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研究生:程微珊
研究生(外文):Wei-Shan Cheng
論文名稱:中臺灣β溶血性G群Streptococcus dysgalactiae subsp. equisimilis臨床分離株毒力因子的分佈
論文名稱(外文):Distributions of virulence factors among β-hemolytic group G Streptococcus dysgalactiae subsp. equisimilis clinical isolates in central Taiwan
指導教授:羅雪霞
指導教授(外文):Hsueh-Hsia Lo
學位類別:碩士
校院名稱:中臺科技大學
系所名稱:醫學檢驗生物技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:62
中文關鍵詞:毒力因子G群鏈球菌
外文關鍵詞:virulence factorsStreptococcus dysgalactiae subsp. equisimilis
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G群鏈球菌 (group G streptococci, GGS) 引起多種疾病,臨床重要性與日俱增,其中又以Streptococcus dysgalactiae subsp. equisimilis (SDSE) 為最重要之人類病原菌。近年來中臺灣SDSE的感染率有增加的趨勢,甚至比A群鏈球菌 (group A streptococci, GAS) 出現的更加頻繁。
許多文獻顯示,SDSE可能藉由水平轉移,獲得一些GAS的毒力因子基因。本研究分析行政院衛生署豐原醫院及台中林新醫院細菌室收集之291株β溶血GGS,經16S rRNA基因定序,其中246株菌(84.5%) 為SDSE,22株 (7.6%) 為S. anginosus,5株 (1.7%) 為S. constellatus,其餘18株 (6.2%) 為non-GGS,顯示SDSE是中臺灣地區臨床感染GGS中最常見的一種。經emm分型,SDSE的emm型別主要為stG10.0,佔39.8% (98/246),其次依序為stG245.0,佔15.4% (38/246)、stG840.0佔12.2% (30/246)、stG6.1佔7.7% (19/246)、stG652.0佔4.1% (10/246),在中臺灣SDSE分離株emm型盛行率異於其它地區。本研究亦發現兩種新emm亞型,分別為stG840.1及stG351.1。利用PCR及Multiplex real-time PCR技術進行毒力因子基因片段增幅,lmb (指令laminin-binding protein)、sagA (指令streptolysin S) 與gapC (指令glyceraldehyde 3-P dehydrogenase) 的基因存在於所有SDSE分離株,帶有slo (指令streptolysin O) 與ska (指令streptokinase)基因的SDSE則分別佔99.2% (244/246) 和72.8% (179/246)。但是,除了74.4% (183/246) 之分離株帶有spegg (指令streptococcus pyrogenic exotoxin Gdys) 之外,所有分離株均未攜帶有其它superantigen基因。經統計分析發現,帶有stG10.0、stG840.0之分離株和spegg的存在為正相關,帶有stG245.0、stG6.1、stG652.0之分離株和spegg的存在則為負相關,帶有stG6.1、stG652.0之分離株和ska為正相關,帶有stG10.0之分離株和ska則為負相關,帶有stG840.0之分離株和cbp的存在為正相關,帶有stG10.0、stG245.0之分離株和cbp的存在則為負相關,帶有stG6.1、stG652.0之分離株和cbp的存在則無顯著性差異。而非無菌部位之分離株和spegg的存在為正相關,無菌部位之分離株和ska的存在為正相關。
這是第一篇在中臺灣進行之β溶血性G群SDSE分離株毒力因子之研究,有助於了解其致病性。
Group G streptococci (GGS) are causative agents of bacteremia, pharyngitis, cellulitis, endocarditis, and streptococcal toxic shock syndrome. The clinical importance of GGS is increasing. Among GGS, Streptococcus dysgalactiae subsp. equisimilis (SDSE) is the major human pathogen. In recent years, the frequency of GGS infections in central Taiwan was higher than that of group A streptococci (GAS). Many studies have revealed that SDSE probably obtained genes encoding virulence factors of GAS via horizontal transfer.
