Kubiet M, Ramphal R
Department of Medicine, University of Florida College of Medicine, Gainesville 32610.
Infect Immun. 1995 Mar;63(3):899-902. doi: 10.1128/iai.63.3.899-902.1995.
Nontypeable Haemophilus influenzae strains are the most common pathogens encountered in patients with chronic bronchitis. These organisms chronically colonize the airways of patients and occasionally cause bacteremia. Nontypeable H. influenzae strains have been demonstrated microscopically to bind to mucus, but quantitative studies of adhesion have not been published to date. We have therefore developed a reproducible microtiter plate assay to study mucin binding and have examined the adhesion of sputum and blood strains of nontypeable H. influenzae. The assay is similar to that described for Pseudomonas aeruginosa (S. Vishwanath and R. Ramphal, Infect. Immun. 45:197-202, 1984), but notably 2% Tween 20 is used to desorb bacteria from the wells to quantitate bacterial binding. Using a standard strain, we have established that 1 h of incubation is optimum with an inoculum of < or = 5 x 10(8) CFU/ml. The standard strain binds to bronchitic and cystic fibrosis mucins equally well but binds less to bronchiectasis mucins. It does not bind to bovine serum albumin or fetuin. We have also examined the levels of adhesion of freshly isolated sputum and bacteremia strains and find very significant differences in adhesion. Blood strains bound six to seven times less than sputum strains ([13.8 +/- 7] x 10(2) per well versus [102 +/- 43] x 10(2); P < 0.001). Studies with adhesion to lactoferrin, another glycosylated protein, revealed variable binding of respiratory strains but marked binding of blood strains compared with mucin. An isogenic pair of respiratory and blood isolates was examined by electron microscopy but did not show surface differences. We speculate that bacteremic strains studied may have masked, lost, or downregulated adhesin production to allow them to escape from mucins or upregulated adhesins for lactoferrin to invade the bloodstream.
不可分型流感嗜血杆菌菌株是慢性支气管炎患者中最常见的病原体。这些微生物长期定植于患者气道，偶尔引起菌血症。已通过显微镜观察证实不可分型流感嗜血杆菌菌株可与黏液结合，但迄今为止尚未发表关于黏附的定量研究。因此，我们开发了一种可重复的微量滴定板测定法来研究黏蛋白结合，并检测了不可分型流感嗜血杆菌痰液菌株和血液菌株的黏附情况。该测定法与描述铜绿假单胞菌的方法相似（S. Vishwanath和R. Ramphal，《感染与免疫》45:197 - 202，1984），但值得注意的是，使用2%吐温20从孔中解吸附细菌以定量细菌结合。使用标准菌株，我们确定接种量≤5×10⁸CFU/ml时，孵育1小时为最佳条件。标准菌株与支气管炎和囊性纤维化黏蛋白的结合效果相同，但与支气管扩张症黏蛋白的结合较少。它不与牛血清白蛋白或胎球蛋白结合。我们还检测了新鲜分离的痰液菌株和菌血症菌株的黏附水平，发现黏附存在非常显著的差异。血液菌株的黏附比痰液菌株少六到七倍（每孔[13.8±7]×10²对[102±43]×10²；P<0.001）。对另一种糖基化蛋白乳铁蛋白的黏附研究表明，呼吸道菌株的结合情况各不相同，但与黏蛋白相比，血液菌株对乳铁蛋白有明显结合。通过电子显微镜检查了一对同源的呼吸道和血液分离株，但未发现表面差异。我们推测，所研究的菌血症菌株可能已掩盖、丧失或下调了黏附素的产生，以使它们能够从黏蛋白中逃脱，或者上调了对乳铁蛋白的黏附素以侵入血液。
