The Korean Journal of Microbiology, Vol. 42, No. 3, September 2006, p. 185-194 Copyright 2006, The Microbiological Society of Korea w ³ w w Á½ Ÿ 1 Á Á½ 1 Á û 1 Áyy¼Á» 1, * w e we w yw e ³ wš, 8 w w ³ w» w w. ³ 27 e e e eù r r s p ³ w. e ƒ e r r s p 500 ul 1 PBS ywwš, 5% x sw BHI w (x w ) w 37 o C x»» 2-5 w. x w ù ³ 16S rrna (rdna)» w w. ³ 8 w w d w., 101 ³ š, Streptococcus spp. (29.7%) Actinomyces spp. (21.8%)ƒ ƒ. ³ 9 ³ x ù w x. ƒ w w w, ³ 80(87.0%) ³ ƒ j, v p lp j 69(75.0%)ƒ, p 66(71.7%) ³ ƒ, r G 63(68.5%) ³ ƒ, p 61(66.3%) ³ ƒ, 41(44.6%) ³ ƒ. ù v 29(31.5%) ³. w 8 w w ³ ³ ƒ. w e y e w ƒ v w w ww z w. Key words ý 16S rdna, antibiotics, bacteria, endodontic infection, identification, MIC e (dental pulp) ù e ³ w. e ƒ w k w ƒ e ƒ š, e e ƒ x š (1). ù ³ e ƒ š e e e û. e ³ j ƒ ù (18, 22). e, w e q ù ³ e q, e w w. e y e û ù e û w ³ e w e. e y w ƒ ³ x w x (7). w x» ³ e (endodontic infection, endodontitis) w w w š (2, 8, 28). ü 500 ³ w (21), e ³ ³ r (13, 23, 14). w wùƒ x ³» ü ³ 50% ƒ w (21, 16). ³ *To whom correspondence should be addressed. Tel: 062-230-6877, Fax: 062-224-3706 E-mail: jkkook@chosun.ac.kr w» w w w e y ³ w ƒ (16). ù w w e ³ w, e y w w w w ³ w p, ³- y w ³ ³. e y w ³» w ³ v w. e y e ¾ w e ù w» w ƒ e. w x ù wz š e y ³ w w w w. w, w e y ³, w w ƒ w» w w w ƒ. w, w ü ³ ³ wš» w ³ wš w w v w. w w ww š w w x z š w ü ³ x. w e ³ w 185
186 Sang-Soo Lim et al. Kor. J. Microbiol, š 8 w w wš ww. v ³ w e e w ü w y w w ù e w 27 y l w 27 e w. v w e wš, e 3% y, 5% 1 wš, 5% p y ùp e iodine y y k wš, e e ³ w. e r r s p w ü ü wš 1ml 1 PBS yw ü x» ³. w v 1,000 w 3% tryptic soy broth, 0.5% yeast extract, 0.05% cysteine HCl, 1.5% bacto-agar, 0.5 µg/ml hemin 2µg/ml vitamin K 1 w w w 85% N 2, 5% H 2, 10% CO 2 ywƒ ƒ œ 37 o C x»» (Model Bactron I, Sheldon Manufacturing Inc., Cornelius, USA) 2-5 w. w ³,, j» š w ƒƒ w 5ml w x»» 1-2 w. ³ DNA ³ 3ml 12,000 rpm w z wš, G-spin TM Genomic DNA Extraction Kit (intron Co., Seoul, Korea) w ³ DNA w. ³ z w 50 µl 3µl š yww 37 C 1 o w.» 250 µl G- š yww 65 C o 15 jš, 250 µl w š» w. w s w G-spin column š TM 13,000 rpm 1 w. G-spin column TM 500 µl A š 1 w.» 500 µl B š 1 wš, G- spin column TM eppendorf tube 100 µl š 1 ew,» 12,000 rpm w 1 w. PCR w 16S rdna s j 16S rdna sw universal PCR v (27F 1492R) (12), AccuPower Premix (Bioneer Corp., Seoul, Korea) PTC-200 PCR machine (MJ Research Inc., Watertown, USA) w 16S rdna sw. PCR ww. PCR yw 20 µlƒ, 20 pmoles 27F 1492R v 100 pg ³ DNA š 94 o C 2» w 94 o C 1, 55 o C 1 w, 72æ 1 w 30z w k z, w 72 o C 10 w g. PCR óù z 20 µl 2µl Trisacetate buffer (0.04 M Tris-acetate, 0.001 M EDTA, [ph8.0]) w wš 1.5% ƒ w 100 V 30» w. s ethidium bromide w UV transilluminator g s y w. sw 16S rdna pgem-t easy vector (Pormega Corp., Madison, USA) w j w. v DNA E. coli DH5α x yw ƒƒ w v DNA AccuPrep TM Plasmid Extraction Kit (Bioneer Corp.) w z w. w, ³ 1ml 30 (10,000 g)wš, ³ 250 µl xk š t, 250 µl s w ƒw yww, 350 