Gerwin J. Puppels

IDENTIFICATION OF MEDICALLY RELEVANT MICROORGANISMS BY VIBRATIONAL SPECTROSCOPY

In the recent years, vibrational spectroscopies (infrared and Raman spectroscopy) have been developed for all sorts of analyses in microbiology. Important features of these methods are the relative ease with which measurements can be performed. Furthermore, in order to obtain infrared or Raman spectra, there is only a limited amount of sample handling involved without the need for expensive chemicals, labels or dyes. Here, we review the potential application of vibrational spectroscopies for the use in medical microbiology. After describing some of the basics of the techniques, considerations on reproducibility and standardisation are presented. Finally, the use of infrared and Raman spectroscopy for the (rapid) identification of medically relevant microorganisms is discussed. It can be concluded that vibrational spectroscopies show high potential as novel methods in medical microbiology.

Combined In Vivo Confocal Raman Spectroscopy and Confocal Microscopy of Human Skin

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.

Prospective Study of the Performance of Vibrational Spectroscopies for Rapid Identification of Bacterial and Fungal Pathogens Recovered from Blood Cultures

ABSTRACT Rapid identification of microbial pathogens reduces infection-related morbidity and mortality of hospitalized patients. Raman spectra and Fourier transform infrared (IR) spectra constitute highly specific spectroscopic fingerprints of microorganisms by which they can be identified. Little biomass is required, so that spectra of microcolonies can be obtained. A prospective clinical study was carried out in which the causative pathogens of bloodstream infections in hospitalized patients were identified. Reference libraries of Raman and IR spectra of bacterial and yeast pathogens highly prevalent in bloodstream infections were created. They were used to develop identification models based on linear discriminant analysis and artificial neural networks. These models were tested by carrying out vibrational spectroscopic identification in parallel with routine diagnostic phenotypic identification. Whereas routine identification has a typical turnaround time of 1 to 2 days, Raman and IR spectra of microcolonies were collected 6 to 8 h after microbial growth was detected by an automated blood culture system. One hundred fifteen samples were analyzed by Raman spectroscopy, of which 109 contained bacteria and 6 contained yeasts. One hundred twenty-one samples were analyzed by IR spectroscopy. Of these, 114 yielded bacteria and 7 were positive for yeasts. High identification accuracy was achieved in both the Raman (92.2%, 106 of 115) and IR (98.3%, 119 of 121) studies. Vibrational spectroscopic techniques enable simple, rapid, and accurate microbial identification. These advantages can be easily transferred to other applications in diagnostic microbiology, e.g., to accelerate identification of fastidious microorganisms.

In Vitro and In Vivo Raman Spectroscopy of Human Skin

Noninvasive techniques that provide detailed information about molecular composition, structure, and interactions are crucial to further our understanding of the relation between skin disease and biochemical changes in the skin, as well as for the development of penetration enhancers for transdermal drug administration. In this study we present in vitro and in vivo Raman spectra of human skin. Using a Raman microspectrometer, in vitro spectra were obtained of thin cross sections of human skin. They provided insight into the molecular composition of different skin layers. Evidence was found for the existence of a large variation in lipid content of the stratum corneum. A simple experimental setup for in vivo confocal Raman microspectroscopy of the skin was developed. In vivo Raman spectra of the stratum corneum were obtained at different positions of the arm and hand of three volunteers. They provided evidence for differences in the concentration of natural moisturizing factor at these positions.

Loss-of-Function Mutations in the Filaggrin Gene Lead to Reduced Level of Natural Moisturizing Factor in the Stratum Corneum

TO THE EDITOR Filaggrin is a key protein required for the formation of the stratum corneum (SC) barrier. Filaggrin is also essential for SC hydration, as it acts as a source of hygroscopic amino acids and their derivatives, known as natural moisturizing factor (NMF). The human gene encoding filaggrin (FLG) is highly polymorphic and to date, 15 null mutations have been detected of which four (R501X, 2282del4, R2447X, and S3247X) are prevalent at varying frequencies in the white European population (Sandilands et al., 2007). Homozygous or compound heterozygous FLG mutations underlie the common skin-keratinizing disorder ichthyosis vulgaris, and have been shown to be a major genetic predisposing factor for atopic dermatitis (AD) (Sandilands et al., 2006). Diminished filaggrin expression has been demonstrated in both ichthyosis vulgaris and AD skin (Seguchi et al., 1996; Sugiura et al., 2005; Smith et al., 2006). As filaggrin is the precursor protein for the amino-acid-derived components of the NMF, we hypothesized that carriers of FLG-null mutations have reduced level of NMF in the SC. To measure NMF in the SC of the palm (thenar eminence) and forearm skin, we used confocal Raman microspectroscopy (3510 Skin Composition Analyzer; River Diagnostics, Rotterdam, The Netherlands). The principles of this method and the procedure have extensively been described elsewhere (Caspers et al., 2001, 2003). The reference spectrum of NMF was constructed from a superposition of the spectra of pyrrolidone-5-carboxylic acid, ornithine, serine, proline, glycine, histidine, and alanine. In addition to NMF, skin barrier function as measured by transepidermal water loss was assessed on the volar forearm (Tewameter 210; Courage and Khazaka Electronic GmbH, Cologne, Germany). One hundred and forty-nine volunteers recruited by public advertisement, as well as 10 AD patients, were screened for four FLG mutations (R501X, 2282del4 R2447X, and S3247X). All subjects filled in a questionnaire on the history of skin diseases and allergies, and the Erlangen atopy questionnaire that also included a question on skin dryness. Signs of active disease (erythema, crusting, weeping, and lichenification) were assessed by a dermatologist. Having visible skin changes on the forearm was the exclusion criterion. Written informed consent was obtained from all subjects. The experimental protocol followed the Declaration of Helsinki Principles and was approved by the Ethical Committee of the Academic Medical Centre. Genomic DNA was extracted from buccal swab samples (Puregene DNA isolation kit; Gentra Systems, Minneapolis, MN). Polymorphisms were genotyped as reported previously (Sandilands et al., 2007). To compare data from two groups, we used twotailed Student’s t-test for unpaired samples. Sixteen carriers (12 female) of an FLG mutation and 23 individuals (15 female) wild type with respect to these mutations were included in the study. Of the 16 carriers, five were heterozygous for R501X, eight were heterozygous for 2282del4, and one was heterozygous for R2447X. One individual was homozygous for Abbreviations: AD, atopic dermatitis; FLG, human filaggrin-encoding gene; NMF, natural moisturizing factor; SC, stratum corneum

