TAP CHi CONG THIfDNB
HOAT TINH KHANG KHUAN STAPHYLOCOCCUS AUREUS KHANG METHICILLIN (MRSA) CUA CAO CHIET
ETHANOL THlJC VAT BAN DIA TAI BINH DITONG
• MAI THI NGOC LAN THANH - HOANG ANH HOANG - TRl/ONG VU THANH
TOM TAT:
Khang sinh la nhffng chat do vi sinh vat tie't ra hoac nhiJng chS't hda hoc ban tdng hdp, tdng hdp, vdi nong do ra't tha'p, cd icha nang dac hieu Idm ham stf phat tridn hoac di6t dtfdc vi Ithuan in vitro. Vancomycin du'dc xem la tieu chuan vang trong dieu tri cac benh nhiem khuan Staphylococcus aureus khang methicillin, nhtfng da xua't hien chung MRSA khang vancomycin. Bai vie't xac dinh kha nang khang MRSA ctia cao chiet ethanol thtfc vat ban dja va khii nang ke't hop ciia cao chie't thtfc vat vdi khang sinh chuan vancomycin bang phifdng phap khuech tan dia. K6't qua khao sat da ttm ra 5 cao thifc vat cho hoat tinh khang MRSA, dong thdi chi ra kha nang ke't hdp cua cac cao thtfc vat vdi vancomycin bang phifdng phap tinh theo ban kinh vong khang khua'n. Trong 5 cao thifc vat cho hoat tinh khang MRSA, thi cao Xang Ma lan dau tien difdc cdng bo'.
Tijf khoa: MRSA, cao chie't thifc vat, Rhizophoraceae, khlng khua'n.
1. Gi(Ji thieu
Staphylococcus aureus khang methicillin (MRSA) la mot chiing S.aureus khang penicillin xua't hien cd che khang methicillm trong vong 2 nam tif khi khang sinh methicillin difdc gidi thieu nam 1959 [1]. S. aureus khang methicillin xua't hien 2 cd che khang pho bie'n: Sieu bieu hien p- lactamases vis thay ddi dang binh thifdng ciia Protein gan Penicillin (PBPs). PBP2a hien dien tren chiing MRSA cd ai life tha'p vdi nhdm khang sinh p-lactam, do dd co khii nang thay the chti"c nang sinh tdng hdp ctia PBPs thdng thtfdng khong
sif hien di6n ciia p-lactam, bang each nay te bao se tranh difdc stfly giai. PBP2a difdc bieu hien tif gene mecA khdng Men dien trong cac chung S.aureus nhay khang sinh [2]. MRSA khong gid'ng nhifng benh nhidm khua'n khac ma nd la mdt chiing dang cpng dong, mac dit ngudi benh chifa hd tiep can vdi cac trung tam cham sdc stfc khde [3]. Nam 2013 d My, cac benh nhidm khuSn cd hen quan dd'n MRSA cd ti le gan 80.000 va 11,000 ngifdi chet trong sd dd ti le chdt do nguyen nhan vi khu£n khang thudc chiem 50% mdi nam [41 Trong cac bao cao gan day cua chinh phii Anh. ONeill va cac
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cong sif da tinh toan den nam 2050 co khoang 10 trieu stf s6'ng bi de doa ma nguyen nhan la do sif gia tang cua cac b6nh nhilm khuan khang thuoc [5].
Vancomycin du'dc xem la tieu chuan vang trong dieu tri cac b8nh nhilm khuan do Staphylococcus aureus khang methieUlin. Khang vancomycin la d di\i C cua tien chat tong hdp vach peptidoglyean D-ala-D-ala difcfc thay the' bang D-lactate hoac D-Serrne, do do lam thay ddi vi tri gan vancomycin vapha huy ai life cao cua tien chat v6i vancomycin, do do xoa bo dich thuoc. Tuy nhien do qua trinh su"
dung vancomycin lau dai nen d6 nhay cua ehung S.aureus vdi khang sinh nay eung giam xuo'ng [6].
