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研究生:湯惠玲
研究生(外文):Tang Hui Ling
論文名稱:探討臨床分離克雷伯氏肺炎桿菌的基因分型和臨床疾病之關聯性
論文名稱(外文):The Genotypes of Klebsiella pneumoniae and Clinical Disease Manifestation
指導教授:林靜茹林靜茹引用關係盧敏吉盧敏吉引用關係
指導教授(外文):Lin Ching JuLu Min Chi
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:118
中文關鍵詞:肺炎克雷伯氏菌糖尿病敗血症肝膿瘍眼內炎脈衝式電泳腦膜炎黏性試驗
外文關鍵詞:Klebsiella Pneumoniaediabetespneumoniasepsisliver abscessEndophthalmitisPulsed-Field Gel ElectrophoresismeningitisString-test
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克雷伯氏肺炎桿菌(Klebsiella pneumoniae;K. pneumoniae )是常見的致病原,在台灣K. pneumoniae的感染,好發於糖尿病病人且併發其它多種的臨床病症,如原發性肺炎、肝膿瘍、腦膜炎、眼內炎等;附著於細胞壁周圍而成的莢膜,組織嚴密含有多醣類或多胜肽或兩者成分所組成之膠狀聚合物,可以保護細菌免受宿主細胞吞噬。已知K. pneumoniae具有莢膜與否影響其毒性反應,但莢膜黏性對於K. pneumoniae可能造成臨床病徵的機轉目前仍是不清楚,因此本研究收集自中國醫學大學附設醫院病患之痰液、血液、稠膿及患部沖洗液培養之K. pneumoniae檢體共485個,來自糖尿病、非糖尿病人,患有肺炎、肝膿瘍、眼內炎等院內或社區感染症之病患,以臨床病例調查及分子生物學的方法,對連續收集的K. pneumoniae菌株及來源病人進行分析,探討不同臨床病症患者所分離出的K. pneumoniae,其基因型態與毒性因子的關係。本實驗使用脈衝式電泳(Pulsed field gel electrophoresis;PFGE)進行基因分型,發現K. pneumoniae的基因型是多樣性的;經電腦軟體精確比對基因型後可將所有菌株分為八個群組(cluster),即A、A’、B、C、D、D’、E、F;所分離出的菌株以黏液性試驗(string test)分析,3/4以低黏液性表現,cluster內的菌株的來源病人以罹患院外感染為多但這和菌株的黏性無關;病患是否為糖尿病與所分離菌株的黏性、cluster分類、或是否為院外感染等並無統計上的差異,但院內感染病患無糖尿病者明顯多於糖尿病者。血液及體液分離出的K. pneumoniae以肝硬化患者感染佔多數,則可能是由於宿主全身系統性的免疫反應低下所導致,但由於菌株數太少有待更進一步的研究。
In Taiwan, Klebsiella pneumoniae (K. pneumoniae) infection is presented with diverse clinical manifestations, including liver abscess and community-acquired pneumonia. The infection tends to occur in diabetic patients and causes a high morbidity and mortality. From April 2002 to June 2003, we collected sequential K. pneumoniae clinical isolates of sputum, blood, pus and fluid, and other samples from Microbiology Laboratory of China Medical University Hospital, Taichung, Taiwan. Clinical data was obtained and K. pneumoniae was harvested in BAP agar plate at 37℃ for 18 hr. Subsequently, mucoviscosity phenotype and genotype was determined by String test and pulse field gel electrophoresis (PFGE) analysis, respectively. Clinical features of these patients were thereafter correlated with the mucoid types and PFGE patterns of K. pneumoniae. The results showed that 144/485 isolates (29.7%) were String test positive, i.e., hypermucoid, and the rest 341/485 (70.3%) were hypomucoid. The Xba I - digested K. pneumoniae displayed a differential PFGE pattern and 33.6% of these K. pneumoniae isolates were among 8 distinct clusters, i.e., clusters A, A’, B, C, D, D’, E, and F. The clinical presentations of infected patients and the isolated K. pneumoniae were further investigated for the elucidation of possible virulence-related factors. The mucoviscosity of intra-cluster K. pneumoniae was not correlated with the resulted community-acquired infection. As well, diabetes mellitus (DM) or non-DM patients did not contract K. pneumoniae of particular mucoid patterns or clusters. In our samples, blood and body fluid isolated-K. pneumoniae were largely from patients with liver diseases, including liver cirrhosis. This might be due to the dysfunction of host immunity. However, due to limited patient number, the above result needs further investigation.
