臺灣博碩士論文加值系統

克雷白氏肺炎菌（K. pneumoniae）屬於腸內菌，是一種革蘭氏陰性菌。它是人類腸道內常見的細菌，可以無症狀的出現在人類的皮膚，呼吸道和胃腸道等地方。一般來說它被視為一種伺機性感染的病原菌，可以在人類身上造成肺炎、傷口、軟組織或尿路感染。克雷白氏肺炎菌除了常見於各種院內感染之外，也偶爾在一些社區感染中被發現。它能在原本健康的個體上造成嚴重的感染，包括化膿性肝膿瘍、肺炎、腦膜炎等。它在我們生活的環境如土壤、水中都普遍存在，它不僅能造成人類的感染，也可以感染其他數種動物和植物。由於近年來抗藥性株與高毒性株的產生與散播，克雷白氏肺炎菌已經成為嚴重的健康威脅。莢膜被認為它重要的毒性因數之一，同時也是一個被用以分類的常見方法。基於莢膜的差異，克雷白氏肺炎菌可被分為約80種不同的K型，其中在台灣盛行的高毒性株多為K1和K2型，這些高毒性株在化膿性肝膿瘍佔據了很高的比例。過去我們發現了台灣有顯著比例的克雷白氏肺炎菌攜帶了一個新的pks基因毒性，能夠造成宿主的DNA破壞。我們用PCR的方法篩選2012年來自全台不同醫院的共400多株克雷白氏肺炎菌，發現帶pks基因的菌株中有10株屬於K62型，並且這些K62同時攜帶iucA（Aerobactin）、rmpA（Hypermucoidy）和ybtA（Yersiniabactin）這3種毒性基因，我們認為K62型極可能是一個新興的高毒性株，並且已經廣泛的存在台灣的各地。然而由於過去並無K62型的基因體研究，我們挑選了一株K62型的克雷白氏肺炎菌HLH157，完成了它的全基因體定序及註解，並且利用比較基因體學的方法，參照資料庫裡已知其他克雷白氏肺炎菌K型的基因序列找出了K62 特異的遺傳序列，設計了基於這些獨特序列的PCR快速鑑定方法，並與目前的其他已知方法進行比較，我們設計的方法能夠100%鑒定K62型，錯誤率0.24%。本次測試的克雷白氏肺炎菌中26株是K62型（6.5%）。除了已知的11株pks-positive占全部K62型的38.5%，其餘15株都是pks-negative（61.5%）。除此之外，比較基因體學的分析也找到了HLH157中攜帶了pks及其他毒性基因的高毒性基因小島。未來應能利用本研究所開發的快速篩檢K62的PCR方法，在近幾年中克雷白氏肺炎菌臨床菌中進行篩檢，並且進行基因體學分析以深入研究這個新興毒性株在台灣的臨床菌群體組成和盛行率。

Klebsiella pneumoniae is a Gram-negative bacterium belonging to the family Enterobacteriaceae. It is originally considered an opportunistic pathogen that causes hospital-acquired infections. Reports of community-acquired infection caused by virulent K. pneumoniae strains were increasing worldwide. K. pneumoniae can be classified into about 80 K-types based on the capsular polysaccharide (CPS). Among these, K1 and K2 are the dominant K-types in Taiwan and were reported in invasive infections such as pyogenic liver abscesses. Previously we have identified a genotoxic phenotype among the K. pneumoniae clinical isolates in a hospital. Subsequent analysis revealed a K62 clone among the genotoxic K. pneumoniae strains, which is likely a emerging virulent clone widespread in Taiwan. Since current information on the genome of K62 is scarce, we have conduct complete sequencing and analyses on the genome of a K. pneumoniae K62 genotoxic strain HLH157. Annotation and comparative genomics analyses with other K. pneumoniae genomes were carried out to identify virulence genes and the pathogenic genomic island components. Moreover, comparative analyses on the genomic region of the CPS genes were carried out to identify K62-specific sequences. A rapid PCR detection method of K62 were developed and the test on our K. pneumoniae clinical isolates appeared to surpass the current K-typing methods. The PCR detection developed in the present study will build a solid basis for future epidemiological study on K. pneumoniae K62 and enhance the study on this emerging virulence clone.

