Destruction-free procedure for the isolation of bacteria from sputum samples for Raman spectroscopic analysis
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Lower respiratory tract infections are the fourth leading cause of death worldwide. Here, a timely identification of the causing pathogens is crucial to the success of the treatment. Raman spectroscopy allows for quick identification of bacterial cells without the need for time-consuming cultivation steps, which is the current gold standard to detect pathogens. However, before Raman spectroscopy can be used to identify pathogens, they have to be isolated from the sample matrix, i.e., sputum in case of lower respiratory tract infections. In this study, we report an isolation protocol for single bacterial cells from sputum samples for Raman spectroscopic identification. Prior to the isolation, a liquefaction step using the proteolytic enzyme mixture Pronase E is required in order to deal with the high viscosity of sputum. The extraction of the bacteria was subsequently performed via different filtration and centrifugation steps, whereby isolation ratios between 46 and 57 % were achieved for sputa spiked with 6·107 to 6·104 CFU/mL of Staphylococcus aureus. The compatibility of such a liquefaction and isolation procedure towards a Raman spectroscopic classification was shown for five different model species, namely S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. A classification of single-cell Raman spectra of these five species with an accuracy of 98.5 % could be achieved on the basis of a principal component analysis (PCA) followed by a linear discriminant analysis (LDA). These classification results could be validated with an independent test dataset, where 97.4 % of all spectra were identified correctly.
KeywordsRaman spectroscopy Single-cell pathogen identification Sputum Isolation
Funding of the research projects FastDiagnosis (13N11350) and “JBCI 2.0” (03IPT513Y—Unternehmen Region, InnoProfile Transfer) from the Federal Ministry of Education and Research, Germany (BMBF) and FastTB (2013FE9057) from Free State of Thuringia and the European Union (EFRE) are gratefully acknowledged. We thank Prof. Dr. Wolfgang Pfister from the Institute of Medical Microbiology (University Hospital Jena) for providing the used bacterial strains. The authors thank Bernd Kampe for the help with the program GnuR and Prof. Dr. Michael Schmitt, Dr. Susann Meisel, and Dr. Stephan Stöckel for the critical reading of the present manuscript.
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