Tap chi Cong ngh¢ Sink hoe 5(3): 291-297, 2007 PHAN LAP VA NHAN DANG MOT SO CHUNG VI KHUAN STREPTOCOCCUS MUTANS TU NGUOI VIET NAM Neguyén Quang Huy, Phing Thj Thu Huong, Phan Tun Nghia Teng Dai hoc Khoa hoc Tue nhién, Dai hoe Quéc gia, Ha Ngi TOM TAT ‘S4u ring la mét trong nhitng bénh phé bién nhét hign nay, ac biét 14 & nhing nude dang phat trién trong d6 cb Vigt Nam, Streptococcus mutans dug xem 14 mét trong nhimg nguyén nhén chinh gay su ring bdi kha nang sinh acid va chju acid tét cua ching. Te céc mu ly tir ngudi Viet nam, bing viée nudi ody trén moi trang chen Ioc Mitis saliverius, moi trrémg Tryptic soy agar (TSA) 6 pH 5 cing nh ic diém hinh thai t8 bao ket hgp vi vigc sir dung va kit thir API 20 Strep chiing tdi 4 phan lp, xte inh duge 6 Yoai thuge chi Streptococcus va duge ky higu lan lugt la Streptococcus sp. HI, Streptococcus sp. H2, Streptococcus sp. H3, Streptococcus sp. H4, Streptococcus sp. H5 va Streptococcus sp. H6. Két qua phan tich phd bang DNA sau khi nhan ban bang PCR cia dogn gen ma héa cho 16S rRNA, sau 46 edt bing enzyme Haelll va Hpall (ky thugt PCR - RFLP) cho thdy phd bang cua 3 ching Streptococcus Sp.HI, Streptococcus sp. H2 va Streptococcus sp. H3 hoan toan tuong déng véi phd bang cia ching S. mutans GS-5 chudn va khéc véi S. sanguis NCTC10904. Tuong ty, sir dung phuong phép PCR véi cap méi ciia gen dextranase dic higu cia S. mutans cia cé 3 ching trén déu cho mét bang duy nhét véi kich thude. 1,3 kb nhu ching chudn. Cac két qua thu duge cho phép khiing dinh 3 ching Streptococcus. sp.H1, Streptococcus. sp. H2 va Streptococcus. sp. H3 la thugc loai Sretococcus mutans. Bang thi nghiém lam gidm pH va giét chét bang acid, ca 3 vi khudn S. mutans dugc phan lap déu chimg t6 c6 kha nang sinh acid va chju acid t6t, tuong qr nhu ching S. mutans GS-5 chudn, Tie kh6a: Streptococcus mutans, PCR-RFLP, gen dextranase, Mitis salivarius, 16S rRNA M6 DAU ‘Tap doan vi sinh vat trén mang bam ring rit phong phi véi hon 500 loai 43 duge xac dinh trong 6 Streptococcus mutans duge xem la tac nhén chinh gay sau ring 6 ngudi. Streptococus mutans 1a chiin| vi khudn Gram duong cé kha ning bam dinh trén mit ring sinh acid va chju acid cao, ching thing xuyén duge phan lp tir mang bém rang noi ching tng hop polysaccharide ngoai bao (Hamada, Slade, 1980; Belli et al., 1995). Viée phat hign, nhan dang s6m su c6 mit cita S. mutans sé gdp phin trong vige digu tri va phong trir bénh séu rang 6 nguoi. Trén thé gidi da cé nhigu nghién ciru v8 phan lip, dinh logi cdc ching S. mutans & ngudsi dya trén ac phuong phap khac nhau nhu dic diém hinh thai Khuan lac (Holt ef al., 1994), dic diém sinh ly sinh héa trén ede mdi tnréng chon loc (Gold ef al., 1973) va gin day 14 cdc ky thugt sinh hoc phan tt (Igarashi et al., 1996; Oho et al., 2000; Sato et al., 1997; 2003). G Vigt Nam, hién nay cac nghién ciru vé S. mutans méi chi dimng lai 1a cée nghién etru ban dau vé sy c6é mat cia ching vi khudn nay trong thanh phan trén mang bém ring, hay dich Igi cba ngudi Vigt Nam (BS Quang Trung ef al., 1999; Nguyén ‘Thj Mai Phuong er al., 2003). Nghién ci cia ching 161 nhim gép phan tim hiéu, nhgn dang Streptococcus mutans phan lap & ngudi Viét Nam bing cdc phuong phdp phan tich hinh thai, héa