µl y ƒw wš z 5 ew. 10 (12,000 g)w d w» p»š, 1 (12,000 g) w. š, w» p 700 µl (80% k ) z 1 (12,000 g)w. w» p û k w» w š, 30 (13,000 g) w. w» p»š,» 100 µl š 1», 1 (12,000 g)w -70 o C wš w» w. w» w» w» (Bioneer Corp.) w w. w w» v ChDC- GEM-F (5'-TTC CCA GTC ACG ACG TTG TAA AA-3'), Seq-F1 (5'-CCT ACG GGA GGC AGC AG-3'), Seq-R2 (5'-GAC TAC CAG GGT ATC TAA TCC-3'), Seq-F16 (5'-TAG ATA CCC YGG TAG TCC-3') ChDC-GEM-R (5'-GTG TGG AAT TGT GAG CGG ATA AC-3') w, SeqMan v (Version 5.00; DNASTAR, Inc., Madison, USA) w w. w» œw Blastn v w wš, 98% t ³ (species) š q w. w x Penicillin G (r G), amoxicillin ( ), tetracycline (lp j ), erythromycin ( p ) clindamycin (j ) Sigma (St. Louis, USA) w w. w, Augmentin (amoxycillin + clavulanic
Vol. 42, No. 3 ³ w 187 acid, 5:1)( p) SmithKline Beecham (Brentford, UK), ciprofloxacin ( v ) z (Seoul, Korea) š, Cefuroxime axetil ( v p, v ) z (Seoul, Korea) œ w. w w (minimum inhibitory concentration; MIC) Murray Jorgensen(17) d w. w, ƒƒ w ƒ 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0 µg/ml 0.1 ml w.» 450 nm q w Ÿ (A450) ƒ 0.05 w xk ³ ƒƒ 0.1ml w š, ƒƒ ³ 48 w z Microplate Autoreader (Model; EL311SX, BIO-TEX Instruments Inc., Cortland, USA) w 450 nm Ÿ d w. ³ Ÿ w ±0.05 w MIC w. National Committee for Clinical Laboratory Standards (NCCLS) «šw w t (Table 1, 19, 20). e ³ 27 e y ³ w 101 ³ (Table 2). w v s³ 3.7 ³ ƒ 1 ³ 12 ³ ƒ. ³ 16S rdna j w,» w w, ³(29.7%) Actinomyces spp. (21.8%) ³ ƒ (Table 3). ³ mitis ³, salivarius ³, anginosus ³ S. constellatus S. intermedia 16S rdna». w A. naeslundii A. viscosus, P. cyclohexanicum P. freudenreichii, P. propionicus P. avidum, S. pasteuri S. warneri, C. sporogenes C. botulinum w (Table 3). 93/94 v w y w e e r r s p w v w. Actinomyces naeslundii/viscosus mitis ³ 7 (25.9%) v ƒ w (Table 2, 3). ³ 8 w w MIC ³ 8 w w MIC Table 4 w. 101 ³ 9 ³ (ChDC B634, ChDC B744) ù w ù, (ChDC B632, ChDC B633, ChDC B647, ChDC B700, ChDC B708, ChDC B750, ChDC B751) w x w w. ³ 80(87.0%) ³ ƒ j, v p lp j 69(75.0%)ƒ, p 66 Table 1. Interpretive standards for dilution susceptibility testing (19, 20) Antibiotics MIC (µg/ml) Susceptible Intermediate Resistant Penicillin G, amoxicillin* Anaerobes 0.5 1 2 Staphylococci 0.12-0.25 Streptococci 0.12 0.25-2 4 Enterococci 8-16 Amoxicillin + clavulanic acid 4 Anaerobes 4 8 16 Staphylococci 4-8 Other aerobes 8 16 32 Tetracycline 4 8 16 Ciprofloxacin 1 2 4 Erythromycin 0.5 1-4 8 Clindamycin Anaerobes 2 4 8 Aerobes 0.5 1-2 4 Cefuroxime axetil 1 2 4 *Amoxicillin is considered to have an MIC similar to ampicillin. (71.7%) ³ ƒ, r G 63(68.5%) ³ ƒ, p 61(66.3%) ³ ƒ, 41(44.6%) ³ ƒ. w v 29(31.5%) ³ (Table 4). š e ³ w 2-13 ³ š (10, 13, 23). v 1-12 ³ ƒ. w 27 e e ³ w s³ 3.7 ³. x ¾ ³» ü ³ w» š, v wù ƒ. w ³ w 16S rdna libraries» w e ³ w š (16, 29). e ³ 16S rdna libraries» ww z w ³ w ³. w, 16S rdna libraries» w ³ ³ w» w. w, 16S rdna libraries» ³ w, ù ³ ³ p w ³ w. e w ³ 8 w w w» w ³ w e ³ w.
