Gen fibrin chứa kháng sinh ức chế sự phát triển của vi khuẩn có khả năng hình thành biofilm

Tap chi Cong nghe Sinh hpc 12(2): 229-233, 2014 GEL FIBRIN CH(JA KHANG SINH (JC CHE Str PHAT TRIEN CUA VI KHUAN CO ICHA NANG HINTH THANH BIOFILM Doan Nguyen VD, Train Le Ban Ha Trufmg Doi hpc Khoa hpc Tic nhien, Dai hpc Qudc gia Thanh phd Ha Chi Minh Ngay nhan bai: 10.5.2014 Ngay nhan Clang: 18.6.2014 TOM TAT Nhient truing la nguyen nhan gay tin vong tucmg dining tai nan giao th8ng, AIDS va ung thu vii Khi cac to bao vi khuan CO ca hei dinh cu tren be mat vat lieu ghep, chung se thay doi kieu hinh de hinh nen clang mai &roc goi la biofilm, bao ve chung cluing lai he mien dich caa co the va tic dung dm kiting sinh Gel fibrin la loai vat lieu co nhieu dac tinh ly cho he thong phan Oat khang sinh co kiem sofit via ming dich de ngan chan su hinh biofilm Trong nghien dm nay, chung tai tien hanh tap gel fibrin tmc tiep tin huyet tucmg nguai; sau do, dinh gia gel tao bang quan sat dual kinh hien vi dien tin pet (SEM) va six phan huY in vitro Sau do, gel fibrin chira khang sinh duqc (faith gia thong qua nang phan phat khang sinh in vitro, nang Lac dung len eking vi khan hinh thinh biofilm, khao sat tinh dac ten to bao nguai in vitro., Ket qua cho thay, gel fibrin chCra khang sinh gay d6c cho to bac) nguai in vitro va co nang Crc che stn phat trien dm cluing vi khuan c6 nang hinh biofilm in vitro De tai nhu buac khai dau dat nen mang cho viec Oat trien phuang phap dieu tri hieu qua nhiem truing va ngan chan stn hinh biofilm Tir khogi: Gel fibrin, biofilm, nhiem trimg, he thong phan phdi thuac, huyat mong nguai M O DA U Hang nam co den 103.000 benh nhan 61 nhiem thing benh vien tin vong Theo To chirc giam sat nhiem thing benh vien quilt gia cua My (NNIS), 38% nhiem truing benh vien la nhiem truing vet me) anh hue:mg tai khoing 1.500.000 benh nhan V ien phi dm nhiing benh nhan 1)1 mac loai nhiem khan cao han 39.000 USD so v6i benh nhan binh thuerng va th6i gran nam vien ding keo dai them 7,5 Nhang hao ton cua nhiem tiling vet m6 chiem tai 15 ti USD mill nam My (Smith, 2008; Mandavyapuram, 2010) Trong cac guy trinh phau thuat, benh nhan thuemg dirge cung cap kiting sinh; dac biet, phltu thuat chinh hinh vi vet mei rOng va di kern veri viec cay ghep cac vat lieu sinh hoc Ngoai ra, hien Wong nhiem truing b nhring vi tri xa (mat de mach man that)), doi hoi lieu khang sinh cao va dieu tri thin gian keo dai, dieu tang nguy co gay dee cho ca the benh nhan va khang thuoc vi khuan Vi khuan san xuat ming nhay pi la biofilm bao ve chung thong lai he mien dich cila co the, lam giam ding ke tic dung cua khang sinh to bio vi khuan va gay nhiem truing man tinh vat lieu nam co the (Pye, 2009; Archer, 2011) fle khic phut nhring nhtroc diem ming nhu ngan chan str hinh thinh biofilm can co met he thong phan phat khang sinh có kiern,soat va truing dich Trong he thong nay, khang sinh can duqc bao b9c beri cac vat lieu c6 nang to phan huS/; nhien, viec sir dung nhang loai vat lieu kitting to than se gay phan Ong viem va thai !coal cna co the benh nhan Fibrin la met nhang foal vat lieu tu than dau tien duqc su citing