sinh eding nhur sinh hgc phan ti va xéc dinh mét s6 age trumg cia ching. NGUYEN LIEU VA PHUONG PHAP. Nguyén ligu Cae mau vi sinh vat duge lay bang tim bong vd tring tir céc hé ring bi sdu rang cdc bénh nhan bj su ring 6 Khoa Ring Ham Mat, Bénh vign Bach Mai, sau d6 duge chuyén vao mdi tring mudi sinh ly 0,9% dé phan lap tiép. Ching Streptococcus mutans GS-5 va Streptococcus sanguis NCTC 10904 la qua ting cia Gido su Robert E. Marquis (Bai hoc Rochester, M§). Céc oligonucleotide, céc thang chudin DNA duge 291mua tir hang Invitrogen (Mf), che dNTP cia hing Promega (Mf), cdc enzyme han ché cia hing New England Biolabs (NEB), méi truéng Mitis salivarius va Tryptic Soy Agar (TSA) cia hang Diffco (MY), kit thir API20 Strep oiia hing Bio Mérieux, Marey- Tetoile (Phap). Céc héa chét khéc déu dat 6 tinh khiét ding cho nghién ctu sinh hoc phan ti. Phwong phap nghién eu Phicong phap phan lép chiing vi khudn Mu thu thap duge edy trai trén méi truémg chon loc Mitis salivarius c6 bd sung tellurite 1% 6 nhiét 6 37°C trong thoi gian 2 ngay. Cac khudn lac sau khi mgc trén mdi trudmg Mitis salivarius sau dé duge cy chuyén sang m6i trudng TSA 6 pH 5,0 voi nhigt 46 37°C. Hinh thai timg khuan lac duge quan sat, méi khuan lac durge tach nudi cdy riéng ré trén mdi trutmg ling TYG (tryptone 3%, cao nim men 0,5% va glucose 1%). Hinh thai t8 bao cia cdc ching vi khudn duge quan sat va chup anh duéi kinh hién vi dién ti quét ISWLV5410 voi sy gitp d& cua Vign 69. Nghién citu mét sé dic diém héa sinh duge tién hanh theo kit thir API20 Strep va két qua duge xit ly theo phan mém do hang cung cp. Tink sach, nhan ban gen mi héa 165 rRNA bing phwong phdp PCR va kj thugt RFLP DNA cila hé gen durge tinh sgch theo phuong phap mé ta bai Sambrook va Rusell (2001) duge xéc dinh ham Iueng qua a> hap thu anh séng & bude séng 260 nm. Céc mau DNA duge kiém tra bing dign di trén gel agarose 1%, gel sau 46 durge nhuém véi ethidium bromide, soi dui anh sng ti ngoai va chyp anh. Cap méi sir dyngnhén ban gen ma héa 16S 1RNA_ bao gdm mdi xudi 8UA (5’- AGA GTT TGA TCC TGG CTC) va mdi nguge 1492R (5'- TAC GGG TAC CTT GTT ACG ACT T). Mét phan tng 25 ul bao gdm 100 ng DNA khuén, 2,5 pl dém 10x Taq DNA polymerase, 50 ng mi logi mdi xuéi va nguge, 25 mM dNTP, 0,5 don vj Tag. PCR duge thy hién theo chu trinh: 15 phut ¢ 95°C dé bién tinh. DNA thanh hai sgi don, 35 chu ky lip tai ciia cfc bude 94°C 1 phut dé tach chudi, 60°C, 1 phut dé gin mdi va 72°C trong | phit 30 gidy dé kéo dai chudi. Hén hop sin phdm nhan lén duge i tiép 72°C trong 10 phat va sau d6 gitr & 4°C dén khi phan tich (Sato et al., 2003). 292 Nguyén Quang Huy e¢ af. San phim PCR sau khi duge nan Ién, tinh sach bing kit cia héing Bioneer (Han Quéc). Dogn gen ma héa 16S rRNA duge ct bdi hai enzyme c&t han ché Hpall va Haelll. Tiép theo, dugc phan tach va kiém tra bing dign di tén gel agarose 2%, nhugm ethidium bromide, soi dudi anh sing tr ngoai va chyp anh. Nhéan ban dogn gen dextranase bang phicong phap PCR _ Doan gen dextranase duge tién hinh nhan ban bing PCR voi cap mdi dic higu SDI (5’- TAT GCT GCT ATT GGA GGT TC)’ va SD2 (S°- AAG GTT GAG CAA TTG AAT CG). San phim PCR duge kiém tra bing dién di trén gel agarose 1% nhuém ethidium bromide quan sat