Nghien
CLPU- Ky thuat
Khao sat hoat tinh khang khuin va nam da I cua cac dan xuat cloro salicylanilid acetat
Ly Th| Thuy Trang, Thai Thj Quynh Nhu^, TrSn Manh Hung Le Thi Ngoc Hue*, Tru-ong Phu-ong Dai hpc Y-Duac TP. Ho Chi Minh 'E-mail: [email protected]
Summary
With regard the occurence of cutaneous pathogenic fungi and bacteria becomes more and more complicated, especially in immunocompromised patients with significant increase in drug resistance, to find new substances of strong antimicroorganism activity, this work was focused on combination of 2 antimicroorganism materials to increase therapeutic efficiency and decrease their concentration. All the 3 tested antidemiatophyte mixtures 5,4'-dielorosalicylanilid acetat and 4,4'-diclorosalicylanilid acetat (B3-B5) exhibited antibacterial activities without initation. At doses of (0.03 + 0.02) pg/ml, the B3-B5 mixture exerted significant activities against Shigela aureus and MRSA. In short, due to lowered concentration, the mixture showed safe, without skin initation. These results could be used for further studies.
Keywords: Chlorosalicylanilid acetat, MIC, antibacteria, antifungi, vancomycin.
Dat van de
Viec diiu tri benh nim da tin rat nhiiu thai gian va d i bj tai phat. Benh ngoai da do nim thuang boi nhiem vi khuan. Hon nti-a, vi khuan va vi nim d i khang thuic ngay cang nhieu ''-^l Dae biet benh ngoai da do vi nim/vi khuan hien nay thuang dien tiin phu'c tap 6' nhu'ng nguai bj suy giam miin djch. De do, viec dieu tri benh nhiim vi nim/vi khuan ngoai da gap khp khan.
Nhieu bao eae khea hpc a treng va ngoai nuac v i viec tim ra thuic mai co heat tinh khang khuin va khang nim. Nhu'ng chat co nguin g i c hoa hpc nhu d i n xuit halogen salicylanilid va dan xuit acetat ciia chiing co heat tinh khang nam tot f-^l
Vai muc tieu nham do tim ra chit thir nghiem khang nim, khang khuin mai gop phin khae phue su khang thuic nhu hien nay d i tai duge thue hien bang each phii hgp 2 chit nham de gia tang hieu lue khang vi sinh vat, giam ning dp chit sir dLing giiip giam bat tac dung phu ciia thuic.
Odi tu'O'ng va phu'O'ng phap nghien cii-u Do! tu'ong thu' nghiem
- 5 din xuit acetat ciia cloro salicylanilid (81- B5)
•5-elorosalicylanilid acetat (Bl)
•4'-cloresalieylanilid acetat (B2)
•5,4'-dielerosalieylanilid acetat (B3)
•4-cleresalicylanilid acetat (B4)
•4,4'-dielorosallcylanilid acetat (B5)
Cac chat tren da duge chung toi tong hgp va e5ng b i truac day '^l
3 chung nim da: Trichophyton rubrum.
Trichophyton mentagrophytes, Microsporum gypseum.
5 chiing vi khuan: Staphylococcus aureus ATCC 29213, MRSA ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Proteus sp..
The trang truo-ng thanh, due heac eai (khOng dung the eo thai heac dang che eon bu), khoe manh, can nang khOng dual 2 kg.
Phu'ang phap nghien cu'u ''•"i
Khao sat hoat ti'nh khang vi sinh vat bang phu'ang phap dat dTa giiy
Chuan bj huyin djch che thir nghiem: Ciy vi sinh vat trong moi truang SDA treng 7 ngay (vi nim), TSA treng 2 ngay (vl khuan) a nhiet dp phpng. Tao djch treo vi sinh vat trong nuac muoi sinh ly vai 0,05% tween 80, do djeh vi sinh vat vol OD = 0,1,aX = 530 nm ehe vi n i m , X = 625 nm che vi khuan. Trai vl sinh vat len dTa moi truang thich hgp (SDA cho vi n i m , MHA cho vi khuan), dat dTa giiy tam 100 pg chit thip len dTa. U d nhiet dp phong (vi nim), 37°C (vi khuan). Dpc kit qua sau 7 ngay (vi nam), 16-18 gia (vi khuan). Do duang kinh vong khang vi sinh vat.