188 Sang-Soo Lim et al. Kor. J. Microbiol Table 2. The bacterial strains isolated in this study Sample' No. Clinical isolates 81 5 ChDC B631*, ChDC B632*, ChDC B633*, ChDC B634*, ChDC B635* 82 2 ChDC B636*, ChDC B637 84 1 ChDC B638* 85 2 ChDC B639*, ChDC B640* 86 4 ChDC B664, ChDC B665, ChDC B666, ChDC B667 88 2 ChDC B641, ChDC B642 89 3 ChDC B643*, ChDC B644*, ChDC B645* 90 1 ChDC B646* 93/94 6 ChDC B668*, ChDC B669*, ChDC B670*, ChDC B671*, ChDC B672*, ChDC B673 95 3 ChDC B674*, ChDC B676*, ChDC B677* 97 1 ChDC B709* 99 2 ChDC B678, ChDC B679* 100 2 ChDC B707, ChDC B708 102 2 ChDC B680*, ChDC B681* 103 2 ChDC B682*, ChDC B683* 104 5 ChDC B647*, ChDC B648*, ChDC B649*, ChDC B650*, ChDC B651* 105 6 ChDC B652*, ChDC B653*, ChDC B654*, ChDC B655*, ChDC B656*, ChDC B657* 106 6 ChDC B658*, ChDC B659, ChDC B660, ChDC B661, ChDC B662, ChDC B663 107 7 ChDC B684*, ChDC B685*, ChDC B686*, ChDC B687*, ChDC B710, ChDC B711, ChDC B712 112 7 ChDC B688, ChDC B689, ChDC B690, ChDC B691, ChDC B692, ChDC B693, ChDC B694 114 4 ChDC B713, ChDC B714, ChDC B715, ChDC B716 115 ChDC B695, ChDC B696, ChDC B697, ChDC B698*, ChDC B699*, ChDC B700*, ChDC B701*, ChDC B702*, ChDC 12 B703*, ChDC B704*, ChDC B705*, ChDC B706* 117 2 ChDC B717, ChDC B718 119 2 ChDC B735, ChDC B736 120 4 ChDC B737, ChDC B741, ChDC B743, ChDC B744 123 4 ChDC B749, ChDC B750 ChDC B751, ChDC B752 180 4 ChDC B730, ChDC B731, ChDC B732, ChDC B733 Total 101 ChDC; Department of Oral Biochemistry, College of Dentistry, Chosun University *Those strains were isolated in a previous study. (14) ChDC B631: This strain doesn't exist at present. 27 e v Lee (14) w 17 v sw., -70 o C þ š v(15% glycerol ) w k ³ w w 6 v 14 ³ w œw (Table 1). w Blastn ww ³ w. ³ 16S rdna» mw ³, t ³ 16S rdna», Lee (14) w e g». p 16S rdna» y w ƒ ƒ¾»w š, ƒ»w (Table 3). ³(viridans streptococci) 16S rdna» 5 (mitis, salivarius, anginosus, mutans, bovis) ù ƒ(9), 6 p w p mitis, mutans, anginosus, sanguinis, salivarius ù (4). 16S rdna» ³ w» Kawamura (9) w ³ w. Kawamura (9) š w, mitis ³, S. mitis, S. pneumoniae, S. oralis, S. gordonii 16S rdna» 99%» w»ƒ š w. w salivarius w S. salivarius, S. vestibularis S. thermophilus, anginosus w S. intermedia S. constellatus. mitis ³, salivarius ³ y S. intermedia / constellatus š» w. 8 w w w, w ƒ w ü ³ 4ƒ w ü ³. w, w ü ³ ³ ƒ., 86, 115, 180 v ³ ³ ƒ, w ü (Table 4). w y ƒ w û w ù w w» w
Vol. 42, No. 3 ³ w 189 Table 3. Summary of detection frequency of bacteria from endodontic infection lesions Phylum/ Class Genus or species Detection frequency Case (total = 27) Strain (total = 101) n % n % Actionobacteria Actinomyces georgiae 1 3.7 1 1.0 Actinomyces israelii 1 3.7 1 1.0 Actinomyces naeslundii / viscosus 7 25.9 10 9.9 Actinomyces odontolyticus 3 11.1 8 7.9 Actinomyces sp. 1 3.7 2 2.0 Atopobium parvulum 1 3.7 1 1.0 Propionibacterium acnes 2 7.4 3 3.0 Propionibacterium cyclohexanicum / freudenreichii 1 3.7 1 1.0 Propionibacterium propionicus / avidum 1 3.7 3 3.0 Firmicutes/ Bacilli Abiotrophia para-adiacens 2 7.4 2 2.0 Bifidobacterium dentium 1 3.7 1 1.0 Granulicatella adiacens 1 3.7 2 2.0 Lactobacillus frumenti 1 3.7 1 1.0 Lactobacillus mucosae 1 3.7 1 1.0 Lactobacillus sp. 2 7.4 4 4.0 Lactobacillus vaginalis 1 3.7 1 1.0 Staphylococcus aureus 1 3.7 2 2.0 Staphylococcus epidermidis 3 11.1 6 5.9 Staphylococcus pasteuri / warneri 1 3.7 1 1.0 Staphylococcus sp. 2 7.4 2 2.0 Streptococcus anginosus 5 18.5 6 5.9 Streptococcus constellatus / intermedius 2 7.4 3 3.0 Streptococcus cristatus 1 3.7 1 1.0 Streptococcus gordonii 1 3.7 1 1.0 Streptococcus sp. (mitis group) 7 25.9 16 15.8 Streptococcus sp. (salivarius group) 1 3.7 3 3.0 Firmicutes/ Clostridia Clostridium sporogenes / botulinum 1 3.7 1 1.0 Dialister invisus 1 3.7 1 1.0 Eubacterium infirmum 1 3.7 1 1.0 Finegoldia magna 1 3.7 1 1.0 Micromonas micros 1 3.7 1 1.0 Shuttleworthia satelles 1 3.7 1 1.0 Proteobacteria Methylobacterium fujisawaense 1 3.7 1 1.0 Pseudomonas aeruginosa 1 3.7 2 2.0 Fusobacteria Fusobacterium nucleatum 1 3.7 1 1.0 Bacteroides Porphyromonas asaccharolytica 1 3.7 2 2.0 Prevotella buccae 1 3.7 1 1.0 Prevotella denticola 1 3.7 4 4.0 Prevotella nigrescens 1 3.7 1 1.0 Total - - 101 100.4 w ƒ w w ww x w ƒ. r w β-lactam ƒ š, ³ s peptidoglycan w w transpeptidases y w w ³ (26). w ü 92 ³ 29 ³ ƒ r G ü w, 51 ³ ƒ ü. w, r Gƒ x š» w ü ³ ƒ ùkù ƒ. ƒƒ ³ ƒ β-lactam r w w ü» ƒ» ƒ w
