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2 Table 1 Bacteroides strains * The isolates were tested f or their ability to transfer clindamycin resistance to the sensitive recipient, B. fragilis The limit of detectable transfer was 10-8 resistant transconjugants per recipient eclls; (+) indicates that the isolate was able to transfer resistance and (-) indicates that the isolate was uable to transfer resistance.
3 VOL.34 C H E M O T H E R A P Y NO.5 Probe DNAと して た8) plasmid はplasmid pmjs100を pmjs100を 有 す るEscherichia RVSmc/100-2は,F.P.TALLYよ り 恵 pmjs100はchimeric coli さ れ た 1B DNAの EtBr超 標 識 and HELINSKIの translation Nuclear. 1,pMJS100 し た Boston)を hybridizationは,cold ratoryのmanuatに 用 い て 行 な っ (0.45μm Spring filterは,membrane φ 82mm,Schloicher & を用 い た 被 験 菌 の 純 培 養 菌 の1 定の 位 置 に2mm 3mm滅 GAM寒 man 天 培 地 上 3MM)上 NaClか 5分 間,最 BA85/20 w Germany) 間 嫌 気 培 養 さ れ た ず10%Sodium 間.次 い で0.5M 後 に1.5M NaCl,0.5M た こ の よ う に 処 理 さ れ た 炉 紙 は,30 60分 間 の 乾 燥 され.次 で2時 mus Na して tetracycline感,陰 4)耐 で65 18時 AR studyに 間 行 な filmを 性2つ お い て は,常 のcontrotsを,pBFTM10を 用 い, 受 性 のB.fragilis GAI-7001を に 一つ の 置 い た 陽 性 有 す るclindamycin耐 trolと し てclindamycinお B.fragilis thydna 間 露 出 さ れ た な お,hybridization controlと Ficol400, geratin,cat 浄 後,Kodak 7日 用 い,0.9M ラ ベ ル し たProbe solution中 われ た filterは,洗 され た,0.2% pで か ら な るhybridization filter上 に 陽 性 bagを,0.02% DNA25mg/ml,32 で2日 使 用 meat citrate(6 SSC) Polyvinylpyrrolidone -80 studyに,plastic NaCl,0.09M 0.2% 間 真 空 オ ー ブ ン 中 でbaking のhybridization hybridizationは GAI-6064 よ びtetracyctineに 用 い た,陰 性 性con感 受 性 の 性 伝 達 実 験 一部 の 菌 株 に つ い て,clindamycineとtetracycline の耐性伝 達 能 の有 無 を 調 査 耐性 の伝 達 は,前 報 に 示 probeの 示 し た clindamycin耐 性 の菌 株 は 対 して 強 いhomologyを 示 した が, 受 性 の 菌 株 は 全 くhomologyを に 耐 性(100μg/ml以 京)の10施 示 さなか 受 性 の 菌 株 の 中 にerythromycin 上)の 株 が,3株 のhomologyは homology 玉,東 ら れ た 特 異性 を検 討 した probeに clindamycin感 して 用 い,得 存在 し た が, 認 め ら れ な か っ た study 阜,長 崎,福 岡,愛 設 由 来 のBacteroides42株 媛,富 中 Tris Cl(pH8.0) 間 置 か れ 後.80 性controlと 受 性 のGAI-7001を pmjs100 山,埼 してclindamycin耐 有 す るGAI-6064,陰 わ が 国 の 異 な る 地 域(岐 か ら な る 中 和 用 溶 液 で 飽 和 さ れ た 炉 紙 上 に5分 (室温)の よび 2.DNA NaOH,1.5M 3 受 性 で に 陽 性controlと pmjs100と 性 用 溶 液 で 飽 和 さ れ た 炉 紙 上 で 性 のB.fragilis 10015).更 っ た clindamycin感 液 で 飽 和 さ れ た 炉 紙(What- で3分 特異性 よ び6068)とclindamycin感 成 績 はFig.1に の 所 の 接 種 面 を 上 に し て, のfilterは,ま ら な るDNA変 め られ な か った もの を陰 性 の6株(GAI-7034,7100,7154,8139,10001お pmjs100 filter 菌 つ ま よ う じ を 用 い て 画 線 sulfonate(sds)溶 株(GAI-6286,6346お clindamycin感 Schul, 天 平 板 上 に 密 着 さ せ,18時 dedoxy cellsで,伝 績 probeの pmftm10を Labo- colonyをfilter上 した 菌 を 接 種 し たfilterは,菌 GAM寒 Harbor 準 じ て 行 な っ た12) Nitrocellulose donor 供 試 菌 株 の 中 か らclindamycin耐 system[32p] hybridization Colony ブ イ ヨ ン で 前 処 理 した II.成 方 法 に た17) 3)Cotony μg/mlのtetracycline加 GAI- の 交 配 に 先 立 ら1 達 が 認 め られ た も の を 陽 性.認 fragmentが 遠 心 分 離 法 に よ っ て 精 製 は,Nick England して は,B.fragilis 用 い,donorは,recipientと 性 と した DNAは,CLEWELL 準 じて,CsCl で 行 な わ れ た12) Recipientと 7001を 耐 性 伝 達 の 検 出 限 界 は,10-8/input れ た も の で あ る13) pmjs100 (New 与 い plasmidで.e.coliのplasmid pmc1403にpbftm10のecor cloningさ 用 395 し た し た よ う にfilter mating法 Fig. 1 32P labelled pmjs 100 was used as a probe. 1, 2, 3: clnr, eryr, tetr strains; GAI-6068 (= TMP 10), GAI-6286, GAI-6346, 3, 5, 6: clns, eryr, tetr strains; GAI-7034, GAI-7154, GAI7100, 7, 8, 9: clns, erys, tets strains; GAI-10001, GAI-8139, GAI-10015,+: positive control which contains pbftm 10; GAI-6064 (=TM 4003),-: negative control; GAI-7001 (=JC101)
