Kusić Dragana, Rösch Petra, Popp Jürgen
Institut für Physikalische Chemie and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, D-07743 Jena, Germany.
Institut für Physikalische Chemie and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, D-07743 Jena, Germany; InfectoGnostics Forschungscampus Jena e.V., Zentrum für Angewandte Forschung, Philosophenweg 7, D-07743 Jena, Germany.
Syst Appl Microbiol. 2016 Mar;39(2):132-40. doi: 10.1016/j.syapm.2016.01.002. Epub 2016 Feb 11.
Legionellae colonize biofilms, can form a biofilm by itself and multiply intracellularly within the protozoa commonly found in water distribution systems. Approximately half of the known species are pathogenic and have been connected to severe multisystem Legionnaires' disease. The detection methods for Legionella spp. in water samples are still based on cultivation, which is time consuming due to the slow growth of this bacterium. Here, we developed a cultivation-independent, label-free and fast detection method for legionellae in a biofilm matrix based on the Raman spectroscopic analysis of isolated single cells via immunomagnetic separation (IMS). A database comprising the Raman spectra of single bacterial cells captured and separated from the biofilms formed by each species was used to build the identification method based on a support vector machine (SVM) discriminative classifier. The complete method allows the detection of Legionella spp. in 100 min. Cross-reactivity of Legionella spp. specific immunomagnetic beads to the other studied genera was tested, where only small cell amounts of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli compared to the initial number of cells were isolated by the immunobeads. Nevertheless, the Raman spectra collected from isolated non-targeted bacteria were well-discriminated from the Raman spectra collected from isolated Legionella cells, whereby the Raman spectra of the independent dataset of Legionella strains were assigned with an accuracy of 98.6%. In addition, Raman spectroscopy was also used to differentiate between isolated Legionella species.
军团菌可在生物膜中定殖，能自行形成生物膜，并在供水系统中常见的原生动物细胞内繁殖。已知的军团菌物种中约有一半具有致病性，并与严重的多系统军团病有关。水样中军团菌属的检测方法仍基于培养法，由于这种细菌生长缓慢，该方法耗时较长。在此，我们基于通过免疫磁珠分离（IMS）对单个分离细胞进行拉曼光谱分析，开发了一种无需培养、无标记且快速检测生物膜基质中军团菌的方法。利用一个包含从每种军团菌形成的生物膜中捕获并分离的单个细菌细胞拉曼光谱的数据库，构建基于支持向量机（SVM）判别分类器的鉴定方法。完整方法可在100分钟内检测出军团菌属。测试了军团菌属特异性免疫磁珠与其他研究属的交叉反应性，结果发现，与初始细胞数量相比，免疫磁珠仅分离出少量的铜绿假单胞菌、肺炎克雷伯菌和大肠杆菌细胞。然而，从分离出的非目标细菌收集的拉曼光谱与从分离出的军团菌细胞收集的拉曼光谱能很好地区分，据此，军团菌菌株独立数据集的拉曼光谱分配准确率达98.6%。此外，拉曼光谱还用于区分分离出的军团菌物种。
