臺灣博碩士論文加值系統

本研究是要確定超廣譜乙型內醯胺?“J雷白氏肺炎桿菌(ESBL-KP)
的blaSHV 基因密碼子238 和240 多型性，使用高解析度熔解曲線(HRM)
和DNA 序列分析技術，HRM 技術使用PCR 擴增產物和飽和DNA 染
料結合的方式記錄高解析度熔解曲線對樣品進行檢測。2010 年1 月
至2011 年12 月從高雄市立小港醫院分離出超廣譜乙型內醯胺??
(ESBL)的克雷白氏肺炎桿菌中blaSHV 基因，在114 株分離出之
ESBL-KP 菌株中有85 株(74.56％)是野生株( GGC- GAG→ GGCGAG)
、3 株(2.63％) G238S (GAG-GAG→AAG- GAG)突變、4 株(3.51
％) G238S 240 E240K (GGC- GAG→GGC- AAG) 突變、22 株(19.30
％)密碼子238 和240 (GGC- GAG→AGC- AAG )具有雙重突變。本實
驗HRM 方法敏感度100.0％、特異性100.0％、陽性預測值100.0％、
陰性預測值100.0％。我們的結論是在分子流行病學研究上以高解析
度熔解曲線為基礎的方法檢測也是另一種基因型鑑定的工具。

The aim of this study was to investigate the blaSHV gene codon 238 and 240 polymorphisms in extended-spectrum beta-lactamase(ESBL) Klebsiella pneumoniae. In this study, the high-resolution melting (HRM) and DNA sequencing were used as the research tools. In the technology of HRM, the PCR products and the saturated DNA dye were used to record the result of the test of the sample by using the high-resolution melting curves.The blaSHV gene was isolated from ESBL- Klebsiella pneumoniae at Kaohsiung Municipal Hsiaokang Hospital from January, 2010 to December, 2011. Among 114 isolated ESBL-KP, there were 85strains (74.56％) wild type ( GGC- GAG ) mutant, 3 strains (2.63％) codon 238 G238S ( GGC→AGC) mutant, 4 strains (3.51％) codon 240 E240K ( GAG→AAG) mutant, and 22 strains (19.30％) codon 238 and 240 (GGC- GAG →AGC- AAG ) with double mutants. The technology has 100.0% of sensitivity, 100.0% of specificity, 100.0% of positive predictive value, and 100.0% of negative predictive value.We can conclude that in molecular epidemiological studies, the high-resolution melting (HRM) is a good identification tool for bacteria genotyping.

第一章 緒論……………………………………………………………..1
第二章 文獻回顧……………………………………………………….4
第一節 腸道桿菌科細菌…………………………………………..4
第二節 細菌抗藥性機轉…………………………………………..6
第三節 beta-lactam 抗藥性機轉…………………………………....8
第四節 ESBLs ………………………………………………………9
第五節 ESBL 表現型測定方法 …………………………………..13
第六節 HRM 分析………………………………………………….14
第三章 研究動機 …………………………………………………...15
第四章 研究目的……………………………………………………16
第五章 研究方法 ………………………………………………….17
第一節 研究設計 ………………………………………………17
第二節 方法和材料 ……………………………………………17
VI
1. 研究對象和樣本收集 …………….………………18
2. ESBL 表現型測定方法…………………………….19
3. 藥物感受性試驗 …………………………………. ..20
4. PCR 分析方法 ……………………………………...21
5. 內部控管方法 ……………………………………...21
6. DNA 序列分析方法…………………………….…..22
7. HRM 分析方法……………………………………..26
第六章 研究結果………………………………………………….27
第一節 菌株之收集資料 ………………………………….…..27
第二節 ESBL 表現型測定結果……………………………….27
第三節 藥物感受性試驗結果 …………………………….….27
第四節 PCR 分析結果 ………………………………………..28
第五節 內部控管結果…………………………………………29
第六節 DNA 序列分析結果……………………………….…..29
第七節 HRM 分析結果………………………………………..30
第七章 結論與討論 ………………………………..…………….32
第一節 本實驗優點…………………………………………….32
第二節 本實驗限制…………………………………………….33
第三節 與其他文獻藥物感受性比較 ………………..……….34
VII
第四節 與國內其他文獻ESBL-KP SHV gene 比較………….35
第五節 與亞太地區ESBL-KP 陽性率比較……………………36
第六節 亞太地區、中國和印度 ESBL-KP 的頻率在社區和醫院性
感染………………………………………………………..37
第七節與國外文獻ESBL-KP其他基因基因型比較…………….38
第八節 總結和未來展望….……………………………………...39
第八章 參考文獻 ………………………………………………40
附錄
流程圖1 HRM結果判定流程圖…………………………………….82
流程圖2 實驗流程圖……………………………………………....