4 396 CHEMOTHERAPY MAY 1986 Fig. 2 Colony hybridization with the pmjs 100 probe. The clnr; tetr B. fragilis strains tested were as followed : 1, GAI-6225; 2, GAI-6235; 3, GAI-6250; 4, GAI-6281; 5, GAI-6286; 6, GAI-6346; 7, GAI-6354; 8, GAI-6355; 9, GAI- 6357; 10, GAI-6359; 11, GAI-6423; 12, GAI- 6425; 13, GAI-7001 (=JC-101, negative control); 14, GAI-6064 (=TM 4003, positive control containing pbftm 10) 1) SALAKI, J. S.; R. BLACK, F. P. TALLY & J. W. KISLAK: Bacteroides fragilis resistant to clin damycin. Am. J. Med. 60:426 `428, ) PRIVITERA, G.; A. DUBLANCHET & M. SEBALD: Transfer of multiple antibiotic resistance be tween subspecies of Bacteroides fragilis. J Infect. Dis. 139: 97 `101, 1979
5 3) TALLY, F. P.; D. R. SNYDMAN, S. L. GORBACH & M. H. MALAMY: Plasmid mediated transfer resistance to clindamycin and erythromycin in Bacteroides fragilis. J. Infect. Dis. 139: 83 `88, ) WELCH, R. A.; K. R. JONES & F. L. MACRINA: Plasmid-mediated transfer of lincosamide macrolide and tetracycline resistance in Bacteroides. Plasmids 2: 261 `268, ) TALLY, F. P.; D. R. SNYDMAN., M. J. SHIMELL & M. H. MALAMY: Characterization of pbftm 10, a clindamycin-erythromycin resistance transfer factor from Bacteroides fragilis. J. Bacteriol. 151: 686 `691, ) WELCH, R. A. & F. L. MACRINA: Physical characterization of Bacteroides fragilis R plasmid pbf 4. J. Bacteriol. 152: 867 `372, ) SHIMELL, M. J.; C. J. SMITH., F. P. TALLY, F. L. MACRINA & M. H. MALAMY: Hybridization studies reveals homology between pbf 4 and pbftm 10, two clindamycin-erythromycin resistance transfer plasmids of Bacteroides fragilis. J. Bacteriol. 152: 950 `953, ) MARSH, P. K.; M. H. MALAMY., M. J. SHIMEL & F. P. TALLY: Sequence homology of clindamycin resistant determinants in clinical isolates of Bacteroides spp. A AC 23(5): 726 `730, ) GUINEY, D. G. JR.; P. HASEQAWA & C. E. DA- VIS: Homology between clindamycin resistance plasmid in Bacteroides. Plasmids 11: 268 `271, ) GUINEY, D. G. JR.; P. HASEQAWA, D. STALKER & C. E. DAVIS: Genetic analysis of clindamycin resistance in Bacteroides species. J. Infect. Dis. 147: 551 `558, ) SMITH, C. J. & F. L. MACRINA: Large transmissible clindamycin resistance plasmid in Bacteroides ovatus. J. Bacteriol. 158(2): 739 `741, ) MANIATIS, T., et al.: Molecular cloning-a Laboratory manual cold spring Harbor Laboratory Cold Spring Harbor, New York, ) MALAMY, M. H. & F. P. TALLY: Mechanisms of drug-resistance transfer in Bacteroides fragilis. J. A. C. 8(suppl. D): 59 `75, ) MACRINA, F. L.; T. D. MAYS., C. J. SMITH & R. A. WELCH: Non-plasmid associated transfer of antibiotic resistance in Bacteroides. J. of Antimicrobial Chemotherapy 8 (suppl. D): 79 `86, 1981 Bacteroides fragilis Bacteroides thetaiotaomicron clindamycin, erythromycin, josamycin Bacteroides fragilis 17) RIGBY, P. W. J.; M. DIECKMANN, C. RHODES & P. BERG: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113: 237 `251, 1977
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