本研究係針對台灣常見的桿菌性痢疾及百日咳等兩項傳染病病原菌進行分子流行病學分析。
關於痢疾桿菌部分，本研究收集台灣東部某大型長期精神照護機構持續3年的桿菌性痢疾案件及自社區病人分離的痢疾桿菌進行分子流行病學分析。經利用脈衝場電泳(PFGE, pulsed-field gel electrophoresis)、質體型別(plasmid profile analysis)、抗藥性譜型(antibiogram typing)等方法分析，結果發現該照護機構痢疾桿菌菌株分子次型(molecular subtype)與社區分離株有所不同，且在其相互分隔之數個病房也有其不同分子次型各自衍生並伺機感染致病，而這些菌株可能潛藏在未被偵測到的帶菌者或慢性病人身上。
關於百日咳菌部分，在收集1992~1997年份共114株百日咳菌臨床分離株，進行PFGE分子流行病學分析後，發現1997年台灣出現的百日咳病例流行高峰，是分別由17種PFGE次型所引起，因此排除了台灣出現高傳染性強毒株或抗疫苗株而造成大流行的疑慮。同時因大量病例(71%)接續出現並集中在人口密集的台北都會區，推測擁擠的都市人口有助於疾病的傳播及病原體的演化。

(一) In Taiwan, the number of pertussis cases has been increasing in the recent years, especially in 1997. In addition to the sporadic cases, pertussis also occurred in outbreaks of household, elementary school, and nosocomial infections. Since 71% of reported cases concentrated in the Taipei metropolitan area, it elicited a great deal of concern that a highly contagious strain of Bordetella pertussis might have appeared in Taipei. Therefore, a total of 114 strains of B. pertussis were collected and typed by the molecular method of pulsed-field gel electrophoresis (PFGE). These examined strains include 61 clinical isolates in 1997 and 53 control strains isolated in the previous years (1992~1996). All these strains were classified into 21 subtypes and among them the 1997 collection showed 17 subtypes with 9 new subtypes as compared to those collected in the previous years. This result indicated that the pertussis outbreaks in 1997 were not results of a sole specific virulent strain. It also implies that highly dense population may facilitate the spread and accelerate the genetic divergency of this pathogen.
(二) With 6 separate wards accommodating over 1,600 patients, VNC is a long-stay psychiatric nursing center in eastern Taiwan. During 2001-2003, 39 shigellosis cases occurred in VNC. Different from the notion that most of shigellosis cases are caused by Shigella sonnei, these strains were all S. flexneri except one S. sonnei isolate. O-antigen serotyping showed that the 38 S. flexneri strains were either type 1a (n=20) or 4a (n=18), the two less prevalent serotypes in Taiwan. NotI-PFGE analyses, using 8 1a and 9 4a non-VNC strains isolated during 1996-2003 for comparison, divided the 28 1a and 27 4a strains into 7 and 10 subtypes, designated P1A to P1G and P4A to P4J, respectively. P1A and P4A, appearing in three consecutive years in VNC as well as outside of VNC, are the most prevalent subtypes. Relatedness analyses of the VNC strains based on banding patterns grouped the 1a and 4a strains into 4 and 5 clusters, respectively. The 1a strains, except one, share 95% similarity indicating a common parent, whereas the 4a strains share similarities ranging from 77 to 93% suggesting diverse origins. In two of the outbreaks, less related subtypes of 4a strains were found in the same VNC wards in consecutive years, suggesting possible existence of different subtypes in VNC all the time. Antibiotic susceptibility test showed that the S. flexneri strains are sensitive to at least 7 antibiotics, except one isolate which is sensitive to 3 antibiotics. The data of the latter tests should be helpful for treating S. flexneri infections in Taiwan.

