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研究生： 徐峰陽
Hsu, Feng-Yang
論文名稱： 台灣豬鏈球菌血清型別、毒力相關基因譜型及藥物敏感性之綜合分析
The comprehensive analysis between serotype, virulence-associated genes profile, and antimicrobial susceptibility of Streptococcus suis in Taiwan
指導教授： 邱明堂
Chiou, Ming-Tang
林昭男
Lin, Chao-Nan
學位類別： 碩士
Master
系所名稱： 獸醫學院 - 獸醫學系所
Department of Veterinary Medicine
畢業學年度： 109
語文別： 中文
論文頁數： 194
中文關鍵詞： 豬鏈球菌 、血清型別 、毒力相關基因譜型 、最小抑制濃度試驗 、常態化抗藥性解釋模型
外文關鍵詞： Streptococcus suis, serotype, virulence-associated genes profile, minimum inhibitory concentration assay, normalized resistance interpretation
DOI URL： http://doi.org/10.6346/NPUST202100160
相關次數： 點閱：41  下載：18 
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    • 豬鏈球菌（Streptococcus suis, S. suis）為重要人畜共通傳染病病原之一，在豬隻可造成腦膜炎、胸膜炎、關節炎或肺炎病變，導致豬隻生長遲滯及死亡。S. suis共區分成29種血清型別（serotype），特定型別具有較強致病能力，例如第2血清型別。此外，其致病機轉牽涉多種毒力相關因子，例如：溶血素（suilysin）、胞壁酸酶釋放蛋白質（muramidase-released protein）及細胞外蛋白質因子（extracellular protein factor）等，可用以評估菌株侵害能力。因此，在S. suis感染症之臨床病例診斷流程中，檢測血清型別及毒力相關基因（virulence-associated genes, VAGs）皆為重要指標。本疾病尚無商業化疫苗可供有效防護，臨床上多使用抗生素進行預防性投藥或病豬治療，故應例行檢測菌株藥物敏感性並追蹤抗藥性變化，以提供謹慎用藥的選擇依據。為了解台灣病豬分離之S. suis菌株血清型別、毒力相關基因譜型與藥物敏感性，本研究自2015至2019年針對S. suis相關病灶之檢體進行菌株分離，以分子生物學方法檢測血清型別和毒力相關基因（sly、mrp、epf），並將相同牧場來源的菌株以17種抗生素進行最小抑制濃度試驗（minimum inhibitory concentration, MIC）。本研究共收集406株S. suis樣本，檢出的22種血清型別中，主要盛行型別為第3型（20.0%）、第8型（11.3%）、第2型（10.3%）及第4型（10.1%），與亞洲主流型別類似，其中第8型比例較其他國家研究多，為台灣菌株的特點之一；sly－/mrp－/epf－（40.9%）、sly－/mrp＋/epf－（29.6%）及sly＋/mrp＋/epf－（18.7%）譜型較盛行。將血清型別對應毒力相關基因譜型，serotype 3 - sly－/mrp＋/epf－（17.4%）和serotype 8 - sly－/mrp－/epf－（10.6%）的組合模式最具代表性（p < 0.01、p < 0.05）。比較菌株於不同感染部位之分離率，血清型別第1型及epf基因與全身性感染病灶較有關聯，血清型別第2、3、4、8型及sly、mrp基因則與呼吸系統病灶較有關聯。46株菌株完成MIC試驗，對amoxicillin、cefotaxime、ceftiofur及florfenicol較具敏感性，而oxytetracycline、doxycycline、tilmicosin、tylosin及lincospectin則不適用於治療感染S. suis之病豬，與各國研究結論相符。本篇為全球首先使用常態化抗藥性解釋模型（normalized resistance interpretation, NRI）分析S. suis菌株的研究，部份菌株對β-內醯胺類藥物具敏感性但為非野型（non-wild type）分類，可能已具有潛在抗藥性能力，須特別留意未來田間盛行率變化。總結而言，本研究提供台灣S. suis菌株血清型別、毒力相關基因譜型及藥物敏感性等豐富資料，並進行多項綜合分析，有助於未來釐清S. suis致病機轉及相關疫苗開發等研究，並提供臨床治療藥物選擇之依據。

    Streptococcus suis (S. suis) is an important zoonotic pathogen in porcine industry over worldwide, causing meningitis, septicemia, arthritis, pneumonia, and even acute death. It has been classified into 29 serotypes based on the differentiation of capsule polysaccharide structures, and some specific serotypes have higher pathogenicity, for instance, serotype 2. Multiple virulence-associated factors are involved in S. suis pathogenesis mechanism such as suilysin (SLY), muramidase-released protein (MRP), and extracellular protein factor (EF). These factors could be key indicators to pathogenicity of the isolates. Thus, serotyping and virulence-associated genes (VAGs) screening are meaningful in diagnosis routine for clinical cases. Without