Vi vay, viec tim ra cac khang sinh mdi hoac heu phap mdi thay the la mpt yeu cau can thiet. Thu'c vat la nguon phong phu cac hdp chat thu" ca'p, viec sil dung du'dc heu trong dieu tri cac benh nhiem khuan trong y hpc co truyen da cd tijf lau. Binh Difdng CO nguon thife vat ban dia va cac bai thuoc coUiiyen trong dieu tri cac benh nhiem khuan nhif tieu chay, ly, ghe 16 da du'dc sif dung tif lau. O Binh Difdng, nguon thife vat ban dia lam thuoc da dang [7], nhUng ed it eong trinh nghien ciJu ve khang sinhthUc vat ma chii yeu la cac cong trinh nghien ciiu v^ phan loai thu'c vat. So' lu'dng eong trinh nghidn ciJu ve eac bai thuoc xua't phat tCf ngu6n thifc vat dac biet la eae bai thuoc khang khuan hau nhif khong co. Hdn nifa, trong v^'n de khang khang sinh cua cac chung vi khuan ngay cang nhieu va viec tim khang sinh mdi ngay eang kho khan, vdi nghien ci?u trifde ma chung toi da thifc hi$n la sang loc khang sinh thifc vat tan trong dung moi nifdc khoang Staphylococcus aureus [8], ihi cau hoi dat ra la cd hay khong viee siJ dung eac nguon difde lieu ban dia hay cac bai thuoc eo truyen co hoat dnh khang cac chung khang khang sinh nhu MRSA?
Xua't phat txt can hoi nghien ciJu nay, muc tieu cua bai bao la thu nhan cao thu'c v | t tiJf 20 loai thu'c v£it bSn dia Binh DifOng. Sau do, khao sat boat tinh khang MRSA cua 20 loai cao thifc vat, trong do chon ra cao 1 loai thifc vat eo hoat linh khang MRSA cao nhat.
2. vat li&u va phifdng phap 2.1. Nudi cay vi khudn
Chijng vi khuan Staphylococcus aureus nhay methieilhn (MSSA) ATCC 6538 du'dc sif dung nhif chung doi chUng, chung Staphylococcus aureus
khang methieilhn (MRSA) ATCC 33591 du'dc cung cap bdi Cong ty Microbiologics (USA) va MSSA difcfc nuoi tren moi tnfdng long Trypton Soy (Himedia, An Do) va moi tnfdng Trypton Soy agar (Himedia, An Do). Chung vi khuan dUde nuQi trong tli am (DaiHan, Han Quoc), 6 nhiet do 37"C, trong vong 18 gid.
2.2. Phi&fngphdp thu nhan cao ethanol thiic vat Sinh khoi tifdi thife vat (!a, hoa, canh non) sau khi thu hai ve difdc ru'a bang nu'dc ca't sach bui ban, triJ tu lanh trong vong 24 gid, cho nitd long de pha v9 te bao nghien bang may xay FY 130, toe do may 34.000 vong/phut, kich thddc mat lifdi 0.074- 0.25nim. Sau do, hot thifc vat se du'dc diing de chie't cao theo phu'dng phap ngam dam trong ethanol d 50°C, lac d 100 vong/phut trong may lac Ika KS 40001 (DiJc), sau thdi gian 24 gid se thu mot lan va chiet kiet trong vong 1 man. Ty le ehife't (ethanol:
sinh khoi thUc vat kho) 2 lit: 50g, chiet lap lai 3 iln.
Dung dich thu difdc se difdc loc qua giay loc Whatman difdng kinh 110mm, vdi kich thifdc 16 lgc 20-25pm, dich loc thu du'dc se difdc co cao bang may c6 quay d 50''C, trong vong 2-3 gid, sau do cao set se difde dl trong tu sS'y d SO^C den khoi lifdng khong doi.
2.3. PhiMng phdp thu nh&n dung dich cao thUc vdt khdo sdt hoat tinh khdng MRSA
Cao thdc vat thu difdc se difdc can khd'i lifdng bang can phan tich Sartorius (Dtfc) Khdi lifdng can difdc se difdc hda tan trong dung dieh DMSO (Merck, DiJc) 10%, Dich hoa tan se thu va dem ly tam cf tdc do 14.000 vong/phut trong vong 4 phlit bang may Hermle (Bu'c). Sau do, loai can va thu dieh. Dung dich nay se du'dc loc qua phm Ipe c6 kich thifdc 16 loc 0.22pm va xac dmh ndng dp thong qua hai phifdng phap gian tidp va trifc tiep. Phu'dng phap gian tiep thong qua khdi lifdng can ddde phdi hong tu say d 50"C va can de'n khdi lifdng khong ddi, lay khoi lifdng eao ban dau tnf cho khoi lifdng can se ra khdi lifdng cha't tan boa trong dung djch DMSO 10%.