78 TAP CHi DirgfC HOC - 4/2016 (SO 480 NAM 56)
• Nghien
CLPU- Ky thuat
Xac dinh MIC ciia chaf thir nghiem
Pha ning dp chit thir vol DMSO va cho vae dia moi truang d i cho nong dp thip nghiem mong muin. Chuin bj huyen djch vi sinh vat nhu tren.
Cho 10 pi djeh vi sinh vat len dTa moi truang (10"
CFU/ml). Lam song song vai dTa chtmg khOng chira chit thir nghiem. 0 6' nhiet dp phong (vi nim), 37°C (vi khuan). Dpe kit qua sau 7 ngay (vi nim), 16-18 gia (vi khuan).
Phirong phap phoi hgp 2 chat thip nghiem Thee phuang phap ban co dua theo chi so FIC (Fractional Inhibitory Concentration):
FIC < 0,5 0,5<FIC<1 1<F1C Hiep luc Cpng lue D i l khang
Mue dich dc tim hgp chit cho heat tinh khang vi sinh vat manh ban so vai chit dung don le, vi thi tap tmng 6" nhu'ng phii hgp eho kit qua hiep lycva cpng luc.
Thuc hien tren 10 cap phii hgp:
B1-B2 B1-B3 B1-B4 B1-B5 B2-B3 B2-B4 B2-B5 B3-B4 B3-B5 B4-B5
Scf bg thir tinh kich iing da tho: La phuang phap sinh hpc dua vap mirc dp phan ipng ciia da tho vol chit thip sp vai phin da k i ben kh6ng dip chit thip, thii' nghiem dting phim mau xanh lam Evans.
Kit qua va ban luan Hoat tinh khang n i m da
Kef qua djnh tinh
Ning dp cac chit thip nghiem la 100 pg/ml.
Bang 1: Buang kinh vdng khang nim da ciia 5 chat B (mm) Chat thir T. rubrum T. mentagrophytes M. gypseum
B1 B2 B3 B4 B5
24-25 20-22 21-23
>30 23-24
25-26
>30 15-16 15-17 22-24
24-26 15-17 23-25 26-27 25-26
Duong kinh vong khang nam ciia cac chat thip tren cac ehiing nim da s i diu rpng (> 20 mm), chp thay hpat tinh khang nim manh.
Kef qua xac djnh MIC
Bang 2: Hoat tirih khang 3 chiing nim da ciia 5 chit B Nam da
Iwbnim T. mentagrophytes U. gypseum
81 7,8 15,6 15,6
B2 15,6 31,2 31,2
MIC (pg/ml) B3 B4 85 15,6 3,9 7,8 31,2 125 7,8 15,6 125 7,8
Ketoconazo!
0,5 32 32
Bl va B5 CO hoat tinh khang nam t i t ban 2 - lin keteeonazel a 2 chiing T. mentagrophytes, M.
gypseum.
Kef qua phoi hgp
Bang 3: Hoat tinh khang nim da ciia 10 phoi hgp
TT 1 2 3 4 5 6 7 8 9 10
Cap phoi hOi B1-B2 B1-B3 B1-B4 B1-B5 B2-B3 82-84 B2-B5 83-84 B3-B5 84-85
Nonq do chat thip nahiem (uo/ml)
^ T. mbaim 1,95+3,9 1,95+1,95 1,95+0,98 1,95+1,95 7,8+7,8 3,9+1,95 1,95+0,98 1,95+1,95 3,9+0,98 0,98+1,95
T mentagrophyt(
1,95+7,8 1,95+7,8 1,95+15,6 1,95+1,95 15,6+7,8 3,9+15,6 3,9+0,98 3,9+15,6 3,9+0,98 15,6+0,98
3S M. gypseum 3,9+7,8 1,95+3,9 1,95+15,6 1,95+0,98 15,6+7,8 3,9+15,6 3,9+0,98 1,95+15,6 3,9+0,98 15,6+0,98
Viec phii hgp 2 chat thir nghiem mang lai ket qua cpng luc va hiep luc bpi tang. Tpan bp 10 cap phii hgp tang hieu qua khang nim, giam neng dp chat thip 2 - 8 lin. Hgp chit eo nong dp sip dung thip nhit la B1-B5 vol (1,95 + 1,95) pg/ml.
