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1 Jurnal f Dental Hygiene Science Vl. 9, N. pp. 49~55 (009) 치면세균막내의 Fusbacterium nucleatum 과 Actinbacillus actinmycetemcmitans 의동정을위한세균배양법및 Multiplex 법의비교 ½y Á û w w e Cmparisn between Bacterial Methd and Multiplex fr Identificatin f Fusbacterium nucleatum and Actinbacillus actinmycetemcmitans frm the Dental Plaques Hwa-Sk Kim and Sun-A Lim Department f Dental Hygiene, Chunnam Techn Cllege, Chunnam , Krea "CTUSBDU This study was carried ut fr the purpse f cmparing bacterial culture methd, single, and multiplex fr identificatin f F. nucleatum and A. actinmycetemcmitans in subgingival plaque f adult peridntitis. Targeting 0 patients with adult peridntitis, the subgingival plaque was cllected in teeth, respectively, fr #16, #36, #44. A bacillus was cultivated by painting it ver the slid selective media f F. nucleatum and A. actinmycetemcmitans. Bacterial species were detected in 0 tth with 1 pieces, respectively. Thrugh single and multiplex, the psitive reactin was indicated in 43 teeth with 45 pieces, respectively, as fr F. nucleatum, and in 1 tth with 4 pieces, respectively, as fr A. actinmycetemcmitans. In the cmparative analysis between bacterial identificatin methds, F. nucleatum shwed the mre statistically significant difference(p = 0.000) in cmparisn between single and multiplex. Even A. actinmycetemcmitans was indicated significantly(p = 0.067) in a case that is based n 0.1 in significant level in the cmparisn between single and multiplex. In cnclusin, as a result f cmparing the bacterial identificatin methds, the detectin frequency was indicated t be higher in than in bacterial culture methd. and multiplex shwed the mutually similar detectin frequency. Accrdingly, given thinking f ecnmic efficiency, quickness, and reductin in labr frce, it is thught t be mre efficient methd t use single as the bacterial identificatin methd.,fz XPSET Fusbacterium nucleatum, Actinbacillus actinmycetemcmitans, methd, Multiplex, 서 론 e y e w, e, e e w y, e ³ w x» ³ w. ü 700 ³ wš 1), e e w e ³ ü Prphyrmnas gingivalis, Prevtella intermedia, Fusbacterium nucleatum, Campylbacter rectus, Trepnema denticla, Actinbacillus actinmycetemcmitans Bacterides frsythus ³ ¾ š ). Crrespnding authr Tel: Fax: F. nucleatum x» ³ nuclease, phsphatase, lipplysaccharide(lps) w e q w ³. w e ù xk e y ƒ w ³3-5) e y x» ³ w x» y w w wš 6), ü ³ (caggregate)w»( ³) z»( ³) e ³ x yw ³ w w 7). A. actinmycetemcmitans m ³ enw LPS leuktxin w» q wš e q e y w. e ƒ w e w e e ³

