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1995 Fiscal Year Annual Research Report

薬剤耐性に係わる細胞膜の機能及びそれと連係的に作用する因子の総合的研究

Research Project

Project/Area Number 05304028
Research InstitutionTokai University

Principal Investigator

中江 太治  東海大学, 医学部, 教授 (50102851)

Co-Investigator(Kenkyū-buntansha) 平松 啓一  順天堂大学, 医学部, 教授 (10173262)
後藤 直正  京都薬科大学, 薬学部, 講師 (30121560)
山口 明人  大阪大学, 産業科学研究所, 教授 (60114336)
井上 松久  北里大学, 薬学部, 教授 (10008336)
伊豫部 志津子  群馬大学, 医学部, 助教授 (90008318)
Keywords薬剤 / 耐性 / 細胞膜 / 透過性 / 排出 / 膜蛋白 / 抗生物質 / 消毒薬
Research Abstract

本年度も1993及び1994年度に続いて大きく分けて薬剤のグラム陰性菌の外膜透過性、内膜からの排出、それに係わる薬剤修飾酵素の役割及びブドウ球菌の耐性の問題解決のための研究を行った。1.先ず緑膿菌外膜のポーリンの抗生物質透過に果たす役割を明らかとするためポーリン欠損変異株を作成して薬剤のMICを測定したところ、OprDのカルバペネム透過に果たす役割を除いてほとんどの抗生物質の透過に役立っていないことが明らかとなった。2.このOprDを介してのイミペネム透過性はすでに確認されていることであるが、今回、我々はOprD蛋白がプロテアーゼ活性を有する新しい蛋白であることをモノクローン抗体を用いて明らかとした。3.外膜透過性とβ-ラクタマーゼの連係はβラクタム耐性の成立に重要な鍵を握る。われわれは高度イミペネム耐性臨床分離株ではOprDの欠損とカルバペナマ-ゼもしくはセファロスポリナーゼの発現によるものであることを明らかとした。4.βラクタマーゼの関わりに関してはβラクタマーゼの発現を制御することができれば膜透過性との連係も低下するので都合がよい。Enterobactorなどを用いた実験からβラクタマーゼ阻害剤では耐性化が抑制されることがわかった。5.薬剤の細胞からの排出は多剤同時耐性の重要な因子である。我々は緑膿菌の薬剤排出蛋白をコードするmex遺伝子をクローン化し、この遺伝子の発現が示す耐性様式を調べたところmexオペロンの発現が亢進した菌ではnalB変異型の耐性を示すことを明らかとした。。6.0prM蛋白質はmexオペロンにコードされていることを示した。別の変異であるnfxBによって発現するOprJも緑膿菌の多剤耐性に係わっていることを明らかとした。7.薬剤排出と耐性に関しては現在テトラサイクリン排出系が最も進んでいる。テトラサイクリン系の新しい誘導体であるグリシルサイクリンはテトラサイクリン排出蛋白の基質として確認されず排出の対象外であることがわかった。8. MRSAを含むブドウ球菌の薬剤耐性に関しては消毒剤の排出に係わるqacEが知られているが、これと相同性のある遺伝子が緑膿菌及び大腸菌にも存在することを明らかとした。9.ブドウ球菌の病原因子のロイコシジン及びγ-ヘモリジンはそのサブユニットの一つを有している。LuksとHγll成分のキメラ蛋白を解析することによってロイコシジンとしての活性部位をヘモリジンとしての部位を明らかとすることができた。10. MRSAにおけるメチシリン耐性を規定する外来遺伝子の押入機構の研究を行った。その結果押入部位にはinverted repeat構造が見いだされた。
このように本研究班では細胞膜の係わる薬剤耐性を多角的にそして深く追求することによって多くの成果を得た。紙面の都合で割愛したが、研究者間の菌種、抗体、プラスミド及び情報の交換が盛んに行われ、共同研究の意義は大いに上がった。

  • Research Products

    (38 results)

All Other

All Publications (38 results)

  • [Publications] Asaka, K., Inaba, Y., Tateda-Suzuki, E., Kuwahara-Arai, K., Ito, T., and Hiramatsu, K.: "Evolution and resistance expression of MRSA: evaluation of β-lactam antibiotics against a set of isogenic strains with different types of phenotypic expression" Pharmacol. Therapeutics.62. (in press). (1995)

  • [Publications] Choorit, W., J. Kaneko, K. Muramoto and Y. Kamio: "Existence of a new protein component with the same function as LukF component ofleukocidin or gamma hemolysin and its gene in Staphylococcus aureus P83" FEBS Lett.357. 260-264 (1995)

  • [Publications] Fukuda, H., Hosaka, M., Iyobe, S., Gotoh, N., Nishino, T., and Hirai, K: "NfxC type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa" Antimcrob. Agents Chemother.39. 790-792 (1995)

  • [Publications] Fukuda, Hideyuki., Masaki Hosaka, Shizuko Iyobe, Naomasa Gotoh, Takeshi Nishino, and Keiji Hirai: "The nfxC-type of quinolone-resistance in a clinically isolated Pseudomonas aeruginosa" Antimicrob. Agents Chemother. 39. 790-792 (1995)

