バンコマイシン耐性腸球菌VREの新たな高頻度接合伝達性プラスミドの研究

• Project Area: Matrix of Infection Phenomena
• Project/Area Number: 21022008
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Gunma University
• Principal Investigator: 富田 治芳 群馬大学, 大学院・医学系研究科, 准教授 (70282390)
• Co-Investigator(Kenkyū-buntansha): 池 康嘉 群馬大学, 大学院・医学系研究科, 教授 (60125820)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥6,600,000 (Direct Cost: ¥6,600,000); Fiscal Year 2010: ¥3,300,000 (Direct Cost: ¥3,300,000); Fiscal Year 2009: ¥3,300,000 (Direct Cost: ¥3,300,000)
• Keywords: 腸球菌 / バンコマイシン耐性 / VRE / 伝達性プラスミド / 病原性 / 多剤耐性菌 / 院内感染症 / 日和見感染

Research Abstract

バンコマイシン耐性腸球菌VREから分離したpMG1型高頻度接合伝達性プラスミドpHTβの高頻度伝達機構と接合凝集性についての遺伝学的解析を行った。これまでの解析によって決定したpHTβの凝集遺伝子群の最上流に存在するORF9のin-frame欠失変異体を作製分離した。ORF9変異体は凝集性と同時に伝達能も完全に失い、ORF9自身の転写活性もほぼ消失していた。この変異はORF9のクローンによって相補されたことから、ORF9は接合伝達と凝集を正に調節する遺伝子であることが明らかとなり新たにtraDと命名した。
これまでのpHTβを用いた遺伝学的解析からpMG1型プラスミドの高頻度接合伝達機構には負の調節因子が存在することが推測されていた。その未知の因子の同定を、凝集遺伝子内へTn917-lacが挿入され転写活性がLacZの発現により測定可能で、かつtraBに変異を持つことにより転写活性の消失した二重変異プラスミドを用い行った。これを保持する株を供与菌とし固形培地上での接合伝達実験を行い、得られた接合伝達株のLacZ発現を調べる方法により、凝集遺伝子領域の転写活性が復帰した変異体を複数個分離した。得られた復帰変異プラスミドは全てORF2内に変異が存在した。ORF2のin-frame欠失変異を作製分離し解析を行ったところ、ORF2変異体は親株に比べ凝集性が強まり、伝達頻度も上昇していた。またTraDの転写活性も増強していた。これらはORF2のクローンのよって相補されたことから、ORF2は接合伝達を負に調節していることが示され、新たにtraFと命名した。
同定した2つの調節遺伝子は共にこれまで報告されている遺伝子との相同性は認めず、pMG1型プラスミドの高頻度接合伝達機構は他の遺伝子発現調節機構とは全く異なる特異的なものであることが示された。

Research Products

• [Journal Article] Assessment of antibiotic resistance in probiotic strain Lactobacillus brevis KB290 (2009)
  • Author(s): Fukao M, Tomita H, Yakabe T, Nomura T, Ike Y, Yajima N
  • Journal Title: Journal of Food Protection 72 Pages: 1923-1929
  • Peer Reviewed
• [Journal Article] Nationwide epidemiological study revealed the dissemination of meticillin-resistant Straphylococcus aureus carrying a specific set of virulence-associated genes in Japanese hospitals (2009)
  • Author(s): Ohkura T, Yamada K, Okamoto A, Baba H, Ike Y, Arakawa Y, Hasegawa T, Ohta M
  • Journal Title: Journal of Medical Microbiology 58 Pages: 1329-1336
  • Peer Reviewed
• [Journal Article] 腸球菌の高頻度接合伝達性プラスミド (2009)
  • Author(s): 富田治芳
  • Journal Title: 日本細菌学雑誌 64 Pages: 343-355
  • NAID: 10027208714
• [Presentation] High conjugative pMG1-like plasmids in enterococci. (2010)
  • Author(s): Tomita H, Ike Y.
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Novel enterococcal bacteriocins. 3^<rd> international ASM conference on enterococci (2010)
  • Author(s): Ike Y, Inoue T, Yamashita H, Tomita H
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Genetic analysis of vancomycin resistance pMG1-like conjugative plasmid pHTβ : identification of traD and traF, the key genes for plasmid transfer and cell aggregation. (2010)
  • Author(s): Tomita H, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Complete nucleotide sequence and analysis of the vanA-type vancomycin resistance highly conjugative plasmid pZB18 found in the Chinese clinical Enterococcus faecium isolate. (2010)
  • Author(s): Tomita H, Zheng B, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Structural and comparative analysis of the pMG1-like plasmids isolated from clinical Enterococcus faecium strains in the world (2010)
  • Author(s): Tomita H, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Genetic analysis of the bactericoin 41 encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14. (2010)
  • Author(s): Tomita H, Todokoro D, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Characterization of the vancomycin-resistant enterococci isolated from human patients and livestock in Taiwan : high incidence of multiple-drug resistant pheromone responsive plasmids and evidence of transmission of VRE from livestock to human. (2010)
  • Author(s): Tomita H, Lu J, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Comparative analysis of the sec-dependent class IIa bacteriocins of enterococci and construction of the conditional expression vector plasmid pHT7343A. (2010)
  • Author(s): Tomita H, Clewell DB, Ike Y.
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Genetic analysis of the ultraviolet resistance determinants encoded on the Enterococcus faecalis pheromone responsive conjugative plasmids. 3^<rd> international ASM conference on enterococci (2010)
  • Author(s): Asano Y, Tomita H, Ozawa Y, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Diversity of activator and immunity gene of bacteriocin 41-type bacteriocin, a novel bacteriocin complemented by two extracell ular components (lysine and activator). (2010)
  • Author(s): Inoue T, Tomita H, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Mode of action of bacteriocin 32 : a widely disseminated bacteriocin among clinical isolates of Enterococcus faecium. (2010)
  • Author(s): Inoue T, Tomita H, Ichimonji I
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Characterization of mobilization of bacteriocin plasmid pTI(12.5kbp) isolated from vancomycin-resistant Enterococcus faecium. (2010)
  • Author(s): Inoue T, Tomita H, Ichimonji I, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Characterization of Tn1546-like elements of vanA-type vancomycin-resistant enterococci(VRE) in Japan. (2010)
  • Author(s): Nomura T, Tomita H, Nagasawa M, Arakawa Y, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Genetic analysis of a novel bacteriocin 51 determinant of vanA type vancomycin resistant Enterococcus faecium. (2010)
  • Author(s): Yamashita H, Tomita H, Ike Y
  • Organizer: International ASM conference on enterococci
  • Place of Presentation: Portland, USA
• [Presentation] Highly conjugative pMG1-like plasmids in enterococci. (2010)
  • Author(s): Tomita H, Ike Y
  • Organizer: 10^<th> Awaji international forum on infection and immunity
  • Place of Presentation: Hyogo, Japan
• [Presentation] Genetic analysis of Enterococcus vancomycin-resistance conjugative plasmid pHTβ (2009)
  • Author(s): Tomita H, Ike Y
  • Organizer: Awaji international forum
  • Place of Presentation: 兵庫県・淡路
• [Presentation] バンコマイシン耐性腸球菌VREの高頻度接合伝達性プラスミド (2009)
  • Author(s): 富田治芳
  • Organizer: 日本細菌学会 関東支部総会
  • Place of Presentation: 東京・四谷