available and efficient commercial vaccine, antibiotic treatment to prevent or control the outbreak of S. suis is still the primary choice in field. Therefore, it is necessary to perform the antimicrobial susceptibility tests and track the resistance dynamic for prudent use references. To investigate serotypes, VAGs profiles, and antimicrobial susceptibility of S. suis from diseased pigs in Taiwan, totally 406 clinical isolates were obtained from S. suis-associated lesions from 2015 to 2019. Serotypes and VAGs (sly, mrp, epf) were both detected by multiplex PCR assays. The isolates from the same farms were subsequently tested for antimicrobial susceptibility against 17 antibiotics by determining minimum inhibitory concentration (MIC) assay. Amount 22 detected serotypes, the predominant serotypes in Taiwan were serotype 3 (20.0%), 8 (11.3%), 2 (10.3%), and 4 (10.1%). This result is similar to those of other Asian countries. In addition, the prevalence of serotype 8 is higher that is one of features in serotype distribution in Taiwan. Main VAGs profiles showed as: sly－/mrp－/epf－ (40.9%), sly－/mrp＋/epf－ (29.6%), and sly＋/mrp＋/epf－ (18.7%). Combined together, serotype 3 - sly－/mrp＋/epf－ (17.4%, p < 0.01) and serotype 8 - sly－/mrp－/epf－ (10.6%, p < 0.05) were dominant patterns as compared to others. Comparing the isolation rates of strains in different lesions, serotype 1 and epf gene were more related to systemic lesions; by contrast, serotype 2, 3, 4, 8 and sly, mrp genes were related to respiratory lesions. Forty-six isolates were performed MIC tests. The results show that isolates are susceptible for amoxicillin, cefotaxime, ceftiofur, and florfenicol; however, resistant to oxytetracycline, doxycycline, tilmicosin, tylosin, and lincospectin, which are not recommended to cure ill pigs affected by S. suis. The tendency is also similar to the previous studies. The present study is the first one to apply normalized resistance interpretation (NRI) to analyze MIC distribution of clinical S. suis isolates over the world. It is necessary to follow up the prevalence of those non-wild type isolates with susceptibility to β-lactam antibiotics, which may have equipped potential resistant abilities. In general, this study provides multiple information on serotype, VAGs profile, and also antimicrobial susceptibility of S. suis isolates in Taiwan. It is valuable for further pathogenesis investigation, vaccine developing, and medication reference establishment.

    中文摘要 I
    Abstract III
    謝誌 V
    目錄 VII
    圖目錄 XII
    表目錄 XIV
    第1章 前言 1
    第2章 文獻回顧 4
    2.1 豬鏈球菌 4
    2.1.1 歷史背景與分類命名 4
    2.1.2 病原形態學及微結構 6
    2.1.3 流行病學 7
    2.1.3.1 自然宿主與棲息部位 7
    2.1.3.2 傳染途徑 8
    2.1.3.3 環境中存活能力 10
    2.1.4 公共衛生意義 11
    2.1.4.1 歐美國家感染概況 12
    2.1.4.2 亞洲國家感染概況 14
    2.1.5 致病機轉 18
    2.1.5.1 呼吸道感染途徑 18