Voi muc tieu do tim cap phoi hgp ire ehi dugc 3 chiing nim da va che ning dp chat thip thip. Qua kit qua phii hgp tren, da chpn duge 3 cap phil hgp la B1-B5 vai (1,95 + 1,95) pg/ml, B2-B5 vol (3,9 + 0,98) pg/ml, B3-B5 vai (3,9 + 0,98) pg/ml.
Scrbg thir finh kich irng da tho
Bang 4: Tinh kich img ciia 3 cap phdi hgp khang nim da
Tho Vk B1-B5 B2-B5 S3-S5
tfii> (1,95*1,95)iiglml (3,9*0,98)iig/ml (3,S*0,98)fig/ml Iviiu Diem Nteu Diem Nteu Diem 1 Khong
2 Khong 3 Khong 4 Khong 5 Khong 6 Khong TrisaiB
Kich i)ng
Khong Khong Khong Khong Khong Khong Khong Khong
Khong Khong Khong Khong Khong Xanh lam
nhat Khgng
0 0 0 0 0 2 0,33
Ba cap phoi hgp (eo heat tinh khang nam da) dugc chpn lua cho thip tinh kich ipng da che kit qua tit. Vol ning dp chit thir eiia 3 cap phii hgp nay khong gay kich irng da the.
Hoat tinh khang khuan Kef qua djnh tinh
Nong dp cac chit thip nghiem la 100 pg/ml.
Treng 5 ehung vi khuan thir nghiem, chi oo 2 ehiing (S. aureus, MRSA) nhay vai chit thir B. Kit qua nay phu hgp vai cac bae cao eho biit din xuit halogen salicylanilid acetat co heat tinh tren vi khuan gram duang.
Bang 5 : Budng kinh vong khang khuin ciia 5 chit B (mm) Chit thir
B1 B2 B3 B4 85
S. aureus 24-25,5 25-26,5 30-32 23-25 35-38
MRSA 24-25 24-25,5
29 22-24 37-38
TAP CHI D U O C HOC - 4/2016 (SO 480 N A M 56) 79
• Nghien
CIFU- Ky thuat
T i t ea d u a n g kinh vong khang khuan eua 5 c h i t thir tren 2 chiing vi khuan d i u rpng (>20 mm), dieu nay ehp thay hoat tinh khang khuan manh.
Dac biet la B3 va B5.
So sanh hoat tinh khang khuan ciia chat thir va vancomycin
Bang 6: Dudng kinh vdng khang khuin ciia 5 chit B va vancomycin
Chat thir (30ug/ml) 81 82 83 84 B5 Vancomvcin
S. aureus 22 23 27 16 29 21
MRSA 23 24 25 15 25 20 D u o n g kinh vong khang k h u i n c h i t thir B (tru"
B4) lan ban eiia Vancomycin. K i t qua nay ehe thay heat tinh khang khuan ciia c h i t thir kha manh.
Ket qua xac dinh MIC
Bang 7: Hoaf tinh khang 2 ehiing vi khuin cita 5 chit thie B
J
S. aureus MRSA
B1 4 4
62 4 4
MIC(pq/ml) 63 0,5 0,5
84 4 4
65 0,5 0,5 Heat tinh khang khuan ciia B3, B5 rat manh vol MIC r i t t h i p (0,5 pg/ml).
5 c h i t thir B eo heat tinh khang khuan manh hon n i m da.
Kit qua phoi hgp
Bang 8: Kei qua hoat tinh khang khuan ciia 10 phoi hgp
TT 1 2 3 4 5 6 7 8 9 10
Cap phoi hop -
B1-82 81-B3 81-84 B1-B5 82-B3 B2-84 B2-B5 B3-B4 B3-B5 B4-85
Nonq do chat thir nghiem (uQ/ml) S. aureus
2+1 0,25+0,03
2+1 0,25+0,03 0,25+0,06 1+2 0,25+0,06 0,03+0,25 0,03+0,02 0,25+0,03
MRSA 2+1 0,25+0,03
2+1 0,25+0,03 0,25+0,06 1+2 0,25+0,06 0,03+0,25 0,03+0,02 0,25+0,03 Viee p h i i hgp 2 c h i t thir nghiem mang lai k i t qua cpng luc va hiep lue b5i tang. Toan b5 10 cap tang hieu qua khang k h u i n , giam n i n g dp c h i t thip tu' 16 - 32 lan. Hgp c h i t co n i n g dp sip dung t h i p n h i t la B3-B5 vai nong dp (0,03+0,02) pg/ml.