190 Sang-Soo Lim et al. Kor. J. Microbiol Table 4. Minimal inhibitory concentration of several antibiotics for species isolated from the endodontic infection Sample' MIC (µg/ml) Strains No. PEN 1 AMX 2 AUG 3 TET 4 CIP 5 ERY 6 CLI 7 CMX 8 107 Abiotrophia para-adiacens ChDC B692 0.25 0.25 0.25 8 4 > 32 > 32 0.25 81 Actinomyces georgiaae ChDC B633 ND 9 - ND ND ND ND ND ND 81 Actinomyces israelii ChDC B632 ND - ND ND ND ND ND ND 104 Actinomyces naeslundii / viscosus ChDC B649 0.125 0.25 0.25 0.125 4 0.125 0.125 0.25 85 Actinomyces naeslundii /viscosus ChDC B639 0.125 0.5 0.25 0.125 4 0.125 0.125 0.25 88 Actinomyces naeslundii / viscosus ChDC B642 0.125 0.5 0.25 0.125 2 0.5 0.125 0.25 89 Actinomyces naeslundii / viscosus ChDC B644 0.25 0.25 0.25 0.5 8 0.125 0.125 0.25 89 Actinomyces naeslundii /viscosus ChDC B645 0.25 0.5 0.25 0.5 8 0.125 0.5 0.25 104 Actinomyces naeslundii / viscosus ChDC B650 0.25 0.25 0.25 0.125 4 0.125 0.125 0.25 107 Actinomyces naeslundii / viscosus ChDC B690 0.5 1 0.25 1 8 0.125 0.125 0.25 107 Actinomyces naeslundii / viscosus ChDC B694 0.25 0.5 0.25 4 4 > 32 > 32 0.25 120 Actinomyces naeslundii / viscosus ChDC B735 0.125 0.25 0.25 1 8 0.125 0.5 0.25 120 Actinomyces naeslundii /viscosus ChDC B736 0.25 0.25 0.25 0.125 4 0.125 0.25 0.25 90 Actinomyces odontolyticus ChDC B646 0.5 2 0.5 4 16 0.125 0.125 0.25 104 Actinomyces odontolyticus ChDC B651 0.125 0.5 0.25 0.125 8 0.125 0.125 0.25 106 Actinomyces odontolyticus ChDC B658 0.125 2 0.5 0.125 8 0.125 0.125 0.25 106 Actinomyces odontolyticus ChDC B659 0.125 1 0.25 0.125 4 0.125 0.125 0.25 106 Actinomyces odontolyticus ChDC B660 0.125 0.25 4 0.125 8 0.125 0.125 0.25 106 Actinomyces odontolyticus ChDC B661 0.125 1 0.25 0.125 8 0.125 0.125 0.25 106 Actinomyces odontolyticus ChDC B662 0.125 1 0.25 0.125 4 0.125 0.125 0.25 106 Actinomyces odontolyticus ChDC B663 0.125 1 0.25 0.125 4 0.125 0.125 0.25 81 Actinomyces sp. ChDC B631 0.125 1 0.5 0.25 2 1 0.125 1 81 Actinomyces sp. ChDC B634 ND - ND ND ND ND ND ND 115 Atopobium parvulum ChDC B703 > 32 2 32 0.125 1 2 0.125 4 97 Clostridium sporogenes / botulinum ChDC B709 1 8 2 0.125 0.25 0.5 8 8 120 Dialister invisus ChDC B744 ND 9 - ND ND ND ND ND ND 115 Eubacterium infirmum ChDC B706 > 32 64 64 32 0.5 32 8 64 105 Finegoldia magna ChDC B654 32 64 64 32 0.5 32 16 64 115 Fusobacterium nucleatum ChDC B705 0.125 0.25 2 0.125 1 8 0.125 2 107 Granulicatella adiacens ChDC B711 0.125 0.25 0.25 0.125 4 0.125 0.125 0.25 107 Granulicatella adiacens ChDC B712 0.125 2 0.5 32 4 0.125 0.125 0.5 123 Lactobacillus frumenti ChDC B750 ND - ND ND ND ND ND ND 120 Lactobacillus mucosae ChDC B743 0.125 0.25 4 0.25 16 2 0.125 16 103 Lactobacillus sp. ChDC B682 > 32 64 16 8 > 32 0.25 0.25 > 64 103 Lactobacillus sp. ChDC B683 2 8 2 0.25 8 0.125 0.25 4 123 Lactobacillus sp. ChDC B749 1 8 2 0.25 4 0.125 0.125 4 123 Lactobacillus sp. ChDC B752 > 32 32 8 8 32 0.125 0.125 > 64 123 Lactobacillus vaginalis ChDC B751 ND - ND ND ND ND ND ND 81 Methylobacterium fujisawaense ChDC B635 0.125 0.25 0.25 0.125 8 0.125 0.125 0.25 100 Micromonas micros ChDC B708 ND - ND ND ND ND ND ND 105 Porphyromonas asaccharolytica ChDC B656 32 64 64 32 1 32 > 32 64 105 Porphyromonas asaccharolytica ChDC B657 32 64 64 32 1 32 > 32 64 115 Prevotella buccae ChDC B697 0.125 0.25 1 0.125 0.5 0.125 0.125 0.25 115 Prevotella denticola ChDC B696 32 8 16 0.125 1 2 0.125 16 115 Prevotella denticola ChDC B698 > 32 4 8 0.125 1 2 0.125 8 115 Prevotella denticola ChDC B702 > 32 2 32 0.125 1 0.5 0.125 4 115 Prevotella denticola ChDC B704 > 32 2 16 0.125 1 2 0.125 4 120 Prevotella nigrescens ChDC B737 32 8 32 0.5 0.25 0.125 0.125 16 1, Penicillin G; 2, Amoxicillin; 3, Augmentin; 4, Tetracycline; 5, Ciprofloxacin; 6, Erythromycin; 7, Clindamycin; 8, Cefuroxime axetil; 9, Not determined.
