Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
Cluster for Molecular Imaging, Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
Pathog Dis. 2019 Feb 1;77(1). doi: 10.1093/femspd/ftz013.
Bacterial biofilm infections often involve aggregates of bacteria heterogeneously distributed throughout a tissue or on a surface (such as an implanted medical device). Identification of a biofilm infection requires direct visualization via microscopy, followed by characterization of the microbial community by culturing or sequencing-based approaches. A sample, therefore, must be divided prior to analysis, often leading to inconsistent results. We demonstrate a combined approach, using scanning electron microscopy and next-generation shotgun sequencing, to visually identify a biofilm and characterize the microbial community, without dividing the sample. A clinical sample recovered from a patient following a dental root-filling procedure was prepared and visualized by scanning electron microscopy. DNA was then extracted from the sample several years later and analyzed by shotgun sequencing. The method was subsequently validated on in vitro cultures of Pseudomonas aeruginosa biofilm. Between 19 and 21 different genera and species were identified in the clinical sample with an estimated relative abundance greater than 1% by two different estimation approaches. Only eight genera identified were not associated with endodontic infections. This provides a proof-of-concept for a dual, microscopy and sequencing-based approach to identify and characterize bacterial biofilms, which could also easily be implemented in other scientific fields.
细菌生物膜感染通常涉及在组织内或表面（如植入的医疗设备）上不均匀分布的细菌聚集体。生物膜感染的鉴定需要通过显微镜直接观察，然后通过培养或基于测序的方法对微生物群落进行特征描述。因此，在分析之前，样本必须进行分割，这通常会导致结果不一致。我们展示了一种联合方法，使用扫描电子显微镜和下一代鸟枪法测序，在不分割样本的情况下，对生物膜进行可视化识别和微生物群落进行特征描述。从患者根管填充手术后恢复的临床样本进行准备并通过扫描电子显微镜进行可视化。几年后，从样本中提取 DNA 并通过鸟枪法测序进行分析。该方法随后在体外培养的铜绿假单胞菌生物膜上进行了验证。在临床样本中，通过两种不同的估计方法，鉴定出了 19 到 21 种不同的属和种，估计相对丰度大于 1%。只有 8 种鉴定出的菌属与牙髓感染无关。这为一种基于显微镜和测序的双重方法来识别和描述细菌生物膜提供了概念验证，该方法也可以很容易地应用于其他科学领域。
