Establishment of experimental models for persistence of Klebsiella pneumoniae in the cells

• Main Authors: YU, WEI CHEN, 余威辰
• Other Authors: CHEN, YA-LEI
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/37d332

碩士 === 國立高雄師範大學 === 生物科技系 === 107 === Klebsiella pneumoniae (Kp) ED2 and ED23, serotype K1, ST (sequencing type) 23, are causative agents of liver and lung abscess with bacteremia. However, strain ED23 was isolated from patients with meningitis, but strain ED2 from patients with localized infection. In this project, both strain are ultilized to test the presistance in macrophage. Between ED2 and ED23 cells, this study first established Kp GFP (Green fluorescent protein) strain. Firstly, nitrogen fixation related genes malM, narH and nirD were inserted by GFP driven by promoter pGAP (Promoter Glyceraldehyde-3-phosphate dehydrogenase) using triparental conjugation [donor strain： E. coli π (pKNOCK/pGAP/GFP/malM、narH、nirD::Cm::tetA)； helper strain： E. coli pRK2013； recipient strain： Kp ED2 and ED23]. At our result, Kp ED2 (GFP/narH) and Kp ED23 (GFP/malM) was easy for observation because their GFP intensities. Further, Acanthamoeba lenticulate and mouse peritoneal exudate cells (PEC) models were performed to test the intracellular survivals. In case of A. lenticulate model, Kp ED23 (GFP/malM) is more latent than strain ED2 (GFP/narH). In a co-culture for 20h, 44% of amoeba harboring GFP cells were present in the case of strain ED23 (GFP/malM), but 7% observed on the case of strain ED2 (GFP/narH). In the case of PEC model, 7% of PEC harboring GFP cells were found in the case of strain ED2 (GFP/narH), while 9% found in the case of strain ED23 (GFP/malM).