2 Cmparisn between Bacterial Methd and Multiplex fr Identificatin f Fusbacterium 막에서도 매우 적은 수로 존재하는 세균이다. 또한 구강 외 여러 부위로 전파되어 감염성 심내막염의 원인이 되기 도 한다. 치주질환의 진단, 치료 및 예후를 평가하기 위해 다양 한 치주 병원성 세균들을 검사하는 방법들이 있는데 세균 배양법은 가장 고전적이면서 정확성이 높은 방법이다. 하 지만 많은 시간, 노동력, 경제력을 필요로 하기 때문에 현 실적으로 세균 동정에 이용하기에는 매우 어려움이 크다. 반면에 multiplex plymerase chain reactin()법 은 individual 법 을 포함한 기존의 세균 검출 방법들 에 비해 좀 더 신속하고, 경제적이며, 노동력 절감과 함께 단시간에 다양한 세균을 동시 다발적으로 검출할 수 있다 는 장점을 가지고 있다. Kim 등 은 16S rrna 유전자(rDNA) 염기서열을 바탕 으로 F. nucleatum과 A. actinmycetemcmitans를 동시에 동정할 수 있는 multiplex 프라이머를 개발하여, 특 이성 및 민감도 조사를 실시하여 우수성을 보고하였고, 성인성 치주염 환자의 치은연하 치면세균막 샘플를 이용 해 multiplex 법과 single 법을 비교하였다. 본 연구는 Kim 등 의 연구를 바탕으로 성인성 치주염 의 치은연하 치면세균막에서 F. nucleatum과 A. actinmycetemcmitans의 동정을 위한 세균배양법, single 법 및 multiplex 법을 비교하여 효율적인 세균 8) 9-1) 13-15) 10) 10) 동정법을 알아보고자 실시하였다. 재료 및 방법 1. 치면세균막 채취 C 대학교 치과병원에 내원한 0명의 성인성 치주염[판 정기준: pcket depth 4 mm, bleeding n prbing] 환자를 대상으로 각각 #16, #36, #44 치아의 치은연하 치 면세균막을 멸균된 paper pint를 이용하여 채취한 뒤 1 PBS 500 µl에 담아 다음의 실험에 사용하였다.. 세균 배양 각각의 부위에서 채취한 샘플은 즉시 실험실로 옮겨 혐 기성 세균배양기 안에서 원액 50 µl를 1 PBS로 10,000배 희석한 다음 멸균된 면봉을 이용하여 F. nucleatum에 대 한 고형 선택배지(1% trytic sy brth, 0.5% yeast extract, 0.5% NaCl, 0.% glucse, 0.0% L-tryptphan, 1.5% Bact agar, 5% defibrinized sheep bld, 4 µg/ml erythrmycin, 5 µg/ml crystal vilet)와 A. actinmycetemcmitans에 대한 고형 선택배지[tryptic sy brth(tsb; Difc, USA); 0.6% yeast extract, 5% hrse serum, 1.5% Bact agar, 75 µg/ml bacitracin, 5 µg/ml vancmycin]에 각각 도말하였다. 37 C 혐기성배양기(85% N, 5% CO, Fig. 1. Phase-cntrast micrscpy f (A) F. nucleatum subspecies nucleatum ATCC 5586, (B) F. nucleatum subsp. plymrphum ATCC 10953, (C) F. nucleatum subsp. vincentii ATCC 51190, (D) A. actinmycetemcmitans ATCC

3 Jurnal f Dental Hygiene Science Vl. 9, N., pp. 49~55 (009) 10% H, Mdel Bactrn I, Crnelius, OR, USA) ~3 w 16-18). ƒƒ k w» w F. nucleatum crystal vilet w š, w, ñ, A. actinmycetemcmitans z n, ñ, ü k w y wš, x mw F. nucleatum j»ƒ w, ó w(fused) xk, A. actinmycetemcmitans j»ƒ š, (0.1~1 µm), š ù x k y w (Fig. 1). ³ wš, yw w ƒ clny w F. nucleatum A. actinmycetemcmitans q w. 3. e w e ³ w v DNA Lee 19) w. 50 µl Lysis buffer( mm EDTA, 1% Tritn X-100) yww 10 ò DNA. F. nucleatum w» w All-F6(5'-CGG GAG GCA GCA GTG GGG AAT-3') Fn-R6(5'-TTG CTT GGG CGC TGA GGT TC-3') v w š, A. actinmycetemcmitans w» w All-F6(5'-CGG GAG GCA GCA GTG GGG AAT-3') ChDC-AaR(5'-CAT CGC TGG TTG GTT ACC CTC TG-3') v w, AccuPwer Premix[5 nmle dexynucleside triphsphate, 0.8 umle KCl, 0. umle Tris-HCl(pH 9.0), 0.03 umle MgCl, 1 unit Taq DNA plymerase](bineer C., K0rea) PTC-00 machine(mj Research Inc., USA) w 16S rdna sw.. 0 µl yw 0 pmles v 4µl ³ DNA š, F. nucleatum w» w 16S rdna s 94» w 94 C 1, annealing, 7 C 45 extensin 30z w z, 7 C 10 extensinw. A. actinmycetemcmitans w» w 16S rdna s 94 C» w 94 1, 68 C 1 annealing, 7 C 1 extensin 3z w z, 7 C 10 extensinw. š 4µl 1.5% ƒ» w s y w. F. nucleatum A. actinmycetemcmitans 'JH Cincidence test f the multiplex and single s perfrmed with the plaque samples. (A) Multiplex fr simultaneus detectin f F. nucleatum and A. actinmycetemcmitans, (B) s fr the detectin f A. actinmycetemcmitans, and (C) F. nucleatum. Lane; S, 100base pair DNA ladder; PC, F. nucleatum subsp. nucleatum ATCC 5586 and A. actinmycetemcmitans ATCC (psitive cntrl) The pen and filled arrwheads indicate the prducts f A. actinmycetemcmitans(95 bp) and F. nucleatum(495 bp), respectively.