Classification and identification of enterococci: a comparative phenotypic, genotypic, and vibrational spectroscopic study

ABSTRACT 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 18Enterococcus 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 Enterococcusspecies 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.

Investigating Microbial (Micro)colony Heterogeneity by Vibrational Spectroscopy

ABSTRACT 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.

Automated depth‐scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin

An automated confocal Raman microspectrometer for rapid measurement of molecular concentration profiles in the skin is described. It permits the successive collection of Raman spectra at a range of depths below the skin surface. The axial resolution of the confocal Raman microspectrometer is 5.1±0.2 µm. The setup was applied to determine water concentration profiles of the stratum corneum and to determine changes therein as a result of hydration of the skin. Copyright

Discriminating Vital Tumor from Necrotic Tissue in Human Glioblastoma Tissue Samples by Raman Spectroscopy

Vital and necrotic glioblastoma tissues were studied by Raman microspectroscopy to identify possibilities for the development of an in vivo Raman method for real-time intraoperative brain biopsy guidance. The histologic malignancy grade of gliomas depends on the presence of parameters such as endothelial proliferation and necrosis, which are often not evenly distributed within the tumor. Because tissue samples obtained by stereotactic surgery are relatively small, sampling errors may easily occur by missing these crucial features. Although necrosis is important for grading, specimens containing only necrosis are diagnostically useless. Raman microspectroscopic mapping experiments were performed on unfixed cryosections of glioblastoma, obtained from 20 patients. After spectral acquisition, a clustering analysis was performed, resulting in groups of similar spectra. Each cluster was assigned a color, and pseudo-color Raman maps of the tissue sections were constructed. After the Raman experiments, the tissue sections were stained for histopathologic analysis, enabling identification of the histologic origin of the Raman spectra and assignment of the Raman spectral clusters to either vital or necrotic tissue. A classification model for discrimination between vital and necrotic tumor tissue based on linear discriminant analysis was developed. The classification model was evaluated using independent Raman data obtained from nine other tissue sections and yielded 100% accuracy. Information about the biochemical differences between necrosis and vital tumor was obtained by the analysis of difference spectra. Necrotic tissue was found to consistently contain higher levels of cholesterol (-esters). This in vitro result indicates that Raman spectra contain the information to distinguish vital glioblastoma from necrosis and makes Raman spectroscopy a powerful candidate for guidance of stereotactic brain biopsy.

Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency

Background Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (ADFLG) compared with that seen in patients with AD without these mutations (ADNON-FLG). Objectives We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with ADFLG versus patients with ADNON-FLG. We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flgft/Flgft [FlgdelAPfal] mice). Methods One hundred thirty-seven patients were studied. NMF levels were ascertained using confocal Raman spectroscopy; transepidermal water loss and skin surface pH were measured. IL-1α, IL-1β, IL-18, IL-1 receptor antagonist (IL-1RA), and IL-8 levels were determined in SC tape strips from 93 patients. All subjects were screened for 9 FLG mutations. Flgft/Flgft (FlgdelAPfal) mice, separated from maFlgft/maFlgft (flaky tail) mice, were used for the preparation and culture of primary murine keratinocytes and as a source of murine skin. RT-PCR was performed using primers specific for murine IL-1α, IL-1β, and IL-1RA. Results SC IL-1 levels were increased in patients with ADFLG; these levels were inversely correlated with NMF levels. NMF values were also inversely correlated with skin surface pH. Skin and keratinocytes from Flgft/Flgft mice had upregulated expression of IL-1β and IL-1RA mRNA. Conclusions ADFLG is associated with an increased SC IL-1 cytokine profile; this profile is also seen in a murine homologue of filaggrin deficiency. These findings might have importance in understanding the influence of FLG mutations on the inflammasome in the pathogenesis of AD and help individualize therapeutic approaches.