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- ItemEosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy(Basel : MDPI, 2020) Dorosz, Aleksandra; Grosicki, Marek; Dybas, Jakub; Matuszyk, Ewelina; Rodewald, Marko; Meyer, Tobias; Popp, Jürgen; Malek, Kamilla; Baranska, MalgorzataLeukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.
- ItemComparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates(Birmingham, Ala. : ASM, 2022) Pistiki, Aikaterini; Monecke, Stefan; Shen, Haodong; Ryabchykov, Oleg; Bocklitz, Thomas W.; Rösch, Petra; Ehricht, Ralf; Popp, JürgenMethicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation. IMPORTANCE Label-free Raman spectra capture the high chemical complexity of bacterial cells. Many different Raman approaches have been developed using different excitation wavelength and cell analysis methods. This study highlights the major importance of selecting the most suitable Raman approach, capable of providing spectral features that can be associated with the cell mechanism under investigation. It is shown that the approach of choice for differentiating MRSA from MSSA should be single-cell analysis with 532 nm excitation since it captures the difference in the overall biochemical composition. These results should be taken into consideration in future studies aiming for the development of label-free Raman spectroscopy as a clinical analytical tool for antimicrobial resistance determination.
- ItemBacterial community and PHB-accumulating bacteria associated with the wall and specialized niches of the hindgut of the forest cockchafer (Melolontha hippocastani)(Lausanne : Frontiers Media, 2017) Alonso-Pernas, Pol; Arias-Cordero, Erika; Novoselov, Alexey; Große, Christina; Rybak, Jürgen; Kaltenpoth, Martin; Westermann, Martin; Neugebauer, Ute; Boland, WilhelmA characterization of the bacterial community of the hindgut wall of two larval and the adult stages of the forest cockchafer (Melolontha hippocastani) was carried out using amplicon sequencing of the 16S rRNA gene fragment. We found that, in second-instar larvae, Caulobacteraceae and Pseudomonadaceae showed the highest relative abundances, while in third-instar larvae, the dominant families were Porphyromonadaceae and Bacteroidales-related. In adults, an increase of the relative abundance of Bacteroidetes, Proteobacteria (γ- and δ- classes) and the family Enterococcaceae (Firmicutes) was observed. This suggests that the composition of the hindgut wall community may depend on the insect’s life stage. Additionally, specialized bacterial niches hitherto very poorly described in the literature were spotted at both sides of the distal part of the hindgut chamber. We named these structures “pockets.” Amplicon sequencing of the 16S rRNA gene fragment revealed that the pockets contained a different bacterial community than the surrounding hindgut wall, dominated by Alcaligenaceae and Micrococcaceae-related families. Poly-β-hydroxybutyrate (PHB) accumulation in the pocket was suggested in isolated Achromobacter sp. by Nile Blue staining, and confirmed by gas chromatography–mass spectrometry analysis (GC-MS) on cultured bacterial mass and whole pocket tissue. Raman micro-spectroscopy allowed to visualize the spatial distribution of PHB accumulating bacteria within the pocket tissue. The presence of this polymer might play a role in the colonization of these specialized niches.