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- ItemSurface-Enhanced Raman Spectroscopy to Characterize Different Fractions of Extracellular Vesicles from Control and Prostate Cancer Patients(Basel : MDPI, 2021) Osei, Eric Boateng; Paniushkina, Liliia; Wilhelm, Konrad; Popp, Jürgen; Nazarenko, Irina; Krafft, ChristophExtracellular vesicles (EVs) are membrane-enclosed structures ranging in size from about 60 to 800 nm that are released by the cells into the extracellular space; they have attracted interest as easily available biomarkers for cancer diagnostics. In this study, EVs from plasma of control and prostate cancer patients were fractionated by differential centrifugation at 5000× g, 12,000× g and 120,000× g. The remaining supernatants were purified by ultrafiltration to produce EV-depleted free-circulating (fc) fractions. Spontaneous Raman and surface-enhanced Raman spectroscopy (SERS) at 785 nm excitation using silver nanoparticles (AgNPs) were employed as label-free techniques to collect fingerprint spectra and identify the fractions that best discriminate between control and cancer patients. SERS spectra from 10 µL droplets showed an enhanced Raman signature of EV-enriched fractions that were much more intense for cancer patients than controls. The Raman spectra of dehydrated pellets of EV-enriched fractions without AgNPs were dominated by spectral contributions of proteins and showed variations in S-S stretch, tryptophan and protein secondary structure bands between control and cancer fractions. We conclude that the AgNPs-mediated SERS effect strongly enhances Raman bands in EV-enriched fractions, and the fractions, EV12 and EV120 provide the best separation of cancer and control patients by Raman and SERS spectra.
- ItemComparison of Multiscale Imaging Methods for Brain Research(Basel : MDPI, 2020) Tröger, Jessica; Hoischen, Christian; Perner, Birgit; Monajembashi, Shamci; Barbotin, Aurélien; Löschberger, Anna; Eggeling, Christian; Kessels, Michael M.; Qualmann, Britta; Hemmerich, PeterA major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections.
- 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.
- ItemLong-Term Sinonasal Carriage of Staphylococcus aureus and Anti-Staphylococcal Humoral Immune Response in Patients with Chronic Rhinosinusitis(Basel : MDPI, 2021) Thunberg, Ulrica; Hugosson, Svante; Ehricht, Ralf; Monecke, Stefan; Müller, Elke; Cao, Yang; Stegger, Marc; Söderquist, BoWe investigated Staphylococcus aureus diversity, genetic factors, and humoral immune responses against antigens via genome analysis of S. aureus isolates from chronic rhinosinusitis (CRS) patients in a long-term follow-up. Of the 42 patients who provided S. aureus isolates and serum for a previous study, 34 could be included for follow-up after a decade. Clinical examinations were performed and bacterial samples were collected from the maxillary sinus and nares. S. aureus isolates were characterized by whole-genome sequencing, and specific anti-staphylococcal IgG in serum was determined using protein arrays. S. aureus was detected in the nares and/or maxillary sinus at both initial inclusion and follow-up in 15 of the 34 respondents (44%). Three of these (20%) had S. aureus isolates from the same genetic lineage as at inclusion. A low number of single-nucleotide polymorphisms (SNPs) were identified when comparing isolates from nares and maxillary sinus collected at the same time point. The overall change of antibody responses to staphylococcal antigens over time showed great variability, and no correlation was found between the presence of genes encoding antigens and the corresponding anti-staphylococcal IgG in serum; thus our findings did not support a role, in CRS, of the specific S. aureus antigens investigated.
- ItemCharacterization of PVL-Positive MRSA Isolates in Northern Bavaria, Germany over an Eight-Year Period(Basel : MDPI, 2022) Szumlanski, Tobias; Neumann, Bernd; Bertram, Ralph; Simbeck, Alexandra; Ziegler, Renate; Monecke, Stefan; Ehricht, Ralf; Schneider-Brachert, Wulf; Steinmann, JoergPurpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.