Giamarellou, H., Multidrug resistance in Gram-negative bacteria that produce
extended-spectrum beta-lactamases (ESBLs). Clin Microbiol Infect, 2005. 11 Suppl
4: p. 1-16.
2. Babic, M., A.M. Hujer, and R.A. Bonomo, What''s new in antibiotic resistance? Focus
on beta-lactamases. Drug Resist Updat, 2006. 9(3): p. 142-56.
3. Paterson, D.L. and R.A. Bonomo, Extended-spectrum beta-lactamases: a clinical
update. Clin Microbiol Rev, 2005. 18(4): p. 657-86.
4. Schooneveldt, J.M., G.R. Nimmo, and P. Giffard, Detection and characterisation of
extended spectrum beta-lactamases in Klebsiella pneumoniae causing nosocomial
infection. Pathology, 1998. 30(2): p. 164-8.
5. Woksepp, H., et al., High-resolution melting-curve analysis of ligation-mediated
real-time PCR for rapid evaluation of an epidemiological outbreak of
extended-spectrum-beta-lactamase-producing Escherichia coli. J Clin Microbiol,
2011. 49(12): p. 4032-9.
6. Turner, M.S., et al., Plasmid-borne blaSHV genes in Klebsiella pneumoniae are
associated with strong promoters. J Antimicrob Chemother, 2009. 64(5): p. 960-4.
7. 蔡文城, ed. 實用臨床微生物診斷學. 第七版 ed. 1993, 九州圖書公司: 台北,台灣.
8. Topaloglu, R., et al., Risk factors in community-acquired urinary tract infections
caused by ESBL-producing bacteria in children. Pediatr Nephrol.
9. Kuster, S.P., et al., Risks Factors for Infections with Extended-Spectrum
Beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae at a Tertiary
Care University Hospital in Switzerland. Infection.
10. Tsai, F.C., et al., Pyogenic liver abscess as endemic disease, Taiwan. Emerg Infect
Dis, 2008. 14(10): p. 1592-600.
11. Chen, S.C., et al., Comparison of Escherichia coli and Klebsiella pneumoniae liver
abscesses. Am J Med Sci, 2007. 334(2): p. 97-105.
12. Mandell, G., B. JE, and D. R, eds. Principles and practice of infectious diseases.
sixth ed. 2005, Elsevier: Philadelphia.
13. Nikaido, H., Molecular basis of bacterial outer membrane permeability revisited.
Microbiol Mol Biol Rev, 2003. 67(4): p. 593-656.
14. Srikumar, R., X.Z. Li, and K. Poole, Inner membrane efflux components are
responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas
aeruginosa. J Bacteriol, 1997. 179(24): p. 7875-81.
15. Malouin, F. and L.E. Bryan, Modification of penicillin-binding proteins as
mechanisms of beta-lactam resistance. Antimicrob Agents Chemother, 1986. 30(1):
p. 1-5.
Poirel, L., et al., Long-term carriage of NDM-1-producing Escherichia coli. J
Antimicrob Chemother, 2011. 66(9): p. 2185-6.
17. Ambler, R., ed. The structure of beta-lactamases. Vol. 289. 1980, Philosophical
Transactions of the Royal Society of London - Series B: Biological Sciences. 321-31.
18. Joris, B., et al., The active-site-serine penicillin-recognizing enzymes as members of
the Streptomyces R61 DD-peptidase family. Biochem J, 1988. 250(2): p. 313-24.
19. Garau, G., et al., Update of the standard numbering scheme for class B
beta-lactamases. Antimicrob Agents Chemother, 2004. 48(7): p. 2347-9.
