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研究生:王書品
研究生(外文):Shu-Ping Wang
論文名稱:台灣北區多重抗藥菌株研究及其檢驗用基因晶片開發
論文名稱(外文):The Investigation of Multidrug Resistant Strains in Northern Taiwan and Application of Clinicl Diagnosis in Gene Chip
指導教授:王成德林如華
指導教授(外文):Cheng-Teh WangJu-Hwa Lin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:95
中文關鍵詞:多重抗藥菌株超廣效乙醯胺酶基因晶片
外文關鍵詞:multidrug resistant strainextended-spectrum beta-lactamasegene chipESBL
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摘 要
超廣效乙醯胺酶 (extended-spectrum β-lactamases,簡稱ESBLs) 基因可藉由質體自由地在菌種間傳播,使細菌獲得額外的抗藥性,而導致具有多重抗藥性的菌株產生。blaTEM和blaSHV是最常見的ESBLs基因,由於基因不斷地發生多點突變,使得blaTEM和blaSHV衍生出的ESBLs基因數目龐大,且數量持續地擴大,因而引發嚴重的抗藥性問題。為了深入探討多重抗藥菌株之抗藥分子機制,本論文針對經常發生多重抗藥性的Klebsiella pneumoniae和Escherichia coli,以抗微生物藥劑敏感性試驗,分析台灣北區臨床多重抗藥菌之抗藥表型。實驗結果發現,台灣北區多重抗藥的E. coli 和K. p.菌有70%為ESBLs菌株,而所有ESBLs菌株對β-lactam類抗生素 (ampicillin、piperacillin、cefazolin、cefuroxime、cefotaxime及aztreonam) 具有抗藥性,但對同屬於β-lactam類的cephamycin (cefoxitin及flomoxef) 卻較敏感。我們也研究blaTEM和blaSHV的基因型及分佈情形,以進一步瞭解其與菌株之抗藥表型間的關聯性。實驗結果發現,蒐集所得之TEM和SHV基因具有多處點突變,且TEM-type以blaTEM-1b (57%) 和新發現的blaTEM-A (28%) 為主,而SHV-type則以blaSHV-12 (63%) 居多。根據實驗發現TEM和SHV基因型與抗藥表型之關聯,我們也利用基因晶片可以檢測單點突變 (single nucleotide mutation) 的優點,設計檢驗TEM-和SHV-type基因型之基因晶片,並調整至最佳化。由於基因晶片具有快速、大量,及同時檢測多種抗藥性菌株之優點,因此適用於臨床醫療檢測,未來希望可以補現有醫療檢測系統之不足,選用正確抗生素,避免細菌產生抗藥性之問題。

ABSTRACT
Extended-spectrum β-lactamases (ESBLs) which predominantly encoded by plasmids can spread among different bacterial strains through conjugative dissemination of ESBL-encoding plasmids. With this, bacteria acquire additional resistance and generate multidrug resistance. ESBLs are predominantly derived from plasmid-mediated TEM- or SHV-type β-lactamases through one or more point mutations that lead to a large number of TEM or SHV derivatives causing serious drug resistance issue. To explore the molecular mechanism concerning multidrug resistance, we adopt an antimicrobial sensitivity testing method to analyze the resistance phenotypes that expressed from multidrug resistant isolates collected in northern Taiwan. We found that 70% of these isolates were ESBLs strains and all of them were resistant to β-lactam antibiotics (ampicillin, piperacillin, cefazolin, cefuroxime, cefotaxime and aztreonam), but most of them were sensitive to another kind of β-lactam antibiotics─cephamycin (cefoxitin and flomoxef). We also analyzed TEM and SHV genotypes and their distribution in order to understand the correlations between resistance genotypes and phenotypes. We found that the TEM and SHV isolates collected in this study contain multiple point mutations and most of the TEM isolates belong to the TEM-1b (57%) and another TEM-A (28%) types, however, most of SHV-12 isolates were the SHV-type (63%). According to the available TEM and SHV genotypes, we designed a gene chip that can detect most of the genotypes of TEM and SHV-ESBLs in clinical isolates, because gene chip has the advantage of detecting single nucleotide mutation. For the purpose of clinical diagnostics, we optimized the hybridization and washing conditions, and also probe design. Gene chips are suitable for use in clinical diagnosis because of the advantage of detecting many resistance strains at the same time. It is likely, in the future, that clinical diagnostic gene chip will be able to make up a deficiency of existing diagnostic system and help us to choice correct antibiotics, then avoid bacteria producing drug resistance.