誌謝辭 i
中文摘要 ii
Abstract iv
目錄 v
表目錄 viii
圖目錄 viii
第一章 前言 1
1.1克雷白氏肺炎菌 1
1.2克雷白氏肺炎菌的分類方法 1
1.2.1 為什麼要建立分類 1
1.2.2 K抗原分型（K type） 2
1.3.3 多位點序列分型（MLST） 3
1.3 克雷白氏肺炎菌的分佈 4
1.3.1 克雷白氏肺炎菌感染是全球性的問題 4
1.3.2 在台灣克雷白氏肺炎菌的感染的問題 5
1.4 克雷白氏肺炎菌的抗藥性 6
1.4.1 克雷白氏肺炎菌的抗藥性 6
1.4.2 克雷白氏肺炎菌抗藥性與K types的關係 8
1.5 克雷白氏肺炎菌的毒性 9
1.5.1　克雷白氏肺炎菌的毒性因子 9
1.5.2 克雷白氏肺炎菌毒性與 K types的關係 11
1.6 克雷白氏肺炎菌K62型 12
1.6.1 克雷白氏肺炎菌K62的發現 12
1.6.2克雷白氏肺炎菌K62的已知研究 13
1.6.3 克雷白氏肺炎菌K62現有的鑒定方法 14
1.7 研究目的 14
第二章 材料與方法 15
2.1 測試菌株的來源 15
2.2 HLH157的定序 15
2.3 基因體學比較分析軟體 15
2.4 PCR的材料和方法 16
2.4.1聚合酶鏈鎖反應（Polymerase Chain Reaction, PCR） 16
2.4.2 配製電泳膠 17
2.4.3 瓊脂膠體電泳（Agarose Gel Electrophoresis） 18
第三章 實驗結果與討論 19
3.1 HLH157（K62）全基因體分析 19
3.1.1 基因組拼接與註解 19
3.1.2全基因組比對 20
3.2 HLH157（K62）毒性基因島分析 20
3.3 K62 cps region相關研究 22
3.3.1 K62的cps組成分析 22
3.3.2 K62 特異性探針設計思路 23
3.3.3 K62 primer泛性測試結果與討論 24
3.4 未來方向 26
參考文獻 28




 

參考文獻
1.Shon AS, Bajwa RP, Russo TA. Hypervirulent (hypermucoviscous) Klebsiella
pneumoniae: a new and dangerous breed. Virulence. 2013 Feb 15;4(2):107-18.
2.Struve C, Krogfelt KA. Role of capsule in Klebsiella pneumoniae virulence: lack of correlation between in vitro and in vivo studies. FEMS Microbiol Lett. 2003 Jan 21;218(1):149-54.
3.Wyres KL, Holt KE. Klebsiella pneumoniae Population Genomics and Antimicrobial-Resistant Clones. Trends Microbiol. 2016 Dec;24(12):944-56.
4.Chun J, Rainey FA. Integrating genomics into the taxonomy and systematics of
the Bacteria and Archaea. Int J Syst Evol Microbiol. 2014 Feb;64(Pt 2):316-24.
5.Shu HY, Fung CP, Liu YM, Wu KM, Chen YT, Li LH, Liu TT, Kirby R, Tsai SF. Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates. Microbiology. 2009 Dec;155(Pt 12):4170-83.
6.Julianelle LA. a biological classification of encapsulatus pneumoniae (friedlander's bacillus). J Exp Med. 1926 Jun 30;44(1):113-28.
7.Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology,
taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998 Oct; 11(4):589-603.
8.Yu WL, Ko WC, Cheng KC, Lee CC, Lai CC, Chuang YC. Comparison of prevalence of virulence factors for Klebsiella pneumoniae liver abscesses between isolates with capsular K1/K2 and non-K1/K2 serotypes. Diagn Microbiol Infect Dis. 2008 Sep;62(1):1-6.
9.Pan YJ, Lin TL, Chen YH, Hsu CR, Hsieh PF, Wu MC, Wang JT. Capsular types of Klebsiella pneumoniae revisited by wzc sequencing. PLoS One. 2013 Dec 9;8(12):e80670.
10.Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3140-5.
11.Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005 Aug;43(8):4178-82.
12.Urwin R, Maiden MC. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 2003 Oct;11(10):479-87.
13.Chen L, Mathema B, Chavda KD, DeLeo FR, Bonomo RA, Kreiswirth BN. Carbapenemase-producing Klebsiella pneumoniae: molecular and genetic decoding. Trends Microbiol. 2014 Dec;22(12):686-96.