Vol. 42, No. 3 ³ w 191 Table 4. Continued Sample' MIC (µg/ml) Strains No. PEN 1 AMX 2 AUG 3 TET 4 CIP 5 ERY 6 CLI 7 CMX 8 97 Clostridium sporogenes / botulinum ChDC B709 1 8 2 0.125 0.25 0.5 8 8 120 Dialister invisus ChDC B744 ND 9 - ND ND ND ND ND ND 115 Eubacterium infirmum ChDC B706 > 32 64 64 32 0.5 32 8 64 105 Finegoldia magna ChDC B654 32 64 64 32 0.5 32 16 64 115 Fusobacterium nucleatum ChDC B705 0.125 0.25 2 0.125 1 8 0.125 2 107 Granulicatella adiacens ChDC B711 0.125 0.25 0.25 0.125 4 0.125 0.125 0.25 107 Granulicatella adiacens ChDC B712 0.125 2 0.5 32 4 0.125 0.125 0.5 123 Lactobacillus frumenti ChDC B750 ND - ND ND ND ND ND ND 120 Lactobacillus mucosae ChDC B743 0.125 0.25 4 0.25 16 2 0.125 16 103 Lactobacillus sp. ChDC B682 > 32 64 16 8 > 32 0.25 0.25 > 64 103 Lactobacillus sp. ChDC B683 2 8 2 0.25 8 0.125 0.25 4 123 Lactobacillus sp. ChDC B749 1 8 2 0.25 4 0.125 0.125 4 123 Lactobacillus sp. ChDC B752 > 32 32 8 8 32 0.125 0.125 > 64 123 Lactobacillus vaginalis ChDC B751 ND - ND ND ND ND ND ND 81 Methylobacterium fujisawaense ChDC B635 0.125 0.25 0.25 0.125 8 0.125 0.125 0.25 100 Micromonas micros ChDC B708 ND - ND ND ND ND ND ND 105 Porphyromonas asaccharolytica ChDC B656 32 64 64 32 1 32 > 32 64 105 Porphyromonas asaccharolytica ChDC B657 32 64 64 32 1 32 > 32 64 115 Prevotella buccae ChDC B697 0.125 0.25 1 0.125 0.5 0.125 0.125 0.25 115 Prevotella denticola ChDC B696 32 8 16 0.125 1 2 0.125 16 115 Prevotella denticola ChDC B698 > 32 4 8 0.125 1 2 0.125 8 115 Prevotella denticola ChDC B702 > 32 2 32 0.125 1 0.5 0.125 4 115 Prevotella denticola ChDC B704 > 32 2 16 0.125 1 2 0.125 4 120 Prevotella nigrescens ChDC B737 32 8 32 0.5 0.25 0.125 0.125 16 114 Propionibacterium acnes ChDC B715 0.125 0.5 0.25 0.25 4 0.125 0.125 0.25 114 Propionibacterium acnes ChDC B716 0.125 0.25 0.25 1 2 0.125 0.125 0.25 104 Propionibacterium cyclohexanicum / freudenreichii ChDC B647 ND 9 - ND ND ND ND ND ND 105 Propionibacterium propionicus / avidum ChDC B655 0.125 0.25 0.25 0.125 0.25 0.125 0.125 0.25 105 Propionibacterium propionicus / avidum ChDC B652 0.125 0.5 0.25 0.125 2 0.125 0.125 0.25 105 Propionibacterium propionicus / avidum ChDC B653 0.125 0.25 1 0.125 0.25 0.125 0.125 0.25 82 Pseudomonas aeruginosa ChDC B636 > 32 > 64 > 64 32 1 > 32 > 32 > 64 82 Pseudomonas aeruginosa ChDC B637 > 32 > 64 > 64 16 0.125 > 32 > 32 > 64 115 Shuttleworthia satelles ChDC B700 ND - ND ND ND ND ND ND 86 Staphylococcus aureus ChDC B664 > 32 > 64 > 64 0.125 1 > 32 0.125 1 86 Staphylococcus aureus ChDC B665 > 32 > 64 > 64 0.125 1 > 32 0.25 1 114 Staphylococcus epidermidis ChDC B714 > 32 > 64 > 64 0.25 1 1 0.25 0.5 180 Staphylococcus epidermidis ChDC B730 > 32 > 64 > 64 32 2 0.5 0.5 2 180 Staphylococcus epidermidis ChDC B731 > 32 > 64 > 64 1 2 1 0.5 1 180 Staphylococcus epidermidis ChDC B732 > 32 > 64 > 64 1 1 1 0.25 1 180 Staphylococcus epidermidis ChDC B733 > 32 > 64 > 64 1 2 1 0.25 2 120 Staphylococcus epidermidis ChDC B741 > 32 > 64 64 0.125 0.5 0.5 0.125 0.5 114 Staphylococcus pasteuri / warneri ChDC B713 0.125 1 0.5 0.25 1 1 0.125 1 88 Staphylococcus sp. ChDC B641 > 32 > 64 > 64 > 32 1 0.5 0.25 0.25 89 Staphylococcus sp. ChDC B643 0.125 0.5 0.25 8 1 1 0.125 0.5 85 Streptococcus anginosus ChDC B640 0.125 0.25 0.25 0.125 2 0.125 0.125 0.25 107 Streptococcus anginosus ChDC B684 0.125 0.25 0.25 16 4 0.125 0.125 0.25 112 Streptococcus anginosus ChDC B688 0.125 0.5 0.25 4 2 0.125 0.125 0.25 115 Streptococcus anginosus ChDC B695 0.125 0.25 0.25 0.125 2 0.125 0.125 0.25