4 Cmparisn between Bacterial Methd and Multiplex fr Identificatin f Fusbacterium... s j» ƒƒ 495 bp 93 bp š, 1.5% ƒ» w s y w. 4. Multiplex e y y e w e ³ F. nucleatum A. actinmycetemcmitans» w All-F6, Fn-R6, ChDC-AaR v w. A. actinmycetemcmitans w» w 16S rdna s w w. 94 C 1, 68 C 30 annealing, 7 C 1 extensin 30z w z, final extensin w 7 C 10 ww. 5. m F. nucleatum A. actinmycetemcmitans w ³, single multiplex SPSS 1.0 w χ -test(fisher's exact test) w w. 결 과 e y 0 60 e w F. nucleatum A. actinmycetemcmitans w ³, single multiplex w, F. nucleatum ƒƒ 1 (0.0%), 45 (75.0%), 43 (71.7%) e, 48 (80%), 15 (5.0%), 17 (8.3%) e. A. actinmycetemcmitans ƒƒ 0 (0.0%), 4 (6.7%), 1 (1.7%) e, 60 (100%), 56 (93.3%), 59 (98.3%) e ùkû (Fig., Table 1). ³, single multiplex m, F. nucleatum single multiplex m w š 5BCMF The detectin f F. nucleatum and A. actinmycetemcmitans using multiplex, single, and bacterial culture methds Pt's Tth Fn 1 Aa Pt's Tth Fn Aa N. Site (#) CC 3 SP 4 MP 5 CC SP MP N. Site (#) CC SP MP CC SP MP Fn 1, Fusbacterium nucleatum; Aa, Actinbacillus actinmycetemcmitans; CC 3, culture methd; SP 4, methd; MP 5, Multiplex methd.

5 5BCMF Cmparisn between single and cell culture Species Jurnal f Dental Hygiene Science Vl. 9, N., pp. 49~55 (009) culture N. f sample(%) culture Detectin frequency f the bacteria(%) culture Fn 1 11(18.3) 1(1.7) 34(56.7) 14(3.3) 45(75.0) 1(0.0).6 Aa 0(0) 0(0) 4(6.7) 56(93.3) 4(6.7) 0(0) * *Nn available, Fn 1 ; Fusbacterium nucleatum, Aa ; Actinbacillus actinmycetemcmitans P- value 5BCMF Cmparisn between single and multiplex Species Multiplex N. f sample(%) Multiplex Detectin frequency f the bacteria(%) Multiplex Fn 1 39(65.0) 4(6.7) 6(10.0) 11(18.3) 45(75.0) 43(71.7).000 Aa 1(1.7) 0( 0) 3(5.0) 56(93.3) 4(6.7) 1(1.7).067 Fn 1 ; Fusbacterium nucleatum, Aa ; Actinbacillus actinmycetemcmitans P- value 5BCMF Cmparisn between cell culture and multiplex Species Multiplex N. f sample(%) Multiplex Detectin frequency f the bacteria(%) Multiplex Fn 1 11(18.3) 3(53.3) 1(1.7) 16(6.7) 1(0.0) 43(71.7).151 Aa 0(0) 1(1.7) 0(0) 59(98.3) 0(0) 1(1.7) * * Nn available, Fn 1 ; Fusbacterium nucleatum, Aa ; Actinbacillus actinmycetemcmitans P- value (p = 0.000), A. actinmycetemcmitans single multiplex 0.05 ƒ 0.1» w ùkû (p = 0.067)(Table 3). F. nucleatum A. actinmycetemcmitans ³ single, ³ multiplex m w (Table, 4). 