目錄 (Contents) 頁數
目錄 (Contents) 2
摘要 (Abstract) 5
緒言 (Introduction) 7
一. 桿菌性痢疾分子流行病學 9
(一)、摘要 (Abstract) 10
英文 10
中文 12
(二)、前言 (Introduction) 14
(三)、材料與方法 (Materials and Methods) 17
1. 菌種收集 17
2. PFGE操作及親源樹狀圖分析 17
3. 質體型別分析 18
4. 抗藥性譜型分析 19
5. 流行病學資料 19
(四)、結果與討論(Results and Discussion) 20
(五)、結論(Conclusion) 33
(六)、誌謝 (Acknowledgement) 34
(七)、參考資料 (Reference List) 35
(八)、圖表 (Tables and Figures) 41
圖一、病例台灣地理分佈圖 41
圖二、S. flexneri 1a NotI-PFGE親源樹狀分析圖 42
圖三、S. flexneri 4a NotI-PFGE親源樹狀分析圖 43
圖四、質體型別分析圖 44
表一、VNC 2001~2003年病例數及PFGE統計表 45
表二、所有菌株基本資料及分析結果 46
表三、抗藥性譜型結果 48
二. 百日咳菌分子流行病學 49
(一)、摘要 (Abstract) 50
英文 50
中文 51
(二)、前言 (Introduction) 52
(三)、材料與方法 (Materials and Methods) 55
1. 細菌菌株及培養 55
2. PFGE操作及親源樹狀圖分析 55
3. 活體攻擊試驗 56
4. 毒力因子測試 56
5. 南方墨點雜交 56
(四)、結果與討論 (Results and Discussion) 58
(五)、結論 (Conclusion) 62
(六)、誌謝 (Acknowledgement) 63
(七)、參考資料 (Reference List) 64
(八)、圖表 (Tables and Figures) 70
表一．1992~1997年台灣百日咳菌株數及次型分佈 71
圖一．各型百日咳PFGE次型在台灣分佈圖 70
圖二．1992-1994年菌株XbaI-PFGE親源樹狀分析圖 71
圖三．南方墨點雜交結果 72
圖四．1992-2001年菌株XbaI-PFGE親源樹狀分析圖 73

(一)
1. Ahmed, K., F. R. Shakoori, and A. R. Shakoori. 2003. Aetiology of shigellosis in northern Pakistan. J. Health Popul. Nutr. 21:32-39.
2. Ashkenazi, S., I. Levy, V. Kazaronovski, and Z. Samra. 2003. Growing antimicrobial resistance of Shigella isolates. J. Antimicrob. Chemother. 51: 427-429.
3. Centers for Disease Control and Prevention. 1996. Shigella sonnei outbreak associated with contaminated drinking water--Island Park, Idaho, August 1995. JAMA 275:1071.
4. Centers for Disease Control and Prevention. 2003. Shigella surveillance: Annual summary, 2002. Atalanta, Georgia: US Department of Health and Human Services, Centers for Disease Control and Prevention.
5. Chen, J. H., C. S. Chiou, P. C. Chen, T. L. Liao, T. L. Liao, J. M. Li, and W. B. Hsu. 2003. Molecular epidemiology of Shigella in a Taiwan township during 1996 to 2000. J. Clin. Microbiol. 41:3078-3088.
6. Chiou, C. S., W. B. Hsu, H. L. Wei, and J. H. Chen. 2001. Molecular epidemiology of a Shigella flexneri outbreak in a mountainous township in Taiwan, Republic of China. J. Clin. Microbiol. 39:1048-1056.
7. Department of Health, Taiwan. 2003. Statistics of Communicable Diseases and Surveillance Report in Taiwan Area. Department of Health, The Executive Yuan, Taipei, Taiwan.
8. Egah, D. Z., E. B. Banwat, E. S. Audu, J. A. Allanana, M. L. Danung, J. G. Damen, and B. P. Badung. 2003. Multiple drug resistant strains of Shigella isolated in Jos, central Nigeria. Niger. Postgrad Med. J. 10:154-156.
9. Gautom, R. K. 1997. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day. J. Clin. Microbiol. 35:2977-2980.
10. Horan, M. A., R. S. Gulati, R. A. Fox, E. Glew, L. Ganguli, and M. Kaeney. 1984. Outbreak of Shigella sonnei dysentery on a geriatric assessment ward. J. Hosp. Infect. 5:210-212.
