臺灣博碩士論文加值系統

沙門氏菌是造成多起食物中毒事件的革蘭氏陰性菌。台灣近幾年研究家禽肉品發現在市售的肉雞有超過五成分離到的沙門氏菌都隸屬於血清型Schwarzengrund。而在人類臨床案例，在2004－2012年期間S. Schwarzengrund為排名第12之常見血清型。本研究收集了2000－2012期間分離的15株來自雞、鴨、豬、寵物飼料與野鳥等流行病學不相關的菌株，外加一株來自同一次爆發病例的菌株共16株，想評估傳統基因分型工具脈衝式電泳（PFGE）與多基因座序列分析（MLST）的分型能力，但發現鑑定效果均不足。PFGE方面，在使用沙門氏菌最常用的限制酶XbaI，其D-value值僅0.79。而MLST僅產生一種Sequence Type 96 (ST96)，則完全無法辨別細菌。因此尋求其他基因分型方法並與傳統分型方式比較。其中CRISPR分析，其鑑定能力優於MLST和使用XbaI-PFGE，能產生8型CRISPR type，其D-value值為0.87，雖不足以單獨運用但推論可以與PFGE併用增加分型能力。而全基因體序列（WGS）分析，可再進一步依據分析策略分為MUMi、ND與SNV親緣關係樹等三種不同分析方式。而SNV、ND、MUMi個別D-value值為0.90、0.94與0.79。三種方法中僅SNV與ND的分析能力高於標準的0.90。MUMi對於核酸序列的差異最為敏感但鑑別力不足，因此，本研究中推論其不適合作為血清型內的分型工具。結論上，目前基因定序工具越來越優化，但不同親緣性分析方法的選擇，將對於親緣關係的決定產生影響。本研究發現CRISPR、SNV與ND分析都可以提供優於XbaI-PFGE的鑑別力，其中以ND分析效果最好。

Salmonella infections are a public health concern. Several species of animals can potentially transmit these pathogens to humans and molecular typing is a useful tool in epidemiological investigation. To evaluate a plausible genotyping method for Salmonella Schwarzengrund, one of the prevalent serotypes in Taiwan, 16 strains with 15 of them being epidemiologically unrelated were genotyped using different methods. Conventional typing methods (XbaI-PFGE and MLST) were found to be inappropriate, with discrimination indexes of 0.79 and 0, respectively. Only PFGE with combined results of multiple restriction enzymes (AvrII + SfiI) can all unrelated strains be differentiated. For alternative typing schemes, clustered regularly interspaced palindromic repeats analysis generated eight types for the 15 strains, with a discrimination index of 0.87, which coordinated well with the XbaI-PFGE phylogenic results and increased the discrimination power. For the whole genome sequence-based analysis, the discrimination indexes of the three approaches utilized (a single nucleotide polymorphism tree, a nucleotide difference tree, and a maximum unique matches index tree) were 0.90, 0.93, and 0.79, respectively. Only the single nucleotide polymorphism tree and the nucleotide difference tree analyses provided sufficient discrimination power (discrimination index>0.90). The maximum unique matches index tree obtained a lower discrimination index value, even though there is no requirement for a reference genome. In conclusion, the clustered regularly interspaced palindromic repeats analysis, the single nucleotide polymorphism tree, and the nucleotide difference tree all performed better than conventional methods, with the nucleotide difference tree being the best approach for S. Schwarzengrund phylogenic analysis.

