克雷白氏肺炎桿菌在小鼠模式中引起肝膿瘍的致病機轉研究

Determination of the pathogenesis mechanism of Klebsiella pneumoniae-caused liver abscess in a mouse model

劉靜(Ching Liu)

指導教授 ： 賴怡琪

中山醫學大學/醫學院/醫學應用微生物研究所/碩士(2011年)

摘要

Klebsiella pneumoniae (克雷白氏肺炎桿菌)屬於腸內菌科的一員，為造成社區和院內感染的常見病原菌，可引起肺炎、敗血症 、腦膜炎、肝膿瘍、眼內炎、泌尿系統發炎，或是傷口感染等，治療不易且易產生抗藥性。 分析台灣地區肝膿瘍糖尿病患者合併肝膿瘍的致病菌株發現K. pneumoniae是主要的致病菌，這個現象與西方國家常見的由大腸桿菌，鏈球菌（Streptococcus spp.）和厭氧菌引起的肝膿瘍有極大差異。目前我們對於K. pneumoniae 導致肝膿瘍的詳細機制仍不清楚。本研究的主要目的在於瞭解K. pneumoniae在腸道菌落化後擴散到肝臟組織的詳細機制。沿用先前已建立的小鼠模式，本研究運用組織免疫染色法偵測小鼠在口餵感染後的不同時間點組織內K. pneumoniae的分布狀態來推測該菌由腸部擴散到肝臟的路徑。結果顯示在感染早期，K. pneumoniae大量分布在腸繫膜淋巴結 (Mesenteric lymph nodes; MLN)、Peyer’s patches (PP) 與肝臟，暗示腸道菌落化後的K. pneumonia可能是經由PP-MLN dependent 的路徑擴散至肝臟組織。以非侵入式活體分子影像系統(IVIS)追蹤腹腔注射進入小鼠的K. pneumoniae亦發現該菌大量分布在腹腔左右兩側的淋巴結內。為瞭解經由淋巴系統擴散的K. pneumoniae是否可進入特定種類的淋巴細胞內，本研究使用FACS分析由感染GFP-K. pneumoniae小鼠的脾臟與MLN中分離的T細胞、B細胞和吞噬細胞後發現部分的B細胞可能可以攜帶GFP-K. pneumoniae。小鼠受到K. pneumoniae感染後2小時與6小時，可在肝臟組織偵測到IL-10上升，暗示該菌的感染可能與Immune evasion有關。然在感染的48小時，受到K. pneumoniae感染的肝臟以及MLN, PP等淋巴組織有明顯的發炎以及細胞凋亡現象。

關鍵字

克雷白氏肺炎桿菌 ； 肝膿瘍 ； 小鼠模式

並列摘要

Klebsiella pneumoniae belongs to the Enterobacteraceae family. It is a community-acquired and nosocomial pathogen which can causes pneumonia, sepsis, meningitis, liver abscess, endophthalmitis, urinary tract infection, and wound infections. Liver abscess in Taiwan is mostly caused by K. pneumoniae. This is different from Western countries, where liver abscess is mainly attributed to mixed infections of Escherichia coli, Streptococcus and anaerobes. Although K. pneumoniae-caused liver abscess (KLA) has its clinical importance and uniqueness in Taiwan, our knowledge about its pathogenesis mechanism is still limited. The goal of this study is to determine the detailed mechanism of how the intestine-colonized K. pneumoniae disseminate into the liver tissues. Based on the oral infection model established in our lab, this study determined the bacterial loads of mouse tissues by using a CFU counting method and an immunohistochemistry staining method. At the early stage of infection, a large quantity of K. pneumoniae was detected in mesenteric lymph nodes (MLN), Peyer's patches (PP) and the liver. Similarly, the intraperitoneal-injected K. pneumoniae were observed in the right and left abdominal lymph nodes with the aid of in vivo imaging system (IVIS). The results suggested that the lymph system might provide K. pneumoniae a pathway to spread from intestines to the liver. To further investigate whether a specific subset of immune cells can deliver K. pneumoniae, FACS analysis of T cells, B cells, and macrophages, which were isolated from the GFP-K. pneumoniae infected mice showed that this bacterium might be carried inside B cells. As compared to the control mouse, an elevated level of IL-10 was detected in the K. pneumoniae-infected mice at 2 and 6 hour post-inoculation (hpi), suggesting that immune evasion might have a role in the early stage of K. pneumoniae infections. At 48 hpi, inflammation and apoptosis were detected in the K. pneumoniae-infected lymph nodes and liver tissues.

並列關鍵字

Klebsiella pneumoniae ； liver abscess ； mouse model

參考文獻

1. Autenrieth, S. E., T. R. Linzer, C. Hiller, B. Keller, P. Warnke, M. Koberle, E. Bohn, T. Biedermann, H. J. Buhring, G. J. Hammerling, H. G. Rammensee, and I. B. Autenrieth. Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo. PLoS Pathog 6:e1001212.

2. Befus, D. 1986. Immunity in intestinal helminth infections: present concepts, future directions. Trans R Soc Trop Med Hyg 80:735-741.

3. Blanco, L. P., and V. J. DiRita. 2006. Bacterial-associated cholera toxin and GM1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system. Cell Microbiol 8:982-998.

4. Bloom, P. D., and E. C. Boedeker. 1996. Mucosal immune responses to intestinal bacterial pathogens. Semin Gastrointest Dis 7:151-166.

5. Chaubey, V. P., J. D. Pitout, B. Dalton, T. Ross, D. L. Church, D. B. Gregson, and K. B. Laupland. Clinical outcome of empiric antimicrobial therapy of bacteremia due to extended-spectrum beta-lactamase producing Escherichia coli and Klebsiella pneumoniae. BMC Res Notes 3:116.