In this study, 291 GGS isolates were collected from the Fong-Yuan Hospital and Lin-Shin Hospital. By 16S rRNA gene sequencing, 246 isolates (84.5%) were identified as SDSE, 22 (7.6%) as S. anginosus, 5 (1.7%) as S. constellatus, while 18 isolates (6.2%) were non-GGS. SDSE is the most common GGS in central Taiwan. By emm typing, the most prevalent emm types were stG10.0 (98/246, 39.8%), stG245.0 (38/246, 15.4%), stG840.0 (30/246, 12.2%), stG6.1 (19/246, 7.7%), and stG652.0 (10/246, 4.1%), respectively. The prevalence of emm types among SDSE isolates in central Taiwan differed from other countries or regions. Two new emm subtypes, stG840.1 and stG351.1, were first found in this study. By PCR or multiplex real-time PCR, it was found that lmb (encoded laminin-binding protein), sagA (encoded streptolysin S) and gapC (encoded glyceraldehyde 3-P dehydrogenase) genes existed in all SDSE isolates. The slo (encoded streptolysin O) gene detected in 99.2% (244/246) of isolates and the ska (encoded streptokinase) gene existed in 72.8% (179/246) of isolates. Except for 74.4% (183/246) of isolates with spegg (encoded streptococcus pyrogenic exotoxin Gdys), whereas all isolates were negative for other superantigens. By chi square test the presence of the spegg gene was positive correlations with emm types stG10.0 and stG840.0. The presence of the spegg gene was negative correlations with emm types stG245.0, stG6.1 and stG652.0. The presence of the ska gene was positive correlations with emm types stG6.1 and stG652.0. The presence of the ska gene was negative correlation with emm type stG10.0.
This is the first study concerning the virulence factors distribution among β-hemolytic group G SDSE isolates in central Taiwan.
目錄
一、前言………………………………………………………………9
(一) β溶血性G群鏈球菌……………………………………………9
(二) SDSE之醫學重要性……………………………………………9
(三) SDSE毒力因子……………………………………………11
(四) 研究目的…………………………………………………………16
二、材料方法……………………………………………………………17
(一) 菌種、培養基及保存……………………………………………17
(二) 染色體DNA抽取…………………………………………………17
(三) 16S rRNA基因定序………………………………………………18 
(四) emm分型…………………………………………………………19
(五) 毒力因子引子(Primer)的設計……………………………………20
(六) 利用PCR測定毒力因子基因……………………………………20
(七) 利用multiplex real-time PCR測定superantigen………………21
(八) 統計分析 (Statistical analysis)…………………………………22
三、結果…………………………………………………………………23
(一) 中臺灣各種GGS之盛行率………………………………………23
(二) 檢體來源分析……………………………………………………23
(三) 病人資料分析……………………………………………………23
(四) SDSE分離株之分子流行病學……………………………………24
(五) SDSE毒力因子的分佈……………………………………………24
(六) SDSE emm型與分離部位之相關性……………………………26
(七) SDSE分離部位與毒力因子之相關性……………………………27
(八) SDSE emm型與毒力因子之相關性………………………………28
四、討論………………………………………………………………30
(一) 中臺灣不同GGS菌種分離率之探討……………………………30
(二) 中臺灣不同emm型SDSE分離株之分佈………………………30
(三) 中臺灣SDSE分離株 emm型與分離部位之相關性…………31
(四) 中臺灣SDSE分離株毒力因子攜帶之情形……………………32
(五) 中臺灣SDSE分離株分離部位與毒力因子之相關性…………32
(六) 中臺灣SDSE分離株emm型與毒力因子之相關性……………33
五、結論…………………………………………………………………34
六、參考文獻……………………………………………………………36





表目錄
表一、使用之各種毒力因子引子序列…………………………………40
表二、使用於multiple real-time PCR增幅superantigen之引子序列
………………………………………………………………………41
表三、中臺灣291 GGS分離株之分子鑑定…………………………43
表四、SDSE感染病人之年齡分佈情形………………………………44
表五、246株 SDSE分離部位與emm型之關係……………………45
表六、SDSE分離株攜帶毒力因子之比例……………………………46
表七、GAS分離株攜帶superantigen毒力因子之比例………………47
表八、S. anginosus group分離株攜帶毒力因子之比例………………48
表九、SDSE emm型與分離部位之間的關係………………………49
表十、SDSE分離部位與毒力因子spegg之間的關係………………50
表十一、SDSE分離部位與毒力因子ska之間的關係………………51
表十二、SDSE分離部位與毒力因子cbp之間的關係………………52
表十三、SDSE emm型與毒力因子spegg之間的關係………………53
表十四、SDSE emm型與毒力因子ska之間的關係…………………54
表十五、SDSE emm型與毒力因子sic之間的關係…………………55


圖目錄
圖一、PCR反應增幅之毒力因子片段…………………………………56
圖二、利用multiplex real-time PCR偵測GAS之superantigen……57
圖三、利用multiplex real-time PCR偵測SDSE之superantigen……58
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