| 摘要: | 在台灣，糖尿病(diabetes mellitus, DM)病人容易感染克雷伯氏肺炎桿菌(Klebsiella pneumoniae, KP)，由KP造成的院內感染與社區性肺炎具有快速的臨床病程與高死亡率，而KP的黏性特質(mucoviscosity)被認為是重要的致病因子。肺臟中常駐的肺泡巨噬細胞(alveolar macrophage, AM)，具有吞噬與殺菌的功能，是肺臟抵抗外來微生物的第一道防線。為探討黏性特質(mucoviscosity)與糖尿病在不同群(cluster) KP間之致病作用，本研究中，將6週齡的C57BL/6J小鼠腹腔注射streptozotocin，以誘發糖尿病，並持續飼養至30週，將不同cluster低黏性(hypo-mucoid)與高黏性(hyper-mucoid)，且基因相似度高達98 % 成對的KP，分別以菌量1×104 cfu感染正常與糖尿病小鼠，於20或40小時將其犧牲。為研究KP在肺部與血液中清除的情形，將小鼠左肺均質(homogenize)，同時收集小鼠心臟血液，進行細菌培養；將小鼠右肺進行支氣管灌流術(bronchoalveolar lavage)分離巨噬細胞，探討KP入侵肺部後引起AM的凋亡(apoptosis)與吞噬(phagocytosis)情形。結果顯示，在hypo-mucoid KP的感染中，DM小鼠肺部或血液內的KP菌量比在正常小鼠的肺或血液內明顯增多；正常小鼠分別感染成對KP時，hyper-mucoid KP在肺中的菌量比hypo-mucoid KP顯著增加。而在宿主防禦方面，DM小鼠的巨噬細胞對KP具有正常的吞噬能力，凋亡現象也與正常小鼠一致。為進一步研究AM與hypo-或hyper-mucoid KP之間是否存在著不同的交互作用(interaction)，因此利用老鼠的巨噬細胞株(RAW 264.7 cell line)感染hypo-或hyper-mucoid KP，比較KP對巨噬細胞的存活力(viability)與凋亡(apoptosis)之作用，同時也比較KP對巨噬細胞的附著(adhesion)與在巨噬細胞內的KP存活量(macrophage-associated KP)，結果發現，hyper-mucoid KP對巨噬細胞具較高的黏著量，甚至可以進入巨噬細胞中存活約一小時；而利用轉移子突變(mini-Tn10 transposon mutagensis)方式將hyper-mucoid KP的黏性去除，再予以感染巨噬細胞，則失去其原先的高黏著量，也無法在巨噬細胞內存活。經由本篇的研究結果，說明了黏性特質影響細菌與巨噬細胞間的交互作用，KP的黏性特質是一個獨立致病因子，hyper-mucoid KP具有較高的致病力；然而，DM此因子一旦存在，便使hypo-mucoid KP的致病力加成，不僅如此，hypo-mucoid KP可能存在著有別於黏性特質的致病因子。; Diabetic (DM) patients are susceptible to Klebsiella pneumoniae (KP) infection, resulting in community- and hospital-acquired pneumonia and a high mortality rate in Taiwan. In the lungs, alveolar macrophages (AM) are pivotal for the defense against KP invasion. To investigate the effect of mucoviscosity and diabetes on virulence of KP between divergent cluster, in this study, C57BL/6J male mice was intraperitoneally injected with streptozotocin at 6 wk and DM was successfully induced with a blood sugar of ≧ 300 ml/dl at 8 wk. At 30 wk, the diabetic mice were instilled intratracheally with hypo- or hyper-mucoid KP from divergent cluster, and sacrificed 20 hr or 40 hr later. To study the clearance of KP in the lung and blood, the KP burden was determined by culturing the homogenate of left lung and the intra-cardiac blood. The lung burden of hyper-mucoid KP, after 20 hr of infection, was greater than that of hypo-mucoid one in control mice. Besides, the hypo-mucoid KP burden in the lung or blood of infected diabetic mice was more than that of control ones. The phagocytic ability and apoptosis of AM from right lung was assessed by Gram stain and FACScan analysis, respectively. The results showed that AM from diabetic mice possess normal phagocytic ability and their percentage of apoptosis was similar to that of control mice. Furthermore, to study the interaction between hypo-/hyper-mucoid KP and macrophage cell line, RAW 264.7 cells were infected with hyper- or hypo-mucoid KP. It showed that there was no difference in the percentages of cell apoptosis. However, hyper-mucoid KP adhere and invade into RAW 264.7 cells easier than hypo-mucoid one, and it survived longer in the host cell. Additionally, mini-Tn10 transposon mutagensis was introduced into wild type KP and resulted in a mutant KP with hypo-mucoid phenotype. RAW 264.7 cells were infected by this mutant KP, and thereafter, the adhesion and invasion of the mutant KP was decreased. These results suggested that the mucoid property was not only a key factor for KP to adhere and to invade macrophages, but also an independent KP virulent factor. On the other hand, DM played a important role for the infection by hypomucoid KP possibly making animals susceptible to hypo-mucoid KP infection. |
