2023, Monecke, Stefan, Bedewy, Amira K., Müller, Elke, Braun, Sascha D., Diezel, Celia, Elsheredy, Amel, Kader, Ola, Reinicke, Martin, Ghazal, Abeer, Rezk, Shahinda, Ehricht, Ralf

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt’s second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.

2020, Litvinova, Karina, Chernysheva, Maria, Stegemann, Berthold, Leyva, Francisco

Wound healing and other surgical technologies traditionally solved by suturing and stapling have recently been enhanced by the application of laser tissue welding. The usage of high energy laser radiation to anastomose tissues eliminates a foreign body reaction, reduces scar formation, and allows for the creation of watertight closure. In the current work, we show that an ultrafast pulsed fibre laser beam with 183 µJ·cm−2 energy fluence at 1550 nm provides successful welding of dissected chicken heart walls with the tensile strength of 1.03±0.12 kg·cm−2 equal to that of native tissue. The welding process was monitored employing fluorescence spectroscopy that detects the biochemical composition of tissues. We believe that fluorescence spectroscopy guided laser tissue welding is a promising approach for decreasing wound healing times and the avoiding risks of postoperative complications.

2020, Bottek, Jenny, Soun, Camille, Lill, Julia K., Dixit, Akanksha, Thiebes, Stephanie, Beerlage, Anna-Lena, Horstmann, Marius, Urbanek, Annett, Heuer, Heike, Uszkoreit, Julian, Eisenacher, Martin, Bracht, Thilo, Sitek, Barbara, Hoffmann, Franziska, Vijitha, Nirojah, von Eggeling, Ferdinand, Engel, Daniel R.

The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CX3CL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CX3CL1 signaling in Cx3cr1gfp/gfp mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CX3CL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder. © 2020, The Author(s).

2022, Marquet, Mike, Hölzer, Martin, Pletz, Mathias W, Viehweger, Adrian, Makarewicz, Oliwia, Ehricht, Ralf, Brandt, Christian

Phages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage"(WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus supporting the user's decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage).

2021, Osei, Eric Boateng, Paniushkina, Liliia, Wilhelm, Konrad, Popp, Jürgen, Nazarenko, Irina, Krafft, Christoph

Extracellular 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.

2020, Rujas, Edurne, Insausti, Sara, Leaman, Daniel P., Carravilla, Pablo, González-Resines, Saul, Monceaux, Valérie, Sánchez-Eugenia, Rubén, Garcıá-Porras, Miguel, Iloro, Ibon, Zhang, Lei, Elortza, Félix, Julien, Jean-Philippe, Saéz-Cirión, Asier, Zwick, Michael B., Eggeling, Christian, Ojida, Akio, Domene, Carmen, Caaveiro, Jose M.M., Nieva, José L.

The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes. © 2020 The Author(s)Rujas et al. describe the site-selective chemical modification of antibodies to improve the molecular recognition of epitopes at membrane surfaces. The modification using aromatic compounds dramatically enhanced the virus neutralization potency and native antigen binding efficiency of HIV-1 antibodies directed against the membrane-embedded MPER epitope. © 2020 The Author(s)

2021, Press, Adrian T., Babic, Petra, Hoffmann, Bianca, Müller, Tina, Foo, Wanling, Hauswald, Walter, Benecke, Jovana, Beretta, Martina, Cseresnyés, Zoltán, Hoeppener, Stephanie, Nischang, Ivo, Coldewey, Sina M., Gräler, Markus H., Bauer, Reinhard, Gonnert, Falk, Gaßler, Nikolaus, Wetzker, Reinhard, Figge, Marc Thilo, Schubert, Ulrich S., Bauer, Michael

Jaundice, the clinical hallmark of infection-associated liver dysfunction, reflects altered membrane organization of the canalicular pole of hepatocytes and portends poor outcomes. Mice lacking phosphoinositide 3-kinase-γ (PI3Kγ) are protected against membrane disintegration and hepatic excretory dysfunction. However, they exhibit a severe immune defect that hinders neutrophil recruitment to sites of infection. To exploit the therapeutic potential of PI3Kγ inhibition in sepsis, a targeted approach to deliver drugs to hepatic parenchymal cells without compromising other cells, in particular immune cells, seems warranted. Here, we demonstrate that nanocarriers functionalized through DY-635, a fluorescent polymethine dye, and a ligand of organic anion transporters can selectively deliver therapeutics to hepatic parenchymal cells. Applying this strategy to a murine model of sepsis, we observed the PI3Kγ-dependent restoration of biliary canalicular architecture, maintained excretory liver function, and improved survival without impairing host defense mechanisms. This strategy carries the potential to expand targeted nanomedicines to disease entities with systemic inflammation and concomitantly impaired barrier functionality.

2021-1-28, Schleusener, Johannes, Guo, Shuxia, Darvin, Maxim E., Thiede, Gisela, Chernavskaia, Olga, Knorr, Florian, Lademann, Jürgen, Popp, Jürgen, Bocklitz, Thomas W.

Psoriasis is considered a widespread dermatological disease that can strongly affect the quality of life. Currently, the treatment is continued until the skin surface appears clinically healed. However, lesions appearing normal may contain modifications in deeper layers. To terminate the treatment too early can highly increase the risk of relapses. Therefore, techniques are needed for a better knowledge of the treatment process, especially to detect the lesion modifications in deeper layers. In this study, we developed a fiber-based SORS-SERDS system in combination with machine learning algorithms to non-invasively determine the treatment efficiency of psoriasis. The system was designed to acquire Raman spectra from three different depths into the skin, which provide rich information about the skin modifications in deeper layers. This way, it is expected to prevent the occurrence of relapses in case of a too short treatment. The method was verified with a study of 24 patients upon their two visits: the data is acquired at the beginning of a standard treatment (visit 1) and four months afterwards (visit 2). A mean sensitivity of ≥85% was achieved to distinguish psoriasis from normal skin at visit 1. At visit 2, where the patients were healed according to the clinical appearance, the mean sensitivity was ≈65%.

2020, Schmidt, Franziska, Thywißen, Andreas, Goldmann, Marie, Cunha, Cristina, Cseresnyés, Zoltán, Schmidt, Hella, Rafiq, Muhammad, Galiani, Silvia, Gräler, Markus H., Chamilos, Georgios, Lacerda, João, Campos, António, Jr., Eggeling, Christian, Figge, Marc Thilo, Heinekamp, Thorsten, Filler, Scott G., Carvalho, Agostinho, Brakhage, Axel A.

Schmidt el al. show that lipid rafts in phagolysosomal membranes of macrophages depend on flotillins. Lipid rafts are required for assembly of vATPase and NADPH oxidase. Conidia of the human-pathogenic fungus Aspergillus fumigatus dysregulate assembly of flotillin-dependent lipid rafts in the phagolysosomal membrane and can thereby escape phagolysosomal digestion. © 2020 The Author(s)Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions and that inhibition of Ca2+-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity. © 2020 The Author(s)

2020, Monecke, Stefan, König, Elisabeth, Earls, Megan R., Leitner, Eva, Müller, Elke, Wagner, Gabriel E., Poitz, David M., Jatzwauk, Lutz, Vremerǎ, Teodora, Dorneanu, Olivia S., Simbeck, Alexandra, Ambrosch, Andreas, Zollner-Schwetz, Ines, Krause, Robert, Ruppitsch, Werner, Schneider-Brachert, Wulf, Coleman, David C., Steinmetz, Ivo, Ehricht, Ralf

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.