Confocal raman microspectroscopy : a novel diagnostic tool in medical microbiology

The aim of the research described in this thesis was to develop confocal Raman microspectroscopy techniques for the rapid identification and characterisation of clinically relevant microorganisms. Chapter 2 describes a study in which the accuracy of the identification of Enterococcus spp., based on Raman spectroscopy, is exemplified by a comparison with identification results based on phenotypic and genotypic methods. This chapter clearly shows the high accuracy that can be obtained when applying vibrational spectroscopies for microbial identifications. Chapter 3 reports on the unique method that was developed to analyse microbial microcolonies, directly on the solid culture medium. With this approach it is possible to "record" Raman spectra from very young cultures, where the colonies are typically between 10 and 100 microns in diameter. Culturing times therefore can be decreased to several hours, allowing rapid identification schemes to be developed. Chapter 4 describes the possibilities of this technique to probe the heterogeneity of microbial colonies directly on the solid culture medium. It was shown that microbial colonies after 6 hours incubation were most homogeneous as compared to older (12 and 24 hours) colonies, and consequently these 6 hour old colonies are better suited for the composition of a database. From the results obtained in this study, standardized protocols were derived, based on which reproducible Raman spectra can be obtained. With the methodology for rapidly performing measurements now developed, and the culturing conditions optimized for obtaining reproducible Raman spectra. the potential ofthe technique to identifY microbes by their Raman spectrum was evaluated in chapters. A collection of yeast strains from the genus Candida was used to arrive at the most appropriate manner of analysing large amounts of data. Based on a library of Raman spectra from known Candida strains, an identification method was developed to identify these strains based on their Raman spectrum. Finally, to test the methodology developed in all previous studies, a prospective study of clinical isolates is described in chapter 6. Parallel to the routine analysis of positive blood samples from patients in intensive care units and a random selection of other wards, Raman identification was performed of these samples. A summary of all results is provided in chapter 7

Raman spectroscopy-based identification of nosocomial outbreaks of the clonal bacterium Escherichia coli

DNA-based techniques are frequently used to confirm the relatedness of putative outbreak isolates. These techniques often lack the discriminatory power when analyzing closely related microbes such as E. coli. Here the value of Raman spectroscopy as a typing tool for E. coli in a clinical setting was retrospectively evaluated