11. Hunter, P. R. and P. G. Hutchings. 1987. Outbreak of Shigella sonnei dysentery on a long stay psychogeriatric ward. J. Hosp. Infect. 10:73-76.
12. Kotloff, K. L., J. P. Winickoff, B. Ivanoff, J. D. Clemens, D. L. Swerdlow, P. J. Sansonetti, G. K. Adak, and M. M. Levine. 1999. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. World Health Organ. 77:651-666.
13. Lee, T. M., C. Y. Chang, L. L. Chang, W. M. Chen, T. K. Wang, and S. F. Chang. 2003. One predominant type of genetically closely related Shigella sonnei prevalent in four sequential outbreaks in school children. Diagn. Microbiol. Infect. Dis. 45:173-181.
14. Maguire, H. C., C. Seng, S. Chambers, T. Cheasty, G. Double, N. Soltanpoor, and D. Morse. 1998. Shigella outbreak in a school associated with eating canteen food and person to person spread. Commun. Dis. Public Health 1:279-280.
15. Maraki, S., A. Georgiladakis, Y. Tselentis, and G. Samonis. 2003. A 5-year study of the bacterial pathogens associated with acute diarrhoea on the island of Crete, Greece, and their resistance to antibiotics. Eur. J. Epidemiol. 18:85-90.
16. McCall, B., R. Stafford, S. Cherian, K. Heel, H. Smith, N. Corones, and S. Gilmore. 2000. An outbreak of multi-resistant Shigella sonnei in a long-stay geriatric nursing centre. Commun. Dis. Intell. 24:272-275.
17. MoezArdalan, K., M. R. Zali, M. M. Dallal, M. R. Hemami, and S. Salmanzadeh-Ahrabi. 2003. Prevalence and pattern of antimicrobial resistance of Shigella species among patients with acute diarrhoea in Karaj, Tehran, Iran. J. Health Popul. Nutr. 21:96-102.
18. National Committee for Clinical Laboratory Standards. 1997. Performance standards for antimicrobial disk susceptibility tests, 6th ed. Approved standard M2-A6. National committee for Clinical Laboratory Standards, Wayne, Pa.
19. Olive, D.M. and P. Bean. 1999. Principles and application of methods for DNA-based typing of microbial organisms. J. Clin. Microbiol. 37:1661-1669.
20. Sansonetti, P. J. 1992. Molecular and cellular bases of the virulence of Shigella flexneri. Ann. Gastroenterol. Hepatol.(Paris) 28:44-47.
21. Shiferaw, B., S. Shallow, R. Marcus, S. Segler, D. Soderlund, F. P. Hardnett, and T. Van Gilder. 2004. Trends in population-based active surveillance for shigellosis and demographic variability in FoodNet sites, 1996-1999. Clin. Infect. Dis. 38 Suppl 3:S175-S180.
22. Surdeanu, M., L. Ciudin, E. Pencu, and M. Straut. 2003. Comparative study of three different DNA fingerprint techniques for molecular typing of Shigella flexneri strains isolated in Romania. Eur. J. Epidemiol. 18:703-710.
23. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.
24. Totaro, J., C. Tan, V. Reddy, K. Dail, M. Davies, P. Jenkins, J. M. Maillard, D. M. Toney, A. Beall, E. Mintz, M. Drees, and A. Shane. 2004. Day care-related outbreaks of rhamnose-negative Shigella sonnei--six states, June 2001-March 2003. Morb. Mortal. Wkly. Rep. 53:60-63.
25. van Pelt, W., M. A. de Wit, W. J. Wannet, E. J. Ligtvoet, M. A. Widdowson, and Y. T. van Duynhoven. 2003. Laboratory surveillance of bacterial gastroenteric pathogens in The Netherlands, 1991-2001. Epidemiol. Infect. 130:431-441.
26. Yamada, S., K. Ogata, R. Kato, K. Morimoto, Y. Hayashi, T. Ito, S. Matushita, N. Konishi, A. Kai, and M. Endoh. 1999. Outbreak case caused by different colicin type of Shigella sonnei in a day nursery in Tokyo (1998). Kansenshogaku Zasshi. 73:1130-1139.