Ket luan
Da s i c h i t thip d i u co heat tinh khang vi sinh vat manh ban ketoconazol va vancomycin. De tai da do tim d u g e nhu'ng hgp c h i t mai vol heat tinh khang vi sinh vat manh va n i n g dp sip dung t h i p .
Hon nO'a, chung khOng gay kich ipng da. Ket qua thu d u g e 4 hgp c h i t :
- 3 hgp chat eo tac dung khang 2 chiing vi khuan va 3 ehiing n i m da.
1 hgp c h i t CO tae dung khang 2 chung vi khuan.
Tip k i t qua nay co the d u a vao nghien c u u voi mong m u i n tim ra c h i t khang khuan va n i m da mai.
Tai lieu tham khao
1. Mendez-Tovar LJ1, Manzano-Gayosso P (2007),
"Resistance to azolic compounds in clinical Trichophyton spp. strains", Rev Iberoam MicoL, 24(4), p. 320-322.
2. Pranab K Mukherjee, Steven D Leidich (2003),
"Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine", Antimicrobial Agents Chemotherapy, 47(1), p.82-86.
3. Bill NghTa Thjnh, Pham Anh Tuan, Khao sat tinh hinh de khang khang sinh cua vi khuan tai khoa Hdi sire tich cue va chdng doc ciia benh vien cip ciru Trung Vuang, Hpi nghj khoa hpc ky thuat Benh vien Cap ciru Trung Vuong nam 2010, ngay 10/10/2013. http://www.
bvtrungvuong.vn/Default.aspx?tabid=73&ctl=ViewNews Detail&mid=402&Nevi^sPK=451 ngay 9/3/2016.
4. Jarmila Vinsova, Ales Imramovsky, Vladimir Buchta, IVlartina Ceckova, Martin Dolezal, Frantisek Staud, Josef Jampiiek, Jarmila Kaustova (2007),
"Salicylanilide Acetates: Synthesis and Antibacterial Evaluation", Molecules, 12(1), p. 1-12.
5. Trin Thj Thu Hiln, Le Thj Ngoc Hue, Truang Phuang (2014), "Khao sat hoat tinh mpt so d i n xuit cloro va acetat ciia salicylanilid tren mot vai chiing nam m i c thuang gap trong khong khi". Tap chi Duac hoc, 2, tr. 34-41.
6. Truang Phuang, Mai Phuang Mai, Tran Thanh Bao, NguySn Dinh Nga, Nguyen Thj Van Ha va Nguy Thj Thiiy Nhung (1998), "Tong hgp va thif hoat tinh sinh hoc ciia cac d i n chit Clorosalicylanilid", Tap chi Dugc /7pc, 5, tr. 8-12.
7. ISO 10993-10:2010, part 10, 6.3.
8. Laurence L. Bruron, John S. Lazo, Keith L.
Parker (2006), Chapter 44. Antimicrobial agents In Goodman and Oilman's The pharmacological Basic of Therapeutics, 11'" ed, p. 1102-1104.
9. Michael A. Pfaller, Chairholder Chaturvedl, Ana Espinel Ingroff, Mahmoud A. Ghannoum, Linda L.
Gosey Frank C. Odds, John H. Rex, Michael G. Rinaldi, J. Sheehan, Thomas J. Walsh, David W. Warnock (2002), Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard- Second Edition, 22 (15), M27-A2.
10. Patrick R.Murray (2005), Manual of clinical microbiology B"" edition, ASM press, Washington DC p 1887-1889.
{Ngay nhan bar 11/02/2016 - Ngay duyet dang: 04/04/2016)
80 TAP CHI DirgiC HOC - 4/2016 (SO 480 NAM 56)
![NGHIEN ClaU XAC BINH HIEU LUr CUA CAC GIEN KHANG V[11 CAC CHUNG [ISOLATE] NAM LIAO ON THU THAP](data:image/gif;base64,R0lGODlhAQABAIAAAP///wAAACH5BAEAAAAALAAAAAABAAEAAAICRAEAOw==)