192 Sang-Soo Lim et al. Kor. J. Microbiol Table 4. Continued Sample' MIC (µg/ml) Strains No. PEN 1 AMX 2 AUG 3 TET 4 CIP 5 ERY 6 CLI 7 CMX 8 107 Streptococcus anginosus ChDC B710 0.125 0.25 4 32 2 0.25 0.25 0.5 100 Streptococcus constellatus / intermedius ChDC B707 0.125 1 0.25 0.125 2 0.125 0.125 0.25 117 Streptococcus constellatus / intermedius ChDC B718 0.125 0.25 2 8 1 0.125 0.125 0.25 117 Streptococcus constellatus / intermedius ChDC B717 0.125 1 0.5 8 1 0.125 0.125 0.25 107 Streptococcus cristatus ChDC B687 0.25 2 0.5 0.125 4 0.125 0.125 0.25 99 Streptococcus gordonii ChDC B679 0.125 0.25 0.25 8 16 > 32 > 32 0.25 104 Streptococcus sp. ChDC B648 (mitis group) 0.25 1 0.25 4 4 2 0.125 0.25 86 Streptococcus sp. ChDC B666 (mitis group) 0.125 0.25 0.25 8 8 2 0.125 0.25 93/94 Streptococcus sp. ChDC B669 (mitis group) > 32 > 64 > 64 > 32 2 > 32 0.25 0.5 93/94 Streptococcus sp. ChDC B670 (mitis group) 0.125 2 0.5 0.125 4 0.125 0.125 0.25 93/94 Streptococcus sp. ChDC B671 (mitis group) 0.5 2 0.5 8 8 0.125 0.125 0.25 95 Streptococcus sp. ChDC B674 (mitis group) 0.125 0.25 0.25 0.125 4 0.125 0.125 0.25 95 Streptococcus sp. ChDC B676 (mitis group) 0.125 0.5 0.25 8 8 2 0.125 0.25 95 Streptococcus sp. ChDC B677 (mitis group) 0.125 0.25 0.25 1 8 0.125 0.125 0.25 99 Streptococcus sp. ChDC B678 (mitis group) 0.25 8 1 0.25 16 0.125 0.125 1 102 Streptococcus sp. ChDC B680 (mitis group) 1 2 0.5 4 8 4 32 0.25 102 Streptococcus sp. ChDC B681 (mitis group) 0.5 2 0.5 0.125 8 0.125 0.125 0.25 107 Streptococcus sp. ChDC B685 (mitis group) 0.5 4 1 0.125 4 0.125 0.125 2 107 Streptococcus sp. ChDC B686 (mitis group) 0.125 0.25 0.25 4 8 0.125 0.125 0.25 107 Streptococcus sp. ChDC B689 (mitis group) 0.125 0.25 0.25 0.5 4 0.125 0.125 0.25 107 Streptococcus sp. ChDC B691 (mitis group) 0.25 0.5 0.25 0.25 8 0.125 0.125 0.5 107 Streptococcus sp. ChDC B693 (mitis group) 0.125 0.5 0.25 8 8 8 16 0.5 93/94 Streptococcus sp. ChDC B668 (salivarius group) 0.25 1 0.25 0.125 2 0.125 0.125 0.25 93/94 Streptococcus sp. ChDC B672 (salivarius group) 0.5 2 1 0.125 4 0.125 0.125 0.25 93/95 Streptococcus sp. ChDC B673 (salivarius group) 0.5 4 1 0.25 8 0.25 0.125 0.25 1, Penicillin G; 2, Amoxicillin; 3, Augmentin; 4, Tetracycline; 5, Ciprofloxacin; 6, Erythromycin; 7, Clindamycin; 8, Cefuroxime axetil; 9, Not determined. ƒ., r w w ü» w transpeptidase r w ù ƒ transpeptidase w, β-lactamase w β-lactam q g w z, ³ s w w n j ù, periplasm l w s rü y (27) w» ³» ƒ š ÿ z ƒ ƒ. s ³, ü ³(P. aeruginosa, Lactobacillus sp., E. infirmum) w ³ r w w ü š. w ü ³, r w β-lactam q w ³» r w clavulanate, tazobactam, sulbactam β-lactamase n w š (25). x p t clavulanic yww ƒ š. r ù ü ³ P. denticola ChDC B698 w ³ p ü. w β-lactamase w ü ³ w β-lactamase w w w z (25). wz r w ü ³ ü» w wwš w. p j r w»ƒ ù ü w. p j (macrolides) w š, j g (licosamids) w. w w w³. r w ü ³ p j ü ³ (ChDC B636, ChDC B637, ChDC B654, ChDC B656, ChDC B707) w. Jenssen (6) ³ ermb 23S rrna py j, g y type B streptogramins w ƒ w w w ü» š šw. p j ü ³ ü» w ermb wz w wš w. v p 2 q s w,
Vol. 42, No. 3 ³ w 193 β-lactam w. r w w³ ƒ r, ³, Pseudomonas ü ³ z ƒ (15). v p w ü ³ P. aeruginosa, Clostridium sp., P. nigrescens, Lactobacillus sp.. v i (fluoroquinolones) w v p - w (methicillin-resistant) S. aureus (MRSA) x» ³ w w³ w, y» ³ w w³ ù (5, 3). v ƒ ü ³ ƒ, q s w v p w ³ v. e y x» ³ y» ³ wš, r w ü ³ ƒ w q s w v i w w w z ƒ. r, p j ü ³ v p ù lp j. lp j ³ 30S ww aminoacyl transfer ribonucleic acidƒ 50S A-site w w w w» Ÿ w. w, lp j ³ š w w» w ³ ü z w y. w, e ³ lp j w (69%). p, r w ü s ³ ³ (ChDC B641, ChDC B730) wš z w³. x lp j w ü š (11, 24). wz w w lp j ü ³ ü» wš w. ww, 27 e y ³ ³ ³ ƒ, w 8 w w w, p w ü w ü ƒ, w ü ³. e w n w v ƒ w w ww v w ƒ. : w BK21. š x 1. Andreasen, F.M. 1986. Transient apical breakdown and its relation to color and sensibility changes after luxation injuries to teeth. Endod. Dent. Traumatol. 2, 9-19. 2. Bergenholtz, G. 1974. Micro-organisms from necrotic pulp of traumatized teeth. Odontol. Revy. 25, 347-358. 