title: Fast label-free detection of Legionella spp. in biofilms by applying immunomagnetic beads and Raman spectroscopy.
abstract: 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.
authors.affiliation: Institut für Physikalische Chemie and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, D-07743 Jena, Germany.
authors.forename: Dragana
authors.initials: D
authors.lastname: Kusić
authors.affiliation: 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. Electronic address: petra.roesch@uni-jena.de.
authors.forename: Petra
authors.initials: P
authors.lastname: Rösch
authors.affiliation: Institut für Physikalische Chemie and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, D-07743 Jena, Germany; Leibniz Institute of Photonic Technology (IPHT), Albert-Einstein-Straße 9, D-07745 Jena, Germany; InfectoGnostics Forschungscampus Jena e.V., Zentrum für Angewandte Forschung, Philosophenweg 7, D-07743 Jena, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Popp
mesh_terms: D018441:Biofilms; D018189:Immunomagnetic Separation; D007875:Legionella / Q000145:classification* / Q000302:isolation & purification / Q000502:physiology*; D013059:Spectrum Analysis, Raman
keywords: Biofilms; Immunomagnetic separation; Legionella spp.; Raman spectroscopy