  • [Publications] Gotoh, Naomasa., Hideto Tsujimoto, Keith Poole, Jun-ichi Yamagishi and Takeshi Nishino: "The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by the oprK gene in the mexA-mexB-oprK multidrug resistance operon. Antimicrob" Agents Chemother. 39. 2567-2569 (1995)

  • [Publications] Hanaki, H., akagi, H., Masaru, Y., Otani, T., Hyodo, A., and IHiramatsu, K.: "TOC-39, a novel parenteral broad-spectrum cephalosporin iwth an excellent activity against methicillin-resistant Staphylococcus aureus" Antimicrob. Agents Chemother. 39. 1120-1126 (1995)

  • [Publications] Higashitani, F., K. Nishida, A. Hyodo and M. Inoue: "Effects of tazobactam on the emrgence of resistant strains from Enterobacter cloacae, Citrobacter freundii, and Proteus vulgaris" J. Antibiotics. 48. 1027-1033 (1995)

  • [Publications] Hiramatsu, K.: "Molecular evolution f MRSA Microbiol" Immunol. 39. 531-543 (1995)

  • [Publications] Hosaka, Masaki., Naomasa Gotoh and Takeshi Nishino: "Purification of a 54 kilodalton protein (OprJ) in NfxB mutants of Pseudomonas aeruginosa and production of a monoclonal antibody specific to OprJ" Antimicrob. Agents Chemother. 39. 1731-1735 (1995)

  • [Publications] Inoue,M., A. Fuse, K. Yamaguchi, H. Kanno, M. Kaku, T. I namatsu, T. Nagahama, M. Yoshida, M. Nonoyama, R. Okamoto: "Characterization of an extended-spectrum β-lactamase from Escherichia coli mediated by a plasmid" J. Infet. Chemother. 1. 70-72 (1995)

  • [Publications] Ishii, J and Nakae, T: "Specific interaction of he protein D2 porin of Pseudomonas aeruginosa with antibiotics" FEMS Microbiol. Letts. In press. (1996)

  • [Publications] Iyobe, S: "Nosocomial infections caused by Pseudomonas aeruginosa strains resistant to multiple β-lactam antibiotics includong carbapenems" ASEAN-Japan Seminor and Workshop on Hospital Infection. 63-69 (1995)

  • [Publications] Kanazawa, Katsunori., Yoriko Kobayashi, Masayuki Nakano, Maki Sakurai, Naomasa Gotoh and Takeshi Nishino: "Identification of three porins in the outer membrane of Bacteroides fragilis" FEMS Microbiol. Lett. 127. 181-186 (1995)

  • [Publications] Kazama, H., Kizu, K., Iwasaki, M., Hamashima, H., Sasatsu, M. and Arai, T: "Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa" FEMS Microbiol. Lett. 134. 137-141 (1995)

  • [Publications] Kimura, T.., Y. Inagaki, T. Sawai and A. Yamaguchi: "Substrate-induced acceleration of N-ethylmaleimide binding to the Cys-65 mutant of the transposon Tn10-encoded metal-tetracycline/H^+ antiporter depends on the interaction of ASP-66 with the substrate" FEBS Lett. 362. 47-49 (1995)

  • [Publications] Kojima, T., J. Yamagishi, Y. Oyamada, H. Yoshida, H. Hatta, M. Inoue and S. Nakamura: "Analysis of quinolone resistance genes in a clinical isolates of quinolone-resistant methicillin-resistant Staphylococcus aureus" Drugs. 49. 182-184 (1995)

  • [Publications] Kondo, N., Ito, T., Tateda-Suzuki,E., Yoshida, T., Abu-Hanifah, Y., and Hirmatsu, K.: "MRSA: World-wide epidemiology and resistance mechanism" In ASEAN-JAPAN Seminar and Workshop on Hospital Infection, ed. By Yun-Fong Ngeow and Hiramatsu. 25-32 (1995)

  • [Publications] Kunugita, C., F. Higashitani, A. Hyodo, N. Unemi and M. Inoue: "Characterization of a new plasmid mediated extended-spectrum β-lactamase from Serratia marcescens" J. Antibiotics. 48. 1453-1459 (1995)

  • [Publications] Matsuda, K., K. Nakamura, Y. Adachi, M. Inoue and M. Kawakami: "Autolysis of methicililn-resistant Staphylococcus aureus involved in synergism between imipenem and cefotiam" Antimicrob. Agents Chemother. 39. 2631-2634 (1995)

  • [Publications] Morshed, S. R. M., Lei, Y., Yoneyama, Y., Nakae, T: "Expresion of genes associated with antibiotic extrusion in Pseudomonas aeruginosa" Biochem. Biophys. Res. Commun. 1995. 210. 356-362 (1995)

  • [Publications] Nakae, T: "Role of membrane permeability in determining antibiotic resistance in Pseudomonas aeruginosa" Microbiol. lmmunol. 39. 211-229 (1995)

