Data_Sheet_1_Classification of Clinical Isolates of Klebsiella pneumoniae Based on Their in vitro Biofilm Forming Capabilities and Elucidation of the Biofilm Matrix Chemistry With Special Reference to the Protein Content.pdf

Klebsiella pneumoniae is a human pathogen, capable of forming biofilms on abiotic and biotic surfaces. The limitations of the therapeutic options against Klebsiella pneumoniae is actually due to its innate capabilities to form biofilm and harboring determinants of multidrug resistance. We utilized a newer approach for classification of biofilm producing Klebsiella pneumoniae isolates and subsequently we evaluated the chemistry of its slime, more accurately its biofilm. We extracted and determined the amount of polysaccharides and proteins from representative bacterial biofilms. The spatial distribution of sugars and proteins were then investigated in the biofilm matrix using confocal laser scanning microscopy (CLSM). Thereafter, the extracted matrix components were subjected to sophisticated analysis incorporating Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, one-dimensional gel-based electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and MALDI MS/MS analysis. Besides, the quantification of its total proteins, total sugars, uronates, total acetyl content was also done. Results suggest sugars are not the only/major constituent of its biofilms. The proteins were harvested and subjected to SDS-PAGE which revealed various common and unique protein bands. The common band was excised and analyzed by HPLC. MALDI MS/MS results of this common protein band indicated the presence of different proteins within the biofilm. The 55 different proteins were identified including both cytosolic and membrane proteins. About 22 proteins were related to protein synthesis and processing while 15 proteins were identified related to virulence. Similarly, proteins related to energy and metabolism were 8 and those related to capsule and cell wall synthesis were 4. These results will improve our understanding of Klebsiella biofilm composition and will further help us design better strategies for controlling its biofilm such as techniques focused on weakening/targeting certain portions of the slime which is the most common building block of the biofilm matrix.

肺炎克雷伯菌作为一种人类病原体，能够在非生物和生物表面形成生物膜。针对肺炎克雷伯菌的治疗手段局限性实际上源于其固有的形成生物膜的能力以及携带的多药耐药性决定因子。本研究采用了一种新颖的方法对产生生物膜的肺炎克雷伯菌分离株进行分类，并随后对其中所分泌的粘液，即生物膜的化学成分进行了评估。我们从代表性的细菌生物膜中提取并确定了多糖和蛋白质的含量。利用共聚焦激光扫描显微镜（CLSM）研究了生物膜基质中糖类和蛋白质的空间分布。随后，提取的生物膜基质成分经过包括傅里叶变换红外（FTIR）光谱学、核磁共振（NMR）光谱学、一维凝胶电泳（SDS-PAGE）、高效液相色谱（HPLC）和基质辅助激光解吸电离飞行时间质谱/质谱（MALDI MS/MS）分析的复杂分析。此外，还对其总蛋白质、总糖、尿酸盐和总乙酰含量进行了量化。结果表明，糖类并非其生物膜的唯一或主要成分。提取的蛋白质经过SDS-PAGE分析，揭示了多种常见和独特的蛋白质带。常见的蛋白质带被切下并经HPLC分析。此常见蛋白质带的MALDI MS/MS结果表明，生物膜中存在多种不同的蛋白质。共鉴定出55种不同的蛋白质，包括细胞质和膜蛋白。其中，约22种与蛋白质合成和加工相关，15种与致病性相关。同样，与能量和代谢相关的蛋白质有8种，与荚膜和细胞壁合成相关的蛋白质有4种。这些结果将有助于加深我们对肺炎克雷伯菌生物膜组成的理解，并将进一步帮助我们设计更有效的控制其生物膜的战略，例如针对粘液某些部分的技术，而粘液是生物膜基质最普遍的构建块。