PDF(Pubmed)
Abstract：
In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.
摘要：
在大自然中,细菌通常以混合物种生物膜的形式存在，他们参与一系列协同和拮抗相互作用，增加他们对环境挑战的抵抗力。生物膜是持续感染的主要原因，并且从初始病灶扩散可能会导致远端部位新的感染，因此需要进一步研究。由于难以原位鉴定不同的细菌物种，因此对混合物种生物膜中的发育和空间相互作用的研究可能具有挑战性。这里,我们将CellTrace染料应用于生物膜细菌的研究，并为多重标记提供了新的应用，允许鉴定混合物种中的不同细菌，体外生物膜模型。用CellTrace染料标记的口腔细菌（远红色，黄色,紫罗兰,和CFSE[绿色])用于创建单物种和混合物种生物膜，用共焦旋转圆盘显微镜(CSDM)分析。用流式细胞术(FC)研究生物膜上清液。革兰氏阳性细菌和革兰氏阴性细菌均被良好标记,并且CSDM显示生物膜具有清晰的形态和稳定的染色长达4天。使用FC对上清液中的CellTrace标记的细胞的分析显示细菌物种之间的生物膜分散的差异。通过对CSDM图像进行分割，可以显示混合物种生物膜中细菌之间的空间关系以及不同物种的相对覆盖率。这个新颖的应用程序，因此，为体外研究混合物种生物膜的结构和组成提供了强大的工具。重要提示虽然大多数慢性感染是由混合物种生物膜引起的，我们的大部分知识仍然来自单一细菌物种的浮游培养。混合物种生物膜的形成和发展的研究是，因此,required.这项工作描述了一种适用于细菌标记的方法，用于生物膜结构和分散的体外研究。严重的,可以使用共聚焦旋转圆盘显微镜对标记的细菌进行多路复用，以鉴定混合物种生物膜中的不同物种，促进不同环境条件下生物膜发育和空间相互作用的研究。这项研究是增加这种复杂和具有挑战性的研究可用工具的重要一步。