14.Bowers JR, Kitchel B, Driebe EM, MacCannell DR, Roe C, Lemmer D, de Man T, Rasheed JK, Engelthaler DM, Keim P, Limbago BM. Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic. PLoS One. 2015 Jul 21;10(7):e0133727.
15.Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Livermore DM, Maya JJ, Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV, Wang H, Woodford N, Quinn JP. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis. 2013 Sep;13(9):785-96.
16.Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing
Enterobacteriaceae. Emerg Infect Dis. 2012 Sep;18(9):1503-7.
17.Lee CR, Lee JH, Park KS, Kim YB, Jeong BC, Lee SH. Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods. Front Microbiol. 2016 Jun 13;7:895.
18.Lai YC, Lin AC, Chiang MK, Dai YH, Hsu CC, Lu MC, Liau CY, Chen YT. Genotoxic Klebsiella pneumoniae in Taiwan. PLoS One. 2014 May 22;9(5):e96292.
19.Chen YT, Lai YC, Tan MC, Hsieh LY, Wang JT, Shiau YR, Wang HY, Lin AC, Lai JF, Huang IW, Lauderdale TL. Prevalence and characteristics of pks genotoxin gene cluster-positive clinical Klebsiella pneumoniae isolates in Taiwan. Sci Rep. 2017 Feb 24;7:43120.
20.Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL.
Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving
crisis of global dimensions. Clin Microbiol Rev. 2012 Oct;25(4):682-707.
21.Kuskucu MA, Karakullukcu A, Ailiken M, Otlu B, Mete B, Aygun G. Investigation of carbapenem resistance and the first identification of Klebsiella pneumoniae carbapenemase (KPC) enzyme among Escherichia coli isolates in Turkey: A prospective study. Travel Med Infect Dis. 2016 Nov - Dec;14(6):572-76.
22.Landman D, Babu E, Shah N, Kelly P, Olawole O, Bäcker M, Bratu S, Quale J.
Transmission of carbapenem-resistant pathogens in New York City hospitals:
progress and frustration. J Antimicrob Chemother. 2012 Jun;67(6):1427-31.
23.Nordmann P, Poirel L, Walsh TR, Livermore DM. The emerging NDM carbapenemases.Trends Microbiol. 2011 Dec;19(12):588-95.
24.Alevizakos M, Kallias A, Flokas ME, Mylonakis E. Colonization with extended-spectrum beta-lactamase-producing Enterobacteriaceae in solid organ transplantation: A meta-analysis and review. Transpl Infect Dis. 2017 May 4.
25. Haller S, Eller C, Hermes J, Kaase M, Steglich M, Radonić A, Dabrowski PW, Nitsche A, Pfeifer Y, Werner G, Wunderle W, Velasco E, Abu Sin M, Eckmanns T, Nübel U. What caused the outbreak of ESBL-producing Klebsiella pneumoniae in a neonatal intensive care unit, Germany 2009 to 2012? Reconstructing transmission with epidemiological analysis and whole-genome sequencing. BMJ Open. 2015 May 11;5(5):e007397.
26.Sękowska A, Gospodarek E, Kamińska D. Antimicrobial susceptibility and genetic similarity of ESBL-positive Klebsiella pneumoniae strains. Arch Med Sci. 2012 Dec 20;8(6):993-7.
27.Pan YJ, Lin TL, Lin YT, Su PA, Chen CT, Hsieh PF, Hsu CR, Chen CC, Hsieh YC, Wang JT. Identification of capsular types in carbapenem-resistant Klebsiella pneumoniae strains by wzc sequencing and implications for capsule depolymerase treatment. Antimicrob Agents Chemother. 2015 Feb;59(2):1038-47.
28.Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, Jenney A,
Connor TR, Hsu LY, Severin J, Brisse S, Cao H, Wilksch J, Gorrie C, Schultz MB,
Edwards DJ, Nguyen KV, Nguyen TV, Dao TT, Mensink M, Minh VL, Nhu NT, Schultsz C, Kuntaman K, Newton PN, Moore CE, Strugnell RA, Thomson NR. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3574-81.
29. Chiang TT, Yang YS, Yeh KM, Chiu SK, Wang NC, Lin TY, Huang LY, Chang FY, Siu LK, Lin JC, Chen JH. Quantification and comparison of virulence and characteristics of different variants of carbapenemase-producing Klebsiella pneumoniae clinical isolates from Taiwan and the United States. J Microbiol Immunol Infect. 2016 Feb;49(1):83-90.
