臺灣博碩士論文加值系統

Integron被認為是細菌獲得抗藥性基因及散佈抗藥性基因的重要機制，在臨床菌株中之Integron以class 1 integron最普遍。Class 1 integron常被發現於不同菌屬的細菌體內不同位置上，包括plasmid、transposon、chromosome 。而Class 1 integron藉著transposon或plasmid傳遞，也暗示class 1 integron可能具有移動性。因此本研究主旨在探討臨床分離之大腸桿菌株之class 1 integron結構散佈能力。首先選殖帶有class 1 integron結構的基因片段，包含兩端反轉重複序列IRi和IRt及轉位相關基因，然後分析integron發生轉位的頻率。
實驗結果顯示class 1 integron發生轉位的頻率低於1.0×10-9。這可能是因為本研究分析之class 1 integron基因片匣下游結構有較大變異，多數與轉位基因相關的tni基因群tniA的截短和tniB序列有短少，而tniQ和tniR則未存在。另外本研究也進行互補實驗，對於前述轉位頻率極低的class 1 integron，探討在外加具有轉位功能的基因後，是否促使integron發生轉位移動。互補實驗結果發現，在外加包含完整轉位基因tniA、B、Q、R的基因片段後，Class 1 integron發生轉位的頻率可達3.2×10-4到1×10-1，比原class 1 integron轉位頻率明顯增加。另外，只外加tniA、B或tniA基因片段後，Class 1 integron雖有轉位作用的發生，但頻率較低約2.0×10-9到6.3×10-6。
綜合本研究結果顯示臨床E. coli菌株所帶之class 1integron因轉位基因之缺失，自行移動的能力偏低，但細菌菌體內若同時有其他具有轉位活性的轉位子構造存在，能補足轉位所需的蛋白質，則class 1 integron仍有發生轉位移動，這將大大提高integron移動散佈的機會，造成多重抗藥性細菌的產生。

Integrons have been demonstrated to play a significant role in the acquisition and dissemination of antibiotic-resistant genes among bacteria. Most integrons from clinical isolates belong to class 1. Class 1 integrons have been found in different DNA locations, such as plasmids, transposons and chromosome among different bacterial species and genera, suggesting class 1 integrons may be mobile. In this study, we attempt to analyze the movement ability of class 1 integrons in clinical isolates of Escherichia coli. At first, we constructed the test plasmids, which carries the class 1 integrons containing inverted repeats (IRs) at their boundaries and transposition related genes. The transposition frequency of class 1 integrons was then determined by using a transposition assay.
The test plasmids carrying class 1 integrons showed a very low tansposition frequency (<1.0×10-9). This may be resulted from the structural variations of class 1 integrons, such as the loss of part of the transposition module. In addition, we carried out complementing experiments to study whether class 1 integrons could be moved by transposition when functional genes of transposition were provided. In the event, transposition of class 1 integrons was complemented by the plasmid containing the whole set of transposition genes (tni A, B, Q, R) and restored at high frequencies (3.2 × 10-4 to 1.0 × 0-1). However, class 1 integrons transposed at lower frequencies (2.0 × 10-9 to 6.3 × 10-6) when complemented by a plasmid only containing tniA and tniB genes or tniA gene.
The present study reveals that class 1 integrons from clinical isolates are defective in transposition. The movement of class 1 integorns can occur when proteins required for transposition are provided by active transposons present in the same bacterial cell. This will facilitate the dissemination of class 1 integrons and contributed to multiple resistance of bacteria.

