Section for Oral Biology and Pathology, Faculty of Odontology and Biofilms Research Center for Biointerfaces, Malmö University, Malmö, Sweden.
CR Competence, Lund, Sweden.
Microbiol Spectr. 2024 Jul 2;12(7):e0025324. doi: 10.1128/spectrum.00253-24. Epub 2024 May 24.
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 . 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, 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 .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 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.
在自然界中,细菌通常以混合物种生物膜的形式存在,在这种生物膜中,它们会发生一系列协同和拮抗相互作用,从而提高它们对环境挑战的抵抗力。生物膜是持续性感染的主要原因,从初始焦点的分散会导致在远处部位发生新的感染,因此需要进一步研究。由于难以识别不同的细菌物种,混合物种生物膜的发育和空间相互作用的研究可能具有挑战性。在这里,我们将 CellTrace 染料应用于生物膜细菌的研究,并提出了一种多重标记的新应用,允许在混合物种生物膜模型中鉴定不同的细菌。用 CellTrace 染料(远红、黄、紫和 CFSE[绿])标记的口腔细菌被用于创建单种和混合物种生物膜,并使用共焦旋转盘显微镜(CSDM)进行分析。用流式细胞术(FC)研究生物膜上清液。革兰氏阳性和革兰氏阴性细菌都得到了很好的标记,CSDM 显示生物膜具有清晰的形态,并且稳定染色可持续长达 4 天。使用 FC 分析生物膜上清液中 CellTrace 标记细胞显示出不同细菌物种之间生物膜分散的差异。用不同颜色的染料进行多重标记可以可视化混合物种生物膜中细菌之间的空间关系,并通过 CSDM 图像的分割揭示不同物种的相对覆盖率。因此,这种新的应用为研究混合物种生物膜的结构和组成提供了一个强大的工具。
虽然大多数慢性感染是由混合物种生物膜引起的,但我们的大部分知识仍然来自于单一细菌物种的浮游培养物。因此,需要研究混合物种生物膜的形成和发展。这项工作描述了一种适用于标记细菌以研究生物膜结构和分散的方法。关键是,标记的细菌可以通过共焦旋转盘显微镜进行多重标记,以识别混合物种生物膜中的不同物种,从而在不同环境条件下研究生物膜的发育和空间相互作用。这项研究是增加此类复杂和具有挑战性的研究工具的重要一步。