目 錄
名詞縮寫…………………………………………………………………I
中文摘要…………………………………………………………………II
英文摘要…………………………………………………………………III
壹、 緒論……………………………………………………………1
貳、 材料與方法
一、 細菌菌株(Bacteria strains)………………………………16
二、 培養基(Culture media)………………………………………17
三、 抗微生物藥劑敏感性試驗(Antimicrobial sensitivity tests)……17
四、 DNA萃取(DNA extraction)……………………………………17
五、 ESBL基因篩檢(ESBL gene screening)………………………18
六、 DNA定序(DNA sequencing)……………………………………19
七、 探針設計(Probe design)………………………………………19
八、 探針固定(Immobilization)……………………………………21
九、 標的物製備(Target preparation)……………………………21
十、 晶片雜合反應(Hybridization)………………………………23
十一、 雜合訊號讀取及量化(Detection & Quantitation)…………24
參、 結果
一、多重抗藥菌株之研究…………………………………………………26
( 1 ) .多重抗藥菌經鑑定可分為ESBLs和non-ESBLs菌株………………26
( 2 ) .多重抗藥菌株之抗微生物藥劑敏感性試驗分析…………………26
( 3 ) .ESBL基因及AmpC基因之篩選與基因型鑑定………………………28
( 4 ) .分析ESBLs基因型分佈與菌株抗藥表型之關聯性………………32
二、建立TEM-和SHV-type基因型之基因晶片……………………………34
( 1 ) .設計TEM-和SHV-type基因型晶片及最佳化測試………………35
( 2 ) .設定基因晶片的臨床檢測標準…………………………………40
( 3 ) . 運用基因晶片檢驗TEM-和SHV-type基因型及菌種鑑定………41
肆、 討論……………………………………………………………44
伍、 參考文獻………………………………………………………61
陸、 附圖及表………………………………………………………69

參考文獻
Ambler, R. P. The structure of beta-lactamases. Philos. Trans. R. Soc. Lond. B. Bio. Sci. 289, 321-331 (1980).
Ambler, R. P. and Coulson, A. F. W. A standard numbering scheme for the Class A β-lactamases. Biochem. J. 276, 269-272 (1991).
Arlet, G., Brami, G., Décrè, D., Flippo, A., Gaillot, O., Lagrange, P. H. and Philippon, A. Molecular characterization by PCR-restriction fragment length polymorphism of TEM β-lactamases. FEMS Microbiol. Lett. 134, 203-208 (1995).
Arlet, G. and Philippon, A. Construction by polymerase chain reaction and intragenic DNA probes for three main types of transferable β-lactamases (TEM, SHV, CARB). FEMS Microbiol. Lett. 82, 19-26 (1991).
Babini, G. S. and Livermore, D. M. Are SHV-beta-lactamase universal in Klebsiella pneumoniae? Antimicrob. Agents Chemother. 44, 2230 (2000).
Bauernfeind, A. and Horl, G. Novel R-factor borne beta-lactamase of Escherichia coli confering resistance to cephalosporins. Infection 15, 257-259 (1987).
Bauernfeind, A., Stemplinger, I., Jungwirth, R., Wilhelm, R. and Giamarellou, H. Characterization of the plasmidic β-lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrob. Agents Chemother. 40, 221-224 (1996a).
