臺灣博碩士論文加值系統

克雷白氏肺炎菌 (Klebsiella pneumoniae) 為一種革蘭氏陰性菌，常見於人類、動物身上，以及其他自然環境中，被視為是一種伺機型病原菌，不僅能造成院內感染，也跟許多社區型感染疾病有關。近年來，由於其多重抗藥性菌株以及高毒性菌株的出現，被認為是對人類健康的重大威脅。克雷白氏肺炎菌重要的毒性因子包含莢膜、脂多醣體、黏附因子、螯鐵因子 (aerobactin、yersiniabactin) ，以及colibactin等。不同莢膜類型的菌株，毒性也有所差異，其中K1以及K2的菌株毒性最高，而K7與K21的毒性則較不具致病力。Colibactin為一種基因毒素，由pks基因體小島上的多種酵素作用產生，能造成宿主的DNA雙股產生斷裂，進而使細胞週期停滯、細胞凋亡以及染色體不穩定，長期下來可能導致癌症的產生。先前我們團隊調查了pks基因體小島在台灣的克雷白氏肺炎菌中的盛行率，發現pks (＋) 的克雷白氏肺炎菌在台灣的盛行率為25.6%，遠高於國外，此外，經過PFGE電泳所呈現的親緣樹中，除了原本在台灣較為盛行的K1、K2、K20、K57之外，還發現一群相似度非常高的K62型，這個莢膜類型的克雷白氏肺炎菌在過去的研究中很少被報導，但在台灣的臨床株中卻有相當比例的K62型，因此我們推測K62型很可能在台灣發展成了新興毒性株。我們團隊先前完成一株pks (＋) 的K62型，HLH157的全基因體定序與註解，並設計出能透過PCR快速檢測K62型的方法，也找出HLH157上帶有的毒性基因體小島與其他毒性基因。為了解K62型是否在台灣發展成新興毒性株，本研究透過細胞感染模式來驗證pks對K62型毒性的關聯性，並使用PCR分析這些菌株的毒性基因體小島組成，PCR難以判別的部分則利用Nanopore定序對這些區域做進一步的比較基因體學分析，發現pks (＋) 之K62型具有非常相似的毒性基因體小島，這種基因體小島的組成對其毒性可能有很大的關連。除此之外，本研究在pks (＋) 的K62型上都發現具有個大小超過200 kb的質體，經由比對發現與先前研究已知的毒性質體pLVPK、pK2044極為相似，與近期另一篇關於高毒性克雷白氏肺炎菌clonal-group 23 (K1)的研究做比較，可以看到這些高毒性株絕大多數都同時具有pks和毒性質體，可以推測pks以及毒性質體是兩個構成高毒性株的重要因素，而這個現象在本研究的pks (＋) K62型上也能看到，這個發現支持了pks (＋) 的K62型可能在台灣發展成為新興毒性株的假說。至於pks (－) 的K62型，在YTM099的序列資料中發現了多個疑似抗藥性質體的contig，可能也是個未來值得探討的方向。本研究的結果讓我們了解毒性基因體小島上模組的組成差異對K62型毒性的影響，也為未來這個可能為新興毒性菌株的研究奠定重要的群體基因體學基礎。

Klebsiella pneumoniae is a Gram-negative bacterium. It is commonly found in the body of human, animal, and a broad range of the environment. Known as an opportunistic pathogen, it can cause not only nosocomial infections but also community-acquired infections. In recent years, it has been recognized as an urgent threat to human health due to the emergence of the multidrug-resistant strains and hypervirulent strains. Capsule, lipopolysaccharide, adhesins, siderophores (e.g. aerobactin and yersiniabactin), and colibactin are important virulence factors of Klebsiella pneumoniae. Klebsiella pneumoniae can be classified in to different K-types based on their capsules and/or genes responsible for capsular polysaccharide synthesis. Toxicity of Klebsiella pneumoniae were known to be related to several K-types, among these, K1 and K2 are the most virulent. Colibactin is a genotoxin produced by enzymes encoded by the pks island. It can induce DNA double-strand breaks in the host cell, leading to cell cycle arrest, apoptosis and subsequently promotes genomic instability, which poses a potential risk for cancer. By collaboration with NHRI, we have investigated the prevalence of pks in clinical Klebsiella pneumoniae isolates in Taiwan and identified a high prevalence of pks (＋) strains among Klebsiella pneumoniae clinical isolates in Taiwan. Among these is a group of K62 strains with are highly homogenous in genomic PFGE patterns. This K-type of Klebsiella pneumoniae was rarely reported in the past. To verify if K62 is an emerging hypervirulent clone in Taiwan, we tried to confirm the genotoxicity of K62 by using cell infection model. The genetic structure of the pathogenicity genomic island among K62 isolates were examined by loci-specific PCR and Nanopore sequencing. We found that the pathogenicity genomic island among pks (＋) K62 isolates are highly similar. The toxicity in pks (+) K62 may due to the composition of pathogenicity genomic island. Interestingly, in the genomic approach we also identified plasmid contigs in the pks (＋) strains which are similar to the virulence plasmid pLVPK or pK2044. Recently, a research about hypervirulent Klebsiella pneumoniae clonal-group 23 (K1) found that the hypervirulent strain mostly have a pathogenicity genomic island and a virulence plasmid at the same time. The two elements may be the essential factors for hypervirulent strain. This appearance can also be seen in pks (+) K62. In addition, many putative plasmid contigs of the pks (－) strain YTM099 were identified to be multidrug-resistant plasmids. In this study, we not only found the association of pks and the genotoxicity of K62, but also laid a foundation of population genomics studies for this possible emerging hypervirulent strain.

摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vi
第一章 前言 1
1.1克雷白氏肺炎菌 1
1.1.1生物特性 1
1.1.2臨床重要性 1
1.1.3 抗生素耐藥性 1
1.1.4新興毒性株 2
1.2 克雷白氏肺炎菌的毒性 2
1.2.1克雷白氏肺炎菌的毒性基因與相關因子 2
1.2.2莢膜 3
1.2.2.1 超高粘性 (hypermucoviscous) 3
1.2.2.2 K抗原分型 3
1.2.3脂多醣體 4
1.2.4 黏附因子 4
1.2.5螯鐵因子 4
1.2.5.1 Aerobactin 4
1.2.5.2 Yersiniabactin 5
1.2.6 Colibactin 5
1.3 克雷白氏肺炎菌的致病性基因體學 5
1.3.1 毒性基因體小島 Pathogenicity genomic island (PAI) 5
1.3.1.1 pks基因體小島的組成 5
1.3.1.2 克雷白氏肺炎菌1084中KPHPI208的發現與功能的驗證 6
1.3.2 質體 6
1.3.2.1克雷白氏肺炎菌質體的研究 6
1.3.2.2 毒性大質體pLVPK 6
1.4 台灣的克雷白氏肺炎菌 7
1.4.1克雷白氏肺炎菌感染與腫瘤的關聯 7
1.4.2克雷白氏肺炎菌的pks基因毒素在台灣的盛行率 7
1.4.3 K型與colibactin 8
1.4.4 K62型疑似為新興毒性株 8
1.5 研究目的 8
第二章 材料方法 10
2.1 實驗菌株來源、選擇 10
2.2 細菌培養基配置 10
2.3 細胞培養基配置 11
2.4 其他實驗溶液配置 11
2.5 細胞感染模式 12
2.6 細胞蛋白質測定 16
2.7 西方墨點法 (Western blot) 16
2.8 菌株基因組 DNA純化 19
2.9 菌株基因組 DNA定量QC 19
2.10 菌株基因組DNA濃縮 19
2.11聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 20
2.12 Nanopore定序、組裝與後續分析 21
第三章 實驗結果與討論 22
3.1 K62型功能性基因體學分析 22
3.1.1 感染模式的菌株選擇方案 22
3.1.2 細胞感染模式條件測試 23
3.1.3 細胞感染模式結果 23
3.2 PCR偵測毒性基因體小島上模組 24
3.2.1 K62型毒性基因體小島模組的假說 24
3.2.2 K62型HLH157的毒性基因體小島與引子設計 25
3.2.3 PCR偵測結果 25
3.2.3.1 PCR結果的呈現 25
3.2.3.2 進行PCR所遇到的問題 26
3.2.4 Nanopore定序 26
3.3 K62型菌株的定序組裝與比較基因體學分析 26
3.3.1 K62型毒性基因體小島分析 26
3.3.1.1 K62型的毒性基因體小島註解 26
3.3.1.2 比較K62型毒性基因體小島的結構 27
3.3.1.3 K62型的毒性基因體小島模組的細部差異 27
3.3.1.4 K62型與先前已知各種毒性基因體小島比對 28
3.3.1.5 K62型毒性基因體小島的組成與演化的推論 28
3.3.1.6 pks (＋) 的K62型與高毒性K1、K2之間的親緣關係比較 28
3.3.2 K62型質體分析 29
3.3.2.1 pks (＋)中疑似高毒性質體的發現 29
3.3.2.2 pks (－) 中疑似高抗藥性質體的發現 30
第四章 結論 31
第五章 未來方向 32
參考文獻 33
附錄一： 克雷白氏肺炎菌K62型經PFGE電泳所呈現之親緣樹 36
附錄二： 高抗藥性質體pUUH239.2複製區域的骨架 37

1.Martin RM, Bachman MA. Colonization, Infection, and the Accessory Genome of Klebsiella pneumoniae. Front Cell Infect Microbiol. 2018;8:4.
2.Runcharoen C, Moradigaravand D, Blane B, et al. Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae. Genome Med. 2017;9(1):6.
3.Holt KE, Wertheim H, Zadoks RN, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci USA. 2015;112(27):E3574-81.
4.Paczosa MK, Mecsas J. Klebsiella pneumoniae: Going on the Offense with a Strong Defense. Microbiol Mol Biol Rev. 2016;80(3):629-61.
5.Struve C, Roe CC, Stegger M, et al. Mapping the Evolution of Hypervirulent Klebsiella pneumoniae. MBio. 2015;6(4):e00630.
6.Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, Chang SC. Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clin Infect Dis. 2007;45(3):284-93.
7.Pan YJ, Lin TL, Chen CT, et al. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp. Sci Rep. 2015;5:15573.
8.Wyres KL, Wick RR, Gorrie C, et al. Identification of capsule synthesis loci from whole genome data. Microb Genom. 2016;2(12):e000102.
9.Brisse S, Issenhuth-jeanjean S, Grimont PA. Molecular serotyping of Klebsiella species isolates by restriction of the amplified capsular antigen gene cluster. J Clin Microbiol. 2004;42(8):3388-98.
10.Struve C, Roe CC, Stegger M, et al. Mapping the Evolution of Hypervirulent Klebsiella pneumoniae. MBio. 2015;6(4):e00630.
11.Nougayrède JP, Homburg S, Taieb F, et al. Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science. 2006;313(5788):848-51.
12.Lai YC, Lin AC, Chiang MK, et al. Genotoxic Klebsiella pneumoniae in Taiwan. PLoS ONE. 2014;9(5):e96292.
13.Ramirez MS, Traglia GM, Lin DL, Tran T, Tolmasky ME. Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-negatives: the Klebsiella pneumoniae Paradigm. Microbiol Spectr. 2014;2(5):1-15.
14.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;337:189-98.
15.Gu D, Dong N, Zheng Z, et al. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. Lancet Infect Dis. 2018;18(1):37-46.
16.Huang WK, Chang JW, See LC, et al. Higher rate of colorectal cancer among patients with pyogenic liver abscess with Klebsiella pneumoniae than those without: an 11-year follow-up study. Colorectal Dis. 2012;14(12):e794-801.
17.Kuo SC, Chang SC, Wang HY, et al. 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;12:200.
18.Chen YT, Lai YC, Tan MC, et al. Prevalence and characteristics of pks genotoxin gene cluster-positive clinical Klebsiella pneumoniae isolates in Taiwan. Sci Rep. 2017;7:43120.
19.Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722-736.
20.Darling AC, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14(7):1394-403.
21.Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273(10):5858-68.
22.El husseini N, Hales BF. The Roles of P53 and Its Family Proteins, P63 and P73, in the DNA Damage Stress Response in Organogenesis-Stage Mouse Embryos. Toxicol Sci. 2018;162(2):439-449.
23.Goodwin S, Mcpherson JD, Mccombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet. 2016;17(6):333-51.
24.Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005;43(8):4178-82.
25.Wu KM, Li LH, Yan JJ, et al. Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. J Bacteriol. 2009;191(14):4492-501.
26.M C Lam M, Wyres KL, Duchêne S, et al. Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination. Nat Commun. 2018;9(1):2703.
27.Sandegren L, Linkevicius M, Lytsy B, Melhus Å, Andersson DI. Transfer of an Escherichia coli ST131 multiresistance cassette has created a Klebsiella pneumoniae-specific plasmid associated with a major nosocomial outbreak. J Antimicrob Chemother. 2012;67(1):74-83.
28.Löhr IH, Hülter N, Bernhoff E, Johnsen PJ, Sundsfjord A, Naseer U. Persistence of a pKPN3-like CTX-M-15-encoding IncFIIK plasmid in a Klebsiella pneumonia ST17 host during two years of intestinal colonization. PLoS ONE. 2015;10(3):e0116516.