中文摘要 1
英文摘要 3
緒論 5
一、Integron基本結構特徵 6
二、基因片匣的構造與特定部位重組作用 7
三、Class 1 integron與相關抗藥基因 9
四、Integron和轉位子結構的相關性 12
五、研究目的 18
材料和方法
一、Class 1 integron的轉位能力分析
（一）實驗菌株 20
（二）質體 21
（三）測試質體的構築 21
1.聚合酶鏈鎖反應 22
2.瓊脂醣明膠電泳 23
3.PCR產物純化 24
4.增幅片段之選殖 25
（1）增幅片段與載體（vector）之連接作用（ligation） 25
（2）重組質體（recombination）之轉形作用（tansfor
mation）
26
（3）轉形菌株之篩選 27
（4）核酸自動定序分析 29
（四）構築授予菌株（Donor strain construction） 29
1.測試質體之轉形作用 29
2.轉形菌株之篩選 31
3.瓊脂醣明膠電泳 32
（五）轉位試驗（transposition assay） 32
1.接合實驗（conjugation） 33
2.接合轉移菌（transconjugate）之篩選 33
二、互補實驗
（一）互補質體的構築 34
1.聚合酶鏈鎖反應 35
2.增幅片段之選殖（Amplicom cloning） 36
（1）增幅片段與載體（vector）之連接作用（ligation） 36
（2）X-gal plate之製備 38
（3）重組質體（recombination）之轉形作用（tansfor
mation）
38

（二）構築授予菌株（Donor strain construction） 39
1.互補質體之轉形作用 39
（三）轉位試驗（transposition assay） 40
結果
一、Class 1 integron進行轉位作用的能力 41
二、加入互補質體後class 1 integron發生轉位的頻率 42
討論 44
結論 48
表 49
圖 53
參考文獻 59