- ItemThe Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates(Basel : MDPI, 2021) Bernreiter-Hofer, Tanja; Schwarz, Lukas; Müller, Elke; Cabal-Rosel, Adriana; Korus, Maciej; Misic, Dusan; Frankenfeld, Katrin; Abraham, Kerstin; Grünzweil, Olivia; Weiss, Astrid; Feßler, Andrea T.; Allerberger, Franz; Schwarz, Stefan; Szostak, Michael P.; Ruppitsch, Werner; Ladinig, Andrea; Spergser, Joachim; Braun, Sascha D.; Monecke, Stefan; Ehricht, Ralf; Loncaric, IgorEscherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.
- ItemBiocompatible magnetic fluids of co-doped iron oxide nanoparticles with tunable magnetic properties(Basel : MDPI, 2020) Dutz, Silvio; Buske, Norbert; Landers, Joachim; Gräfe, Christine; Wende, Heiko; Clement, Joachim H.Magnetite (Fe3O4) particles with a diameter around 10 nm have a very low coercivity (Hc) and relative remnant magnetization (Mr/Ms), which is unfavorable for magnetic fluid hyperthermia. In contrast, cobalt ferrite (CoFe2O4) particles of the same size have a very high Hc and Mr/Ms, which is magnetically too hard to obtain suitable specific heating power (SHP) in hyperthermia. For the optimization of the magnetic properties, the Fe2+ ions of magnetite were substituted by Co2+ step by step, which results in a Co doped iron oxide inverse spinel with an adjustable Fe2+ substitution degree in the full range of pure iron oxide up to pure cobalt ferrite. The obtained magnetic nanoparticles were characterized regarding their structural and magnetic properties as well as their cell toxicity. The pure iron oxide particles showed an average size of 8 nm, which increased up to 12 nm for the cobalt ferrite. For ferrofluids containing the prepared particles, only a limited dependence of Hc and Mr/Ms on the Co content in the particles was found, which confirms a stable dispersion of the particles within the ferrofluid. For dry particles, a strong correlation between the Co content and the resulting Hc and Mr/Ms was detected. For small substitution degrees, only a slight increase in Hc was found for the increasing Co content, whereas for a substitution of more than 10% of the Fe atoms by Co, a strong linear increase in Hc and Mr/Ms was obtained. Mössbauer spectroscopy revealed predominantly Fe3+ in all samples, while also verifying an ordered magnetic structure with a low to moderate surface spin canting. Relative spectral areas of Mössbauer subspectra indicated a mainly random distribution of Co2+ ions rather than the more pronounced octahedral site-preference of bulk CoFe2O4. Cell vitality studies confirmed no increased toxicity of the Co-doped iron oxide nanoparticles compared to the pure iron oxide ones. Magnetic heating performance was confirmed to be a function of coercivity as well. The here presented non-toxic magnetic nanoparticle system enables the tuning of the magnetic properties of the particles without a remarkable change in particles size. The found heating performance is suitable for magnetic hyperthermia application. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemRaman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges(Basel : MDPI, 2021) Azemtsop Matanfack, Georgette; Pistiki, Aikaterini; Rösch, Petra; Popp, JürgenRaman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.
- ItemCharacterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt(Basel : MDPI, 2020) Ahmed, Wedad; Neubauer, Heinrich; Tomaso, Herbert; El Hofy, Fatma Ibrahim; Monecke, Stefan; Abdeltawab, Ashraf Awad; Hotzel, HelmutThe aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistanceassociated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis. © 2020 by the authors.
- ItemPhenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria(Basel : MDPI, 2020) Achek, Rachid; Hotzel, Helmut; Nabi, Ibrahim; Kechida, Souad; Mami, Djamila; Didouh, Nassima; Tomaso, Herbert; Neubauer, Heinrich; Ehricht, Ralf; Monecke, Stefan; El-Adawy, HosnyStaphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilmassociated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilmassociated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.