he thong phan phat khang sinh nhan' kiem soat so nhiem truing Fibrin co nhieu dac tinh 15, tuemg de tr6 Ong vien tiem nang cho mtic dich nhu tinh tucmg hem sinh hoc, nang to phan hujr mai tru6ng, co the, tinh chat barn dinh va cau truc xop tao dieu kien thuan Iqi cho viec phan phat &roc pham Tuy nhien, nu6c ta, van de rat mai; hau nhu khang co cling trinh nghien ciru khoa hoc nao lien quan doge cong bo Do do, chint tai tien hanh nghien dm nhu buoy kheri dau dat nen m6ng cho viec Oat trier' phrrang phau diet' tri hien qua nhiem truing hatt phau (Tsourvakas, 1995; Mosesson et al., 2001; Canonico, 2003) VAT Lieu VA PHUONG PHAP tuvng nghien aim Mau ngoai vi nguai dai xet nghiem am tinh vii 229 Doan Nguyen Vu & Trail Le Bin Ha cac bEnh truytn nhiem &roc cung cap to Trung tam truyen miu huyet hoc bEnh vier' Cher Ray thinh phi) H6 Chi Minh Phirang phi') tao gel fibrin Mau ngoai vi nguai duqc ly tam 3000 vong/phit 10 phut, thu nhan phan dich n8i, loai b6 can Sau do, mkt bo sung CaCl2 cac nong LIE Ichfic (0,012M; 0,013M; 0,014M), 37°C mot khoing thai gian dE khio sat thai gian tao gel Dinh gia mat so dac tinh can gel fibrin tao Danh gia eau trac be mat Ngam gel fibrin tao thinh qua dem dung djch co djnh Mau glutaraldehyde 2,5% Er 4°C; sau do, khu nu& C; cac nong,d6 giim dan Mau gel sau khir nuac duqc dua den Vin khoa hoc va c8ng nghE ph6 HO Chi Minh tit tiEn hanh chop SEM (scanning electron microscope) Dcinh gici kha nang phcin hay Gel fibrin tao Er nling CaCl khitc duqc lam kh8 nhiEt dO phong qua dem, can de xac djnh khoi luqng ban dau Ngam gel huyet twang ngueri, lac 100 vong/phOt 37°C Can khOi luqng, gel cac thai diem ngiy, va ngiy Huyet twang &rem thay mai hang ngiy suOt qua trinh thi nghiEm Dcinh gici khci ming thai khang sinh caa gel fibrin in vitro Gel fibrin china mg kiting sinh vancomycin &roc ngatn huyet tuong ngtplii, lac 100 yang/phut 37°C Huy& tucmg &roc thu nhan 11161 -20°C; sau do, b6 sung huytt tuong va luu nguai moi N6ng d6 vancomycin duqc gel fibrin giai ph6ng vio huyet tuang mai ngiy &roc bang phuong phip Kirby-Bauer Day la phuang phip xac djnh nong dE kiting sinh dung djch thong qua duErng chuan ttrcmg quan gift duling kinh yang khing lchuan vi dung djch co !tong kiting sinh da Wet Mir nghiem lac clang caa gel fibrin chaa khang sinh len to bao vi khucin Staphylococcus aureus flat gel fibrin chira lmg kiting sinh vancomycin vat) cac eppendorf china vi khuan S aureus yeti mat to bao/ml U eppendorf a 37°C 24 h, sau dO loai moi trinling, them dung djch crystal violet 1%, a 20 phid nhiEt di) phong Loai bo dung Bich crystal violet, hob tan thuoc nhuEm lai to bio vi khuan bang curet diii Do mat (V quang hoc bulic song 570nm Khoo sat dac tinh caa gel fibrin chart khang sinh dal vai nguyen bao sal ngwai NuOi cay nguyen bio sqi ngtrai dEn phil kin 80-90% be met dia nu8i cay Dar gel fibrin chira khing sinh vio tam cila dia nu8i, bo sung moi tnrang nuoi cay, net) tuc 37°C, 5% CO2 Sau 24h, quan sat inh hulling dm gel fibrin china kiting sinh len hinh thai, mat dia, cub to bao ducti kinh hien vi dito nguqc Sir dung cao su thien nhien (latex) lam nhom chimg ducmg Tien chuan