Absolute Quantitation of Bacterial Biofilm Adhesion and Viscoelasticity by Microbead Force Spectroscopy
Absolute Quantitation of Bacterial Biofilm Adhesion and Viscoelasticity by Microbead Force Spectroscopy
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DOI：
10.1016/j.bpj.2008.12.3943
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发表时间：
2009-04-08
影响因子：
3.4
通讯作者：
Lam, Joseph S.
中科院分区：
文献类型：
--
作者：
Lau, Peter C. Y.;Dutcher, John R.;Lam, Joseph S.
细菌生物膜是自然界中最普遍的细菌生长方式。细菌的黏附性和粘弹性在生物膜发育的不同阶段起着重要作用。随着细菌细胞不可逆地附着在表面上，生物膜可以生长，其基质粘弹性有助于保持结构完整性，确定抗逆性，并控制分散的便利性。在这项研究中，开发了一种新的应用力谱来表征细菌细胞在生物膜中的表面粘附和粘弹性。通过用闭环原子力显微镜进行微珠力谱分析，我们在一个确定的接触面积上精确地量化了这些特性。利用模型革兰氏阴性细菌铜绿假单胞菌，我们观察到一种等基因脂多糖突变体wapR生物膜的粘附和粘弹性性能与野生型菌株PAO1生物膜的粘附和粘弹性性能有显著差异。此外，两种菌株的生物膜成熟也导致了粘附性和粘弹性的显著变化。为了最大限度地减少实验参数变化导致的力测量变化，我们开发了微珠力谱的标准化条件，以便对不同实验中获得的数据进行有意义的比较。标准条件下测得的力图显示，PAO1和wapR早期生物膜的粘附压力分别为34 +/- 15 Pa和332 +/- 47 Pa，而PAO1和wapR成熟生物膜的粘附压力分别为19 +/- 7 Pa和80 +/- 22 Pa。将蠕变数据拟合到Voigt标准线性固体粘弹性模型中，发现P. aeruginosa的瞬时弹性模量和延迟弹性模量因脂多糖缺乏和生物膜成熟而急剧降低，而粘度仅因生物膜成熟而降低。总之，我们介绍了一种直接的生物物理方法，可以同时定量细菌生物膜在天然条件下的粘附性和粘弹性。该方法对阐明遗传背景、生长条件和环境胁迫对微生物群落生理的影响具有重要价值。
Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 +/- 15 Pa and 332 +/- 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 +/- 7 Pa and 80 +/- 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.