3. Cohen, M.A., J.M. Embil, and T. Canosa. 2003. Osteomyelitis of the maxilla caused by methicillin-resistant Staphylococcus aureus. J. Oral. Maxillofac. Surg. 61, 387-390. 4. Facklam, R. 2002. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin. Microbiol. Rev. 15, 613-630. 5. Grossman, R.F. 1997. The role of fluoroquinolones in respiratory tract infections. J. Antimicrob. Chemother. 40, 59-62. 6. Jenssen, W.D., S. Thakker-Varia, D.T. Dubin, and M.P. Weinstein. 1987. Prevalence of macrolides-lincosamides-streptogramin B resistance and erm gene classes among clinical strains of staphylococci and streptococci. Antimicrob. Agents Chemother. 31, 883-888. 7. Kakehashi, S., H.R. Stanley, and R.I. Fitzgerald. 1965. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg. Oral Med. Oral Pathol. 20, 340-349. 8. Kantz, W.E. and C.A. Henry. 1974. Isolation and classification of anaerobic bacteria from intact pulp chambers of non-vital teeth in man. Arch. Oral Biol. 19, 91-96. 9. Kawamura, Y., X.G. Hou, F. Sultana, H. Miura, and T. Ezaki. 1995. Determination of 16S rrna sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int. J. Syst. Bacteriol. 45, 406-408. Erratum in: Int. J. Syst. Bacteriol. 1995, 45, 882. 10. Khemaleelakul, S., J.C. Baumgartner, and S. Pruksakorn. 2002. Identification of bacteria in acute endodontic infections and their antimicrobial susceptibility. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 94, 746-755. 11. Lacroix, J.M. and C.B. Walker. 1995. Detection and incidence of tetracycline resistance determinant tet (M) in the microflora associated with adult periodontitis. J. Periodontol. 66, 102-108. 12. Lane, D.J., B. Pace, G.J. Olsen, D.A. Stahl, M.L. Sogin, and N.R. Pace. 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82, 6955-6959. 13. Le Goff, A., L. Bunetel, C. Mouton, and M. Bonnaure-Mallet. 1997. Evaluation of root canal bacteria and their antimicrobial susceptibility in teeth with necrotic pulp. Oral Microbiol. Immunol. 12, 318-322. 14. Lee, Y.-J, M.-K. Kim, H.-K. Hwang, and J.-K. Kook. 2005. Isolation and identification of bacteria from the root canal of the teeth diagnosed as the acute pulpitis and acute periapical abscess. J. Kor. Acad. Cons. Dent. 30, 409-422. 15. Mandel, G.L. and M.A. Sande. 1992. Antimicrobial agents. p. 1085. In Gilman AF, Rall TW, Nies AS, Taylor P. (ed.), The pharmacological basis of therapeutics. Vol. 2, 1st ed. McGraw-Hill, INC. Singapore. 16. Munson, M.A., T. Pitt-Ford, B. Chong, A. Weightman, and W.G. Wade. 2002. Molecular and cultural analysis of the microflora associated with endodontic infections. J. Dent. Res. 81, 761-766. Erratum in: J. Dent. Res. 2003, 82, 69. J. Dent. Res. 2003, 82, 247. 17. Murray, P.R. and J.H. Jorgensen. 1981. Quantitative susceptibility test methods in major united states medical center. Antimicrob. Agents Chemother. 20, 66-70. 18. Nair, P.N. 2004. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit. Rev. Oral Biol. Med. 15, 348-381. 19. National Committee for Clinical Laboratory standards. 2000. Method for dilution antimicrobial susceptibility testing of bacteria
194 Sang-Soo Lim et al. Kor. J. Microbiol that grow aerobically, 5th ed. Approved standard M7-A5. National Committee for Clinical Laboratory standards, Wayne, Pa. 20. National Committee for Clinical Laboratory standards. 2001. Methods for antimicrobial susceptibility testing of anaerobic bacteria. 5th ed. Approved standard M11-A5. National Committee for Clinical Laboratory standards, Wayne, Pa. 21. Paster, B.J., S.K. Boches, J.L. Galvin, R.E. Ericson, C.N. Lau, V.A. Levanos, A. Sahasrabudhe, and F.E. Dewhirst. 2001. Bacterial diversity in human subgingival plaque. J. Bacteriol. 