Bauernfeind, A., Stemplinger, I., Jungwirth, R., Wilhelm, R. and Chong, Y. Comparative characterization of the cephalosporinase blaCMY-1 gene and its relationship with other β-lactamase genes. Antimicrob. Agents Chemother. 40, 1926-1930 (1996b).
Bush, K. Characterization of beta-lactamases. Antimicrob. Agents Chemother. 33, 259-263 (1989).
Bush, K., Jacoby, G. A. and Mederios, A. A. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39, 1211-1233 (1995).
Bush, K. and Jacoby, G. Nomenclature of TEM β-lactamase. Antimicrob. Agents Chemother. 39, 1-3 (1997).
Canica, M. M., Barthelemy, M., Gilly, L., Labia, R., Krishnamoorthy, R. and Paul, G. Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type β-lactamase. Antimicrob. Agents Chemother. 41, 374-378 (1997).
Chang, F. Y., Siu, L. K., Fung, C. P., Huang, M. H. and Ho, M. Diversity of SHV and TEM β-lactamases in klebsiella pneumoniae: gene evolution in northern Taiwan and two novel β-lactamases, SHV-25 and SHV-26. Antimicrob. Agents Chemother. Sept., 2407-2413 (2001).
Chaves, J., Ladona, M. G., Segura, C., Coira, A., Reig, R. and Ampurdanés, C. SHV-1 β-lactamase is mainly a chromosomally encoded species-specific enzyme in klebsiella pneumoniae. Antimicrob. Agents Chemother. 45, 2856-
2861 (2001).
Cuzin, M. DNA chip: a new tool for genetic analysis and diagnostics. Transfus. Clin. Biol. 8, 291-6 (2001).
Fiett, J., Palucha, A., Miacynska, B., Stankiewicz, M., Mordarska, H. P., Hryniewicz, W. and Gniadkowski, M. A novel complex mutant β-lactamase, TEM-68, identified in a Klebsiella pneumoniae isolate from an outbreak of extended-spectrum β-lactamase-producing Klebsiella. Antimicrob. Agents Chemother. 44, 1499-1505 (2000).
Fluit, A. C., Visser, M. R. and Schmitz, F. J. Molecular detection of antimicrobial resistance. Clin. Microbiol. Review. 14, 836-871 (2001).
Fodor, S. P., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T. and Solas, D. Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767-773 (1991).
Guo, Z., Liu, Q. and Smith, L. M. Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization. Nat. Biotechnol. 15, 331-335 (1997).
Horii, T., Arakawa, Y., Ohta, M., Sugiyama, T., Wacharotayankun, R., Ito, H. and Kato, N. Characterization of a plasmid-borne and constisutively expressed blaMOX-1 gene encoding AmpC-type β-lactamase. Gene 139, 93-98 (1994).
Imtiaz, U., Manavathu, E. K., Mobashery, S. and Lerner, S. A. Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 β-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime. Antimicrob. Agents Chemother. 38, 1134-1139 (1994).
Jacoby, G. A. and Carreras, I. Activities of β-lactam antibiotics against Escherichia coli strains producing extended-spectrum β-lactamases. Antimicrob. Agents Chemother. 34, 858-862 (1990).
Jelsch, C., Mourey, L., Masson, J. M. and Samama, J. P. Crystal structure of E. coli TEM-1 β-lactamase at 1.8 Å resolution. Protein: Struct. Funct. Genet. 16, 364-383 (1993).
Katsanis, G. P., Spargo, J. M., Ferraro, J., Sutton, L. and Jacoby, G. A. Detection of Klebsiella pneumoniae and Escherichia coli strains producing extended-spectrum β-lactamases. J. Clin. Microbiol. 32, 691-696 (1994).
Kim, J. and Lee, H. J. Rapid discriminatory detection of genes coding for SHV β-lactamases by ligase chain reaction. Antimicrob. Agents Chemother. 44, 1860-1864 (2000).