1.Partridge, S.R., et al., Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins. Antimicrob Agents Chemother, 2001. 45: 1263-70.
2.Partridge, S.R., et al., Family of class 1 integrons related to In4 from Tn1696. Antimicrob Agents Chemother, 2001. 45: 3014-20.
3.Rowe-Magnus, D.A., A.M. Guerout, and D. Mazel, Super-integrons. Res Microbiol, 1999. 150: 641-51.
4.Hall, R.M., D.E. Brookes, and H.W. Stokes, Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point. Mol Microbiol, 1991. 5: 1941-59.
5.Japoni, A., et al., Assay for integrons and pattern of antibiotic resistance in clinical Escherichia coli strains by PCR-RFLP in Southern Iran. Jpn J Infect Dis, 2008. 61: 85-8.
6.Hsueh, P.R., C.Y. Liu, and K.T. Luh, Current status of antimicrobial resistance in Taiwan. Emerg Infect Dis, 2002. 8: 132-7.
7.Watanabe, T. and T. Fukasawa, Episome-mediated transfer of drug resistance in Enterobacteriaceae. I. Transfer of resistance factors by conjugation. J Bacteriol, 1961. 81: 669-78.
8.Lupski, J.R., Molecular mechanisms for transposition of drug-resistance genes and other movable genetic elements. Rev Infect Dis, 1987. 9: 357-68.
9.Rubens, C.E., W.F. McNeill, and W.E. Farrar, Jr., Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences. J Bacteriol, 1979. 140: 713-9.
10.Watanabe, T. and T. Fukasawa, Episome-mediated transfer of drug resistance in Enterobacteriaceae. III. Transduotion of resistance factors. J Bacteriol, 1961. 82: 202-9.
11.Martinez, E. and F. de la Cruz, Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol Gen Genet, 1988. 211: 320-5.
12.Sundstrom, L., et al., Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21. Mol Gen Genet, 1988. 213: 191-201.
13.Hall, R.M. and H.W. Stokes, Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica, 1993. 90: 115-32.
14.Ploy, M.C., et al., Integrons: an antibiotic resistance gene capture and expression system. Clin Chem Lab Med, 2000. 38: 483-7.
15.Bennett, P.M., Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol, 2008. 153 Suppl 1: S347-57.
16.Hall, R.M. and C.M. Collis, Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist Updat, 1998. 1: 109-19.
17.Hall, R.M. and C.M. Collis, Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol, 1995. 15: 593-600.
18.Rowe-Magnus, D.A. and D. Mazel, The role of integrons in antibiotic resistance gene capture. Int J Med Microbiol, 2002. 292: 115-25.
19.Fluit, A.C. and F.J. Schmitz, Resistance integrons and super-integrons. Clin Microbiol Infect, 2004. 10: 272-88.
20.Recchia, G.D. and R.M. Hall, Gene cassettes: a new class of mobile element. Microbiology, 1995. 141 ( Pt 12): 3015-27.
21.Mazel, D., Integrons: agents of bacterial evolution. Nat Rev Microbiol, 2006. 4: 608-20.
22.Collis, C.M., et al., Site-specific insertion of gene cassettes into integrons. Mol Microbiol, 1993. 9: 41-52.
23.Collis, C.M. and R.M. Hall, Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother, 1995. 39: 155-62.
24.Collis, C.M. and R.M. Hall, Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase. J Bacteriol, 1992. 174: 1574-85.
25.Stokes, H.W. and R.M. Hall, A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol, 1989. 3: 1669-83.
26.Rowe-Magnus, D.A., A.M. Guerout, and D. Mazel, Bacterial resistance evolution by recruitment of super-integron gene cassettes. Mol Microbiol, 2002. 43: 1657-69.
27.Grape, M., et al., Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria. Clin Microbiol Infect, 2005. 11: 185-92.
28.Saenz, Y., et al., Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob Agents Chemother, 2004. 48: 3996-4001.
29.Nandi, S., et al., Gram-positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter. Proc Natl Acad Sci U S A, 2004. 101: 7118-22.
30.Tauch, A., et al., The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100. Plasmid, 2002. 48: 117-29.
31.Nesvera, J., J. Hochmannova, and M. Patek, An integron of class 1 is present on the plasmid pCG4 from gram-positive bacterium Corynebacterium glutamicum. FEMS Microbiol Lett, 1998. 169: 391-5.
32.Stokes, H.W., et al., Class 1 integrons potentially predating the association with Tn402-like transposition genes are present in a sediment microbial community. J Bacteriol, 2006. 188: 5722-30.
33.Partridge, S.R., H.J. Brown, and R.M. Hall, Characterization and movement of the class 1 integron known as Tn2521 and Tn1405. Antimicrob Agents Chemother, 2002. 46: 1288-94.
34.Peters, J.E. and N.L. Craig, Tn7 recognizes transposition target structures associated with DNA replication using the DNA-binding protein TnsE. Genes Dev, 2001. 15: 737-47.
35.Hayes, F., Transposon-based strategies for microbial functional genomics and proteomics. Annu Rev Genet, 2003. 37: 3-29.
36.Kholodii, G.Y., et al., Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron. Mol Microbiol, 1995. 17: 1189-200.
37.Radstrom, P., et al., Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol, 1994. 176: 3257-68.
38.Shapiro, J.A. and P. Sporn, Tn402: a new transposable element determining trimethoprim resistance that inserts in bacteriophage lambda. J Bacteriol, 1977. 129: 1632-5.
39.Toleman, M.A., P.M. Bennett, and T.R. Walsh, Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons. J Antimicrob Chemother, 2006. 58: 1-6.
40.Toleman, M.A., P.M. Bennett, and T.R. Walsh, ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev, 2006. 70: 296-316.
41.Partridge, S.R. and R.M. Hall, In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob Agents Chemother, 2003. 47: 342-9.
42.Doi, Y., et al., Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate. Antimicrob Agents Chemother, 2002. 46: 2427-34.
43.Kado, C.I. and S.T. Liu, Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol, 1981. 145: 1365-73.
44.Kamali-Moghaddam, M. and L. Sundstrom, Transposon targeting determined by resolvase. FEMS Microbiol Lett, 2000. 186: 55-9.
45.Liebert, C.A., R.M. Hall, and A.O. Summers, Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev, 1999. 63: 507-22.
46.Brown, H.J., H.W. Stokes, and R.M. Hall, The integrons In0, In2, and In5 are defective transposon derivatives. J Bacteriol, 1996. 178: 4429-37.
47.Aubert, D., et al., Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of beta-lactam resistance genes. J Bacteriol, 2006. 188: 6506-14.
48.Burkardt, H.J., G. Riess, and A. Puhler, Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol, 1979. 114: 341-8.
49.Jobanputra, R.S. and N. Datta, Trimethoprim R factors in enterobacteria from clinical specimens. J Med Microbiol, 1974. 7: 169-77.
50.Yanisch-Perron, C., J. Vieira, and J. Messing, Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene, 1985. 33: 103-19.