183, 3770-3783. 22. Solomon, C., H. Chalfin, M. Kellert, and P. Weseley. 1995. The endodontic- periodontal lesion: a rational approach to treatment. J. Am. Dent. Assoc. 126, 473-479. 23. Sundqvist, G. 1994. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg. Oral Med. Oral Pathol. 78, 522-530. 24. Villedieu, A., M.L. Diaz-Torres, N. Hunt, R. McNab, D.A. Spratt, M. Wilson, and P. Mullany. 2003. Prevalence of tetracycline resistance genes in oral bacteria. Antimicrob. Agents Chemother. 47, 878-882. 25. Wang, X., G. Minasov, and B.K. Shoichet. 2002. The structural bases of antibiotic resistance in the clinically derived mutant betalactamases TEM-30, TEM-32, and TEM-34. J. Biol. Chem. 277, 32149-32156. 26. Waxman, D.J. and J.L. Strominger. 1983. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu. Rev. Biochem. 52, 825-869. 27. Wilke, M.S., A.L. Lovering, and N.C. Strynadka. 2005. Beta-lactam antibiotic resistance: a current structural perspective. Curr. Opin. Microbiol. 8, 525-533. 28. Wittgow Jr., W.C. and C.B. Sabiston Jr. 1975. Microorganisms from pulpal chambers of intact teeth with necrotic pulps. J. Endod. 1, 168-171. 29. Yoo, S.Y., M.-K. Kim, H.-S. Kim, H.-K. Hwang, P.-S. Kim, S.-Y. Lim, S.-H. OH, J.-B. Min, and J.-K. Kook. 2004. Identification of bacteria from periapical abscess using 16S rdna clone libraries. Kor. J. Microbiol. Biotechnol. 32, 195-198. (Received March 24, 2006/Accepted September 14, 2006) ABSTRACT: Antibiotic Susceptibility of Bacteria Isolated from Infected Root Canals Sang-Soo Lim, Mi-Kwang Kim 1, Jeong Beom Min, Min Jung Kim 1, Soon-Nang Park 1, Ho-Keel Hwang, Joong-Ki Kook 1 * (Department of Conservative Dentistry and 1 Department of Oral Biochemistry, College of Dentistry, Chosun University, Gwangju 501-759, Korea) The aim of this study was to identify the bacteria isolated from endodontic lesions by cell culture and to determine the antimicrobial susceptibility of them against 8 antibiotics. The necrotic pulpal tissues were collected from 27 infected root canals, which were diagnosed as endodontic infection. Samples were collected aseptically from the infected pulpal tissue of the infected root canals using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 µl of 1 PBS. The sample solution was briefly mixed and plated onto a BHI-agar plate containing 5% sheep blood. The agar plates were incubated in a 37 o C anaerobic chamber for 2 to 5 days. The bacteria grown on the agar plates were identified by comparison of 16S rrna gene (rdna) sequencing method at the species level. To test the sensitivity of the bacteria isolated from the infected root canals against 8 antibiotics, minimum inhibitory concentrations (MIC) were determined using broth dilution assay. The data showed that 101 bacterial strains were isolated and were identified. Streptococcus spp. (29.7%) and Actinomyces spp. (21.8%) were predominantly isolated. The 9 strains were excluded in antimicrobial susceptibility test because they were lost during the experiment or were not grown in broth culture. The percentage of bacteria susceptible for each antibiotic in this study was clindamycin, 87.0% (80 of 92); tetracycline, 75.0% (69 of 92); cefuroxime axetil, 75.0% (69 of 92); amoxicillin + clavulanic acid (5:1), 71.7% (66 of 92); penicillin G, 66.3% (61 of 92); erythromycin, 66.3% (61 of 92); amoxicillin, 44.6% (41 of 92); and ciprofloxacin, 31.5% (29 of 92). The susceptibility pattern of 8 antibiotics was dependent on the host of the bacteria strains rather than the kinds of bacterial species. These results indicate that antibiotic susceptibility test should be performed when antibiotics are needed for the treatment of infected root canals.