Kim, J., Kwon, Y., Pai, H., Kim, J. W. and Cho, D. T. Survey of Klebsiella pneumoniae strains producing extended-spectrum β-lactamase: prevalence of SHV-12 and SHV-2a in Korea. J. Clin. Microbiol. 36, 1446-1449 (1998).
Knox, J. R. Extended-spectrum and inhibitor-resistant TEM-type β-lactamases: mutation, specificity, and three-dimensional structure. Antimicrob. Agents Chemother. 39, 2593-2601 (1995).
Korfmann, G. and Wiedemann, B. Genetic control of β-lactamase production in Enterobacter cloacae. Ref. Infect. Dis. 10, 793-799 (1998).
Kuzin, A. P., Nukaga, M., Nukaga, Y., Hujer, A. M., Bonomo, R. A. and Knox, J. R. Structure of the SHV-1 β-lactamase. Biochemistry 38, 5720-5727 (1999).
Leonidas, S. T. and Robert, B. A. SHV-type β-lactamases. Curr. Pharm. Des. 5, 847-864 (1999).
Livermore, D.M., Moosdeen, F., Lindridge, M. A., Kho, P. and Williams, J. D. Behaviour of TEM-1 β-lactamases as a resistence mechanism to mezlocillin, ampicillin and azlocillin in Escherichia coli. J. Antimicrob. Chemother. 17, 139-146 (1986).
Livermore, D.M. and Seetulsingh, P. Susceptibility of Escherichia coli isolates with TEM-1 β-Lactamase to combinations of BRL42715, tazobactam or clavulanate with piperacillin or amoxycillin. J. Antimicrob. Chemother. 27, 761-767 (1991).
Livermore, D.M. Activity of sulbactam combinations against Escherichia coli isolates with known amounts of TEM-1 β-Lactamase. J. Antimicrob. Chemother. 29, 219-232 (1992).
Livermore, D.M. Determinants of the activity of β-Lactamases inhibitor combinations. J. Antimicrob. Chemother. 31 (Suppl. A), 9-21 (1993).
Livermore, D.M. β-Lactamases in laboratory and clinical resistance. Clin. Microbiol. Reviews Oct., 557-584 (1995).
Liu, P. Y. F., Tung, J. C., Ke, S. C. and Chen, S. L. Molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan. J. Clin. Microbiol. 36, 2759-2762 (1998).
Mabilat, C. and Courvalin, P. Development of “oligotyping” for characterization and molecular epidemiology of TEM β-lactamases in members of the family Enterobacteriaceae. Antimicrob. Agents Chemother. 34, 2210-2216 (1991).
Medeiros, A. A. Evolution and dissemination of β-lactamases accelerated by generations of β-lactam antibiotics. Clin. Infect. Dis. 24 (Suppl. 1), S19-S45
(1997).
M’Zali, F.-H., Gascoyne-Binzi, D. M., Heritage, J. and Hawkey, P. M. Detection of mutations conferring extended-spectrum activity on SHV β-lactamases using polymerase chain reaction single strand conformational polymorphism (PCR-SSCP). J. Antimicrob. Chemother. 37, 797-802 (1996).
National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Ninth informational supplement, M100-S9. National Committee for Clinical Laboratory Standards. Wayne, Pa. (1999).
Nester, E. W., Roberts, C. E. and Nester, M. T. Microbiology. Wm. C. Brown Publishers. Oxford, England (1995).
Nicolas-Chanoine, M. H. Inhibitor-resistant β-lactamases. J. Antimicrob. Chemother. 40, 1-3 (1997).
Pease, A. C., Solas, D. E., Sullivan, J. M., Cronin, T., Holmes, C. P. and Fodor, S. P. A. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. USA 91, 5022-5026 (1994).
Philippon, A., Arlet, G. and Lagrange, P. H. Origin and impact of plasmid- mediated extended-spectrum beta-lactamases. Eur. J. Clin. Microbiol. Infect. Dis. 13 (suppl. 1), 17-29 (1994).
