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標題: | 由肝膿瘍之克雷伯氏肺炎桿菌菌株分離生物膜生成相關基因 Isolation of Genes Involved in Biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess |
作者: | Meng-Chuan Wu 吳孟娟 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 克雷伯氏肺炎桿菌,肝膿瘍,生物膜,海藻醣-六-水解酶,競爭性分析, klebsiella pneumoniae,pyogenic liver abscess,biofilm,sugE,treC, |
出版年 : | 2011 |
學位: | 博士 |
摘要: | 克雷伯氏肺炎桿菌(Klebsiella pneumoniae)為革蘭氏陰性桿菌,屬於腸內菌科,常在免疫功能不全的病患造成泌尿道感染、呼吸道感染、及菌血症,為重要的伺機性感染病原菌。近二十多年來,克雷伯氏肺炎桿菌在許多地區造成社區感染症的病例逐年上升,且其症狀顯著不同,經常造成細菌性肝膿瘍合併菌血症及轉移性的眼內炎或腦膜炎。形成生物膜可使細菌對於抗生素及免疫反應有較強的扺抗能力膜,因此細菌形成生物膜的能力在致病過程中扮演了重要的角色。我們利用微量培養盤探討克雷伯氏肺炎桿菌造成的細菌性肝膿瘍與生物膜形成的相關性,發現肝膿瘍菌株生物膜形成的能力明顯較非組織侵襲性菌株的能力高。接著由肝膿瘍菌株的跳躍子突變株庫中篩選出23株生物膜生成下降與4株上升的突變菌株並分析被影響的基因。在生物膜生成改變的跳躍子突變菌株中,treC突變菌株的生物膜形成、黏性與莢膜多醣的產生量都減少;反之sugE的這些表型都上升。treC與sugE突變菌株生物膜形成能力改變的情況在玻片培養時亦與在微量培養盤中相似,而兩基因的基因剔除菌株也驗證了在跳躍子突變菌株的觀察。treC基因剔除菌株利用海藻醣的能力受到損害,在培養基中加入葡萄糖可使treC基因剔除菌株的生物膜形成能力與莢膜多醣表現量回復。體內競爭性分析的結果顯示treC基因剔除菌株在胃內感染老鼠時,拓殖的競爭力下降。另一方面,轉錄表現分析顯示sugE基因剔除菌株中莢膜多醣產生量的增加是由於莢膜生合成基因群(經由莢膜調控基因rmpA)與treC基因的RNA表現量上升所導致。treC基因在腸胃道感染時的重要性顯示生物膜形成應有助於克雷伯氏肺炎桿菌感染的建立與持續。 Background: Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain. Methodology/Principal Findings: The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice. Conclusions/Significance: We have identified 25 genes important for biofilm formation in a K. pneumoniae PLA strain using microtiter plate assay and confirmed by slide cultures. Among these genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66586 |
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