論文口試審定書 I
致謝 II
中文摘要 III
Abstract IV
Abbreviation List VI
Contents VII
Table List IX
Figure List X
Chapter 1. Introduction 1
Chapter 2. Literature Review 3
2.1. Salmonella Serotype Schwarzengrund 3
2.1.1. Salmonella 3
2.1.2. Salmonella Schwarzengrund 4
2.2. Conventional Typing (PFGE and MLST) 6
2.2.1. Pulsed-field gel electrophoresis 6
2.2.2. Multilocus sequence typing 8
2.3. CRISPR 10
2.4. WGS 13
2.4.1. From Sanger to high-throughput Sequencing 13
2.4.2. Genome Phylogenic Analysis Approaches 16
2.4.3. In silico Analysis 18
2.5. Discriminatory power of genotyping methods 19
Chapter 3. Materials and Methods 20
3.1. Strains Identification 20
3.2. DNA Extraction 22
3.3. Pulse Field Gel Electrophoresis 23
3.4. CRISPR 24
3.5. Whole Genome Sequence 25
Chapter 4. Results 28
4.1. Conventional Typing: PFGE and MLST 28
4.2. CRISPR 29
4.3. Whole Genome Sequence 30
4.3.1. SNV tree 30
4.3.2. Nucleotide difference tree 31
4.3.3. MUMi tree 31
4.3.4. In silico MLST and antimicrobial resistance gene detection 32
Chapter 5. Discussion 33
5.1. Conventional typing 33
5.2. CRISPR 35
5.3. WGS phylogenic analysis 37
5.4. In silico analysis 38
Chapter 6. Conclusions 39
References 40
Tables 46
Figures 56

1.Coburn B, Grassl GA, Finlay BB. 2007. Salmonella, the host and disease: a brief review. Immunol Cell Biol 85:112-118.
2.Vugia DJ, Samuel M, Farley MM, Marcus R, Shiferaw B, Shallow S, Smith K, Angulo FJ. 2004. Invasive Salmonella infections in the United States, FoodNet, 1996-1999: incidence, serotype distribution, and outcome. Clin Infect Dis 38 Suppl 3:S149-156.
3.Lin CC, Guo JW, Chang CC, Wang YC, Shien JH, Yeh KS, Cheng TH. 2008. Salmonella Serovars Isolated from Marketing Broilers and simulated native chickens: prevalence and drug resistance Taiwan Vet J 34:217-225..
4.Chiou CS, Liao YS, Liao CH, Tsao CS, Kuo JC. 2015. Salmonellosis surveillance and epidemiological trend in Taiwan. Taiwan Epidemiology Bulletin 31.
5.Kuo HC, Lauderdale TL, Lo DY, Chen CL, Chen PC, Liang SY, Kuo JC, Liao YS, Liao CH, Tsao CS, Chiou CS. 2014. An association of genotypes and antimicrobial resistance patterns among Salmonella isolates from pigs and humans in Taiwan. PLoS One 9:e95772.
6.Prevention Center for Disease Control. 2008. Multistate outbreak of human Salmonella infections caused by contaminated dry dog food--United States, 2006-2007. MMWR Morb Mortal Wkly Rep 57:521-524.
7.Harbottle H, White DG, McDermott PF, Walker RD, Zhao S. 2006. Comparison of multilocus sequence typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolates. J Clin Microbiol 44:2449-2457.
8.Allard MW, Luo Y, Strain E, Li C, Keys CE, Son I, Stones R, Musser SM, Brown EW. 2012. High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach. BMC Genomics 13:32.
9.Fricke WF, Mammel MK, McDermott PF, Tartera C, White DG, Leclerc JE, Ravel J, Cebula TA. 2011. Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution. J Bacteriol 193:3556-3568.
10.Gori A BA, Marchetti G, Esposti AD, Catozzi L, Nardi GP, Gazzola L, Ferrario G, ven Embden JDA, ven Soolingen D, Moroni M, Franzetti F. 2005. Spoligotyping and Mycobacterium tuberculosis. Emerg Infect dis 11:1242-1248.
11.Fabre L, Zhang J, Guigon G, Le Hello S, Guibert V, Accou-Demartin M, de Romans S, Lim C, Roux C, Passet V, Diancourt L, Guibourdenche M, Issenhuth-Jeanjean S, Achtman M, Brisse S, Sola C, Weill FX. 2012. CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS One 7:e36995.