so sfinh theo bang (Theo tieu chuan cub Hip hEi thir nghiEm va vat liEu M9-ASTM) Bang Chi so dQc t6 cap tinh Chi s8 Me to Kheng c6 vung to bac) chat nhan biet xung quanh hoec ben duel vet lieu VOng to bac chat giai han & khu v rc ben &raj vet lieu Viing to bac chat Ian ngoai it nhet han 0,5cm so vai vat lieu Vbrig to bao chat Ian ngoai tin 0,5 — 1cm so vai vet lieu Khu vtyc ti bao chi& Ian ngoai leen han 1cm so vai vet lieu nhung khOng Ian het dia Khu vir to bao chat Ian toan be dia KET QUA VA THAO LUAN biting CaCl2 cang cao thi thai gian to gel cang nhanh (Bing 2) To gel fibrin Kin tang nting 80 CaCl2 them vio huyEt twang, lung ion Ca' theo ding tang len Dieu anh huling den hai buErc qui trinh tao gel fibrin: Theo ktt qui,khio sit, thai gian tao gel to huyEt tucrng vai cac nong di) CaCl2 khic Ia khic 230 Tgp chi Cong nghe Sinh hoc 12(2): 229-233, 2014 thilc day su hoat him thrombin, qua day nhanh qua trinh chuyen tibrinogen thank fibrin, cling nhu thac day qui trinh hoat hoa nhan to &Ong mau XIII, tao lien ket cheo giira cac sqi fibrin mang luOi sqi, day nhanh qua trinh tao gel Dinh gii ciu trtic be mit Cac sqi fibrin dan chang chit v6i tao mat mang 1u6i,vai cac nhanh va cac lien ket ,cheo, tao nen nhieu tren be mat gel (Hinh 1) Ket qua phis hop vai nhieu ding trinh ding bo tnr6c day (Mosesson et al., 2001; Woolverton, 2001; Canonico, 2003) Dinh gia kha nang phan hay Cac Mau gel fibrin tao ter ba n6ng CaCl2 khac c6 so phan huy khac dtra teen mix dO giam khoi luong theo phin tram (Hinh 2) Kh6i luong gel fibrin giam gin 50% &Au nen, nhfing sau la str giam tit tit cua khoi Itrong gel Trong do, gel fibrin tao to ,nong da CaC12 0,014 M c6 so phan hiry nhanh Mat dau tien (mit 42,89% khoi Wong ban diu), gel tao tit n6ng dia CaCl2 0,013 M giam it nhat (mat 23,47% khoi luong ban diu) Sau thi nghigm, khoi Itrong lai ctia gel fibrin tao bang cach them 0,013 M CaC12 la nhieu nhat (68%), gel tao to nong da CaCl2 0,014 M co khoi lutmg lai sau ngity thap Mit (49%) Theo (Woolverton, 2001, Janmey et al., 2009), gel fibrin tao v6i thin gian cang cham thi mat da 16 cang day va kich thuerc 11) cang nh6, gel tucrng dOi chic chin hon so v6i gel fibrin tao voi th6i gian nhanh hon Do d6, gel v6i, nong d6 CaCl2 0,0I4M c6 cau tnic it chac chan nhat, nen bi phan htiy nhit a Bang Th61 gian tao gel fibrin theo n6ng dO CaCI, Nang dO CaCl2 (M) 0,012 0,013 0,014 Th&I gian (phtit) 20 ± 0,471 15 ± 0,428 13 ± 0,340 Hinh Cau trtic dm gel fibrin chop bang kinh hien vi dian tit (pet (SEM) Gel 0.012 ••••• Gel 0,013 Gel 0.014 thiri gran (ngay) Hinh Phan tram khoi krong gel fibrin phan huji huyit twang 231 Doan Nguyen WI & Tran Le Bao HA Ket qua danh gia kha nang thai khang Binh cua gel fibrin in vitro Trong dau fib thai thu6c, gel fibrin too boi nang de CaCl2 0,012M va 0,0I3M cho kat qua thai thuric gan giang (khoang 30%), luong thuoc thai cfm gel too boi the tich CaC12 0,014 M rat thap (khoang 14%) (Bang 3) Tsourvakas (1995) cling cid chimg minh kha nang thai cila gel fibrin &A veri nhieu loai khang sinh khac la khac nhau; nhien, khoang 30% 60% khang sinh se duuc thai 48h dau tien va lucmg Iai se duric thai