目 錄
學位考試委員審定書影本………………………………………. 1
致謝………………………………………………………………. 2
中文摘要…………………………………………………………. 4
英文摘要…………………………………………………………. 6
目錄………………………………………………………………. 8
表目錄……………………………………………………………. 13
圖目錄……………………………………………………………. 14
前言………………………………………………………………. 15
第一章緒論…………………………………………………….… 16
壹、克雷伯氏肺炎桿菌好發在台灣及糖尿病病人,引發臨床多樣性之疾病……………………………………….…… 16
一、克雷伯氏肺炎桿菌是台灣常見的感染菌………… 16
二、克雷伯氏肺炎桿菌感染常見於台灣糖尿病病人… 17
三、臨床上常見的克雷伯氏肺炎桿菌感染………….... 18
1、細菌性肺炎……………………………………...… 18
2、肝膿瘍……………………………………….…..… 20
3、敗血症………………………………………..….… 22
4、腦膜炎………………………………………..….… 24
5、眼內炎………………………………………….….. 24
6、泌尿道感染……………………………………...… 24
7、外科傷口感染………………………………..……. 25
貳、克雷伯氏肺炎桿菌的微生物學………………………… 25
一、細菌的基本結構………………………………….…… 26
1、細胞壁…………………………………………...… 26
2、細胞質膜………………………………………...… 27
3、細胞質…………………………………………...… 27
二、細菌的特殊結構…………………………………..…... 27
1、莢膜…………………………………………...…… 28
2、纖毛…………………………………..……….…… 28
三、克雷伯氏肺炎桿菌造成伺機性感染的致病機轉… 29
參、針對克雷伯氏肺炎桿菌的宿主防禦機轉…………...… 30
一、先天性免疫……………………………………….…… 31
1、表皮及黏膜防禦機轉…………………………...… 31
2、發炎反應…………………………………….…..… 31
3、吞噬作用……………………………..……….…… 32
二、後天性免疫………………………………………….… 32
肆、以實驗室分型區分臨床感染的克雷伯氏肺炎桿菌菌株. 33
一、細菌的血清分型……………………………………… 33
二、菌落黏性分析………………………………………… 34
三、聚合酶連鎖反應……………………………………… 34
四、隨機增幅多型性DNA分析法……………….……… 35
五、脈衝式電泳…………………………………………… 36
伍、研究目的…………………………………………………… 36
第二章材料與方法……………………………………….……… 39
壹、臨床檢體的採檢與接種………………………………….… 40
一、採取檢體時間…………………………………….…… 40
二、檢體接種方法…………………………………….…… 40
1、四區劃線法……………………………………...… 40
2、標準圈環直線劃線法……………………….…..… 41
三、檢體培養條件…………………………………….…… 41
貳、需氧細菌類培養……………………………………….…… 42
一、測試方法與原理………………………………….…… 42
1、判讀時間………………………………………...… 42
2、判讀標準………………………………….……..… 42
叁、需氧革蘭氏陰性桿菌的鑑定………………………….…… 44
一、測試方法與原理………………………………….…… 44
二、測試結果判定………………………………….……… 44
肆、細菌藥物敏感性試驗…………………………………….… 45
一、測試方法與原理…………………………………….… 45
二、測試結果判定……………………………………….… 45
伍、菌株之保存……………………………………………….… 46
陸、黏性試驗……………………………………………….…… 46
柒、脈衝式電泳分型………………………………………….… 47
一、原理及方法………………………………………….… 48
二、圖譜資料庫電腦分析……………………………….… 49
1、電腦軟體分析原理……………………………...… 49
2、電腦軟體分析方法……………………………...… 50
捌、臨床資料收集與分析…………………………………….… 50
第三章結果…………………………………………………….… 51
一、菌株資料分析……………………………………….… 51
二、分析脈衝式電泳圖譜資料庫與黏液性的相關性….… 52
1、Cluster和Non-cluster與黏性的分析……………. 52
2、Cluster A、A’、B、C、D、D’、E、F的黏液性分析…………………………………………………. 52
三、Cluster之黏液特性和感染來源的相關性…………… 53
四、克雷伯氏肺炎桿菌在糖尿病病患的致病因子分析 54
五、克雷伯氏肺炎桿菌在肝硬化、肝炎、肝膿瘍病患的致病因子分析……………………………………….… 55
六、不同Clusters的可能致病因子之分析……….…….… 57
第四章討論…………………………………………………….… 59
第五章結論…………………………………………..……….….. 64
第六章參考文獻……………………………………………….… 65
專有名詞中英文對照與縮寫表…………………………………. 75
附錄………………………………………………………………. 80
壹、儀器設備與分析軟體………………………………………. 80
貳、PFGE試劑及藥品配製…………………………………….. 81
叁、培養基………………………………………………………. 84
表目錄
表一 485株臨床K. pneumoniae菌株黏性分析…….... 86
表二 cluster內的163株菌之基本資料………………….….... 87
表三 K. pneumoniae菌株黏性和院外、院內感染間相關性的分析………………………………………….. 89
表四 從有與無糖尿病的患者所分離出的K. pneumoniae菌株其菌株黏性之分析………….... 90
表五 從有與無糖尿病的患者所分離出的K. pneumoniae菌株在不同clusters間之分佈情形.. 91
表六 從有與無糖尿病的患者所分離出的K. pneumoniae菌株在院外、院內感染的相關性分析.. 93
表七 從肝臟疾病患者分離出的K. pneumoniae在8個cluster的分佈比例……………………………….... 94
表八 臨床上分離出的K. pneumoniae在檢體來源和肝臟疾病間關係的分析………………………….... 96
表九 臨床上分離出K. pneumoniae菌株的檢體來源在8個clusters的分布情形………………..……….. 97
表十 從有與無糖尿病的患者所分離出的K. pneumoniae菌株和肝臟疾病間的關係…………. 98
表十一臨床上分離出的K. pneumoniae在肝臟疾病和院外、院內感染間相關性的分析……..…………… 99
表十二clusters與性別、年齡、檢體來源之關係分析. 101
表十三臨床上分離出的K. pneumoniae在院外、院內感染的血液檢體分離率…………………………….. 103
圖目錄
圖一 PFGE的基因分型與菌株黏液屬性的相關性……. 104
圖二 PFGE和cluster圖例…………………………….….. 105
圖三 不同的Cluster黏液性表現的差異………………... 115
圖四 檢體的K. pneumoniae菌株黏性分析…………….. 116
作者簡介………………………………………………………….. 117
博碩士論文授權書……………………………………………….. 118
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