12.Shariat N, Kirchner MK, Sandt CH, Trees E, Barrangou R, Dudley EG. 2013. Subtyping of Salmonella enterica serovar Newport outbreak isolates by CRISPR-MVLST and determination of the relationship between CRISPR-MVLST and PFGE results. J Clin Microbiol 51:2328-2336.
13.Bachmann NL, Petty NK, Ben Zakour NL, Szubert JM, Savill J, Beatson SA. 2014. Genome analysis and CRISPR typing of Salmonella enterica serovar Virchow. BMC Genomics 15:389.
14.Hommais F, Pereira S, Acquaviva C, Escobar-Paramo P, Denamur E. 2005. Single-nucleotide polymorphism phylotyping of Escherichia coli. Appl Environ Microbiol 71:4784-4792.
15.Kwong JC, Mercoulia K, Tomita T, Easton M, Li HY, Bulach DM, Stinear TP, Seemann T, Howden BP. 2016. Prospective whole-genome sequencing enhances national surveillance of Listeria monocytogenes. J Clin Microbiol 54:333-342.
16.Leekitcharoenphon P, Nielsen EM, Kaas RS, Lund O, Aarestrup FM. 2014. Evaluation of whole genome sequencing for outbreak detection of Salmonella enterica. PLoS One 9:e87991.
17.Deloger M, El Karoui M, Petit MA. 2009. A genomic distance based on MUM indicates discontinuity between most bacterial species and genera. J Bacteriol 191:91-99.
18.Dhanoa A, Fatt QK. 2009. Non-typhoidal Salmonella bacteraemia: epidemiology, clinical characteristics and its'' association with severe immunosuppression. Ann Clin Microbiol Antimicrob 8:15.
19.Asai T, Murakami K, Ozawa M, Koike R, Ishikawa H. 2009. Relationships between multidrug-resistant Salmonella enterica Serovar Schwarzengrund and both broiler chickens and retail chicken meats in Japan. Jpn J Infect Dis 62:198-200.
20.Aarestrup FM, Hendriksen RS, Lockett J, Gay K, Teates K, McDermott PF, White DG, Hasman H, Sorensen G, Bangtrakulnonth A, Pornreongwong S, Pulsrikarn C, Angulo FJ, Gerner-Smidt P. 2007. International spread of multidrug-resistant Salmonella Schwarzengrund in food products. Emerg Infect Dis 13:726-731.
21.Chou CH and Tsai HJ. 2001. The prevalence and antimicrobial susceptibility of Salmonella and Campylobacter in meat-type chickens in Taiwan. Taiwan Vet J 27:27-38.
22.Lauderdale TL, Aarestrup FM, Chen PC, Lai JF, Wang HY, Shiau YR, Huang IW, Hung CL. 2006. Multidrug resistance among different serotypes of clinical Salmonella isolates in Taiwan. Diagn Microbiol Infect Dis 55:149-155.
23.Chen MH, Wang SW, Hwang WZ, Tsai SJ, Hsih YC, Chiou CS, Tsen HY. 2010. Contamination of Salmonella Schwarzengrund cells in chicken meat from traditional marketplaces in Taiwan and comparison of their antibiograms with those of the human isolates. Poult Sci 89:359-365.
24.Zou W, Lin WJ, Foley SL, Chen CH, Nayak R, Chen JJ. 2010. Evaluation of pulsed-field gel electrophoresis profiles for identification of Salmonella serotypes. J Clin Microbiol 48:3122-3126.
25.Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis 7:382-389.
26.Zheng J, Keys CE, Zhao S, Ahmed R, Meng J, Brown EW. 2011. Simultaneous analysis of multiple enzymes increases accuracy of pulsed-field gel electrophoresis in assigning genetic relationships among homogeneous Salmonella strains. J Clin Microbiol 49:85-94.
27.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. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95:3140-3145.
28.Kidgell C, Reichard U, Wain J, Linz B, Torpdahl M, Dougan G, Achtman M. 2002. Salmonella Typhi, the causative agent of typhoid fever, is approximately 50,000 years old. Infect Genet Evol 2:39-45.
