臺灣博碩士論文加值系統

克雷伯氏肺炎桿菌(Klebsiella pneumoniae；K. p)為一種常引起院內感染的重要病原菌，其中有許多屬於ESBL strain，且具多重抗藥性，這在台灣已是嚴重的問題。近年來，由K. p引起的化膿性肝膿瘍的病例無論國內外皆有逐漸增加的趨勢，在台灣地區更高達82.1 %。
本研究為探討除了ESBL所引起的抗藥性外，還有哪些基因與抗藥性相關，故利用Tn5 transposon mutagensis送入對CRO (ceftriaxone)高抗藥的K. p去建立一個Tn5突變株圖庫，從library篩選出13株對CRO- less resistance的菌株，經由southern blot確定有12株只有單一個基因遭受Tn5破壞，進而分析破壞基因名稱。
在2E11、9D4兩株突變菌株中，其破壞基因分別為hfq、及rpmE，再經由pGEM-T載體將完整的基因送入2E11及9D4兩突變株中，可回復對CRO的抗藥性。Hfq 為RNA binding protein，可調控RpoS的轉錄，RpoS為sigma factor，可經由外界環境壓力所誘導的因子，進而去調控下游基因的表現，RpmE為50S ribosomal protein L31，與蛋白質合成相關，也許可以成為抗細菌的新標的。為求何基因受到影響，藉由2D電泳來分析外膜蛋白，發現hfq及rpmE兩基因的突變，皆引起兩個外膜蛋白的減少，包括：conserved hypothetical protein 及 outer membrane protein K 17，這其中的機制還需更進一步的研究。

Klebsiella pneumoniae is a common nosocomial pathogen, characterized by ESBL-producing with multi-drug resistance. In addition, this microorganism is a significant cause of liver abscess in Taiwan. In this study, we are searching for genes, but β-lactamase -encoded gene, or proteins which involving in ceftriaxone (CRO) resistance. A clinical isolate of K. pneumoniae, with resistance to CRO but susceptible to kanamycin (KAN), was chosen to generate insertion mutants using Tn5 transposome mutagenesis. Among 1500 insertion mutants we obtained, the MICs of thirteen mutants have 3-fold reduction of CRO resistance. Twelve out of thirteen mutants had single Tn5 insertion by southern blot assay and their inserted sites in the chromosome were identified by DNA sequencing method.
Insertion mutants 2E11 and 9D4 were selected in this study. Mutant 2E11 has transposon inserted in hfq gene, encoded for a RNA binding protein which can regulate sigma factor RpoS translation. Mutant 9D4 has transposon inserted in rpmE gene, encoded for 50S ribosomal protein L31. The wild type genes hfq and rpmE were inserted into pGEM-T expression vector to construct pGEM-spt-hfq and pGEM-spt-rpmE and transformed in 2E11 and 9D4 mutants, respectively, for complementation experiment. The result showed both of genes were able to restore CRO resistance in their own mutants. To investigate expression of outer membrane protein regulated by hfq and rpmE, the outer membrane protein profile between parental strains and insertion mutant 2E11 or 9D4 was compared using two-dimensional gel electrophoresis(2-DE). Two protein spots with molecular mass about 18kDa were up-regulated in parental strain, but down-regulated in both insertion mutants. These two proteins are further identified by MALDI-TOF as conserved hypothetical protein (18567Da) and outer membrane protein K17 (18452Da), respectively. These two protein involving in CRO resistance or not and how to regulate need further investigation in the future.

Arous, S., C. Buchriese, P. Folio, P. Glaser, A. Namane, M. Hebraud, Y. Hechard. 2004. Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes. Microbiology. 150(Pt5) :1581-1590.
Bagley, S. T., R. J. Seidler, H. W. J. Talbot, and J. E. Morrow. 1978. Isolation of Klebsiellae from within living wood. Appl. Environ. Microbiol. 36:178-185.
Bjork, G. R. 1995. Biosynthesis and function of modified nucleosides, p. 165-205. In D. soll and U. L. RajBhandary(ed.), tRNA: structure, biosynthesis, and function. ASM press, Washington, D.C
Brown, L., T. Elliott. 1996. Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene. J Bacteriol. 178(13):3763-3770.
Bush, L. M., J. Calmon, C. C. Johnson. 1995.Newer penicillins and beta-lactamase inhibitors. Infect Dis Clin North Am. 9(3):653-686.
Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A Functional Classification Scheme for β-lactamases and its Correlation with Molecular Structure. Antimicrobial Agents and Chemotherapy, 39:1211-1233.
Bush, K. 2001. New b-Lactamases in Gram-Negative Bacteria: Diversity and Impact on the Selection of Antimicrobial Therapy. Clinical Infectious Diseases. 32: 1085-1089.