    2.1.5.2 消化道感染途徑 20
    2.1.5.3 毒力相關因子 20
    2.1.6 臨床症狀 22
    2.1.7 病理變化 22
    2.1.7.1 肉眼病變與病灶 22
    2.1.7.2 組織病理學病變 23
    2.1.8 實驗室診斷 24
    2.1.8.1 病原菌分離 24
    2.1.8.2 生化反應鑑別 24
    2.1.8.3 免疫學檢驗 25
    2.1.8.4 分子生物學診斷 26
    2.1.8.5 基質輔助雷射脫附游離飛行時間質譜測定法（MALDI-TOF） 26
    2.1.9 菌株型別測定 27
    2.1.9.1 血清型別 27
    2.1.9.2 基因型別 28
    2.1.10 預防方針 30
    2.1.11 治療方法 31
    第3章 材料與方法 33
    3.1 實驗設計與流程 33
    3.2 樣本採集 33
    3.2.1 臨床病例蒐集 33
    3.2.2 細菌分離 34
    3.2.3 細菌鑑定 34
    3.2.3.1 革蘭氏染色 35
    3.2.3.2 氫氧化鉀試驗 36
    3.2.3.3 過氧化氫酶試驗 36
    3.3 聚合酶鏈鎖反應 36
    3.3.1 細菌核酸萃取 37
    3.3.2 豬鏈球菌菌種鑑別 37
    3.3.2.1 豬鏈球菌16S rRNA基因單管單引子聚合酶鏈鎖反應 37
    3.3.2.2 豬鏈球菌recN基因單管單引子聚合酶鏈鎖反應 38
    3.3.3 分子血清分型 38
    3.3.3.1 血清型別特異性單管多引子聚合酶鏈鎖反應 38
    3.3.3.2 血清型別第1、2、1/2、14型錯配擴增突變測定聚合酶鏈鎖反應 39
    3.3.4 毒力相關基因（VAGs）檢測 40
    3.4 瓊脂膠體電泳分析判讀 40
    3.4.1 電泳瓊脂膠體製備 40
    3.4.2 瓊脂膠體電泳樣本染色劑配製 41
    3.4.3 水平電泳槽電泳 41
    3.4.4 溴化乙錠染色 41
    3.4.5 電泳瓊脂膠體成像攝影 41
    3.5 最小抑制濃度試驗（MIC） 42
    3.5.1 試驗抗生素選用 43
    3.5.2 液態培養基製備 43
    3.5.2.1 陽離子調整Mueller-Hinton液態培養基 44
    3.5.2.2 裂解脫纖馬血 44
    3.5.3 抗生素溶液配製與稀釋 45
    3.5.4 待測菌液製備 46
    3.5.4.1 保菌管復養 46
    3.5.4.2 菌液濃度回歸直線方程式參數計算 47
    3.5.4.3 正式試驗之菌液濃度調整 48
    3.5.4.4 品質管控 48
    3.5.5 培養條件與結果判讀 48
    3.6 統計分析項目 49
    3.6.1 菌株背景資料 49
    3.6.2 血清型別 49
    3.6.3 毒力相關基因（VAGs）譜型 49
    3.6.4 血清型別及VAGs譜型交互對照 49
    3.6.5 最小抑制濃度試驗結果 50
    3.6.5.1 MIC50、MIC90與抗藥性菌株 50
    3.6.5.2 常態化抗藥性解釋模型（NRI） 50
    3.7 統計分析方法 51
    第4章 結果 59
    4.1 菌株樣本收集 59
    4.1.1 各年度菌株樣本數量 59
    4.1.2 縣市地區 59
    4.1.3 豬隻年齡 59
    4.2 分子血清型別分型 60
    4.2.1 各年度型別分布 60
    4.2.2 縣市地區型別分布 60
    4.2.3 豬隻年齡型別分布 61
    4.3 毒力相關基因（VAGs）譜型 61
    4.3.1 各年度譜型分布 61
    4.3.2 縣市地區譜型分布 62
    4.3.3 豬隻年齡譜型分布 62
    4.4 菌株對宿主組織親和性 63
    4.4.1 各年度病灶分離率 63
    4.4.2 豬隻年齡與病灶分離率 63
    4.4.3 血清型別與病灶分離率 63
    4.4.4 VAGs譜型與病灶分離率 64
    4.5 血清型別及VAGs譜型之對應情形 64
    4.6 抗生素最小抑制濃度試驗 65
    4.6.1 牧場與試驗菌株數量 65
    4.6.2 MIC分布、MIC50、MIC90與抗藥性菌株總計 66
    4.6.3 歷年MIC50、MIC90與抗藥性菌株百分比變化 67
    4.6.4 菌株多重抗藥性（MDR）表現形態種類 68
    4.6.5 常態化抗藥性解釋模型（NRI）與野型菌株 68
    4.6.6 抗藥性菌株與非野型菌株之百分比比較 69
    第5章 討論 117
    5.1 菌株樣本來源 117
    5.1.1 採樣豬隻選擇 117
    5.1.2 豬隻來源縣市 118
    5.1.3 不同豬隻年齡階段 119
    5.2 菌株血清型別 119
    5.2.1 歷年型別變化 119
    5.2.2 其他國家分布差異 120
    5.2.3 台灣縣市區域分布 122
    5.2.4 血清型別與感染部位組織親和性之關聯 123
    5.3 菌株VAGs概況 124
    5.3.1 主要VAGs之檢出率 124
    5.3.2 VAGs譜型與感染部位組織親和性之關聯 125
    5.4 菌株血清型別及VAGs譜型之對應情形 126
    5.5 菌株MIC試驗 128
    5.5.1 與其他國家菌株的抗藥性差異 128
    5.5.2 菌株多重抗藥性（MDR）能力與機制 131
    5.5.3 NRI應用與野型菌株判別 132
    5.5.4 不同牧場抗藥性菌株與非野型菌株比較 134
    5.6 其他台灣本土研究之比較 135
    第6章 結論 136
    第7章 參考文獻 138
    附錄 187
    1. 革蘭氏染色原理 187
    2. 氫氧化鉀試驗原理 187
    3. 過氧化氫酶試驗原理 187
    4. 專有名詞中英文對照及縮寫 188
    5. 抗生素藥品名稱中英文對照及縮寫 191
    6. 第4章各節目錄示意圖 192
    7. 第4章各節圖表示意圖 193
    作者簡介 194

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