Phu'dng phap xac dinh ndng do dung dich Qrfc tie'p bang each dilng pipetman lay 1ml dich thu difdc va CO ddn khdi lifdng khong ddi trong tu say DaiHan (Han Qudc) d 50"C. Khdi lifdng can difde cbinh la khdi lifdng chat tan trong Inil dung dich khao sat.
2.4. PhiAfng phdp khdo sdt hoat tinh khdng MRSA , , Thtf nghiem kha nang khang khuan bang
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TAP CHi CONG THIftfNG
phu'dng phap khuech tan tren dia thach (Agar diffusion method): Chuan hi dia moi tnfdng Muller -Hinton Agar (Neogen,USA) la moi tru'dng chuan diing de khao sat kha nang khang khuan cua dich chiet, ed bo sung lOOpl dich mdi tnfdng nuoi vi khuan MRSA mat do 106CFU/ml. Stf dung phifdng phap Kirby-Bauer, cac gieng tren dia thach difdc khoan cd kieh thifdc lo 4mm. Tren moi dia co khoan 3 gieng, gieng 1 la khang sinh chuan vancomycin (Sigma, Dtfc) difdc cho vao gieng vdi the tieh 1001 d nong dO MIC to'i thieu cua khang sinh thii' nghiem. Gieng 2,3 dddc eho vao 1001 dich chiet cao thUc vat. Sau do, cac dia dUdc u 6 ST^C trong vdng 18 gid. Cac thi nghiSm du'dc lap lai 3 lan. Cae kieh thifde vong khang khuan theo milimet (mm) dtfdc dpc sau 18 gid [9] va stf dung tiSu chuan vdng khang khuan ciia CLSI de giai thich [10].
Xdc djnh nong do lic che toi thieu cda cao chie't bdng phuong phdp pha hdng (Broth microdilution method); Nong dp tfc che tdi thieu dtfdc dmh nghia la ndng dp tha'p nha't tfc che' sif phat trien cua vi khuan sau 18 gid nuoi c§'y. Xac dinh nong do tfc che tdi thieu bang phtfdng phap pha Ioang day ndng dp ttong cac dng. Stf dung dia nudi ca'y 96 gieng, moi gieng se gdm cac thanh phan: lOpl dich vi khuan cd ndng dp 106CFU/ml; cha't chiet du'dc pha Ioang 2 lan bd sung vao moi gieng vdi the tieh bang lOOpI,
ehe'stfphat trien eiia vi khuan dtfde nhan la ndng do tfc che tdi thieu (MIC) [11],
2.4. Khdo sdt khd nang ket h(fp bdng phi&fng phdp khuech tdn dia
Stf dung phtfdng phap Kirby-Bauer, trong do cd stf thay ddi cu thd nhu' sau: Tren moi dTa mdi trUdng MHA CO 3 gieng; gieng thtf nhai la cao thifc vat A;
gieng thtf hai la cao thtfc vat B, va gieng thtf ba se bd sung Itfdng ke't hdp giffa cao thifc v§l A va cao thtfc vat B. Mdi gieng du'dc bo sung lOOpl dich, gieng thtf ba se bd sung lOOpl dich cao thtfc vat A va lOOpl dich cao thtfe vat B. Sau dd, cae dia dtfdc u d 37*'C trong vong 18 gicf. Cac thi nghiem dtfdc lap lai 3 iln. Cac kich thu'dc vdng khang khuan theo milimel (mm) dtfdc dpc sau 18 gid [9] va stf dung tieu chuan vdng khang khuS'n cua CLSI de giai thich [10].