Carpenter, E.P., A. R. Hawkins, J. W. Frost, K. A. Brown. 1998. Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis. Nature. 394:299-302 .
Cheng, H. P., F. Y. Chang, C. P. Fung, L. K. Siu. 2002. Klebsiella pneumoniae liver abscess in Taiwan is not caused by a clonal spread strain. J Microbiol Immunol Infect. 35(2):85-88.
Climent, N., S. Ferrer, X. Rubires, S. Merino, J. M. Tomas, M. Regue. 1997 Molecular characterization of a 17-kDa outer-membrane protein from Klebsiella pneumoniae. Res Microbiol. 148(2):133-143.
Coggins, J. R., C. Abell, L. B. Evans, M. Frederickson, D. A. Robinson, A. W. Roszak, A. P. Lapthorn. 2003. Experiences with the shikimate-pathway enzymes as targets for rational drug design. Biochem. Soc. Trans. 31:548-552.
Coudron, P. E., E. S. Moland, and K. S. Thomson. 2000. Occurrence and Detection of AmpC Beta-Lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis Isolates at aVeterans Medical Center. Journal of Clinical Microbiology. 38:1791-1796.
Darget, M., and S. D. Ehrlich. 1979. Prolonged incubation in CaCl2 improves the competence of Escherichia coli cells. Gene. 6:23-628.
Durand, J. M., G. R. Bjork, A. Kuwae., M. Yoshikawa, C. Sasakawa. 1997. The modified nucleoside 2-methylthio-N6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes. J Bacteriol. 179(18):5777-5782.
Enfedaque, J., S. Ferrer, J. F. Guasch, J. Tomas, M. Regue. 1996. Bacteriocin 28b from Serratia marcescens N28b: identification of Escherichia coli surface components involved in bacteriocin binding and translocation. Can. J. Microbiol. 42:19-26.
Franze de Fernandez, M. T. L. Eoyang, and J. T. August. 1968. Factor fraction required for the synthesis of bacteriophage Qb RNA. Nature. 219:588-590.
Haber, C. L., C. L. Heckaman, G. P. Li, D. P. Thompson, H. A. Whaley, V. H. Wiley. 1991. Development of a mechanism of action-based screen for anthelmintic microbial metabolites with avermectinlike activity and isolation of milbemycin-producing Streptomyces strains. Antimicrob Agents Chemother. 35(9):1811-1817.
Hanahan, D. 1983. Studied on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166(4):557-580.
Hasan Yesilkaya, Aras Kadioglu, Neill Gingles, Janet E. Alexander, Tim J. Mitchell, and Peter W. Andrew. 2000. Role of Manganese-Containing Superoxide Dismutase in Oxidative Stress and Virulence of Streptococcus pneumoniae. Infection and Immunity. 68: 2819-2826.
Heffernan, E. J., J. Harwood, J. Fierer, D. Guiney. 1992b. The salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail. J. Clin, Invert. 90:953-964.
Helmann J. D., and M. J. Chamberlin. 1988. Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57:839-872.
Hyunjoo Pai, Eun-Hwa Chio, Hoan-Jong Lee, Jung Yun Hong, and George A. Jacoby.2001.Identification of CTX-M-14 Extended-Spectrum β-Lactamase in Clinical Isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. Journal of clinical microbiology. 39: 3747-3749.
Klein, N. C., and B. A. Cunha, 1995. The selection and use of cephalosporins: a review. Adv-Ther. 12(2):83-101.
Krebs, C., J. N. Agar, A. D. Smith, J. Frazzon, D. R. Dean, B. H. Huynh, M. K. Johnson. 2001. IscA, an alternate scaffold for Fe-S cluster biosynthesis. Biochemistry. 40(46):14069-14080.
Larry Reitzer. 2003. Nitrogen assimilation and global regulation in Escherichia coli. Annu. Rev. Microbiol. 57:155-176.
Lauhon, C. T., R. Kambampati. 2000. The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD. J Biol Chem. 275(26):20096-20103.
LeBlanc, D.J., L. N. Lee, A. Abu-Al-Jaibat. 1992. Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin. Plasmid. 28(2):130-145.
Livermore, D. M. 1995.β-Lactamases in Laboratory and Clinical Resistance. Clinical microbiology reviews 557-584.
Loewen, P. C., and R. Hengge-Aronis. 1994. The role of the sigma factor σS(KatF) in bacterial global regulation. Annu. Rev. Microbiol. 48:53–80.
Loewen, P. C., Bei Hu, Jeanna Strutinsky, Richard Sparling. 1998. Regulation in the rpoS regulon of Escherichia coli Canadian Journal of Microbiology. 8:707-717.