27. World Health Organization. Programme for Control of Diarrhoeal Diseases. Guidlines for the control of epidemics due to shigella dysenteriae 1, Geneva: World Health Organization, 1988;1-15, (WHO/CDD/SER/88.12.)
28. Yurdakok, K., N. Sahin, E. Ozmert, and E. Berkman. 1997. Shigella gastroenteritis: clinical and epidemiological aspects, and antibiotic susceptibility. Acta Paediatr. Jpn. 39:681-684.
(二)
1. 1996. Update: vaccine side effects, adverse reactions, contraindications, and precautions. Recommendations of the Advisory Committee on Immunization Practices (ACIP). Morb. Mortal. Wkly. Rep. Recomm. 45:1-35.
2. 2002. Food and Drug Administration approval of a fifth acellular pertussis vaccine for use among infants and young children--United States, 2002. Morb. Mortal. Wkly. Rep. 51:574.
3. 2002. Multistate outbreak of Escherichia coli O157:H7 infections associated with eating ground beef--United States, June-July 2002. Morb. Mortal. Wkly. Rep. 51:637-639.
4. 2003. Statistics of Communicable Diseases and Surveillance Report in Taiwan Area, 2001. Center for Disease Control, Department of Health, The Executive Yuan, Taipei, Taiwan.
5. Beall, B., P. K. Cassiday, and G. N. Sanden. 1995. Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis. J. Clin. Microbiol. 33:3083-3086.
6. Bisgard, K. M., C. D. Christie, S. F. Reising, G. N. Sanden, P. K. Cassiday, C. Gomersall, W. A. Wattigney, N. E. Roberts, and P. M. Strebel. 2001. Molecular epidemiology of Bordetella pertussis by pulsed-field gel electrophoresis profile: Cincinnati, 1989-1996. J. Infect. Dis. 183:1360-1367.
7. Bopp, D. J., B. D. Sauders, A. L. Waring, J. Ackelsberg, N. Dumas, E. Braun-Howland, D. Dziewulski, B. J. Wallace, M. Kelly, T. Halse, K. A. Musser, P. F. Smith, D. L. Morse, and R. J. Limberger. 2003. Detection, Isolation, and Molecular Subtyping of Escherichia coli O157:H7 and Campylobacter jejuni Associated with a Large Waterborne Outbreak. J. Clin. Microbiol. 41:174-180.
8. Brennan, M., P. Strebel, H. George, W. K. Yih, R. Tachdjian, S. M. Lett, P. Cassiday, G. Sanden, and M. Wharton. 2000. Evidence for transmission of pertussis in schools, Massachusetts, 1996: epidemiologic data supported by pulsed-field gel electrophoresis studies. J. Infect. Dis. 181:210-215.
9. Cherry, J. D. 1996. Historical review of pertussis and the classical vaccine. J. Infect. Dis. 174 Suppl. 3:S259-S263.
10. Chiou, C. S., W. B. Hsu, H. L. Wei, and J. H. Chen. 2001. Molecular epidemiology of a Shigella flexneri outbreak in a mountainous township in Taiwan, Republic of China. J. Clin. Microbiol. 39:1048-1056.
11. Chiu, T. F., C. Y. Lee, P. I. Lee, C. Y. Lu, H. C. Lin, and L. M. Huang. 2000. Pertussis seroepidemiology in Taipei. J. Formos. Med. Assoc. 99:224-228.
12. Christie, C. D., M. L. Marx, C. D. Marchant, and S. F. Reising. 1994. The 1993 epidemic of pertussis in Cincinnati. Resurgence of disease in a highly immunized population of children. N. Engl. J. Med. 331:16-21.
13. Cordova, S. P., M. T. Gilles, and M. Y. Beers. 2000. The outbreak that had to happen: Bordetella pertussis in north-west Western Australia in 1999. Commun. Dis. Intell. 24:375-379.