고 찰 e y ¾ ³ w» w ƒ m ³. ³ šx ³ mw ³ wš, šx, yw ³ w w ³ w. w m ³ w ³ w ƒ š, ¾ š, ¼, ƒ» ƒ š 0). š 1 allenzyme, immunenzymatic assay,» DNA-DNA hybridizatin ³ w» w, (false-psitive) ƒ 1). x m w d ³ (species) w» 16S rdna»,3). š e w e ³ e y ³ w» w ƒ z ³ 16S rdna» k -p v w š w 15). Kim 10) 16S rdna» k F. nucleatum A. actinmycetemcmitans w v w 5' 16S rdna» ƒ œm (All-F6), 3' ƒ ƒ p w Fn-R6 ChDC-AaR v w. Multiplex v p» w 4 F. nucleatum t ³ (ATCC 5586, ATCC 10953, ATCC 4956, ATCC 51190) 3 A. actinmycetemcmitans t ³ (ATCC 43717, ATCC 43718,

6 Cmparisn between Bacterial Methd and Multiplex fr Identificatin f Fusbacterium... ATCC 33384) ü w ³ 7 8³ DNA x w, F. nucleatum 495 bp A. actinmycetemcmitans 95 bp ³ p ƒ š, ³ single multiplex 4 fg¾ ƒ w šw. š e y e w e ³ v 0 w multiplex single e w s j»ƒ w ùkù šw. Kim 10) k w e y e w e ³ F. nucleatum A. actinmycetemcmitans w» w ³, single multiplex wš w. e y w 60 v F. nucleatum A. actinmycetemcmitans w ³, single multiplex w F. nucleatum ƒƒ 1 (0.0%), 45 (75.0%), 43 (71.7%) e, A. actinmycetemcmitans ƒƒ 0 (0.0%), 4 (6.7%), 1 (1.7%) e ùkû. F. nucleatum w ³ single, ³ multiplex w ³ w single multiplex z ³ ƒ m w ƒ. A. actinmycetemcmitans ³ m w š, ³. ³ w m w x v ƒ ùkù ƒ. multiplex ³ j, m w š, F. nucleatum p = ùkû. F. nucleatum ƒ A. actinmycetemcmitans w { ùkû» m ùkü. š Kim 10) single multiplex 4 fg¾ ƒ wš, s j» w š w, F. nucleatum A. actinmycetemcmitans multiplex w single ƒƒ, 3. e w e ³ w v Lee 19) DNA w 4 µl ³ DNA w w, DNA w, ƒ p ùkù š ƒ. ü F. nucleatum 5ƒ F. nucleatum subsp. plymrphum w e, F. nucleatum subsp. nucleatum e y š w. 4), F. nucleatum e k ü x ƒ ³ w. A. actinmycetemcmitans e ³ e q e y ƒ š w. e v F. nucleatum x, A. actinmycetemcmitans û ùkû, w e y ƒ û» A. actinmycetemcmitans x ƒ. ww, w e e w e ³ F. nucleatum A. actinmycetemcmitans w» w ³ w ³ ùkûš, single multiplex w. ³ w y,, ƒw x ¾ multiplex ~3 single w z ƒ. 요 약 e y e w e ³ 60 e w F. nucleatum A. actinmycetemcmitans w ³, single multiplex w š, ³ mw. 1. F. nucleatum A. actinmycetemcmitans w ³, single multiplex w F. nucleatum ƒƒ 1 (0.0%), 45 (75.0%), 43 (71.7%) e, A. actinmycetemcmitans ƒƒ 0 (0.0%), 4 (6.7%), 1 (1.7%) e ùkû.. F. nucleatum ³ w single multiplex z ³ ƒ, m w ƒ. 3. A. actinmycetemcmitans ³ x m w š, ³. 4. F. nucleatum A. actinmycetemcmitans w single multiplex ³ j, m w.