29.Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. 2005. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551-2561.
30.Bhaya D, Davison M, Barrangou R. 2011. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273-297.
31.Shariat N, Timme RE, Pettengill JB, Barrangou R, Dudley EG. 2015. Characterization and evolution of Salmonella CRISPR-Cas systems. Microbiology 161:374-386.
32.Fournier PE, Dubourg G, Raoult D. 2014. Clinical detection and characterization of bacterial pathogens in the genomics era. Genome Med 6:114.
33.Grad YH, Lipsitch M, Feldgarden M, Arachchi HM, Cerqueira GC, Fitzgerald M, Godfrey P, Haas BJ, Murphy CI, Russ C, Sykes S, Walker BJ, Wortman JR, Young S, Zeng Q, Abouelleil A, Bochicchio J, Chauvin S, Desmet T, Gujja S, McCowan C, Montmayeur A, Steelman S, Frimodt-Moller J, Petersen AM, Struve C, Krogfelt KA, Bingen E, Weill FX, Lander ES, Nusbaum C, Birren BW, Hung DT, Hanage WP. 2012. Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011. Proc Natl Acad Sci U S A 109:3065-3070.
34.Hoffmann M, Luo Y, Monday SR, Gonzalez-Escalona N, Ottesen AR, Muruvanda T, Wang C, Kastanis G, Keys C, Janies D, Senturk IF, Catalyurek UV, Wang H, Hammack TS, Wolfgang WJ, Schoonmaker-Bopp D, Chu A, Myers R, Haendiges J, Evans PS, Meng J, Strain EA, Allard MW, Brown EW. 2016. Tracing origins of the Salmonella Bareilly strain causing a food-borne outbreak in the United States. J Infect Dis 213:502-508.
35.Kircher M, Kelso J. 2010. High-throughput DNA sequencing--concepts and limitations. Bioessays 32:524-536.
36.Kaas RS, Leekitcharoenphon P, Aarestrup FM, Lund O. 2014. Solving the problem of comparing whole bacterial genomes across different sequencing platforms. PLoS One 9:e104984.
37.Compeau PE, Pevzner PA, Tesler G. 2011. How to apply de Bruijn graphs to genome assembly. Nat Biotechnol 29:987-991.
38.Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81-91.
39.Hasman H, Saputra D, Sicheritz-Ponten T, Lund O, Svendsen CA, Frimodt-Moller N, Aarestrup FM. 2014. Rapid whole-genome sequencing for detection and characterization of microorganisms directly from clinical samples. J Clin Microbiol 52:139-146.
40.Hunter PR, Gaston MA. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson''s index of diversity. J Clin Microbiol 26:2465-2466.
41.Grimont PAD and Weill FX. 2007. Antigenic formulae of Salmonella serovars, (9th ed). Paris: WHO Collaborating Center for Reference and Research on Salmonella.
42.Liu F, Barrangou R, Gerner-Smidt P, Ribot EM, Knabel SJ, Dudley EG. 2011. Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica. Appl Environ Microbiol 77:1946-1956.
43.Grissa I, Vergnaud G, Pourcel C. 2007. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8:172.
44.Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. 2004. Versatile and open software for comparing large genomes. Genome Biol 5:R12.
45.Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870-1874.
46.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640-2644.
47.Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Ponten T, Ussery DW, Aarestrup FM, Lund O. 2012. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 50:1355-1361.
48.Chen MH, Hwang WZ, Wang SW, Shih YC, Tsen HY. 2011. 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 28:399-405.
49.Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodriguez-Valera F. 2005. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 43:4382-4390.
50.Achtman M, Wain J, Weill FX, Nair S, Zhou Z, Sangal V, Krauland MG, Hale JL, Harbottle H, Uesbeck A, Dougan G, Harrison LH, Brisse S, Group SEMS. 2012. Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog 8:e1002776.