20. Bush, K., G.A. Jacoby, and A.A. Medeiros, A functional classification scheme for
beta-lactamases and its correlation with molecular structure. Antimicrob Agents
Chemother, 1995. 39(6): p. 1211-33.
21. Bradford, P.A., Extended-spectrum beta-lactamases in the 21st century:
characterization, epidemiology, and detection of this important resistance threat. Clin
Microbiol Rev, 2001. 14(4): p. 933-51, table of contents.
22. Philippon, A., G. Arlet, and G.A. Jacoby, Plasmid-determined AmpC-type
beta-lactamases. Antimicrob Agents Chemother, 2002. 46(1): p. 1-11.
23. Walsh, T.R., et al., Metallo-beta-lactamases: the quiet before the storm? Clin
Microbiol Rev, 2005. 18(2): p. 306-25.
24. Erali, M., R. Palais, and C. Wittwer, SNP genotyping by unlabeled probe melting
analysis. Methods Mol Biol, 2008. 429: p. 199-206.
25. Erali, M., K.V. Voelkerding, and C.T. Wittwer, High resolution melting applications for
clinical laboratory medicine. Exp Mol Pathol, 2008. 85(1): p. 50-8.
26. Galloway, R.L. and P.N. Levett, Application and validation of PFGE for serovar
identification of Leptospira clinical isolates. PLoS Negl Trop Dis, 2010. 4(9).
27. Chen, M.H., et al., Pulsed field gel electrophoresis (PFGE) analysis for multidrug
resistant Salmonella enterica serovar Schwarzengrund isolates collected in six years
(2000-2005) from retail chicken meat in Taiwan. Food Microbiol, 2011. 28(3): p.
399-405.
28. Kilian, M., C. Scholz, and H.B. Lomholt, Multilocus Sequence Typing (MLST) and
Phylogenetic Analysis of Propionibacterium acnes. J Clin Microbiol, 2011.
29. Bennett, D.E. and M.T. Cafferkey, Multilocus restriction typing: a tool for Neisseria
meningitidis strain discrimination. J Med Microbiol, 2003. 52(Pt 9): p. 781-7.
30. Ereqat, S., et al., Rapid differentiation of Mycobacterium tuberculosis and M. bovis
by high-resolution melt curve analysis. J Clin Microbiol, 2010. 48(11): p. 4269-72.
31. Tong, S.Y., et al., High-resolution melting genotyping of Enterococcus faecium based
on multilocus sequence typing derived single nucleotide polymorphisms. PLoS One,
2011. 6(12): p. e29189.
32. Chia, J.H., et al., Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of
Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan. J Clin
Microbiol, 2005. 43(9): p. 4486-91.
33. Andersson, P., et al., Analysis of bla(SHV) codon 238 and 240 allele mixtures using
Sybr green high-resolution melting analysis. Antimicrob Agents Chemother, 2009.
53(6): p. 2679-83.
34. Cerqueira, G.M., et al., Monitoring Leptospira strain collections: the need for quality
control. Am J Trop Med Hyg, 2010. 82(1): p. 83-7.
35. M.R.Roozbhani, et al., PCR-Based Detection of Yersinia ruckeri Infection in
Rainbow Trout Fish. Asian Journal of Animal and Veterinary Adances
4(5):258-262,2009, 2009(1683-9919): p. 258-262.
36. Chaves, J., et al., SHV-1 beta-lactamase is mainly a chromosomally encoded
species-specific enzyme in Klebsiella pneumoniae. Antimicrob Agents Chemother,
2001. 45(10): p. 2856-61.
37. Ambler, R.P., et al., A standard numbering scheme for the class A beta-lactamases.
Biochem J, 1991. 276 ( Pt 1): p. 269-70.
38. de Juan, I., et al., High-resolution melting analysis for rapid screening of BRCA1 and
BRCA2 Spanish mutations. Breast Cancer Res Treat, 2009. 115(2): p. 405-14.
39. Wittwer, C.T., High-resolution DNA melting analysis: advancements and limitations.
Hum Mutat, 2009. 30(6): p. 857-9.