cham o cac sau Tu nhOng kat qua tren, c6 the lila), gel fibrin too vei ming do, CaCl2 0,0I3M co do, ben va kha nang thai khang sinh tot nhat;Do d6, cluing Col quyet dinh chon ming di tien hinh cac thin nghiim flap theo Tic di:Ing ciia gel fibrin cilia khang sinh len to bac) vi khuin S aureus Kat qua kiem tra tac dens clia gel fibrin chin khang sinh len ti bao vi khufin S aureus cho thay sau 24 h, chi s6 OD dm mei trueng vi khufin cao rat nhieu so voi nh6m lai (Bang 4) Ben canh do, chi se OD cua mai trutmg vi khuan co gel chtra khang sinh va moi truong vi khuan chi co khang sinh tying dung (khang khac vi mat thong ki) Chfrng to gel fibrin china khang Binh vi khuan S co tac dung frc chi so phat trim aureus nuei cay mai trutrng giau dinh du6ng D6c tinh ciia gel fibrin chum khing sinh dei veri nguyen bao soi ngittri Ket qua hinh cho thiy mit do, hinh thai cita to bao phat trien xung quanh va ca ben dual gel deu binh thizong, khong quan sat thay to bao chat hay bi bong troc Ngtrqc lai, vat lieu cao su thien nhien lam cho toan,bt) to bao &fa nuoi cay chat va bong tric kh6i be mit TO kit qua tren c6 the ket luin gel fibrin chda kiting sinh khong gay dee tinh cap tinh tren nguyen bao soi ngutri lulu Bing Phan tram khoi luang vancomycin gel fibrin thai day tien Nang d6 CaCl2 (M) Phan tram kh6i krcrng vancomycin thai (%) 0,012 , 34,022 t 0,026 0,013 30,264 t 0,019 0,014 14,195 t 0,024 Bang Chi s6 OD clia cac nhorn thi nghiem sau 24 glee Chi at:, OD Mei trtrong vi khuin Moi trutmg vi khuan ce gel china vancomycin Moi truirng vi khuin co vancomycin 0,204 t 0,02 0,063 t 0,03 0,07 t 0,02 b Gel fibrin china khang s• Hinh Ket qui khio sat dec tinh hen nguyen baO scri ngutri a Cao su thien nhien (latex) 232 Tap chi Gong nghe Sin!: hoc 12(2): 229-233, 2014 KET LUAN Chang Vol da tao cong gel fibrin co kha rang phan phat khang sink Ngoai ra, gel fibrin chira khang sinh, co kha rang tic the six tang sinh to bao vi khufin S aureus in vitro Nghien dm ma met huerng di mai, ,met phucmg phap dieu tri hieu qua ham yeti van de nhibn thing hat: phau Uri cam an: Nghien ctiu &roc tai tro poi BO Khoa hoc va Gong nghe khuon kW de tai ma so DTDL 2012-G34 TAI LIEU THAM KHAO Archer NK, Mazaitis JM, Costerton JW, Leid JG, Powers ME, Shirtliff ME (2011) Staphylococcus aureus biofilms: properties, regulation, and roles in human disease Virulence 2: 445-459 Canonico S (2003) The use of human fibrin glue in the surgical operations Acta Biomed 74: 21-25 Janmey PA, Winer JP, Weisel JW (2009) Fibrin gels and their clinical and bioengineering applications J R Sac Interface 6: 1-10 Mandavyapuram HB (2010) Antibiotic delivery system for surgical site infection prevention in spinal implant surgery Disertation of Master of Science Case Western Reserve University Mosesson MW, Siebenlist KR, Meh DA (2001) The structure and biological features of fibrinogen and fibrin Ann N Y Acad Sci 936: 11-30 Pye AD, Lockhart DE, Dawson MP, Murray CA, Smith AJ (2009) A review of dental implants and infection J Hosp Infect 72: 104-110 Smith PW, Bennett G, Bradley S, Drinka P, Lautenbach E, Marx J, Mody L, Nicolle L, Stevenson K (2008) SHEA/APIC