  • [Publications] Nariya, H. and Y. Kamio: "Identification of the essential regions for lukS-and Hll-specific function of Staphylococcal leuko-cidin and gamm-hemolysin" Biosci. Biotech. Biochem.59. 1603-1604 (1995)

  • [Publications] Noguchi, N., Emura, A., Matsuyama, H., O'hara, K., Sasatsu, M., and Kono, M: "Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Antimicrob" Agents Chemother.39. 2359-2363 (1995)

  • [Publications] Ozawa, T., J. Kaneko, and Y. Kamio: "Essential binding of LukF of Staphylococcal gamma-hemolysin followed by the binding of H γ ll for the hemolysin of human erythrocytes" Biosci. Biotech. Biochem.59. 1181-1183 (1995)

  • [Publications] Sasatsu, M., Shirai, Y., Hase, M., Noguchi, N., Kono, M., Behr, H., and Freney, J. and Arai, T: "The origin of antiseptic-resistant gene ebr in Staphylococcus aureus" Microbios. (in press). (1995)

  • [Publications] Satake, S., Nakae, T: "Outer membren permeability of β lactamase inibitors in Pseudomonas aeruginosa" FEMS Microbiol. Letts. 29. 251-254 (1995)

  • [Publications] Someya, Y., A. Niwa, T. Sawai and A. Yamaguchi: "Site-specificity of the second-site suppressor mutation of the Asp-285→Asn mutant of metal-tetracy cline/H^+ antiporter of Escherichia coli and the effects of amino acid substitutions at the first and second sites" Biochemistry. 34. 7-12 (1995)

  • [Publications] Someya, Y., Y. Moriyama, M. Futai, T. Sawai, and A. Yamaguchi: "Reconsitution of the metal-tetracycline/H^+ antiporter of Escherichia coli in proteoliposomes including F0F1-ATPase" FEBS Lett. 374. 72-76 (1995)

  • [Publications] Someya, Y., A. Yamaguchi and T. Sawai: "A novel glycylcycline, 9-(N, N-dimethyl glycylamido)-6-demethyl-6-deoxy-tetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H^+ antiporter" Antimicrob. Agents Chemother. 39. 247-249 (1995)

  • [Publications] Sudo, K., W. Choorit, I. Asami, J. Kaneko, K. Muramoto, and Y. Kamio: "Substititution of lysine for arginine in the N-terminal 217th amino acid residue of the H γ ll of Staphylococcal γ-hemolysine lowerws the activity of the toxin" Biosci. Biotech. Biochem.59. 1786-1789 (1995)

  • [Publications] Sumita, Y., M. Fukazawa, S. Mitsuhashi and M. Inoue: "Binding affinities of β-lactam antibiotics for penicillin-binding protein 2' in methicillin-resistant Staphylococcus aureus" J. Antimicrob. Chemother. 35. 473-481 (1995)

  • [Publications] Takahashi, A., S. Yomoda, Y. Ushijima, I. Kobayashi and M. Inoue: "Ofloxacin, norfloxacin and ceftazidime increase the production of alginate and promote the formation of biofilm of Pseudomonas aeruginosa in vitro" J. Antimicrob. Chemother. 36. 743-745 (1995)

  • [Publications] Tanaka, T., Okuzumi, K., Iwamoto, A., and Hiramatsu, K.,: "A retrospective study on methicillin-resistant Staphylococus aureus Cliical strain in Tokyo University Hospital" J. Infect. Chemother. 1. 42-51 (1995)

  • [Publications] Yamaguchi, A., T. Samejima and T. Sawai: "His-257 is a Uniquely lmportant Histidine Residue for Tetracycline/H^+ Antiport Function but Not Mandatory for Full Activity of the Transposon Tn10-Encoded Metal-Tetracycline/H^+ Antiporter" Biochemistry. (in press). (1996)

  • [Publications] Yamaguchi, A., Shiina, T., Fujihira, E., Sawai, T., Noguchi,N, and Sasatsu, M: "The tetracycline efflux protein encoded by the qac (K) gene from Staphylococcus aureus is a metal-tetracycline/H^+ antiporter" FEBS Lett. 365. 193-197 (1995)

  • [Publications] Yamaguchi, A., Y. Inagaki and T. Sawai: "Second-site suppressor mutations for the Asp-66→Cys mutant of the transposon Tn10-encoded metal-tetracycline/H^+ antiporter of Escherichia coli" Biochemistry. 34. 11800-11806 (1995)

  • [Publications] Yamaguchi, A., Y. Shiina, E. Fujihira, T. Sawai, N. Noguchi and M. Sasatsu: "The tetracycline efflux protein encoded by the tet (K) gene from Staphylococcus aureus is a metal-tetracycline/H^+ antiporter" FEBS Lett. 365. 193-197 (1995)

  • [Publications] Yoneyama, H., Yamano, Y., Nakae, T: "Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: Construction of mutants wth deletions in the multiple poin genes Biochem" Biophys. Res. Commun. 213. 88-95 (1995)

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Published: 1997-02-26   Modified: 2016-04-21  

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