Budng kinh vong khang khuan ket hdp theo cong thiic D=rA+rB (mm);
rAla ban kinh vdng khang khuan cua thdc vat A (mm) rB la ban kinh vong khang khuan cua thuc vat B (mm) C la dudng kinh vdng khang khuan md rang (mm)
C > D = Synergism (+), C = D = indifference(). [12]
3. K&'t qua va thao luiin Bang 1. Danh muc thtfc vqt Ihu coo chief ethanol cho hogt tinh khdng MRSA
Stt
1 2 3 4 5
"Fenfhitcvlit Ten thong thifdng
Kim vang Tram Trdn Tram Dal
Coke Xang ma
"Fen khoa hpc Barleria lupulina Lindl Syzygium szetnaoense Merr Perry
Syzygium cumini L Grewia asiatica L Carallia brachiata
B$ ph$n silfdung Toan cay La va canh non La va canh non Lava canh non La va canh non
NongdOCaothitc v$t (gg/ml)
90,12 9.83 50 83
294 31.34
Hoat tfnh khang MRSA
+ + + + + tJinn VI: 1 hanh pho 1 hu Dau Mot, iinh bmn uuang 7 ruusd.OZ i^hia Bac; Wb'^3yvo 37'phia Dong; Do cao Utrti-Str}
lOOpl moi mfcfng long Muller Hinion them viio de CO the tich cuo'i la 210pl. Sii dung hai gieng la mau doi chi?ng, mot chi co moi trudng MHB lam doi chlJug iim va mot gieng chi co moi tru'dng MHB va chiing vi kliuan lam dS'i chu'ng dudng. Cac gieng se dtfdc ii hieu khi 6 37^C trong vdng 18 gid. Sau thdi gian ii, nong do tha'p nhat trong day nong do ma itc
4. Xac dinh kha nang khang S.aureus va MRSA cua cao Xang Ma (Carallia brachiata)
DUa vilo Bang 1 danh muc cao thUc vat cho hoat tmh khang MRSA, da lira chon duoc 4 cao thu'c vat C(S hoat tinh khang MRSA cao nhat la cao Tram Trdn, Cd Ke, Xang Ma, vii Trim Din. Trong do Tram Dili va Tram Trdn lit cting ho Svzyginm hoat
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HdA HOC-CONG NGHE THlfC PHAM
tinh khang khuan cua cao Kim Vang tha'p nha't trong 5 loai. Trong do, eao Tram Trdn dtfdc bao cao irong nghien etfu khae cua nhdm. Trong bai bao nayse trinh bay mot khia canh khac nhtf nong dp tfc che to'i thieu (MIC) ciia cao Xang Ma la cay thupc hp Rhizophoraceae, la mot loai chtfa dtfdc bao eao k6't qua khang MRSA trtfde do. Dong thdi, bao eao ket qua xac dinh kha nang kg't hdp gitfa cao Xang Ma, Tram Dai, Co Ke vdi cao Tr^m Trdn, Kim Vang va kha nang ke't hdp gitfa eao Xang Ma, Tram Dai, Co Ke vdi khang smh vancomycin trong hoat tinh khang MRSA Nong do dung dich cao Xang Ma khao sat la cho boat tinh MRSA 31.34 )ig/ml vdi kich thudc vong khang khuan 13mra tren chung S.aureus va 11mm tren chung MRSA. Khao sat MIC bang khuech tan dTa (Hinli 1) d n6ng dd pha Ioang 8 lan dung dich cao Xang Ma van cho hoat tinh khang S.aureus va MRSA Ian Itfdt vdi kich thu'dc vdng khang la 8mm va 7mm, d nong dd MIC la 3.9175 |Jg/ml nong do nay gan bang nong dO MIC cua khang sinh chuan vancomycin la la 4,65)ig/ml |Jg/m! bang phifdng phap pha Ioang 96 gieng, vdi nong dp 9,766pg/ml khang sinh cho kieh thu'dc vong khang la 5mm do'i vdi chung MSSA, khong xuat hien vong khang vdi MRSA (Hinh 1 va Hinh 2). The hien boat tinh khang S.aureus va MRSA khi pha Ioang day nong dp XM1-XM4, trong do, gieng 1 la XMl eo nong dp ban dau la
Hinh 1: Hogt finh khong S.aureus cua cao Xang Ma
31.34 jjg/ml, XM2, XM3, XM4 ttfdng tfng vdi gieng 2,3,4, mdi gieng each nhau do pha Ioang 2 lan.
Theo Bang 1, ke't qua thi hi6n kich thtfdc vdng khang MSSA va MRSA khi nong dp cao Xang Ma dtfdc pha Ioang, khi nong dp dung dich cao pha Ioang 8 lan ttf nong dp ban dau la 31.34 |ag/ml de'n nong dp 3.9175 |ig/ml thi kich thtfdc vong khang khuan ttf 13mm giam xuo'ng 8mm tren ehung S.aureus va 11mm xuo'ng 7mm do'i vdi ehung MRSA, vi vay kich thtfdc vdng khang khuan giam dan theo nong dp dung dieh cao dieu do chtfng to boat tinh khang khuan eua dung dich cao Xang Ma phu thupc vao nong d6. Kich thudc vong khang khuan eua khang sinh vancomycin d nong dp MIC 2,325149 |Jg/ml la 7mm. Dieu nay rat quan trpng do hoat tinh khang khuin ciia eao Xang Ma chu'a phdi la tinh cha't ma eho hoat tinh khang MRSA ttfdng dtfdng vdi tinh ehat vancomycm. Vl vay, viec stf dung eao se It to'n chi phi vi qua trinh tach tinh chat se giam xud'ng. Hdn ntfa, hoat tinh khang MRSA cua cao Xang Ma chua cd nghien etfu, dac biet Xang Ma ed tai Viet Nam. (Bang 2).