7 Jurnal f Dental Hygiene Science Vl. 9, N., pp. 49~55 (009) 참고문헌 1. Kazr CE, Mitchell PM, Lee AM, Stkes LN, Lesche WJ, Dewhirst FE, Paster BJ: Diversity f bacterial ppulatins n the tngue drsa f patients with halitsis and healthy patients. J Clin Micrbil 41(): , Scransky SS, Haffajee AD: The bacterial etilgy f destructive peridntal disease: current cncepts. J Peridntl 63(4 Suppl): 3-331, Dzink JL, Scransky SS, Haffajee AD: The predminant cultivable micrbita f active and inactive lesins f destructive peridntal diseases. J Clin Peridntl 15(5): , Haffajee AD, Scransky SS: Micrbial etilgical agents f destructive peridntal diseases. Peridntl 000 5: , Blstad AI. Jensen HB, Bakken V: Taxnmy, bilgy, and peridntal aspects f Fusbacterium nucleatum. Clin Micrbil Rev 9(1): 55-71, Diaz PI, Zilm PS, Rgers AH: Fusbacterium nucleatum supprts the grwth f Prphyrmnas gingivalis in xygenated and carbn-dixide-depleted envirnments. Micrbil 148(Pt ): , Rberts GL: Fusbacterial infectins: an underestimated threat. Br J Bimed Sci 57(): , Mandell RL: A lngitudinal micrbilgical investigatin f Actinbacillus actinmycetemcmitans and Eikenella crrdens in juvenile peridntitis. Infect Immun 45(3): , Cnrads G, Flemmig TF, Seyfarth I, Lampert F, Lütticken R: Simultaneus detectin f Bacterides frsythus and Prevtella intermedia by 16S rrna gene-directed multiplex. J Clin Micrbil 37(5): , Kim MK, Kim HS, Kim BO, Y SY, Seng JH, Kim DK, Lee SE, Che SJ, Park JC, Min BM, Jeng MJ, Kim DK, Shin YK, Kk JK: Multiplex using cnserved and species-specific 16S rdna primers fr simultaneus detectin f Fusbacterium nucleatum and Actinbacillus actinmycetemcmitans. J Micrbil Bitechnl 14(1): , Seng JH, Kim DK, Shin KY, Kim BO, Kim MS, Kim MK, Kk JK: Simultaneus detectin f Prevtella intermedia and Prevtella nigrescens by 16S rdna-directed multiplex. J Krean Acad Dent Health 6: , Tran SD, Rudney JD: Multiplex using cnserved and species-specific 16S rrna gene primers fr simultaneus detectin f Actinbacillus actinmycetemcmitans and Prphyrmnas gingivalis. J Clin Micrbil 34(11): , Ashimt A, Chen C, Bakker I, Slts J: Plymerase chain reactin detectin f 8 putative peridntal pathgens in subgingival plaque f gingivitis and advanced peridntitis lesins. Oral Micrbil Immunl 11(4): 66-73, Cnrads G, Mutters R, Fischer J, Brauner A, Lütticken R, Lampert F: reactin and dt-blt hybridizatin t mnitr the distributin f ral pathgens within plaque samples f peridntally healthy individuals. J Peridntl 67(10): , Slts J, Ashimt A, Flynn MJ, Li G, Chen C: Detectin f putative peridntal pathgens in subgingival specimens by 16S ribsmal DNA amplificatin with the plymerase chain reactin. Clin Infect Dis 0(Suppl ): S , Bakken V, Högh BT, Jensen HB: Utilizatin f amin acids and peptides by Fusbacterium nucleatum. Scand J Dent Res 97(1):43-53, Bakken V, Högh BT, Jensen HB: Grwth cnditins and uter membrane prteins f Fusbacterium nucleatum. Scand J Dent Res 98(3):15-4, Hlm A, Rabe P, Kalfas S, Edwardssn S: Imprved selective culture media fr Actinbacillus actinmycetemcmitans and Haemphilus aphrphilus. J Clin Micrbil 5(10): , Lee SE, Kim SY, Kim SJ, Kim HS, Shin JH, Chi SH, Chung SS, Rhee JH: Direct identificatin f Vibri vulnificus in clinical specimens by nested. J Clin Micrbil 36(10): , Savitt ED, Strzempk MN, Vaccar KK, Pers WJ, French CK: Cmparisn f cultural methds and DNA prbe analyses fr the detectin f Actinbacillus actinmycetemcmitans, Bacterides gingivalis, and Bacterides intermedius in subgingival plaque samples. J Peridntl 59(7): , Chen C, Slts J: Micrbilgical tests fr Actinbacillus actinmycetemcmitans and Prphyrmnas gingivalis. Peridntl 000 0:53-64, Amann R, Ludwig W, Schleifer KH: Identificatin f uncultured bacteria: a challenging task fr mlecular taxnmists. ASM News 60: , Jusunues-Sinerm HR, Summanen PH, Finegld SM: Bacterides, Pphyrmnas, Prevtella, Fusbacterium, and ther anaerbic gram-negative rds and ccci,. In: Murray, F.C. and Ylken, R.H.(Eds.), Manual f clinical micrbilgy. 7th ed. ASM press Washingtn pp , Gharbia SE, Shah HN, Lawsn PA, Haapasal M.: Distributin and frequency f Fusbacterium nucleatum subspecies in the human ral cavity. Oral Micrbil Immunl 5(6): 34-37, (Received May 7, 009; Revised June 0, 009; Accepted June, 009)

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