应用原子力学显微术研究壳聚糖抑菌活性

Eaton, P., J. Fernandes, et al. (2008). "Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus." Ultramicroscopy 108(10): 1128-1134.

译者：乔莹

背景介绍：
    Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus，是葡萄牙Faculdade de Cieˆncias da Universidade do Porto大学发表于Ultramicroscopy的最新研究，文章主要应用原子力学显微术来分析不同分子量壳寡/聚糖对两种品系细菌的抑制作用。

    很多的研究表明壳聚糖具有良好的抑菌活性，然而不同实验室的研究却得到相反的结果。一些研究者认为壳聚糖的抑菌活性较壳寡糖更佳，而另一部分的研究却认为高分子量的壳聚糖，在一些情况下降低了抑菌效果。不同分子量的壳聚糖对不同菌种的抑制机制尚有争议，目前最为广泛认可但未被证实的假设是壳聚糖在细菌外表面形成一层不透水层，或者壳寡糖可渗入细菌细胞壁，使内容物泄露，造成细菌死亡。

    作者选用2种典型的细菌--大肠杆菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus），前者为革兰氏阴性细菌，后者为革兰氏阳性细菌作为抑制的对象。选取分子量628kDa的壳聚糖，壳寡糖分子量<3kDa，脱乙酰度为80-85%。运用原子力学显微术（AMF），直观的图像帮助我们理解不同分子量壳聚糖抑菌机理。图1显示的为大肠杆菌的AMF图像，最上图为未经处理的大肠杆菌，经壳寡糖处理4小时后，细菌出现较大集落，并且能观察到细菌细胞壁的破裂，当处理24小时后，出现更大的集落，并且细菌形态与未处理组有很大不同，作者推测为细菌内含物泄露出来所致。右侧壳聚糖处理组与之类似，但并没有壳寡糖效果强。图2为金黄色葡萄球菌的AFM图像，最上图仍为未处理组，左侧图像为壳寡糖处理组，右侧为壳聚糖处理组。处理4小时组出现部分小集落，有部分细胞出现破裂，但大部分细胞没有明显变化。24小时后，与4小时情况变化不大。壳聚糖处理组结果与之相似。
    作者认为壳寡/聚糖的抑菌效果与细菌品系有很大关。壳寡糖对革兰氏阴性细菌有较强的抑制作用，对革兰氏阳性菌则抑制率较低。
Chitosan has been reported to be a non-toxic, biodegradable antibacterial agent. The aim of this work was to elucidate the relationship between the molecular weight of chitosan and its antimicrobial activity upon two model microorganisms, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Atomic force microscopy (AFM) imaging was used to obtain high-resolution images of the effect of chitosans on the bacterial morphology. The AFM measurements were correlated with viable cell numbers, which show that the two species reacted differently to the high- and low-molecular-weight chitosan derivatives. The images obtained revealed not only the antibacterial effects, but also the response strategies used by the bacteria; cell wall collapse and morphological changes reflected cell death, whereas clustering of bacteria appeared to be associated with cell survival. In addition, nanoindentation experiments with the AFM revealed mechanical changes in the bacterial cell wall induced by the treatment. The nanoindentation results suggested that despite little modification observed in the Gram-positive bacteria in morphological studies, cell wall damage had indeed occurred, since cell wall stiffness was reduced after chitooligosaccharide treatment.