食物中毒感染是常見但非常重要的全球性公共健康問題。沙門氏鼠傷寒桿菌乃常被分離出來的細菌性病原體之一。隨著實驗室參考菌株LT2的基因組序列於2001年被發表之後，另外9個沙門氏鼠傷寒菌菌株的基因序列均已陸續進行測序。最近，本實驗室亦對十個本地沙門氏鼠傷寒菌臨床分離菌株的基因序列進行了測序。為了為這些基因組序列提供高品質的註釋，我們把預測的基因組提交到質量控制工具GenePRIMP以識別有潛在錯誤或異常的預測基因。本研究針對血液分離菌株78896和糞便分離菌株1047518的GenePRIMP報告進行人工檢查，並對每個菌株超過270個的基因進行了修訂。此外，本研究亦對上述的10個本地菌株進行了功能註釋。註釋項目包括沙門氏菌致病島（SPIs）、致病因子、tRNA和非編碼小分子RNA、噬菌體和CRISPRs結構等基因組及致病元素。 KEGG通路則提供了進一步的功能註釋。 / 本研究同時對本地的血液和糞便分離菌株，連同國外的臨床分離菌株，進行了廣泛的比對，用以識別全身性沙門氏菌感染的潛在遺傳因素。 本研究進行了以下基因分析：（1）多位點序列分型（MLST）；（2）在小鼠全身性感染中涉及的主調控因子和關鍵元素; 及（3）人類腸胃道感染中涉及的基因。然而，這些分析產生只能對全身性沙門氏菌感染提供有限的見解。然而，透過使用RAST註釋系統，我們於其中三個血液分離菌株中發現了一個的額外的螯鐵蛋白aerobactin鐵採集系統。儘管在體外實驗中，這些血液分離菌株並沒有明顯的生長優勢，但實驗結果表明，在缺乏鐵的培養液中，aerobactin基因的表達水平是比較高的。此外，我們亦於其中四個血液分離菌株中，發現負責細胞色素c熟成(ccm)的基因座均被中斷。這可能改變了這些血液分離菌株中細胞色素c的生物合成途徑。這些鐵採集和同化機制的觀察均為未來全身性沙門氏菌感染的研究提供了可能的發展方向。 / 本研究同時識別了用以分別本地及海外的沙門氏鼠傷寒菌菌株的分子標記，並在鮭魚和生菜的接種實驗中，展現了它們分辨本地及海外菌株的能力。然而，在投入實際應用之前，這些標記尚需要進一步的驗證和測試，以便確定快速檢測方法的有效性。 / Foodborne infection is a common but important public health issue worldwide. Salmonella enterica serovar Typhimurium is frequently isolated from outbreaks as one of the common bacterial causative agents. Following the availability of the genome sequence of the reference lab strain LT2 in 2001, nine genomes of S. Typhimurium had been sequenced since then. Recently, genomes of ten local S. Typhimurium clinical isolates have been assembled in our laboratory. In order to provide high quality annotation of these genome sequences, the predicted gene sets were submitted to the quality control tool GenePRIMP (Gene PRediction IMprovement Pipeline) to identify potentially erroneous and abnormal gene calls. The GenePRIMP reports for the local blood isolate 78896 and stool isolate 1047518 were manually inspected and more than 270 genes were amended individually for each isolate. Functional annotation had also been performed for the 10 local isolates. Genomic and virulent elements including Salmonella Pathogenicity Islands (SPIs), virulence factors, tRNAs and small non-coding RNAs, prophage elements and CRISPRs structures had been annotated. The KEGG pathways provided a further means of functional annotation. / The local blood and stool isolates, together with the sequenced foreign clinical isolates, had also been extensively compared to identify potential genetic determinants of Salmonella systemic infection. (1) Multilocus sequence typing (MLST); (2) Alignment of master regulators and key players of systemic infection in mice; and (3) Analyses of the genes responsible for human gastrointestinal tract infection had been performed. However, these analyses yielded limited insights on systemic infection. Alternatively, using subsystems annotation by RAST, an additional aerobactin siderophore iron acquisition system was shown to be prevalent among three of the blood isolates. Despite no obvious growth advantage was offered to the blood isolates in an in vitro experiment, it was demonstrated that expression of the aerobactin genes was higher in iron-depleted culturing medium. In addition, a disrupted cytochrome c maturation (ccm) locus that may alter the cytochrome c biogenesis pathway was also identified in four of the blood isolates. These observations in iron acquisition and assimilation mechanisms suggest their potential in future direction of Salmonella systemic infection studies. / Molecular markers specific to local and foreign S. Typhimurium isolates were also identified and their utility in differentiating local and foreign isolates was demonstrated in a pilot spiking experiment using raw salmon and lettuce. These markers will require further verification and testing prior to actual application in real-world settings in order to examine the validity of the rapid detection method. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Chi Keung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis entitled --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction and Taxonomy --- p.1 / Chapter 1.2 --- Epidemiology of Salmonella Typhimurium infections --- p.2 / Chapter 1.3 --- Pathogenesis of Salmonella Typhimurium infection --- p.4 / Chapter 1.3.1 --- Infection mechanisms --- p.4 / Chapter 1.3.2 --- Salmonella Pathogenicity Islands --- p.6 / Chapter 1.3.3 --- Regulation of virulence --- p.9 / Chapter 1.4 --- Non-typhoid Salmonella (NTS) systemic infection --- p.11 / Chapter 1.4.1 --- Epidemiology of NTS systemic infection --- p.11 / Chapter 1.4.2 --- Salmonella Typhimurium multidrug resistance --- p.12 / Chapter 1.5 --- Salmonella Typhimurium genomics --- p.15 / Chapter 1.5.1 --- Salmonella Typhimurium genome sequencing --- p.15 / Chapter 1.5.2 --- Comparative studies on Salmonella genomes --- p.17 / Chapter 1.6 --- Aims of project --- p.19 / Chapter Chapter 2 --- Curation and detailed annotation of genomes of local Salmonella Typhimurium clinical isolates --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Manual curation of GenePRIMP results --- p.27 / Chapter 2.2.