dudi dén UV va chyp anh (Sato ef al, 2003). Dinh gié kha nang sinh acid va chiu acid cha cée chiing 8. mutans Kha ning sinh acid cha S. mutans duge danh gid bing phuong phép lam giam pH trong méi trudng du thita glucose theo phuong phap mé ta trong Phan, Marquis (2006). Kha niing chju acid ca céc ching S. mutans dugc danh gid qua mac 6 séng sét cita vi khuan & pH 2,7 nhu duge mé ta trong Phan va dong tae gia (2000). KET QUA VA THAO LUAN Phan Ip va nhan dang so b@ cdc ching vi khudn tir m4u bénh pham sau rang Tir cde mau bénh phim eta bénh nban sau ring ching téi da tién hanh phén lap trén mdi truong Mitis salivarius c6 bd sung 1% tellurite, day 1a mai truémg chon gc ding dé phan lp cae chiing thugc chi Streptococcus va Enterococcus (Gold et al., 1973). Dya trén dic diém Khuan lac cua S. mutans trén méi trudng nay cé mau xanh nhat, mé duc, hoi 18i va lrgn séng ching tdi da phan Idip va tach duge 20 ching vi khuan. Trong sé 20 ching vi khudn durge phan lp trén m6i truémg Mitis salivarius khi chuyén sang nui cy trén méi trudng TSA co pH thip (pH 5) chi c6 6 ching vi khudn phat trién c6 mang cfc dic diém turong déng gidng ching chuan S. mutans GS-5. Két qui nhuém Gram cho thdy cd 6 ching déu la vi khudn Gram duong cé dang hinh cau xép thanh chudi, va céc ching déu gidng nhau khéng cé kha nang sinh catalase. Dac biét, khi quan sat duéi kinhTap chi Cong nghé Sink hoc 5(3): 291-297, 2007 hién vi dign tir céic ching nay déu cé dang lién chu khuan (Hinh 1), Theo Holt va ding téc gia (1994), dya vao dic diém trén 6 ching vi khudn nay cé thé tube chi Streptococcus va ching duge ky higu tir Streptococcus sp. Hl dén Streptococcus sp. H6 . Dé tiép tue nhan dang 6 chiing vi khudn thude chi Streptococcus 6 trén ching tdi da sir dung kit thir API20 Strep dya trén kha mang sir dung céc logi dug va hoat dong cla céc enzyme dac trung. Két qua phan tich thu doe (Bang 1) cho thay trong sé 6 ching vi khudn phén lip duge ching Streptococcus sp. HI gidng S. mutans voi d6 tin c@y 14 99%, T = 0,9. Chiing Streptococcus sp. H1 so véi ching chudn S. mutans GS-5 chi cé mot sai khéc duy nhét vé kha nang déng héa inulin, 2 ching vi_khudn Streptococcus sp. H2 va Streptococcus sp. H3 ¢6 46 tin c€y tong doi thép déu 55,3% voi gid tri T bing, 0,75 06 thé thudc nhém S. equinus. 3. ching Streptococcus sp. H4, Streptococcus sp. HS va Streptococcus sp. H6 06 kha ning thudc S. sanguis véi d6 tin cAy 14 hon 99,5% va véi hé sé tuong im; T véi cde ching 14 0,88, 0,71 va 0,83. Nin vay, két qua phan tich bang kit API20 Strep khang dinh mét lin ntta ca 6 ching phan lap durge déu thude chi Streptococcus. Hinh 4. Anh chyp hién vi cia ching vi khuan Streptococcus sp. H1 (6 phéng dai 15000 lan), Bang 1. Mot s6 dac tinh sinh ly clla 6 ching Streptococcuc sp. phan lp tir benh pham sau rang nguoi Viet Nam. Bae tinh sinh héa ‘S. mutans S. sp. H1 GSS 8. .sp.H2 S.sp.H3 S.sp.H4 S.sp.HS S. sp. H6 Sinh acetoin ‘Thuy phan hippuric acid ‘Thiy phén B-glucoside Pyrrolidony! arylamidase o-Galactosidase B-glucuronidase 8 -Galactosidase Alkaline phosphatase Leucine amino peptidase Arginine dihydrolase ‘Kha nang déng hoa D-ribose L-arabinose D-mannitol D-sobitol D-lactose D-trehalose Inulin D-raffinose Amidon Acid hoa Glycogen thee