Mabilat, C., and S. Goussard. 1993. PCR detection and identification of genes for extended-spectrum -lactamases, p. 553-559. In D. H. Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.), Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, D.C.
Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Habor Laboratory, Cold Spring Habor, New York.
Martinez-Martinez, L., M. C. Conejo, and A. Pascual. 2000. Activities of Imipenem and cephalosporins against clonally related starins of Escherichia coli hyperproducing chromosomal beta-lactamase and showing altered porin profile. Antimicrobial Agents and Chemother. 44(9):2534-6.
Matsen, J. M., J. A. Spindler, and R. O. Blosser. 1974. Characterization of Klebsiella isolates from natural receiving waters and comparison with human isolates. Appl. Microbiol. 28:672-678.
McEvoy, G. K. 1998. Cephalosporins. AHFS Drug Information 98. Bethesda; American Society of Health-System Pharmacists, Inc. 125-205
Miller, V. L. and S. Falkow. 1988. Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of ephithelial cells. Infect. Immun. 56:1242-1248.
Miller, V. L., K. B. Beer, W. P. Loomis, J. A. Olson, S. I. Miller. 1992. An unusual pagC::TnphoA mutation leads to an invasion and virulence defective phenotype in Salmonella. Infect. Immun. 60:3763-3770.
Mitchell, S. J. and M. F. Minnick. 1995. Characterization of a two-gene locus from Bartonella bacilliformis associated with the ability to invade human erythrocytes. Infect. Immun. 63:1552-1562.

Molloy, M. P., B. R. Herbert , M. B. Slade, T. Rabilloud, A. S. Nouwens, K. L. Willians and A. A. Gooley. 2000. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267:2871-2881.
National Committee for Clinical Laboratory Standards. Villanova, Pennsylvania: National Committee for Clinical Laboratory Standards, January 1999: M100-S9.
Nikaido, H. 1994. Prevention of drug access to bacterial targets:permeability barriers and active efflux. Science. 264:382-388.
Nikaido, H., M. Basina, V. Nguyen, and E. Y. Rosenberg. 1998. Multidrug efflux pump AcrAB of Salmonella typhimurium excretes only those beta-lactam antibiotics containing lipophilic side chains. J. Bacteriol. 180:4686-4692.
Patricia, A. B., Wyeth-Ayerst Research, Pearl River. 2001. Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat Clinical Microbiology Reviews. 933-951.
Podschun, R, and U. Ullmann 1998. Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors Clinical Microbiology Reviews. 11(4):589-603.
Rasheed, J. K., C. JAY, B. Metchock, F. Berkowitz, L. Weigel, et. 1997. Evolution of Extended-Spectrum b-Lactam Resistance (SHV-8) in a Strain of Escherichia coli during Multiple Episodes of Bacteremia Antimicrobial agents and chemotherapy. 41:647-653.
Rupp, Mark E. and Paul D. Fey. 2003. Extended Spectrum β-Lactamase-Producing Enterobacteriaceae Considerations for Diagnosis, Prevention and Drug Treatment Drugs. 63(4):353-65.
Sambrook, J., E. F. Fritsch, , and T. Manitis. 1989. Molecular Cloning: a laboratory manual. Cold Spring Habor Laboratory, Cold Spring Habor, New York.
Schimmel, P, J. Tao, J. Hill. 1998. Aminoacyl tRNA synthetases as targets for new anti-infectives. FASEB J. 12(15):1599-609.
Stoorvogel, J., van Bussel, M. J. A. W. M., & van de Klundert, J.A.M. 1997. Cloning of beta-lactam resistance determinant of Enterobacter cloacae affecting outer membrane proteins of Enteobacteriaceae. FEMS Microbiol. Lett. 48. 277-281.
Tsui, H. C., G. Feng, M. E. Winkler. 1996. Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock. J Bacteriol. 178(19):5719-31.
Weiner, L., and P. Model. 1994. Role of an Escherichia coli stress-response operon in stationary-phase survival. Proc. Natl. Acad. Sci. USA 91:2191-2195.
Winokur, P. L., R. Canton, J. M.Casellas, N. Legakis. 2001. Variations in the prevalence of strains expressing an extended-spectrum beta-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis.15 Suppl 2:S94-103.
Wosten, M. M. 1998. Eubacterial sigma-factors. FEMS Microbiol. Rev. 22:127–150.
Yinnon, A. M., A. Butnaru, D. Raveh, Z. Jerassy, and B. Rudensky. 1996. Klebsiella bacteremia: community versus nosocomial infection. Monthly J. Assoc. Physicians 89:933-941.