40. Hammond, D.S., et al., Selection of SHV
extended-spectrum-beta-lactamase-dependent cefotaxime and ceftazidime
resistance in Klebsiella pneumoniae requires a plasmid-borne blaSHV gene.
Antimicrob Agents Chemother, 2008. 52(2): p. 441-5.
41. Hammond, D.S., et al., bla(SHV) Genes in Klebsiella pneumoniae: different allele
distributions are associated with different promoters within individual isolates.
Antimicrob Agents Chemother, 2005. 49(1): p. 256-63.
42. Liao, C.H., et al., In vitro activities of 16 antimicrobial agents against clinical isolates
of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella
pneumoniae in two regional hospitals in Taiwan. J Microbiol Immunol Infect, 2006.
39(1): p. 59-66.
43. Yu, W.L., Y.C. Chuang, and R.N. Jones, A pragmatic approach to identify
extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in Taiwan: in
vitro activity of newer and established antimicrobial agents. Diagn Microbiol Infect
Dis, 2004. 48(4): p. 277-82.
44. Hirakata, Y., et al., Regional variation in the prevalence of extended-spectrum
beta-lactamase-producing clinical isolates in the Asia-Pacific region (SENTRY
1998-2002). Diagn Microbiol Infect Dis, 2005. 52(4): p. 323-9.
Ko, W.C. and P.R. Hsueh, Increasing extended-spectrum beta-lactamase production
and quinolone resistance among Gram-negative bacilli causing intra-abdominal
infections in the Asia/Pacific region: data from the Smart Study 2002-2006. J Infect,
2009. 59(2): p. 95-103.
46. Lin, C.J., et al., Molecular Epidemiology of Ciprofloxacin-Resistant
Extended-Spectrum beta-Lactamase-Producing Klebsiella pneumoniae in Taiwan.
Microb Drug Resist, 2012. 18(1): p. 52-8.
47. Liu, P.Y., et al., Molecular epidemiology of extended-spectrum
beta-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in
Taiwan. J Clin Microbiol, 1998. 36(9): p. 2759-62.
48. Yan, J.J., et al., Prevalence of SHV-12 among clinical isolates of Klebsiella
pneumoniae producing extended-spectrum beta-lactamases and identification of a
novel AmpC enzyme (CMY-8) in Southern Taiwan. Antimicrob Agents Chemother,
2000. 44(6): p. 1438-42.
49. Chang, F.Y., et al., Diversity of SHV and TEM beta-lactamases in Klebsiella
pneumoniae: gene evolution in Northern Taiwan and two novel beta-lactamases,
SHV-25 and SHV-26. Antimicrob Agents Chemother, 2001. 45(9): p. 2407-13.
50. Hawkey, P.M., Prevalence and clonality of extended-spectrum beta-lactamases in
Asia. Clin Microbiol Infect, 2008. 14 Suppl 1: p. 159-65.
51. Hawser, S.P., et al., Emergence of high levels of
extended-spectrum-beta-lactamase-producing gram-negative bacilli in the
Asia-Pacific region: data from the Study for Monitoring Antimicrobial Resistance
Trends (SMART) program, 2007. Antimicrob Agents Chemother, 2009. 53(8): p.
3280-4.
52. Manoharan, A., et al., Phenotypic & molecular characterization of AmpC
beta-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from
five Indian Medical Centers. Indian J Med Res, 2012. 135(3): p. 359-64.
53. Lee, C.H., et al., Spread of ISCR1 elements containing blaDHA-(1) and multiple
antimicrobial resistance genes leading to increase of flomoxef resistance in
extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae. Antimicrob
Agents Chemother, 2011. 55(9): p. 4058-63.
54. Lee, M.Y., et al., High prevalence of CTX-M-15-producing Klebsiella pneumoniae
isolates in Asian countries: diverse clones and clonal dissemination. Int J Antimicrob
Agents, 2011. 38(2): p. 160-3.
55. Nakamura, T., et al., Epidemiology of Escherichia coli, Klebsiella species, and
Proteus mirabilis strains producing extended-spectrum beta-lactamases from clinical
samples in the Kinki Region of Japan. Am J Clin Pathol, 2012. 137(4): p. 620-6.