Tiep theo de danh gia kha nang ket hdp hoat ddng cua cae cao thifc vat. Chung toi stf dimg phtfdng phap khuech tan dia theo cdng thtfc cua Farrukh Aqil va cpng sif [12] nham xac dinh kha nang ke't hdp eua cao Xang Ma, cao Cd Ke, cao Tram Dai vdi khang sinh vancomycin cd nong dp 750 ng/ml cao hdn 75 lan MIC cua khang sinh, do d nong MIC cua khang sinh khdng xuat hien vong khang nen viSc Itfa chpn nong dp cao hdn nham xac dinh vdng khang va kich thtfdc vdng khang md rong mpt each rd rang cd y nghia. Ket qua difdc chi ra d Bang 3 va Hinh 3, trong hinh kieh thtfdc gieng la 4mm, gieng 1 la dung dieh cao thtfc vat, gi6'ng 2 la khang sinh vancomycin, va gieng 3 la ket hdp gitfa eao thtfe vat va vancomycin. Cac dung dich eao Xang Ma, eao Cd Ke, cao Tram Dai cd nong do ttfdng tfng la 31.34|jg/ml, 29.4
|jg/ml, 50.83|jg/nil. (Bang 3).
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77. Wayne, P., Clinical and Laboratory Standards Institute' Performance standards for antimicrobial susceptibility testing: 20th informational supplement. CLSI document MIOO'S20, 2010.
72. Aqil. F.. I Ahmad, and M. Owais, Evaluation of antiIfm€tMcillin#resistant Staphylococcus aureus (MRSA) activity and synergy of some bioaclive plant extracts. Biotechnology Journal: Healthcare Nutrition Technology, 2006.1(10)-p. 1093-1102.
Ngay nh$n bai: 20/3/2019
Ngay phan bi$n danh gia va siifa chffa: 30/3/2019 Ngay chS'p nh$n dang bai: 9/4/2019
Thong lintdc gid:
1. MAI THI NGOC LAN THANH
TrirSng Dai hoc Bach Khoa, Dai hoc Quoc gia TP. H 6 Chi Minh TrifSng Dai hpc Thu D^uMpt
2. HOANG ANH HOANG - TRlJONG VU THANH Trtfcing Dai hpc Bach Khoa, Dai hpc Quo'c gia TP. H 6 Chf IVIinh
THE RESISTANCE OF ETHANOL EXTRACTS FROM NATIVE PLANTS IN BINH DUONG PROVINCE TO METfflCILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)
• MAI THI NGOC LAN THANH Ho Chi Minh City University of Technology, Vietnam National Universit/ - Ho Chi Minh City
Thu Dou Mot University
• HOANG ANH HOANG
• TRUONG VU THANH
Univerist/ hay Ho Chi Minh C i t / Universit/ of Technoiogy, Vietnam Nationai University - Ho Chi Minh City ABSTRACT:
Antibiotics are substances secreted by microorganisms or semi-synthetic and synthetic chemicals with very low concentrations. Antibiotics are able to inhibit the growth or kill bacteria grown in vifro. Vancomycin is considered as a goid standard for the treatment of methicilUn- resistant Staphylococcus aureus (MRSA) infections. However, Methicrllin-resistant Staphylococcus aureus irrfection is caused by a type of staph bacteria that's become resistant to many of the antibiotics. This study is to identify the impact of Ethanol extracts of native plants on the MRSA and also assess the combmation between plant exh-acts with standard vancomycin antibiotics by using the disc diffusion method. The studys results show that there are five nlant extracts which have anti-MRSA activity. The smdy also finds that it is possible to combine the plant extracts with vancomycm by using the diffusion mediod depend on the radius of inhibition zone. Among these five plant extracts, the plant extract of Caralha brachiate is firsdy published
Keywords: MRSA, plant extracts, ]?hizophoraceae, antibacterial.
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