Fourier transform infrared and Raman microspectroscopy are currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as 6 h, followed by the nonsubjective identification of microorganisms through the use of multivariate statistical analyses. To examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12-and 24-h cultures exhibited a significant amount of heterogeneity. Hierarchical cluster analysis of the spectra from the various positions and depths reveals the presence of different layers in the colonies. Further analysis indicates that spectra acquired from the surface of the colonies exhibit higher levels of glycogen than do the deeper layers of the colony. Additionally, the spectra from the deeper layers present with higher RNA levels than the surface layers. Therefore, the 6-h colonies with their limited heterogeneity are more suitable for inclusion in a spectral database to be used for classification purposes. These results also demonstrate that vibrational spectroscopic techniques can be useful tools for studying the nature of colony development and biofilm formation.In recent years, there has been much effort invested into the development of new techniques for the identification of microorganisms. Many of these methods are aimed at providing the clinician with more rapid identification of the microorganism responsible for infection in order to begin the appropriate course of antimicrobial treatment (1,9,15,21,27,31,44,51). The emergence of these novel methods reflects the rise in drug-resistant microorganisms, which requires that antimicrobial treatment be more effectively managed (2, 12, 28, 52). Among the new methods are those based on vibrational spectroscopic techniques, namely Fourier transform infrared (FT-IR) and Raman spectroscopies. Vibrational spectroscopic methods are reagentless procedures in which there is no need to add dyes or labels for spectral measurement. These nondestructive techniques are based on the absorption (FT-IR) or scattering (Raman) of light directed onto a sample. The amount of light absorbed or scattered depends on the molecules found within the sample and the environment in which these molecules are found. With these highly sensitive techniques, the frequency of light in the resulting spectrum provides biochemical information regarding the molecular composition and molecular structure of and molecular interaction in cells and tissues (24,55). Raman and infrared spectroscopies are complementary techniques which together can provide a more complete impression of the biochemical information within a sample. Furthermore, these two methods differ such that each is capable of providing informatio...

Rapid and accurate identification of enterococci at the species level is an essential task in clinical microbiology since these organisms have emerged as one of the leading causes of nosocomial infections worldwide. Vibrational spectroscopic techniques (infrared [IR] and Raman) could provide potential alternatives to conventional typing methods, because they are fast, easy to perform, and economical. We present a comparative study using phenotypic, genotypic, and vibrational spectroscopic techniques for typing a collection of 18 Enterococcus strains comprising six different species. Classification of the bacteria by Fourier transform (FT)-IR spectroscopy in combination with hierarchical cluster analysis revealed discrepancies for certain strains when compared with results obtained from automated phenotypic systems, such as API and MicroScan. Further diagnostic evaluation using genotypic methods-i.e., PCR of the species-specific ligase and glycopeptide resistance genes, which is limited to the identification of only four Enterococcus species and 16S RNA sequencing, the "gold standard" for identification of enterococci-confirmed the results obtained by the FT-IR classification. These results were later reproduced by three different laboratories, using confocal Raman microspectroscopy, FT-IR attenuated total reflectance spectroscopy, and FT-IR microspectroscopy, demonstrating the discriminative capacity and the reproducibility of the technique. It is concluded that vibrational spectroscopic techniques have great potential as routine methods in clinical microbiology.

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Magnetic resonance imaging (MRI) is at the forefront of non-invasive medical imaging techniques. It provides good spatial and temporal resolution that can be further improved by the use of contrast agents (CAs), providing a valuable tool for diagnostic purposes. Ultrasmall SuperParamagnetic Iron oxides (USPIOs) nanoparticles are attractive MRI contrast agents due to their negative (T 2 ) contrast enhancement capability and biocompatibility. Clusters of USPIOs with polymer material are of particular interest since they can sustain additional functionalities like drug delivery and targeting. Aiming to establish a relation between the cluster morphology and their efficacy as MRI contrast agent (relaxometric properties), we preparedby using three different maghemite (γ-Fe 2 O 3 ) USPIOs' diametersa series of hybrid copolymer/iron oxide CAs presenting two different geometries (micellar or vesicular). The NMR relaxometry profiles confirmed the nature of the physical mechanisms inducing the increased nuclear relaxation rates at low (magnetic anisotropy) and high (Curie relaxation) magnetic fields. A heuristic model, first proposed by Roch, Muller, Gillis, and Brooks, allowed the fitting of the whole longitudinal relaxivity r 1 () profile, for samples with different magnetic core sizes. We show that both types of cluster exhibit transverse relaxivity (r 2 ) values comparable or higher than those of common contrast agents, over the whole tested frequency range. Moreover, in-depth analysis revealed substantially a linear relation between r 2 and the number of encapsulated USPIOs divided by the diameter of the clusters (N USPIO /D H ), for each USPIOs size. The cluster structure (i.e. micelle or vesicle) appeared to have a mild influence on the transverse relaxivity value. Indeed, the r 2 value was mainly governed by the individual size of the USPIOs, correlated to both the cluster external diameter and the magnetic material volume fraction.