thudng gap tai Viet Nam nam 2003" Mot so cong trinh nghien cuU ve dp nhay cam cua vi khuan vdi thudc khang smh, Nha Xuat ban Y hoc. Ha Npi, tr: 5 - 88,
2. Didier Hocquet, et al (2003), "Genetic and pheno- typic variations of a resistant Pseudomonas aeruginosa epidemic done", Antimicrobial Agents and Chemotherapy, Vol 47, (No.6), pp: 1887 -1894.
3. Hyam Mouneimne, et al (1999), "Type I I topoiso- merase mutations in ciprofloxacin - resistant strains of Pseudomonas aeruginosa', Antimicrobial Agents and Chemotherapy, Vol 43, (No.l), pp: 62 - 66.
4. Lomholt J. A., et al (2003), "Ciprofloxacin suscep- tibility of Pseudomonas aemginosa isolates from kerati- tis", Br Journal Ophdialmol, (No.87), pp: 1238 -1240.
5. Joaquim Ruiz (2003), "Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA - gyrase protection" Journal of Antimicrobial Chemotherapy, (No.51), pp; 1109 -1117.
6. Jun - Ichiro Sekiguchi, et al (2007), "Outbreaks of multidrug - resistant Pseudomonas aeruginosa in com- munity hospitals in Japan", Journal of Clinical Micmblol- ogy, Vol. 45, (No.3), pp: 979 - 989.
7. Katsumi Shigemura, et al (2013), "Does mutation in gyrA or parC or efflux pump expresaon play the main rale in fluoroquinolone resistant Esdieriehia coll urinary tract infections" The Joumal of Urt^ogy, Vol. 189, (No.4), pp:
471 - 472.
8. Minori Matsumoto, et al (2012), "Mutations in the gyrA and parC genes and in vitro activities of fluoro- quinolones in 114 clinical isolates of Pseudomonas aerug- inosa derived from urinary tract infections and their rapid detection by denaturing high - performance liquid chro- matography" International Journal of Antimicrobial Agents, (No.40), pp: 440 - 444.
9. Minoru Yonezawa, et al (1995), "DNA - gyrase gyrA mutations in quinolone - resistant clinical isolates of Pseudomonas aeruginosa'. Antimicrobial Agents and Chemotherapy, Vol 39, (No.9), pp: 1970 -1972.
10. Neda Gorgani, et al (2009), "Detecting mutations related to antibiotic resistance in Pseudomonas aerugi- nosa" Int Journal Antimicrob Agents, (No.34), pp: 414 - 418.
11. Takashi Deguchi, et al (2000), "Development of a rapid assay for screening point mutations associated with quinolone resistance in the Pseudomonas aeruginosa parZ gene", Joumal Infect Chemother, (No.6), pp; 26 - 29.
MUTAnONS OF gyrA, patC GENES I N PSEUDOMONAS AERUGINOSA RESISTANT
TO CIPROFLOXACIN AND LEVOFLOXACIN
Summary
Pseudomonas aeruginosa are resistant to fluoro- quinolone because of mutations in gyfA and parC genes.
Objective: This study was to detect mutations of gyrA, parC genes in Pseudomonas aemginosa resistant to dprafloxacin and Ievofloxacin. Subjects and Mediod: 107 isolates of R aemginosa (82 resistant, 19 susceptible and 6 intermediate to fluoroquinolone) w/ere tested for Oie mu- ations of gyrA and parC genes by PCR and sequencing.
Results: 57/107 isolates habored mutations in gyiA gene (codon 83 - Thr, 84 - Ala and 85 - Vai). 22/107 isolates ha-
Key words: R aemginosa, mutation of gyrA, paiC genes, fluoroquinolone.
bored mutations in paiC gene (codon 76 - Vai, 87 - Ser, 103 - Arg and 123 - Aia). More mutations have been seen in gy/A, paiC genes in isolates resistant to high MIC of flu- oroquinolone. Conclusion: Mutations in gyrA, paiC genes were mainly detected in resistant isolates and the number of mutations were proportinal to resistance level (MIC).
GyrA gene mutations would play the important role to the resistance to fluoroquinolone for R aemginosa and paK.
gene mutations could be additional in this pathogen.
14 - TRUYENNHiMVierNAM* SO 4 ( 1 2 ) - 2 0 1 5