30. Bossuet-Greif N, Dubois D, Petit C, Tronnet S, Martin P, Bonnet R, Oswald E, Nougayrède JP. Escherichia coli ClbS is a colibactin resistance protein. Mol Microbiol. 2016 Mar;99(5):897-908.
31.de Lorenzo V, Martinez JL. Aerobactin production as a virulence factor: a reevaluation. Eur J Clin Microbiol Infect Dis. 1988 Oct;7(5):621-9.
32.Carniel E. The Yersinia high-pathogenicity island: an iron-uptake island. Microbes Infect. 2001 Jun;3(7):561-9.
33. Perry RD, Balbo PB, Jones HA, Fetherston JD, DeMoll E. Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation. Microbiology. 1999 May;145 (Pt 5):1181-90.
34.Lawlor MS, O'connor C, Miller VL. Yersiniabactin is a virulence factor for Klebsiella pneumoniae during pulmonary infection. Infect Immun. 2007 Mar; 75(3):1463-72.
35.Bachman MA, Oyler JE, Burns SH, Caza M, Lépine F, Dozois CM, Weiser JN. Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2. Infect Immun. 2011 Aug;79(8):3309-16.
36.Fung CP, Chang FY, Lin JC, Ho DM, Chen CT, Chen JH, Yeh KM, Chen TL, Lin YT, Siu LK. Immune response and pathophysiological features of Klebsiella pneumoniae liver abscesses in an animal model. Lab Invest. 2011 Jul;91(7):1029-39.
37.Fung CP, Chang FY, Lee SC, Hu BS, Kuo BI, Liu CY, Ho M, Siu LK. A global emerging disease of Klebsiella pneumoniae liver abscess: is serotype K1 an important factor for complicated endophthalmitis? Gut. 2002 Mar;50(3):420-4.
38.Kuo SC, Chang SC, Wang HY, Lai JF, Chen PC, Shiau YR, Huang IW, Lauderdale TL; TSAR Hospitals. Emergence of extensively drug-resistant Acinetobacter baumannii complex over 10 years: nationwide data from the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program. BMC Infect Dis. 2012 Aug 28;12:200.
39.Kabha K, Nissimov L, Athamna A, Keisari Y, Parolis H, Parolis LA, Grue RM,
Schlepper-Schafer J, Ezekowitz AR, Ohman DE, et al. Relationships among capsular
structure, phagocytosis, and mouse virulence in Klebsiella pneumoniae. Infect Immun. 1995 Mar;63(3):847-52.
40.Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008 Feb 8;9:75.
41. Chen YT, Chang HY, Lai YC, Pan CC, Tsai SF, Peng HL. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. Gene. 2004 Aug 4;337:189-98.
42.Tang HL, Chiang MK, Liou WJ, Chen YT, Peng HL, Chiou CS, Liu KS, Lu MC, Tung KC, Lai YC. Correlation between Klebsiella pneumoniae carrying pLVPK-derived loci and abscess formation. Eur J Clin Microbiol Infect Dis. 2010 Jun;29(6):689-98.
43.Doerks T, Copley RR, Schultz J, Ponting CP, Bork P. Systematic identification of novel protein domain families associated with nuclear functions. Genome Res. 2002 Jan;12(1):47-56.
44.Whitehead NA, Barnard AM, Slater H, Simpson NJ, Salmond GP. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol Rev. 2001 Aug;25(4):365-404.
45.Putze J, Hennequin C, Nougayrède JP, Zhang W, Homburg S, Karch H, Bringer MA, Fayolle C, Carniel E, Rabsch W, Oelschlaeger TA, Oswald E, Forestier C, Hacker J,Dobrindt U. Genetic structure and distribution of the colibactin genomic island among members of the family Enterobacteriaceae. Infect Immun. 2009 Nov;77(11):4696-703.
46.Pan YJ, Lin TL, Chen CT, Chen YY, Hsieh PF, Hsu CR, Wu MC, Wang JT. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp. Sci Rep. 2015 Oct 23;5:15573.
47.Inoue A, Murata Y, Takahashi H, Tsuji N, Fujisaki S, Kato J. Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli. J Bacteriol. 2008 Nov;190(21):7298-301.