Morphological and biochemical changes in Pseudomonas fluorescens biofilms induced by sub-inhibitory exposure to antimicrobial agents
Morphological and biochemical changes in Pseudomonas fluorescens biofilms induced by sub-inhibitory exposure to antimicrobial agents
复制标题
DOI：
10.1139/w08-109
复制
发表时间：
2009-02-01
影响因子：
2.8
通讯作者：
Hitchcock, Adam P.
中科院分区：
文献类型：
--
作者：
Dynes, James J.;Lawrence, John R.;Hitchcock, Adam P.
使用共聚焦激光扫描显微镜 (CLSM) 和扫描透射 X 射线显微镜 (STXM) 检查在亚抑制浓度的 4 种抗菌剂（三氯生、苯扎氯铵、二盐酸氯己定和磷酸三钠）存在下生长的荧光假单胞菌生物膜的形态和生化变化。使用染色剂 SYTO9 和碘化丙啶进行的 CLSM 分析表明，抗菌剂不同程度地影响细胞膜完整性和细胞密度。然而，荧光素二乙酸酯测定和平板计数表明细胞仍然具有代谢活性。荧光凝集素结合测定表明，生物膜的外聚合物基质的排列和组成也发生了变化，并且这些变化取决于抗菌剂。使用 STXM 进行的详细单细胞分析提供的证据表明，每种抗菌剂的细胞形态以及生物膜和细胞内蛋白质、脂质和多糖的空间分布和相对量是不同的。根据其独特的光谱特征确定，生物膜中氯己定的分布主要位于细菌细胞内。每种抗菌剂都会引起独特的反应；荧光假单胞菌细胞和生物膜改变了它们的形态和结构，以及生物大分子的分布和丰度，特别是外聚合物基质。荧光假单胞菌也表现出对 10 μg/mL 苯扎氯铵的适应。我们的观察指出了外聚合物基质的数量和化学变化在抗菌剂反应中的重要性，并表明它们作为控制目标的重要性。
Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of bio-macromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 mu g/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.