14. De Melker, H. E., J. F. Schellekens, M. A. Conyn-Van Spaendonck, and S. E. Neppelenbroek. 1999. The incidence of pertussis in the Netherlands has remained high since an outbreak occurred in 1996. Euro. Surveill 4:133-134.
15. de Moissac, Y. R., S. L. Ronald, and M. S. Peppler. 1994. Use of pulsed-field gel electrophoresis for epidemiological study of Bordetella pertussis in a whooping cough outbreak. J. Clin. Microbiol. 32:398-402.
16. Hallander, H. O. and P. Olin. 1999. Marked decline in pertussis followed reintroduction of pertussis vaccination in Sweden. Euro. Surveill. 4:128-129.
17. Heininger, U. 2001. Pertussis: an old disease that is still with us. Curr. Opin. Infect. Dis. 14:329-335.
18. Heininger, U. 2001. Recent progress in clinical and basic pertussis research. Eur. J. Pediatr. 160:203-213.
19. Hoppe, J. E. 1999. Bordetella, p. 625-631. In P. R. Murray, M. A. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.), Mannual of Clinical Microbiology, 7th ed. American society for microbiology, Washington, DC.
20. Houard, S., C. Hackel, A. Herzog, and A. Bollen. 1989. Specific identification of Bordetella pertussis by the polymerase chain reaction. Res. Microbiol. 140:477-487.
21. Juretzko, P., R. von Kries, M. Hermann, C. H. Wirsing von Konig, J. Weil, and G. Giani. 2002. Effectiveness of acellular pertussis vaccine assessed by hospital-based active surveillance in Germany. Clin. Infect. Dis. 35:162-167.
22. Lai, H. K. and S. F. Tsai. 2000. Epidemiology of pertussis in Taiwan, 1993-1998, and its control measures. Epidemiol. Bull. 16:224-281.
23. Moissenet, D., M. Valcin, V. Marchand, E. Grimprel, P. Begue, A. Garbarg-Chenon, and H. Vu-Thien. 1996. Comparative DNA analysis of Bordetella pertussis clinical isolates by pulsed-field gel electrophoresis, randomly amplified polymorphism DNA, and ERIC polymerase chain reaction. FEMS Microbiol. Lett. 143:127-132.
24. Olive, D. M. and P. Bean. 1999. Principles and applications of methods for DNA-based typing of microbial organisms. J. Clin. Microbiol. 37:1661-1669.
25.Popovic, T., S. Schmink, N. A. Rosenstein, G. W. Ajello, M. W. Reeves, B. Plikaytis, S. B. Hunter, E. M. Ribot, D. Boxrud, M. L. Tondella, C. Kim, C. Noble, E. Mothershed, J. Besser, and B. A. Perkins. 2001. Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C. J. Clin. Microbiol. 39:75-85.
26. Swaminathan, B., T. J. Barrett, S. B. Hunter, and R. V. Tauxe. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis. 7:382-389.
27. Tseng, Y. H., K. T. Choy, C. H. Hung, N. T. Lin, J. Y. Liu, C. H. Lou, B. Y. Yang, F. S. Wen, S. F. Weng, and J. R. Wu. 1999. Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation. J. Bacteriol. 181:117-125.
28. van Loo, I. H., K. J. Heuvelman, A. J. King, and F. R. Mooi. 2002. Multilocus sequence typing of Bordetella pertussis based on surface protein genes. J. Clin. Microbiol. 40:1994-2001.
29. Weber, C., C. Boursaux-Eude, G. Coralie, V. Caro, and N. Guiso. 2001. Polymorphism of Bordetella pertussis isolates circulating for the last 10 years in France, where a single effective whole-cell vaccine has been used for more than 30 years. J. Clin. Microbiol. 39:4396-4403.
30. Weber, S., M. A. Pfaller, and L. A. Herwaldt. 1997. Role of molecular epidemiology in infection control. Infect. Dis. Clin. North Am. 11:257-278.
31. Zanardi, L., F. B. Pascual, K. M. Bisgard, T. Murphy, M. Wharton, and E. Maurice. 2002. Pertussis -- United States, 1997--2000. MMWR CDC Surveill. Summ. 51:73-76.