Pozhitkov, A. E. Identifikazia miskroorganismos s pomoschtschju oligo- nukleotidni mikrochipow. Diploma thesis, Moscow. (in Russian) (1998).
Prinarakis, E. E., Tzelepi, V. E., Gazouli, M. and Tzouvelekis, L. S. Emergence of an inhibitor-resistant β-lactamase (SHV-10) derived from an SHV-5 variant. Antimicrob. Agents Chemother. 41, 838-840 (1997).
Reig, R., Roy, C., Hermida, M., Teruel, D. and Coira, A. A survey of β-Lactamases from 618 isolates of Klebsiella spp. J. Antimicrob. Chemother. 31, 29-35 (1993).
Richmond, M. H. and Sykes, R. B. The β-Lactamases of Gram-negative bacteria and their possible physiological role. Adv. Microb. Physiol. 9, 31-88 (1973).
Schena, M., Shalon, D., Davis, R. W. and Brown, P. O. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray. Science 270, 467-470 (1995).
Sirot, D., Recule, C., Chaibi, E. B., Bret, L., Croize, J., Chanal-Claris, C., Labia, R. and Sirot, J. A complex mutant of TEM-1 β-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an
Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 41, 1322-1325 (1997).
Siu, L. K., Lu, P. L., Hsueh, P. R., Lin, F. M., Chang, S. C., Luh, K. T., Ho, M. and Lee, C. Y. Bacteremia due to extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a pediatric oncology ward: clinical features and identification of different plasmids carrying both SHV-5 and TEM-1 genes. J. Clin. Microbiol. 37, 4020-4027 (1999).
Speldooren, V., Heym, B., Labia, R. and Nicolas-Chanoine, M.-H. Discriminatory detection of inhibitor-resistant β-lactamases in Escherichia coli by single-strand conformation polymorphism-PCR. Antimicrob. Agents Chemother. 42, 879-884 (1998).
Spratt, B. G. and Cromie, K. D. Penicillin-binding proteins of gram-negative bacteria. Rev. Infect. Dis. 10, 699-711 (1988).
Tenover, F. C., Mohammed, M. J., Gorton, T. S. and Dember, Z. F. Detection and reporting of organisms producing extended-spectrum β-lactamases: survey of laboratories in Connecticut. J. Clin. Microbiol. 37, 4065-4070 (1999).
Tham, T. N., Mabilat, C., Courvalin, P. and Guesdon, J.-L. Biotylated oligonucleotide probes for the detection and the characterization of TEM-type extended broad spectrum β-lactamases in Enterobacteriaceae. FEMS Microbiol. Lett. 69, 109-116 (1990).
Vercauteren, E., Descheemaeker, P., Ieven, M., Sanders, C. C. and Goossens, H. Comparison of screening methods for detection of extended-spectrum β-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital. J. Clin. Microbiol. 35, 2191-2197 (1997).
Waley, S. G. β-Lactamase: mechanism of action, p.198-228. In M. I. Page(ed.), The chemistry of β-lactams. A. and P. Blackie, London (1992).
Walsh, C. Molecular mechanisms that confer antibacterial resistance. Nature 406, 775-781 (2000).
Yagi, T., Kueokawa, H., Shibata, N., Shibayama, K. and Arakawa, Y. A preliminary survey of extended-spectrum β-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Japan. FEMS Microbiol. Lett. 184, 53-56 (2000).
Yan, J. J., Wu, S. M., Tsai, S. H., Wu, J. J. and Su, I. J. Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases and identification of a novel AmpC enzyme (CMY-8) in southern Taiwan. Antimicrob. Agents Chemother. 44, 1438-1442 (2000).
Yan, J. J., Ko, W. C. and Wu, J. J. Identification of a plasmid encoding SHV-12, TEM-1, and a variant of IMP-2 metallo-β-lactamase, IMP-8, from a clinical isolate of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45, 2368-2371 (2001).
蔡文城. 微生物學. 藝軒圖書出版社 台北市 (1996).

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