TY - THES A1 - Wilhelm, Hannah T1 - Multiresistenzen in klinischen \(C.glabrata\) Isolaten T1 - Multidrug Resistance in clinical \(C.glabarata\) Isolates N2 - Die Zahl invasiver Pilzinfektionen ausgelöst durch C. glabrata steigt zunehmend und auch die Ausbildung multipler Resistenzen wird immer häufiger registriert. In dieser Arbeit wurden zwei klinische MDR-C. glabrata-Stämme systematisch analysiert, um den Ursprung der Mehrfachresistenz zu finden. Aufgefallen waren jene Isolate in vorhergehenden Untersuchungen von Aldejohann et. al., die 176 Stämme, die dem Referenzzentrum NRZ-Myk zugesandt wurden, auf ihr Resistenzverhalten gegen Echinocandine analysierten und auf FKS-Mutationen untersuchten. Die Isolate CG22 und CG56 zeigten ein Resistenzverhalten gegen Anidulafungin ohne eine FKS-Mutation aufzuweisen. In Mehrfachtestungen wurde das einheitliche Verhalten von CG56 in zehn Einzelkolonien verifiziert, um Mischkulturen oder heterogenes Verhalten innerhalb des Isolates ausschließen zu können. Nach Analyse der gesamten Genomsequenz von CG56 zeigte sich eine Mutation kurz vor der HS-Region von FKS2, die eine Erklärung für das Resistenzverhalten zu liefern scheint. Neben der Mutation in FKS2 wurde ebenfalls eine Mutation in FKS1 und in ERG3 bestätigt. Die Mutation in ERG3 führt zu einer Verschiebung im Sterolsynthesepathway und zu einer Neuverteilung der Zellmembranbestandteile. Das klinische Isolat CG22 fällt mit Resistenzen gegen Azole, Echinocandine und Amphotericin B auf und zeigte ebenfalls eine Mutationen in ERG3. Zusätzlich dazu ergab sich eine Loss-of- Function-Mutation in ERG4 und damit verbunden einen massiv reduzierten Ergosterolgehalt der Zellmembran. Die seltene Kombination aus ERG3 und ERG4 Mutation scheint die Erklärung für die außergewöhnliche Amphotericin B-Resistenz von CG22 zu liefern und wird hier als erstmals bei einem C. glabrata Isolat beschrieben. Dieser besondere Stamm, der sogar als panresistent bezeichnet werden kann, sollte Bestandteil weiterer Forschung werden. Der Sterolsynthesepathway dient als Angriffspunkt vieler Antimykotika und kann durch seine vielen Intermediate und abweichenden Abläufen zu unterschiedlichen Stoffwechselendprodukten führen. Der Ergosterolgehalt der Zellmembran eines C. glabrata-Stammes kann weitere Rückschlüsse auf die Empfindlichkeit des Isolates geben und somit die Chancen des Therapieerfolges der Antimykotikagabe besser vorhersagen und könnte somit einen vielversprechenden Beitrag zur Behandlung lebensbedrohlicher Candidosen leisten. N2 - The number of invasive fungal infections caused by C. glabrata is increasing and the development of resistance to multiple drug classes is also being recorded more frequently. In this study, two clinical MDR C. glabrata strains were systematically analyzed to find the origin of their Multidrug Resistance. These isolates had attracted attention in previous studies by Aldejohann et. al. who studied 176 strains that have been sent to the NRZ-Myk reference center. The strains have been analyzed for their resistance to echinocandins and have been examined for mutations in FKS1 and FKS2 hotspots. The isolates CG22 and CG56 showed resistance to anidulafungin without showing mutation in the FKS hotspots. In multiple testing, the uniform behavior of CG56 was verified in ten individual colonies in order to exclude mixed cultures or heterogeneous behavior within the isolate. Analysis of the entire genome sequence of CG56 revealed a mutation just upstream of the HS region of FKS2, which appears to provide an explanation for the resistance behavior. In addition to the mutation in FKS2, a mutation in FKS1 and ERG3 was also confirmed. The mutation in ERG3 leads to a shift in the sterol synthesis pathway and to a redistribution of the cell membrane components. The clinical isolate CG22 was found to be resistant to azoles, echinocandins and amphotericin B. CG22 showed a mutation in ERG3 and additionally a loss-of-function mutation in ERG4. This leads to a massively reduced ergosterol content of the cell membrane. The rare combination of ERG3 and ERG4 mutation seems to explain the extraordinary amphotericin B resistance of CG22 and is described for the first time in a C. glabrata isolate. This particular strain, which can even be described as panresistant, should become part of further research. The sterol synthesis pathway serves as a target for many antimycotics and can lead to different metabolic products due to its many intermediates and divergent processes. The ergosterol content of the cell membrane of a C. glabrata strain can provide further information on the susceptibility of the isolate. This could possibly predict the chances of therapeutic success of antimycotic treatment better and could therefore make a promising contribution to the treatment of life-threatening candidiasis. KW - Torulopsis glabrata KW - Antimykotikum KW - Anidulafungin KW - Amphotericin B KW - FKS Gene KW - Ergosterolsyntheseweg KW - ERG Gene KW - Multiresistenzen Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-347186 ER - TY - THES A1 - Koch, Thorsten Manfred T1 - Wirt – Pathogen Interaktion bei Hornhautinfektionen durch \(Fusarium\) spp. T1 - Host – Pathogen Interaction in Keratitis caused by \(Fusarium\) spp. N2 - Fusarium (F.)-Infektionen des Auges zeigen oft einen schwerwiegenden Verlauf und sind am häufigsten mit Spezies des Fusarium solani species complex assoziiert. Dabei sind das Tragen von weichen Kontaktlinsen sowie Traumata die wichtigsten prädisponierenden Faktoren. Vorangegangene Untersuchungen des Nationalen Referenzzentrums für invasive Pilzinfektionen hatten ergeben, dass Infektionen durch F. petroliphilum mit der Nutzung von Kontaktlinsen, Infektionen durch F. falciforme jedoch überwiegend traumaassoziiert uns vor allem aus tropischen und subtropischen Ländern bekannt sind. Das Ziel dieser Arbeit war es daher zu untersuchen, ob F. falcifomre und F. petroliphilum physiologische Merkmale aufweisen, die für die Unterschiede in den Risikofaktoren für Keratitiden durch die beiden Arten verantwortlich sein könnten. N2 - Fusarium (F.) - infections of the eye often show a severe course and are most frequentlyassociated with species (spp.) of the Fusarium solani species complex (van Diepeningen et al., 2014, Walther et al., 2017, Walther et al., 2018). Wearing soft contact lenses (CL) and trauma are the most important predisposing factors (Gaujoux et al., 2008, Thomas and Kaliamurthy, 2013, Ong et al., 2016, Bourcier et al., 2017). In previous studies of the National Reference Center for Invasive Fungal Infections it could be shown that infections caused by F. petroliphilum are associated with the use of CL, infections caused by F. falciforme, however, are predominantly trauma-associated and are mainly known from tropical and subtropical countries (Walther et al, 2018). This opposite behaviour of the spp. could be caused by differences in habitat and geographical distribution or by physiological differences such as germination rate, growth rate, tolerance to disinfectants, biofilm formation or cytotoxicity (Walther et al., 2018). Therefore, the aim of this study was to investigate whether F. falciforme and F. petroliphilum have physiological characteristics that could be responsible for the differences in the risk factors for keratitis between the two spp. For this purpose, the germination rate of the conidia and the length of the germ tubes of both Fusarium spp. was shown after staining of the conidia with fluorescein isothiocyanate and incubation at different incubation times in CL cleaning solutions at room temperature. A standardized approval test for CL cleaning solutions was carried out to investigate the behaviour of the conidia towards three different CL cleaning solutions that were available in Germany at the time of the investigations. Furthermore, the tolerance of the fungi to various CL cleaning solutions was examined in realistic conditions by incubating the conidia in CL cleaning solutions with CL overnight. The ability of biofilm formation was assessed by applying the conidia to CL from various manufacturers in Sabouraud-dextrose-broth and various CL cleaning solutions. The in vitro cytotoxicity of the conidia towards human corneal epithelial cells (HCE) was measured indirectly with the aid of a corneal epithelial cell model in aerobic and microaerophilic conditions via the lactate dehydrogenase release of HCE. Establishing a statement about the immune induction was possible due to the measurement of Interleukin-8 in the supernatants of the infection models. After the nightly incubation of the conidia in CL cleaning solutions, a lower germination rate was found for all F. petroliphilum isolates than for the F. falciforme isolates in one of the three cleaning solutions. It was remarkable that F. falciforme isolate 2015-96 was insensitive to this CL cleaning solution in this test setup. By contrast, the isolates did not show a different behaviour in the two other used CL cleaning solutions. It could be also shown that F. petroliphilum isolate 2014-79 germinates more frequently in one of the three tested cleaning solutions than F. falciforme, but at the same time the mycelium formation is effectively inhibited. All other isolates of both spp. behave similarly. After incubation the CL in CL cleaning solutions, no biofilm formation on the CL could be found in any of the two species in any of the test constellations, which could be due to the short incubation time or the inability of biofilm formation of the tested Fusarium spp. In the cytotoxicity and IL-8 experiments, no difference between the two spp. could be found. More realistic conditions in the test setup, such as e. g. a more realistic imitating of the effects of CL on corneal epithelial cells, could possibly reveal any differences. The natural pathogen reservoir could play the crucial role, why F. petroliphilum is more frequently associated of with corneal infections in CL users in moderate latitudes. A possible explanation could be that F. falciforme predominantly occurs in tropical areas and more often in the soil, whereas F. petroliphilum is more common in indoor areas. Another important result of my studies was that one of the tested CL cleaning solutions was proven to be ineffective against both Fusarium spp. Consequently, Fusarium would be able to get into the eyes of the CL wearer in large numbers through contamination of the CL in the cleaning vessel and trigger an infection. A further screening of CL cleaning solutions and the documentation of the cleaning solutions used by infected patients could show whether there is a connection between the occurrence of contact lens-associated eye infections and certain CL cleaning solutions. KW - Fusarium KW - Hornhautinfektionen Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-347774 ER - TY - JOUR A1 - Duske, Helene A1 - Claus, Heike A1 - Krone, Manuel A1 - Lâm, Thiên-Trí T1 - Prevalence of piperacillin/tazobactam resistance in invasive \(Haemophilus\) \(influenzae\) in Germany JF - JAC-Antimicrobial Resistance N2 - Background Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi. Objectives To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms. Methods According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller–Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing. Results Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates. Conclusions Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations. KW - microbiology KW - immunology KW - generalized anxiety disorder KW - haemophilus influenzae KW - agar KW - Germany KW - piperacillin KW - piperacillin/tazobactam KW - tazobactam KW - Haemophilus influenzae Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350424 SN - 2632-1823 VL - 6 IS - 1 ER - TY - THES A1 - Endres, Leo Maximilian T1 - Development of multicellular \(in\) \(vitro\) models of the meningeal blood-CSF barrier to study \(Neisseria\) \(meningitidis\) infection T1 - Entwicklung multizellulärer \(in\) \(vitro\) Modelle der meningealen Blut-Liquor Schranke zur Untersuchung der \(Neisseria\) \(meningitidis\) Infektion N2 - Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies. Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis. N2 - Neisseria meningitidis (der Meningokokkus) ist einer der Hauptursachen bakterieller Meningitis, einer lebensbedrohlichen Entzündung der Hirnhäute. Entscheidend für das für das Voranschreiten der Krankheit ist die Fähigkeit des Erregers, die meningeale Blut-Liquor-Schranke (mBCSFB), bestehend aus spezialisierten Hirnendothelzellen (BECs) und leptomeningealen Zellen (LMCs), zu überwinden und in den submeningealen Raum einzudringen. Da es sich bei N. meningitidis um ein rein humanes Pathogen handelt, beschränkt sich die Erforschung dieser speziellen Interaktion primär auf die Verwendung von in vitro Modellen. Bisher wurden hierfür hauptsächlich periphere oder immortalisierte BECs verwendet, welchen jedoch wichtige Barriere-Eigenschaften fehlen. Um die Interaktion von N. meningitidis mit der mBCSFB in einem physiologisch relevanteren Umfeld zu untersuchen, wurden in dieser Arbeit neuartige BEC-LMC Kokulturmodelle entwickelt. Dabei wurden sowohl BEC-ähnliche Zellen, die aus induzierten pluripotenten Stammzellen generiert wurden (iBECs), als auch hCMEC/D3 Zellen verwendet und zusammen mit LMCs aus Tumorbiopsien kultiviert. Mittels Transmissions-Elektronenmikroskopie und Immunfluoreszenzfärbung konnten die unterschiedlichen Zellschichten und deren Expression charakteristischer zellulärer Marker dargestellt werden. Durchgängige Expression von wichtigen Bestandteilen Barriere-formender Zellverbindungen, sogenannter Tight und Adherens Junctions, wurde in der iBEC-Schicht beobachtet. Die Integrität der zellulären Barriere wurde mittels transendothelialer elektrischer Resistenz (TEER) und Permeabilität gegenüber Natrium-Fluorescein (NaF) bestimmt. Erhöhte TEER-Werte und verringerte NaF-Permeabilität, gemessen über einen Zeitraum von sieben Tagen, zeigten eine durch die Kokultur mit LMCs ausgelöste Steigerung der Dichtigkeit und Stabilität der iBEC-Barriere. Infektionsexperimente mit N. meningitidis zeigten in allen Modellen vergleichbare bakterielle Adhäsion und Invasion der BEC-Schicht. Bakterielle Transmigration durch die gesamten Zellbarriere war im iBEC-LMC Modell kurz nach Infektion nur in geringem Maße detektierbar, nahm jedoch innerhalb von 24 Stunden deutlich zu. Interessanterweise wurde bis zu 24 Stunden nach Infektion noch eine hohe Integrität der Barriere gemessen, welche allerdings im weiteren Verlauf verloren ging. Neben signifikantem TEER-Verlust wurde eine verringerte Expression der Tight und Adherens Junction Proteine ZO-1, claudin-5, und VE-cadherin mittels qPCR festgestellt. qPCR und siRNA Knockdown Experimente deuteten darauf hin, dass dies möglicherweise, aber nicht ausschließlich, auf den Transkriptionsfaktor Snail1 zurückzuführen war. Zusätzlich zu den beobachteten Effekten auf die zelluläre Transkription von Tight Junction Genen, zeigten Immunfluoreszenzfärbungen eine verringerte Expression von Occludin an den Zell-Zell-Verbindungen, was auf eine post-translationale Modulation schließen lässt. Zusammen deuten die Ergebnisse dieser Infektionsstudien auf eine mögliche Kombination aus trans- und parazellulärer bakterieller Transmigration der mBCSFB hin. Zuletzt wurden in dieser Arbeit noch die Immunaktivierung von BECs nach N. meningitidis Infektion in den neuen BEC-LMC Kokulturmodellen untersucht. Hierbei wurde eine erhöhte Expression von Zytokinen, insbesondere Interleukin-8, beobachtet. Insgesamt konnten in dieser Arbeit neue, fortschrittlicher in vitro Modelle der mBCSFB entwickelt werden, welche die humane Physiologie besser widerspiegeln und daher für Infektionsstudien mit Meningitis-verursachenden Erregern wie N. meningitidis von besonderem Nutzen sind. KW - Bakterielle Hirnhautentzündung KW - Blut-Liquor-Schranke KW - Induzierte pluripotente Stammzelle KW - Neisseria meningitidis KW - In-vitro-Kultur KW - Brain endothelial cells KW - Leptomeningeal cells KW - Hirnendothelzellen KW - Leptomeningealzellen Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-346216 ER - TY - THES A1 - Ulrich, Johannes T1 - Molekulare Charaktierisierung einer DyP-Typ Peroxidase des Humanparasiten \(Echinococcus\) \(multilocularis\) T1 - Molecular characterisation of a DyP-type peroxidase of the human parasite \(Echinococcus\) \(multilocularis\) N2 - Die Alveoläre Echinokokkose (AE) ist eine tödliche Infektionserkrankung, die durch den parasitären Plattwurm Echinococcus multilocularis verursacht wird. Genomanalysen von E. multilocularis ergaben ein Gen, das laut Vorhersage für eine DyP-Typ Peroxidase codiere. Ziel dieser Arbeit ist die biologische Funktion des codierten Enzyms besser zu verstehen und Hinweise auf eine mögliche Rolle in der Abwehr von Reaktiven Sauerstoffspezies (ROS) zu erlangen. Das Gen wurde heterolog in E. Coli exprimiert und molekulare Charakteristika des Gens mit bioinformatischen und molekularbiologischen Methoden untersucht. Quantitative RT-PCR Untersuchungen gaben Aufschluss über das Transkriptprofil von emipox in unterschiedlichen Entwicklungsstadien von E. mulitlocularis. Mittels Whole-Mount In Situ-Hybridisierung (WMISH) wurden die Transkripte zudem lokalisiert und ihre Beziehung zum Stammzellsystem von E. multilocularis näher untersucht. Die Zugehörigkeit von EmIPOX zur Gruppe der DyP-Typ Peroxidasen wurde bestätigt. Homologe beim Menschen kommen nicht vor. Es konnte nachgewiesen werden, dass Transkripte von emipox auch, aber keinesfalls ausschließlich, in Stammzellen vorliegen. Überdurchschnittlich viele Transkripte liegen im aktivierten Protoscolex und im Metacestoden ex vivo aus einer infizierten Wirtsleber vor. Untersuchungen zur Enzymaktivität von EmIPOX zeigten neben einer Peroxidase- auch eine Katalaseaktivität. Die vorliegende Arbeit ist die erste Charakterisierung einer DyP-Typ Peroxidase bei Tieren. Sie legt nahe, dass EmIPOX eine Rolle in der Entgiftung von ROS in E. multilocularis spielt und stellt den Charakter von EmIPOX als potenzieller pharmakologischer Zielstruktur heraus. N2 - Alveolar echinococcosis (AE) is a fatal infectious disease caused by the parasitic flatworm Echinococcus multilocularis. Genome analyses of E. multilocularis revealed a gene predicted to encode a DyP-type peroxidase. The aim of this work is to better understand the biological function of the encoded enzyme and to obtain information on a possible role in the defence against reactive oxygen species (ROS). The gene was heterologously expressed in E. Coli and molecular characteristics of the gene were investigated using bioinformatic and molecular biological methods. Quantitative RT-PCR analyses provided information on the transcript profile of emipox in different developmental stages of E. mulitlocularis. Whole-mount in situ hybridisation (WMISH) was also used to localise the transcripts and investigate their relationship to the stem cell system of E. multilocularis. The affiliation of EmIPOX to the group of DyP-type peroxidases was confirmed. There are no homologues in humans. It has been shown that transcripts of emipox are also, but by no means exclusively, present in stem cells. An above-average number of transcripts are present in the activated protoscolex and in the metacestode ex vivo from an infected host liver. Investigations into the enzyme activity of EmIPOX revealed both peroxidase and catalase activity. The present work is the first characterisation of a DyP-type peroxidase in animals. It suggests that EmIPOX plays a role in the detoxification of ROS in E. multilocularis and highlights the character of EmIPOX as a potential pharmacological target. KW - Fuchsbandwurm KW - Oxidativer Stress KW - Peroxidase KW - Parasitäre Krankheit KW - Alveoläre Echinokokkose KW - alveolar echinococcosis KW - oxidative stress KW - DyP-type peroxidase KW - DyP-Typ Peroxidase KW - cestode KW - Bandwurm KW - Plathelminthes KW - flat worms KW - neglected tropical disease KW - katalase KW - Bandwürmer KW - Plattwürmer Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357143 ER - TY - THES A1 - Herrmann, Ruth Magdalena T1 - Molekular- und zellbiologische Untersuchung zur Rolle des kanonischen Wnt-Signalwegs bei der Entwicklung von \(Echinococcus\) \(multilocularis\) T1 - Molecular and cell biological investigations on the role of canonical Wnt signaling in \(E.\) \(multilocularis\) development N2 - Die alveoläre Echinokokkose (AE) ist eine lebensbedrohliche Erkrankung des Menschen, welche durch das infiltrative Wachstum des Metazestoden-Larvenstadiums des Fuchsbandwurms (Echinococcus multilocularis) in der Leber verursacht wird. Das tumorartige Wachstum des Metazestoden beruht auf einer Echinococcus-spezifischen Modifikation der anterior-posterioren-Körperachse (AP Achse). Es wird vermutet, dass dabei der anteriore Pol der invadierenden Oncospären-Larve zunächst abgeschaltet wird und sich der Metazestode anschließend asexuell als vesikuläres, posteriorisiertes Gewebes im Wirt vermehrt. Nach massiver Proliferation wird der anteriore Pol reetabliert und führt zur Bildung zahlreicher Bandwurm-Kopfanlagen (Protoskolizes). Da die Ausbildung der AP Körperachse evolutionsgeschichtlich konserviert über den wingless-related (Wnt)-Signalweg gesteuert wird, wurde in dieser Arbeit die Rolle von Wnt-Signaling bei der Musterbildung von E. multilocularis über molekular- und zellbiologische Studien näher beleuchtet. Zentraler methodischer Ansatz der vorliegenden Arbeit war ein E. multilocularis Stammzell-Kultursystem, das Primärzellsystem, welches die in vitro-Generierung von Metazestoden-Vesikeln durch Proliferation und Differenzierung von germinativen Zellen (Stammzellen) erlaubt. Über RNA-Sequenzierung wurde zunächst gezeigt, dass in Primärzellkulturen sowohl Markergene für posteriore Entwicklung in Richtung Metazestode wie auch für Anterior-und Protoskolexmarker exprimiert werden. Unter Verwendung von RNA-Interferenz (RNAi) wurde anschließend ein erfolgreicher Knockdown des vermuteten Hauptregulators des kanonischen Wnt-Signalwegs, β Catenin (em-bcat1), erreicht und führte zu einem charakteristischen, sogenannten ‚red dot‘ Phänotyp, dem ersten jemals beschriebenen RNAi Phänotyp für E. multilocularis-Primärzellen. Primärzellkulturen nach em-bcat1 RNAi zeigten eine stark verminderte Fähigkeit, Metazestoden-Vesikel zu bilden sowie eine Überproliferation von germinativen Zellen. Zusätzliche RNA-Seq-Analysen des Transkriptoms von RNAi(em-bcat1)-Kulturen zeigten eine signifikant verringerte Expression von Posterior- und Metazestodenmarkern, während Anterior- und Protoskolexmarker deutlich überexprimiert wurden. Durch umfangreiche Whole-mount-in-situ-Hybridisierung (WMISH)-Experimente wurden diese Daten für eine Reihe ausgewählter Markergene für posteriore (Metazestode; em-wnt1, em-wnt11b, em-muc1) und für anteriore Entwicklung (Protoskolex; em sfrp, em-nou-darake, em npp36, em-frizzled10) verifiziert. In allen genannten Fällen zeigte sich durch Änderung der Polarität eine verminderte Genexpression von Posteriormarkern, während Anteriormarker deutlich erhöht exprimiert wurden. Ähnlich wie bei den verwandten, freilebenden Planarien, führt demnach ein Knockdown des zentralen Wnt-Regulators β-Catenin bei E. multilocularis zu einer anteriorisierten, Anterior- und Protoskolexmarker dominierte Genexpression, welche der posteriorisierten Entwicklung zum Metazestoden entgegenwirkt. Neben Markergenen für die Ausbildung der AP-Achse wurden in dieser Arbeit auch solche für die medio-laterale (ML)-Körperachse bei Zestoden erstmals beschrieben. So zeigte sich, dass ein Slit-Ortholog (em slit) im E. multilocularis Protoskolex im Bereich der Körper-Mittellinie exprimiert wird und lieferte Hinweise darauf, dass, ähnlich zur Situation bei Planarien, die ML Achse von E. multilocularis durch Morphogengradienten aus slit (Mittellinie) und wnt5 (lateral) definiert wird. Im Metazestoden wird hingegen nur em-slit exprimiert. Der Metazestode besitzt damit als posterior-medianisiertes Gewebe Anlagen zur Polarität zur AP- und ML-Achse, welche erst mit Bildung von Protoskolizes vollständig etabliert werden. Schließlich deuten die Ergebnisse dieser Arbeit darauf hin, dass bei der Wiederherstellung der Körperachsen während der Entwicklung von Protoskolizes Hedgehog (Hh)-Signale entscheidend mitwirken. Zusammenfassend wurde in dieser Arbeit der zentrale Faktor des kanonischen Wnt Signalwegs, β-Catenin, als Hauptregulator der Entwicklung des tumorartig wachsenden E. multilocularis-Metazestoden identifiziert. Zudem wurde gezeigt, dass zur Metazestodenbildung neben einer Echinococcus-spezifischen Modifikation der AP Körperachse auch eine solche der ML Achse beiträgt. In humanen malignen Tumoren sind der Wnt-, Slit-Robo- und Hh-Signalweg gut erforschte Wirkstofftargets und könnten in Zukunft in ähnlicher Weise für eine zielgerichtete Therapie von AE dienen. N2 - Alveolar echinococcosis (AE) is a life-threatening human disease caused by the infiltrative growth of the metacestode larval stage of the fox tapeworm (Echinococcus multilocularis) within the host liver. According to previous research, the tumor-like growth of the metacestode is due to an Echinococcus-specific modification of the anterior-posterior (AP)-body axis formation. It is thus assumed that the invading oncosphere larva transiently represses the anterior pole, giving rise to metacestode vesicles which proliferate within the host as posteriorized tissue. Upon massive proliferation, the anterior pole is re-established at numerous sites within the metacestode tissue, yielding large numbers of tapeworm heads (protoscoleces). Since the formation of the AP-body axis is evolutionarily conserved and regulated by canonical wingless-related (Wnt) signaling, the present work investigated in detail the role of the Wnt-pathway in Echinococcus metacestode formation via molecular and cell biological studies. Methodologically, this work focussed on an Echinococcus stem cell cultivation system, called the primary cell system, which allows the in-vitro generation of mature metacestode vesicles through proliferation and differentiation of germinative cells (stem cells). By genome-wide RNA-Seq transcriptomics it is shown that primary cell cultures express marker genes for both posterior development towards the metacestode as well as anterior development of head organizers. By RNA interference (RNAi), successful knockdown of the presumed central regulator of canonical Wnt-signaling, β-catenin (em-bcat1), was achieved, yielding a striking phenotype ('red dot'), the first RNAi phenotype described for E. multilocularis primary cells. Primary cell cultures after em-bcat1 RNAi showed a greatly reduced ability to form metacestode vesicles as well as an overproliferation of germinative cells. Additional RNA-Seq analysis of the transcriptome of RNAi(em-bcat1) cultures indicated significantly decreased expression of posterior and metacestode markers whereas anterior and protoscolex markers were markedly overexpressed. These data were verified using whole-mount-in-situ-hybridization (WMISH) for several selected marker genes for posterior (metazestode; em-wnt1, em-wnt11b, em-muc1) and for anterior development (protoscolex; em-sfrp, em-nou-darake, em npp36, em frizzled10). In all cases, a change in polarity showed decreased gene expression of posterior markers whereas anterior markers were significantly increased in expression. Similar to the situation in related planarians, knockdown of β Catenin in E. multilocularis lead in anteriorized, anterior- and protoscolex marker-dominated gene expression and antagonized the formation of the posteriorized metacestode. In addition to marker genes for AP-axis formation, this work also established marker genes for the medio-lateral (ML)-body axis in cestodes for the first time. In particular, a slit orthologue (em slit) was shown to be expressed in the E. multilocularis protoscolex at the body midline and provided evidence that, similar to the situation in planarians, the ML-axis of E. multilocularis is defined by morphogen gradients consisting of slit (midline) and wnt5 (lateral). In contrast, only em-slit is expressed in the metacestode. Thus, the metacestode tissue is indeed posterior-medianized and the AP- and ML-axes are established only with formation of protoscoleces. Finally, the results of this work suggest that Hedgehog (Hh) signaling plays a critical role in the reestablishment of body axes during protoscoleces development. In conclusion, this work identified the central regulator of the canonical Wnt-signaling pathway, β-catenin, as a master regulator of E. multilocularis metacestode development. Furthermore, it is herein established that metacestode formation not only involves Echinococcus-specific modification of the AP-axis but also of the ML-axis. In human malignant tumors, the Wnt, Slit-Robo, and Hh-pathway are well-studied drug targets and may similarly serve for AE targeted therapy in the future. KW - Fuchsbandwurm KW - Transkriptomanalyse KW - foxtapeworm KW - Echinococcus KW - Wnt-Signalweg KW - Körperachsen KW - Germinative Zellen KW - beta-Catenin KW - Wnt-pathway KW - body axis KW - Stammzelle Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-271937 ER - TY - JOUR A1 - Soundararajan, Manonmani A1 - Marincola, Gabriella A1 - Liong, Olivia A1 - Marciniak, Tessa A1 - Wencker, Freya D. R. A1 - Hofmann, Franka A1 - Schollenbruch, Hannah A1 - Kobusch, Iris A1 - Linnemann, Sabrina A1 - Wolf, Silver A. A1 - Helal, Mustafa A1 - Semmler, Torsten A1 - Walther, Birgit A1 - Schoen, Christoph A1 - Nyasinga, Justin A1 - Revathi, Gunturu A1 - Boelhauve, Marc A1 - Ziebuhr, Wilma T1 - Farming practice influences antimicrobial resistance burden of non-aureus staphylococci in pig husbandries JF - Microorganisms N2 - Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms. KW - non-aureus staphylococci KW - NAS KW - alternative pig farming KW - antimicrobial resistance KW - one-health approach KW - intervention strategies KW - livestock-associated staphylococci KW - organic farming KW - pig farming methods Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312750 SN - 2076-2607 VL - 11 IS - 1 ER - TY - THES A1 - Ebner, Sebastian Manfred T1 - Antimykotikaresistenzen bei deutschen \(Candida\) \(auris\) Isolaten T1 - Antimycotic resistance in German \(Candida\) \(auris\) isolates N2 - Bei dem 2009 erstbeschriebenen Hefepilz C. auris handelt es sich um einen Keim, welcher aufgrund von nosokomialen Ausbrüchen und hohen Antimykotikaresistenzen Aufmerksamkeit erregte. Ziel dieser Arbeit war es in Deutschland gesammelte Isolate bezüglich vorhandener Resistenzen und Mutationen in Resistenzregionen zu testen und das epidemiologische Geschehen hierzulande mit dem globalen Auftreten des Keims zu vergleichen. Bezüglich der durchgeführten Resistenztestungen wiesen die CLSI-konformen Testarten (YO-Platten und E-Test-Verfahren) meist vergleichbare Ergebnisse auf. Für das EUCAST-konforme Mikrodilutionstestverfahren kann aufgrund eines stark ausgeprägten paradoxen Wachstumseffekts nur Anidulafungin, nicht jedoch Caspofungin, zur Testung empfohlen werden. Insgesamt erwiesen sich 25 % der Isolate als Caspofungin-resistent. Zwei Isolate zeigten eine Resistenz gegenüber allen getesteten Echinocandinen (16,7 %). Die höchsten Resistenzraten wurden gegenüber Fluconazol (92 %) beobachtet. Zwei der Isolate zeigten sich gegenüber Voriconazol resistent (16,7 %). Für Amphotericin B konnte eine Resistenzrate von 33,3 % festgestellt werden. Für die Wirkstoffe Posaconazol und Itraconazol erwiesen sich alle untersuchten Isolate als sensitiv. Dies konnte auch mit Ausnahme eines Isolates für 5-Flucytosin beobachtet werden. Die durch eine Sanger-Sequenzierung erhaltenen Sequenzen der Gene FKS1 und ERG11 wurden auf Mutationen untersucht, welche zu Aminosäuresubstitutionen im Gesamtprotein führten. Hierbei ergaben sich für zwei Isolate (16,7 %) Mutationen im FKS1-Hot Spot 1 (Typ S639F und S639Y). Beide Isolate zeigten sich in den AFST Echinocandin-resistent. Bei allen untersuchten Isolaten lagen Mutationen im ERG11 Gen vor. So fand sich in 8 Fällen eine Mutation des Typen Y132F (66,7 %), in 3 Fällen der Typ K143R (25 %) und in einem Fall der Typ F126L (8,3 %). Im Rahmen eines anderen Projekts wurde mit den hier gewonnenen PCR-Produkten ein WGS durchgeführt, um die Isolate durch SNPs-Vergleich mit Referenzstämmen phylogenetischen Clades zuzuordnen. Dabei konnten 91,7 % der Isolate dem südasiatischen Clade I und ein Isolat dem südafrikanischen Clade III zugeordnet werden. Aufgrund der geringen epidemiologischen Fallzahlen in Deutschland scheint gegenwärtig keine Bedrohung von C. auris auszugehen. Berichte aus anderen Ländern konnten allerdings eine rasche, ausbruchartige Zunahme von C. auris Fällen nachweisen. So kann nur angeraten werden das infektiologische Geschehen in Deutschland weiterhin zu beobachten. N2 - The fungus C. auris was first described in the year 2009. Because of a high number of nosocomial outbrakes and high antimycotic resistance rates the fungus attracted great media attention. The aim of this dissertion was to test German isolates for antimycotic resistance and mutations in resistance genes. Additionally, the epidemiological occurrence in Germany was compared to the global outspread. In this context CLSI-conform methods for resistance testing (YO-Plates and E-Test-Plates) generated comparable results. The testing of EUCAST-conform microdilution plates showed a strong paradoxical growth for Caspofungin. Because of this only Anidulafungin can be recommended for testing. In summary 25 % of the isolates were resistant against Caspofungin. Two isolates showed resistance against all tested Echinocandines (16,7 %). The highest rates were detected for Fluconazol (92 %). Furthermore, two of the isolates (16,7 %) showed resistance against Voriconazol. There was a resistance rate of 33,3 % to Amphotericin B. No isolate showed resistance against Posaconazol or Itraconazol. And only one isolate was resistant against 5-Flucytosin. Sanger-Sequencing was used to detect mutations in resistance genes FKS1 und ERG11, which could lead to a substitution of amino acids in the protein. There were two isolates (16,7 %) with mutations in FKS1-Hot Spot 1 (type S639F and S639Y). Both isolates showed a Echinocandin resistance in AFST. All tested isolates showed a mutation in ERG11. There were eight cases of type Y132F (66,7 %), three cases of K143R (25 %) and in one case type F126L (8,3 %). The PCR products of this study were used in a different project for WSG. This made it possible to group the isolates into phylogenetic clades. In summary 91,7 % of the isolates were related to Clade I (South Asia) and one isolate was related to Clade III (South Africa). Because of low epidemiologic occurence in Germany, there is little threat of servere health care issues at the moment. Reports from diffferent countries all over the world however, showed a quick, outbrake-like increase of C. auris cases. Therefore, further observation of German epidemiology is highly recommended. KW - Candida KW - Resistenz KW - Wirkstoff KW - Behandlung KW - Antimykotikaresistenz KW - Candida auris KW - Resistance mechanism C. auris KW - Mutation FKS Hot Spot 1/ERG11 KW - Nosokomiale Infektion KW - E-Test KW - Mikrodilutionstest KW - Hospitalismus KW - Pilz Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318068 ER - TY - THES A1 - Nürnberg, Sebastian T1 - Invasive \(Haemophilus\) \(influenzae\)-Isolate in Deutschland: Methodenvalidierung des VITEK MS IVD MALDI-TOF-MS und Untersuchung von Resistenzen gegen Imipenem und Cefotaxim T1 - Invasive \(Haemophilus\) \(influenzae\) Isolates in Germany: Method Validation of the VITEK MS IVD MALDI-TOF-MS and Investigation of Imipenem and Cefotaxime Resistance N2 - Die Inzidenz invasiver H. influenzae-Infektionen in Deutschland steigt seit Jahren an. Die akkurate Identifizierung und Resistenztestung dieses Erregers sind von großer klinischer und epidemiologischer Bedeutung. Daher wurden im Rahmen der vorliegenden Promotionsarbeit umfangreiche Untersuchungen zur Diagnostik und zur Epidemiologie von Antibiotikaresistenzen bei H. influenzae durchgeführt. Es konnte gezeigt werden, dass die in der Routinediagnostik mittlerweile weit verbreitete MALDI-TOF-MS-Diagnostik durch das VITEK MS IVD nur eingeschränkt zur sicheren Unterscheidung von H. influenzae und H. haemolyticus einsetzbar ist. H. influenzae-Isolate erkannte das System mit einer Genauigkeit von 100 %. Bei H. haemolyticus-Isolaten wurden dagegen 42 % der untersuchten Stämme fälschlicherweise als H. influenzae erkannt. Dieser Fragestellung wurde mit der bisher umfangreichsten molekularbiologisch charakterisierten Studienpopulation beider Bakterienspezies nachgegangen. Die kalkulierte antibiotische Therapie einer Sepsis oder Meningitis erfolgt häufig mit Carbapenemen, die leitliniengerechte Therapie invasiver H. influenzae-Infektionen mit Drittgenerations-Cephalosporinen. Imipenem und Cefotaxim gehören zu den Hauptvertretern dieser Gruppen. Bezüglich der Antibiotikaresistenztestung wurde erstmalig für H. influenzae herausgefunden, dass die routinemäßig verwendete Gradientenagardiffusion (GAD) bei der Testung von Cefotaxim im Vergleich zum Goldstandard Bouillon-Mikrodilution gleichwertig und bei Imipenem sogar sensitiver in der Detektion von Heteroresistenzen ist. Die Epidemiologie dieser Resistenzen wurde in dieser Arbeit erstmalig für Deutschland systematisch erfasst, indem alle verfügbaren invasiven Isolate gemeldeter H. influenzae-Infektionen der Jahre 2016 (Imipenem) beziehungsweise 2016-2019 (Cefotaxim) untersucht wurden. Es wurde eine hohe Prävalenz einer Imipenem-Resistenz von 13,5 % festgestellt. Die Prävalenz einer Cefotaxim-Resistenz lag bei 0,9 %. Zur molekularen Typisierung wurde bei den Imipenem-resistenten Isolaten eine Multilocus-Sequenztypisierung, bei den Cefotaxim-resistenten Stämmen eine Sequenzierung des vollständigen Genoms durchgeführt. Hierbei wurde eine hohe genetische Diversität der Stämme festgestellt, was die Schlussfolgerung zulässt, dass resistente Mutanten sporadisch entstehen. Die Untersuchung möglicher spatio-temporaler Cluster führte zum Nachweis einer sehr selten vorkommenden Übertragung eines Imipenem-resistenten Stamms. Durch die Sequenzierung von Resistenzgenen wurde die Epidemiologie und Relevanz bekannter Aminosäuresubstitutionen beleuchtet. Unter anderem wurde für die PBP3-Substitutionen L389F und Y557H eine hochsignifikante Korrelation mit dem Auftreten von Cefotaxim-Resistenzen nachgewiesen. Die gewonnenen Genomdaten bieten die Grundlage für die Forschung an weiteren Antibiotikaresistenzdeterminanten von H. influenzae. N2 - Haemophilus influenzae is a fastidious, facultative anaerobic, Gram-negative bacillus that colonizes the respiratory tract and can cause respiratory and invasive infection such as meningitis and sepsis. Invasive H. influenzae infections are potentially life-threatening and incidence rates have been increasing for years. Therefore, fast and accurate diagnostics, reliable testing of antibiotic resistance and a successful antibiotic treatment is of great importance. Therefore, the objective of the first part of this thesis was to evaluate the diagnostic and discriminative potential of the MALDI-TOF-MS system VITEK® MS regarding H. influenzae and H. haemolyticus. H. influenzae can cause invasive infections, whereas H. haemolyticus is mostly apathogenic. The system showed excellent accuracy for the identification of H. influenzae isolates, as 100 % of the 236 isolates were correctly identified. When testing 50 H. haemolyticus strains, however, the system showed significant limitations, since 42 % of these strains were misidentified as H. influenzae. According to the current German guidelines for the treatment of sepsis and meningitis, treatment of invasive H. influenzae infections is carried out using carbapenems, such as imipenem, or third-generation cephalosporins, such as cefotaxime. Therefore, the prevalence of antibiotic resistances to these substances was investigated and possible resistance mechanisms were examined. The two antibiotic susceptibility testing methods Gradient agar diffusion (GAD) and broth microdilution (BMD) were compared. As a result, for the determination of the cefotaxime MIC, the two methods showed an excellent correlation, whereas for imipenem there were significant differences in the measured MIC values. Since strains tested by GAD often showed double or fuzzy inhibition zones, heteroresistances may be more apparent using this method and GAD may be more sensitive at detecting imipenem resistance. The prevalence of imipenem resistance was determined for the year 2016. The analysis of 474 different invasive isolates showed a high prevalence of 5.5 %. If including all resistant isolates according to GAD, the prevalence would be even as high as 13.5 %. MLST was performed on all isolates to investigate the genetic relationship. As a result, however, some sequence types were observed more frequently, it revealed a significant diversity. Both the analysis of ftsI and acrR showed previously described amino acid substitutions. Cefotaxime resistance was investigated for all 2432 invasive H. influenzae for the years 2016-2019. The low prevalence of 0.9 % shows that cefotaxime is still well suited for the treatment of invasive H. influenzae infections. For the investigation of the genetic relationship and possible causes of cefotaxime resistance whole genome sequencing (WGS) was performed on all resistant isolates. The strains showed high genetic diversity and the geographic analysis also showed that the resistant strains were evenly spread throughout the population in Germany. This led to the conclusion that cefotaxime resistance is more likely caused by sporadic mutation events rather than by specific clones spreading in certain areas. The analysis of the ftsI gene showed that the amino acid substitutions L389F and Y557H are significantly associated with elevated cefotaxime MICs. This dissertation provides comprehensive data regarding diagnostics, antimicrobial susceptibility testing and the epidemiology of antibiotic resistances of invasive H. influenzae. It could be shown that the VITEK MS IVD, although established in the routine diagnostics, can only be used to a limited extent for reliably differentiating H. influenzae and H. haemolyticus isolates. Regarding antibiotic susceptibility testing, it was found that GAD showed similar results compared to the gold standard BMD when testing cefotaxime. When testing imipenem, this method was even more sensitive in detecting heteroresistances compared to the gold standard. For the first time, the epidemiology of antibiotic resistance against cefotaxime and imipenem was carried out using a large set of precisely defined invasive H. influenzae isolates to obtain representative data for the prevalence of imipenem and cefotaxime resistance in Germany. By investigating amino acid substitutions in the ftsI and acrR gene the epidemiology and relevance of these substitutions could be shown. A well-founded statement on the relationship of the resistant strains could be made using the state-of-the-art typing methods MLST and whole genome sequencing. The genome data also offers the possibility of examining other genes of these strains in more detail. KW - Haemophilus influenzae KW - MALDI-MS KW - Antibiotikum KW - Cefotaxim KW - Imipenem KW - Haemophilus haemolyticus KW - MALDI-TOF-MS KW - Antibiotikaresistenzen Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-345067 ER - TY - THES A1 - Koike, Akito T1 - Molekular und zellbiologischer Ansatz hin zu neuartigen Medikamenten gegen \(Echinococcus\) \(multilocularis\) T1 - Molecular and cell biological approach towards novel drugs against \(Echinococcus\) \(multilocularis\) N2 - Echinococcosis is an important zoonosis. The causative agent of Alveolar Echinococcosis (AE) is Echinococcus multilocularis. The treatment of human AE is limited to surgery and chemotherapy with albendazole (ABZ). However, ABZ works only parasitostatically and it needs to be taken for long periods, although it causes adverse side effects. Thus, development of new, parasiticidal drug with selective toxicity is required. Because undifferentiated stem cells of E. multilocularis play key role in its longevity and regenerative capacity, targeting stem cells is especially important. In vitro screening of protein kinases inhibitors demonstrated that human PIM kinases inhibitors have detrimental effects on E. multilocularis. Through yeast two hybrid assay, the interaction of parasite PIM kinase (EmPIM) and its CDC25 (EmCDC25) was indicated. Through in situ hybridization, expression of EmPIM in the stem cells was observed. Therefore, EmPim is likely to be a positive regulator of cell cycle progression, the same as human Pim1. In addition, 20 compounds against EmPIM were selected through in silico screening and synthesized. One of them has a detrimental effect on E.multilocularis comparable to human pan-PIM inhibitors, but has much weaker toxicity on human cell lines. Furthermore, triclabendazole (TCBZ) and its metabolite TCBZSX, which are approved for another flatworm disease, Fascioliasis were tried on E. multilocularis. With two stem cell markers, damage to stem cells by TCBZSX was shown. In addition, primary cells from treated vesicles never regenerated and the damage to stem cells proved to be irreversible. Our in silico screening method used in EmPIM research has potential to identify compounds which overcome the side effect problem in ABZ-based chemotherapy. On the other hand, it is expected that my research of TCBZ can lead to development of a practical parasiticidal chemotherapy by combining TCBZ, which damages stem cells, and ABZ, which damages differentiated cells. N2 - Die Echinokokkose ist eine der wichtigsten Zoonosen sowohl für die Human- als auch für die Veterinärmedizin. Der Erreger der alveolären Echinokokkose (AE) ist Echinococcus multilocularis. Metazestode Bläschen, das Larvenstadium dieses parasitären Helminthen, können in die Leber eindringen und ungeschlechtlich wie bösartige Tumore wachsen. Dies kann ohne geeignete Behandlung tödlich sein. Die Behandlung von AE beim Menschen beschränkt sich auf Chirurgie und Chemotherapie, aber die Chirurgie ist nur bei einem kleinen Prozentsatz der Patienten anwendbar, die im Frühstadium diagnostiziert werden. Die meisten Patienten können sich nur auf eine Chemotherapie mit Albendazol (ABZ) verlassen. ABZ wirkt jedoch nur parasitostatisch und kann die Krankheit nicht heilen. Daher muss ABZ über einen längeren Zeitraum eingenommen werden, obwohl es mit Nebenwirkungen einhergeht. Daher ist die Entwicklung eines neuen, parasitentötenden und selektiven Medikaments gegen AE erforderlich. Da die undifferenzierte Stammzellpopulation von E. multilocularis eine Schlüsselrolle für seine Langlebigkeit und Regenerationsfähigkeit spielt, ist eine auf Stammzellen abzielende Chemotherapie wichtig. In dieser Arbeit wurde ein In-vitro-Screening verschiedener Hemmstoffe gegen Kinasen und Tubuline durchgeführt. Das Ergebnis des Screenings zeigte, dass Inhibitoren gegen humane pim-Kinasen starke schädliche Auswirkungen auf E. multilocularis haben. Durch ein Hefe-Zwei- Hybrid-System wurde die Interaktion der Parasiten-Pim-Kinase (EmPIM) mit der Zellteilungszyklus 25 (EmCDC25) nachgewiesen, und durch In-situ-Hybridisierung wurde die teilweise Lokalisierung von EmPIM in den Stammzellen beobachtet. Daher ist es wahrscheinlich, dass EmPim ein positiver Regulator der Zellzyklusprogression ist, genau wie menschliches Pim1. ... KW - Bandwürmer KW - Zellzyklus KW - Benzimidazolderivate KW - tapeworm KW - kinase KW - benzimidazole Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288649 ER - TY - JOUR A1 - Cucher, Marcela A. A1 - Mariconti, Mara A1 - Manciulli, Tommaso A1 - Vola, Ambra A1 - Rosenzvit, Mara C. A1 - Brehm, Klaus A1 - Kamenetzky, Laura A1 - Brunetti, Enrico T1 - Circulating small RNA profiling of patients with alveolar and cystic echinococcosis JF - Biology N2 - Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection. KW - echinococcosis KW - small RNA KW - extracellular KW - circulating KW - microRNA KW - serum KW - tapeworm KW - diagnosis KW - marker KW - Echinococcus Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319270 SN - 2079-7737 VL - 12 IS - 5 ER - TY - JOUR A1 - Weiß, Martin A1 - Gründahl, Marthe A1 - Deckert, Jürgen A1 - Eichner, Felizitas A. A1 - Kohls, Mirjam A1 - Störk, Stefan A1 - Heuschmann, Peter U. A1 - Hein, Grit T1 - Differential network interactions between psychosocial factors, mental health, and health-related quality of life in women and men JF - Scientific Reports N2 - Psychosocial factors affect mental health and health-related quality of life (HRQL) in a complex manner, yet gender differences in these interactions remain poorly understood. We investigated whether psychosocial factors such as social support and personal and work-related concerns impact mental health and HRQL differentially in women and men during the first year of the COVID-19 pandemic. Between June and October 2020, the first part of a COVID-19-specific program was conducted within the “Characteristics and Course of Heart Failure Stages A-B and Determinants of Progression (STAAB)” cohort study, a representative age- and gender-stratified sample of the general population of Würzburg, Germany. Using psychometric networks, we first established the complex relations between personal social support, personal and work-related concerns, and their interactions with anxiety, depression, and HRQL. Second, we tested for gender differences by comparing expected influence, edge weight differences, and stability of the networks. The network comparison revealed a significant difference in the overall network structure. The male (N = 1370) but not the female network (N = 1520) showed a positive link between work-related concern and anxiety. In both networks, anxiety was the most central variable. These findings provide further evidence that the complex interplay of psychosocial factors with mental health and HRQL decisively depends on gender. Our results are relevant for the development of gender-specific interventions to increase resilience in times of pandemic crisis. KW - anxiety KW - depression KW - human behaviour KW - quality of life Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357858 VL - 13 ER - TY - JOUR A1 - Häder, Antje A1 - Schäuble, Sascha A1 - Gehlen, Jan A1 - Thielemann, Nadja A1 - Buerfent, Benedikt C. A1 - Schüller, Vitalia A1 - Hess, Timo A1 - Wolf, Thomas A1 - Schröder, Julia A1 - Weber, Michael A1 - Hünniger, Kerstin A1 - Löffler, Jürgen A1 - Vylkova, Slavena A1 - Panagiotou, Gianni A1 - Schumacher, Johannes A1 - Kurzai, Oliver T1 - Pathogen-specific innate immune response patterns are distinctly affected by genetic diversity JF - Nature Communications N2 - Innate immune responses vary by pathogen and host genetics. We analyze quantitative trait loci (eQTLs) and transcriptomes of monocytes from 215 individuals stimulated by fungal, Gram-negative or Gram-positive bacterial pathogens. We identify conserved monocyte responses to bacterial pathogens and a distinct antifungal response. These include 745 response eQTLs (reQTLs) and corresponding genes with pathogen-specific effects, which we find first in samples of male donors and subsequently confirm for selected reQTLs in females. reQTLs affect predominantly upregulated genes that regulate immune response via e.g., NOD-like, C-type lectin, Toll-like and complement receptor-signaling pathways. Hence, reQTLs provide a functional explanation for individual differences in innate response patterns. Our identified reQTLs are also associated with cancer, autoimmunity, inflammatory and infectious diseases as shown by external genome-wide association studies. Thus, reQTLs help to explain interindividual variation in immune response to infection and provide candidate genes for variants associated with a range of diseases. KW - antimicrobial responses KW - immunogenetics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357441 VL - 14 ER - TY - JOUR A1 - Schreiber, Laura M. A1 - Lohr, David A1 - Baltes, Steffen A1 - Vogel, Ulrich A1 - Elabyad, Ibrahim A. A1 - Bille, Maya A1 - Reiter, Theresa A1 - Kosmala, Aleksander A1 - Gassenmaier, Tobias A1 - Stefanescu, Maria R. A1 - Kollmann, Alena A1 - Aures, Julia A1 - Schnitter, Florian A1 - Pali, Mihaela A1 - Ueda, Yuichiro A1 - Williams, Tatiana A1 - Christa, Martin A1 - Hofmann, Ulrich A1 - Bauer, Wolfgang A1 - Gerull, Brenda A1 - Zernecke, Alma A1 - Ergün, Süleyman A1 - Terekhov, Maxim T1 - Ultra-high field cardiac MRI in large animals and humans for translational cardiovascular research JF - Frontiers in Cardiovascular Medicine N2 - A key step in translational cardiovascular research is the use of large animal models to better understand normal and abnormal physiology, to test drugs or interventions, or to perform studies which would be considered unethical in human subjects. Ultrahigh field magnetic resonance imaging (UHF-MRI) at 7 T field strength is becoming increasingly available for imaging of the heart and, when compared to clinically established field strengths, promises better image quality and image information content, more precise functional analysis, potentially new image contrasts, and as all in-vivo imaging techniques, a reduction of the number of animals per study because of the possibility to scan every animal repeatedly. We present here a solution to the dual use problem of whole-body UHF-MRI systems, which are typically installed in clinical environments, to both UHF-MRI in large animals and humans. Moreover, we provide evidence that in such a research infrastructure UHF-MRI, and ideally combined with a standard small-bore UHF-MRI system, can contribute to a variety of spatial scales in translational cardiovascular research: from cardiac organoids, Zebra fish and rodent hearts to large animal models such as pigs and humans. We present pilot data from serial CINE, late gadolinium enhancement, and susceptibility weighted UHF-MRI in a myocardial infarction model over eight weeks. In 14 pigs which were delivered from a breeding facility in a national SARS-CoV-2 hotspot, we found no infection in the incoming pigs. Human scanning using CINE and phase contrast flow measurements provided good image quality of the left and right ventricle. Agreement of functional analysis between CINE and phase contrast MRI was excellent. MRI in arrested hearts or excised vascular tissue for MRI-based histologic imaging, structural imaging of myofiber and vascular smooth muscle cell architecture using high-resolution diffusion tensor imaging, and UHF-MRI for monitoring free radicals as a surrogate for MRI of reactive oxygen species in studies of oxidative stress are demonstrated. We conclude that UHF-MRI has the potential to become an important precision imaging modality in translational cardiovascular research. KW - ultrahigh-field MRI KW - large animal models KW - translational research KW - research infrastructure KW - heart KW - organoid KW - pig KW - cardiovascular MRI Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-317398 SN - 2297-055X VL - 10 ER - TY - THES A1 - Münstermann, Marcel T1 - The roles of the anaphylatoxin receptors during invasive disease as well as mucosal colonization caused by \(Neisseria\) \(meningitidis\) T1 - Die Rolle der Anaphylatoxinrezeptoren während invasiver Infektion sowie mukosaler Kolonisation verursacht durch \(Neisseria\) \(meningitidis\) N2 - The human specific gram-negative bacterium Neisseria meningitidis (Nme, meningococci) is a common colonizer of the upper respiratory tract. Upon becoming invasive, Nme can cause meningitis and life-threatening sepsis. The most important immune defense mechanism in invasive meningococcal disease (IMD) is the complement mediated killing of bacteria. The complement cascade is activated through different pathogen associated patterns and finally leads to the lysis of the bacteria by the membrane attack complex. In addition to the direct bacterial killing, the complement system is also an important player in different inflammatory processes. A hallmark of IMD is an overreaction of the immune system and the release of the potent anaphylatoxins C3a and C5a by the complement system is an important factor hereby. There are three anaphylatoxin receptors (ATRs), the C3aR, the C5aR1 and the C5aR2, capable of detecting these anaphylatoxins. It has already been shown that blocking the ATR C5aR1 strongly benefitted the outcome of IMD in a murine sepsis model. However, the roles of ATRs C3aR and C5aR2 in IMD are still unclear. This work aims to analyze the role of these ATRs in meningococcal sepsis and to identify possible underlying mechanisms. Furthermore, a possible involvement of the complement system, the ATRs and the type II CRISPR/Cas system on nasopharyngeal colonization is analyzed. In vivo depletion experiments showed that without neutrophils or monocytes/macrophages the complement system alone was not able to clear a low dose Nme infection, which highlights the importance of cellular components in IMD. