臺灣博碩士論文加值系統

克雷伯氏肺炎桿菌為一革蘭氏陰性腸內細菌，自1882年發現以來，至今仍是造成人類疾病的重要病原菌。克雷伯氏肺炎菌透過院內感染以及社區感染所造成之臨床表現隨著不同年代和地域而有所差異，除了肺炎與尿道感染為其傳統病徵之外，同時它也是造成菌血症與抗藥性細菌感染之新興病源。近三十年來，在台灣以及鄰近亞洲各國，出現一種好發於糖尿病病患的侵犯性克雷伯氏肺炎桿菌感染症，能引起原發性肝膿瘍合併菌血症、轉移性內眼炎或腦膜炎的發生。雖然目前對於克雷伯氏肺炎桿菌在亞洲造成嚴重感染症之主要原因仍不清楚，但推測可能與宿主及菌株因子兩者都有相關。
我們對於同一種細菌會因帶有不同基因內涵而造成不同臨床症狀有興趣，因此我們採用散彈槍定序策略完成一個克雷伯氏肺炎桿菌致病性菌株NTUH-K2044之全部基因體序列。此一菌株對人體能造成侵犯性感染並對小鼠具有致死性毒性。與另一株取自肺炎病患並由華盛頓大學所定序的菌株MGH 78578做序列分析比較，發現六處基因叢集有所不同，包含與檸檬酸發酵途徑、脂多醣合成、莢膜多醣合成、噬菌體、纖毛等相關之基因叢集。運用本實驗室自行建立之Genomic Shotgun Array（GSA）技術，將所收集之15株由社區感染或醫院感染而來的臨床菌株與NTUH-K2044菌株進行基因體雜交比較，我們可以區分出三群細菌，其基因體內容型式與臨床病徵、抗藥性種類、小鼠模式所得之毒性特徵相符。
為了瞭解克雷伯氏肺炎桿菌感染之分子機制並發展有效之治療方法，我們進一步研究克雷伯氏肺炎桿菌之基因體演化、致病性及抗藥性。為此，我們挑選三株 (NK8, NK29, NK245) 具有不同抗藥性、臨床特徵與採菌歷史之菌株進行全基因體定序。如同預測，我們發現抗藥菌株帶有多重抗藥性基因的質體：菌株NK245的一個質體pK245帶有qnrS 喹諾酮(quinolone)類抗藥基因與SHV-2乙內醯胺酶基因(beta-lactamase)，而菌株NK29的一個質體pK29帶有CMY-8乙內醯胺酶基因與CTX-M-3 廣效性乙內醯胺酶(extended-spectrum beta-lactamase)基因。
為了快速鑑定克雷伯氏肺炎桿菌的基因體內容，我們利用NimbleGen oligonucleotide array (NOA) 發展第二代基因體雜交技術。微陣列上的探針是設計自五株 (NTUH-K2044, NK8, NK29, NK245 and MGH 78578) 已完成定序的克雷伯氏肺炎桿菌的非重複編碼序列。將所收集之26株臨床菌株(包含GSA所用的14株)與參考菌株NTUH-K2044進行基因體雜交比較並利用階層式分群法進行分析，結果顯示NOA技術不僅能將菌株基因內涵型態與其表現型特徵作良好關聯，NOA技術還具有更高解析度、涵蓋更多基因內涵與降低實驗所需之操作等優點。
使用本人所寫的「蛋白質序列交互比較程式」分析六株已完成定序的克雷伯氏肺炎桿菌，發現不同菌株間相對應的基因在染色體上的排列相當一致。然而在大腸桿菌即使在親緣關係非常接近的K12亞種與O157:H7亞種裡也能發現明顯的基因片段倒置。此結果顯示克雷伯氏肺炎桿菌相較於其他腸內菌科的細菌，如大腸菌，擁有相對穩定的染色體。
為了瞭解親緣關係相近菌種其基因在演化上的動態變化，分別計算克雷伯氏肺炎桿菌與大腸桿菌各自核心基因群的Ka/Ks值，以及核心基因群依照功能分類後所各自計算的Ka/Ks值，結果都分別小於1，顯示克雷伯氏肺炎桿菌與大腸桿菌這兩個菌種都受到強烈的負向天擇作用，使得有害的突變大多都被移除。然而，在依照功能分類後所各自計算的核心基因Ka/Ks值卻在兩個菌種中不盡相同，顯示在克雷伯氏肺炎桿菌與大腸桿菌之不同功能的基因群承受著不同的演化壓力。

Klebsiella pneumoniae is a Gram-negative enteric bacillus that has been a significant human pathogen since its discovery in 1882. The clinical manifestations of K. pneumoniae infection acquired from hospital or community vary in different time periods and geographical areas. Besides its classic manifestation as pneumonia and urinary tract infection, K. pneumoniae is emerging worldwide as a major cause of bacteremia and drug-resistant infection. Moreover, in the past three decades, an invasive form of K. pneumoniae infection, which presents as primary bacteremic liver abscesses, metastatic endophthalmitis or meningitis, has been reported to be associated with diabetes mellitus and occurred almost exclusively in Asia, including Taiwan. Although the reasons for the preponderance of this severe invasive K. pneumoniae infection in Asia are unknown, they are likely to involve both host and microbial factors.
We are interested in understanding how differences in gene content of the same bacterial species result in variable diseases in human infection. Therefore, we have determined the whole genome sequence of K. pneumoniae NTUH-K2044, a virulent strain causing invasive phenotype in humans and lethality in mice, and compared to that of strain MGH 78578, which was isolated from a patient with pneumonia in USA. Major genomic differences were found in six gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization on 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome (GSA method). Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlated with the strains’ clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice.
To further elucidate the genomic contents of drug-resistant K. pneumoniae strains and gain knowledge about the evolution of enteric bacteria, three hospital isolates, NK8, NK29 and NK245, have been selected for whole genome shotgun sequencing based on their isolation history and antibiotics resistance profile. As expected, two plasmids harbor multiple antimicrobial resistance genes were found. The plasmid pK245 contains the qnrS quinolone resistance determinant and the gene encoding SHV-2 beta-lactamase in strain NK245, and the plasmid pK29 contains genes encoding CMY-8 beta-lactamase and CTX-M-3 ESBL in strain NK29.
To rapidly identify the patterns of K. pneumoniae gene contents, we have developed another CGH method using NimbleGen oligonucleotide array (NOA). The probes were designed based on all non-redundant coding sequences from five completed genomes (NTUH-K2044, NK8, NK29, NK245 and MGH 78578). Twenty-six strains (including 14 used in GSA) were analyzed by comparative genomic hybridization using the NTUH-K2044 as the reference strain. The hierarchical clustering results indicate that the NOA method not only can correlate gene contents with phenotype features, but also provide the advantages of higher resolution, better gene-content coverage and less laboriousness.
Using the self-developed “Reciprocal Protein Sequence Comparison” program, we found the coding genes are highly syntenic along the chromosomal coordinates among K. pneumoniae isolates. By contrast, within E. coli, inversions could be found between substrains of K12 and O157:H7. This finding suggests that K. pneumoniae has a relatively stable chromosome compared to E. coli and other species within Enterobacteriaceae.
To understand the evolutionary dynamics of protein-coding sequences across closely related species, the Ka/Ks ratios were calculated for K. pneumoniae and E. coli core genes and analyzed for each COG functional groups. In either case we found an average Ka/Ks ratio of less than 1, suggesting both species were subjected to strong purifying selection to eliminate harmful mutations. However, some functional groups showed different Ka/Ks ratios in K. pneumoniae and E. coli, indicating that genes of different function groups had subjected to different selection pressures for the two related enterobacteria during evolution.

Chinese Abstract ……………………………………………………….…………………………… i
English Abstract …………………………………………………………………………………… iii
Abbreviations ……………………………………………………………………………………… vi
Introduction ………………………………………………………………………………………… 1
Materials and Methods …………………………………………………………………………….. 8
Part 1. Genome Sequencing and Annotation of Selected K. pneumoniae Clinical Strains
1. K. pneumoniae strains for this study ……………………………………………………… 8
2. Shotgun sequencing and assembly of NTUH-K2044 genome …………………………… 9
3. Shotgun sequencing of NK8, NK29 and NK245 genomes ……………………………… 10
4. Genome annotation pipeline ……………………………………………………………… 10
5. Web interface for data access and annotation inspection ………………………………… 11
6. Nucleotide sequence accession number …………………………………………………… 13
Part 2. Genome Analysis of K. pneumoniae Clinical Strains
1. Method for antimicrobial susceptibility testing …………………………………………… 13
2. Detection of extended spectrum β-lactamase (ESBL) producers ………………………… 14
3. Procedures for genome shotgun array (GSA) …………………………………………… 14
4. Genome sequencing of NK5 and CG43 strains by the 454 technology ………………… 16
5. Procedures for NimbleGen oligonucleotide array (NOA) ………………………………… 16
Part 3. Comparative Genome Analysis Between K. Pneumoniae and Non-Klebsiella Enterobacteria
1. Reciprocal protein sequence comparison …………………………………………………… 19
2. Calculating genomic distance matrix based on MUMs and constructing phylogenetic tree of Enterobacteriaceae species ……………………………………………………………… 20
3. Identifying K. pneumoniae and E. coli core genes ……………………………………… 21
4. Calculating Ka/Ks ratios of core genes …………………………………………………… 22
Results ……………………………………………………………………………………………… 23
Part 1. Genome Sequencing and Annotation of Selected K. pneumoniae Clinical Strains
1. General features of the NTUH-K2044 genome …………………………………………… 23
2. Genome comparison with strain MGH 78578 ……………………………………………… 23
3. General features of the NK8, NK29 and NK245 genomes ……………………………… 25
4. Antibiotics resistance gene profiles in NK8, NK29 and NK245 genomes ……………… 26
Part 2. Comparative Genome Analysis of K. pneumoniae Clinical Strains
1. Comparative analysis of various K. pneumoniae clinical strains using GSA ……………… 27
2. Comparative analysis of various K. pneumoniae clinical strains using NOA …………… 30
Part 3. Comparative Genome Analysis Between K. pneumoniae and Non-Klebsiella Enterobacteria
1. Stable genome of K. pneumoniae compared to that of E. coli ……………………………… 33
2. Whole-genome based phylogeny of Enterobacteria …………………………...…………… 35
3. Evolutionary selection on core genes of K. pneumoniae and E. coli …………………….. 36
Discussion ……………………………………………………………………………………….…… 38
References …………………………………………………………………………………………… 43
Tables ………………………………………………………………………………………………… 52
1. (A) Summary of the clinical strains of K. pneumoniae ……………………………………. 53
1. (B) General features of the K. pneumoniae NTUH-K2044 genome ……………………… 53
1. (C) General features of the K. pneumoniae NK8, NK29, NK245 genomes ……………… 53
2. Differences in gene clusters between K. pneumoniae NTUH-K2044 and K. pneumoniae MGH 78578 …………………………………………………………………….…………………. 54
3. Prophages found in completely sequenced K. pneumoniae genomes ……………………… 55
4. Antibiotics resistance gene profiles in completely sequenced K. pneumoniae strains ……… 56
5. Clinical and bacteriological features of the K. pneumoniae isolates ……………………….. 57
6. Genomic features that distinguished the major groups of K. pneumoniae strains ………… 58
7. List of predicted genes for the seven INDEL regions from GSA result ………………… 59
8. Core gene matrix among six K. pneumoniae strains ……………………………………… 62
9. The existence of genes involved in the methionine salvage pathway …………………… 63
Figures …………………………………………………………………………………………...… 64
1. (A) Genomic maps of the K. pneumoniae NTUH-K2044 chromosome and plasmid …….. 65
1. (B) Circular plot of the K. pneumoniae NK8 genome …………………………………….. 66
1. (C) Circular plot of the K. pneumoniae NK29 genome …………………………………….. 67
1. (D) Circular plot of the K. pneumoniae NK245 genome ………………………………….. 66
2. Hierarchical cluster analysis of clinical isolates based on GSA with 813 genomic DNA probes ………………………………………………………………………………………… 69
3. Linear representation of the NTUH-K2044 genome and hybridization patterns of K. pneumoniae isolates ………………………………………………………………………… 70
4. Four major insertion/deletion events that contribute to the difference in length between pK2044 of NTUH-K2044 and pLVPK of CG43 …………………………………………………… 72
5. Genomic structure of the ICEKp1 region in K. pneumoniae NTUH-K2044, compared to Y. pestis CO92 HPI and E. coli ECOR31 ICEEc1 …………………………………………… 73
6. Overview of 26 K. pneumoniae NimbleGen array CGH results that displayed by SignalMap software ……………………………………………………………………………………… 74
7. Hierarchical clustering results of NimbleGen array CGH data with all, chromosome-only and plasmid-only CDS groups in 26 clinical strains …………………………………………… 75
8. Hierarchical clusters of NimbleGen array CGH data with all, chromosome-only and plasmid-only CDS groups in 26 clinical strains ………………………………………… 76
9. Comparison of GSA clustering result with NOA clustering results on all CDS groups …. 77
10. (A) Reciprocal protein sequence comparison in six K. pneumoniae strains ……………… 78
10. (B) Reciprocal protein sequence comparison between NTUH-K2044 and KP342 ……… 78
11. Reciprocal protein sequence comparison in substrains of E. coli K12 and O157:H7 ……… 79
12. Reciprocal protein sequence comparison in 15 E. coli strains …………………………… 80
13. Whole-genome based phylogenic tree of 79 completely sequenced Enterobacteriaceae species using genomic distance matrix …………………………………………………………… 81
14. Ka/Ks ratios of core genes and each COG functional group in K. pneumoniae and E. coli… 82
Appendix …………………………………………………………………………………………….. 83
I. Whole genome shotgun sequencing procedure for strain NTUH-K2044 ………………… 84
II. Assembling & finishing flowchart ………………………………………………………… 85
III. The workflow of annotation processes …………………………………………………… 86
IV. (a) Screenshot of “Annotation Query System”; (b) Screenshot of “Sequence Retrieve System” ……………………………………………………………………………………... 87
IV. (c) Screenshot of “Clone Search System”; (d) Screenshot of “GSA Deletion Map” …… 88
IV. (e) Screenshot of ORF annotations under “Annotation Query System”; (f) Screenshot of ORF sequences (nucleotides and amino acids) …………………………………………… 89
IV. (g) Screenshot of BLASTP results of the ORF against RefSeq/NR/KEGG gene databases; (h) Screenshot of ORF region view around the selected ORF ……………………………… 90
V. Schema of NimbleGen array CGH chip design for groups of paralogs/orthologs CDSs from five K. pneumoniae strains ………………………………………………………………… 91
VI. Schema of NimbleGen array CGH chip layout for groups of CDSs from five K. pneumoniae strains ……………………………………........................................................................... 92
VII. Limited clinical features of CMKa K. pneumoniae isolates ………………………… 93
VIII. The methionine salvage pathway ……………………………………………………… 94
IX. Usage of Klebsiella pneumoniae genome sequences by local scientists ……………… 95
X. Published paper: “Genome Sequencing and Comparative Analysis of Klebsiella pneumoniae NTUH-K2044, a Strain Causing Liver Abscess and Meningitis”.
XI. Manuscript: “Genome Sequencing and High-Density Array Define Genetic Diversity and Evolution of K. pneumoniae Isolates Causing Different Clinical Manifestations”.

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