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.29 / Chapter 2.2.3 --- Small RNA and t-RNA annotation --- p.29 / Chapter 2.2.4 --- Phage elements annotation --- p.30 / Chapter 2.2.5 --- CRISPRs annotation --- p.30 / Chapter 2.2.6 --- KEGG annotation --- p.30 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Manual curation of GenePRIMP results --- p.32 / Chapter 2.3.1.1 --- Short genes --- p.35 / Chapter 2.3.1.2 --- Long genes --- p.35 / Chapter 2.3.1.3 --- Unique genes --- p.36 / Chapter 2.3.1.4 --- Overlapped genes --- p.36 / Chapter 2.3.1.5 --- Broken genes --- p.37 / Chapter 2.3.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.37 / Chapter 2.3.2.1 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.37 / Chapter 2.3.2.2 --- Virulence factors annotation --- p.44 / Chapter 2.3.3 --- Small RNA and t-RNA annotation --- p.44 / Chapter 2.3.4 --- Phage elements annotation --- p.44 / Chapter 2.3.5 --- CRISPRs annotation --- p.50 / Chapter 2.3.6 --- KEGG annotation --- p.51 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- Manual curation of GenePRIMP results --- p.53 / Chapter 2.4.1.1 --- Gene amendment not required --- p.54 / Chapter 2.4.1.2 --- Genes with boundaries relocated --- p.54 / Chapter 2.4.1.3 --- Genes to be discarded --- p.55 / Chapter 2.4.1.4 --- Gene pairs to be fused --- p.55 / Chapter 2.4.1.5 --- Potential pseudogenes formation --- p.56 / Chapter 2.4.2 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.57 / Chapter 2.4.3 --- Virulence factors annotation --- p.57 / Chapter 2.4.4 --- Small RNA and t-RNA annotation --- p.58 / Chapter 2.4.5 --- Phage elements annotation --- p.59 / Chapter Chapter 3 --- Identification of genetic determinants of blood invasiveness in local S. Typhimurium clinical isolates --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods --- p.66 / Chapter 3.2.1 --- Multilocus Sequence Typing (MLST) --- p.66 / Chapter 3.2.2 --- Phage elements annotation for foreign isolates --- p.67 / Chapter 3.2.3 --- Alignment of genes inferred to play important roles in NTS systemic --- p.infection67 / Chapter 3.2.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.68 / Chapter 3.2.5 --- Subsystems assignment using Rapid Annotation using Subsystem Technology (RAST) server --- p.68 / Chapter 3.2.6 --- Growth analysis of local S. Typhimurium clinical isolates in iron-limiting environment --- p.69 / Chapter 3.2.7 --- Reverse transcription and real-time PCR --- p.70 / Chapter 3.2.7.1 --- Primer design and verification --- p.70 / Chapter 3.2.7.2 --- cDNA synthesis and real-time PCR --- p.70 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Multilocus Sequence Typing (MLST) --- p.73 / Chapter 3.3.2 --- Phage elements annotation for foreign isolates --- p.73 / Chapter 3.3.3 --- Alignment of genes inferred to play important roles in NTS systemic infection --- p.74 / Chapter 3.3.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.79 / Chapter 3.3.4.1 --- Acid tolerance response --- p.79 / Chapter 3.3.4.2 --- Epithelial cells attachment --- p.80 / Chapter 3.3.4.3 --- Epithelial cells invasion --- p.83 / Chapter 3.3.4.4 --- Survival within macrophages --- p.83 / Chapter 3.3.5 --- RAST subsystem analysis --- p.86 / Chapter 3.3.6 --- Growth analysis and aerobactin genes expression --- p.87 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Molecular markers identification and testing on selected foodstuff for local S. Typhimurium isolates --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Molecular markers identification --- p.101 / Chapter 4.2.2 --- Primer design and verification --- p.101 / Chapter 4.2.3 --- Spiking experiments on selected food samples --- p.103 / Chapter 4.2.4 --- Quantitative TaqMan real-time PCR --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Molecular markers identification --- p.105 / Chapter 4.3.2 --- Spiking experiments and TaqMan real-time PCR --- p.109 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Molecular markers identification --- p.113 / Chapter 4.4.2 --- Spiking experiments and TaqMan real-time PCR --- p.114 / Chapter Chapter 5 --- General discussion --- p.116 / Chapter 5.1 --- Manual curation of GenePRIMP results --- p.116 / Chapter 5.2 --- Functional annotation of local S. Typhimurium genomes --- p.118 / Chapter 5.3 --- Systemic infection studies --- p.120 / Chapter 5.4 --- Molecular markers identification and spiking experiments --- p.121 / Chapter 5.5 --- Conclusion and future perspectives --- p.122 / References --- p.124
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328218 |
| Date | January 2013 |
| Contributors | Cheng, Chi Keung., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xv, 146 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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