Guideline: infection prevention and control in the long-term care facility Am J Infect Control 36: 504507 Tsourvakas S, Hatzigrigoris P, Tsibinos A, Kanellakopoulou K, Giamarellou H, Dounis E (1995) Pharmacokinetic study of fibrin clot-ciprofloxacin complex: an in vitro and in vivo experimental investigation Arch Orthop Trauma Surg 114: 295-297 Woolverton CJ, Fulton JA, Salstrom Si, Hayslip 1, Haller NA, Wildroudt ML, MacPhee M (2001) Tetracycline delivery from fibrin controls peritoneal infection without measurable systemic antibiotic J Antimicrob Chemother 48: 861-867 FIBRIN GEL CONTAINING ANTIBIOTIC PREVENTS GROWTH OF BIOFILM BACTERIA Doan Nguyen VII', Tran Le Bao Ha University of Science, Vietnam National University Ho Chi Minh City SUMMARY Infeztion diseases cause of death for patients at the same number of patients died by traffic accidents, AIDS and breast cancer When the bacterial cells have a chance to settle on the surface of graft materials, they will change the phenotype and produce mucus to form a new form called biofilm to protect themselveg against the immune system of the body and the effects of antibiotics The fibrin gel has many useful characteristics as biocompatibility, biodegradation in the human body environment, adhesion and spongy structure for development of antibiotic delivery system to prevent the formation of biofilm In this study, human plasma was collected form human peripheral blood After that we created a fibrin gel directly from human plasma by supplement ion Cat*, then gel was evaluated gel formed by scanning electron microscope (SEM) and degradation in vitro Fibrin gel containing antibiotics was assessed through ability to deliver antibiotics in vitro, effect on biofilm bacteria, and poison for human fibroblast cells in vitro Of the investigated concentration of CaCl2 applied for the fibrin gel formation, the gel created by higher concentration of CaCl2 was degraded in vitro lower than the gel generated by lower concentration of CaCl2 Most important, fibrin gels containing antibiotic were not toxic for human fibroblast cells in vitro and inhibited the growth of biofilm bacteria in vitro In Viet Nam, this issue is still very new; virtually no scientific studies published concerning This research as the first step for the development of antibiotic delivery for effective treatments for infection diseases Keywords: Fibrin gel, biofilm, infection, drug delivery, human plasma Author for correspondence: Tel: +84-1648064416; E-mail: dnyua,hcmus.edu.vn 233 ... khing lchuan vi dung djch co !tong kiting sinh da Wet Mir nghiem lac clang caa gel fibrin chaa khang sinh len to bao vi khucin Staphylococcus aureus flat gel fibrin chira lmg kiting sinh vancomycin... se OD cua mai trutmg vi khuan co gel chtra khang sinh va moi truong vi khuan chi co khang sinh tying dung (khang khac vi mat thong ki) Chfrng to gel fibrin china khang Binh vi khuan S co tac dung... cilia khang sinh len to bac) vi khuin S aureus Kat qua kiem tra tac dens clia gel fibrin chin khang sinh len ti bao vi khufin S aureus cho thay sau 24 h, chi s6 OD dm mei trueng vi khufin cao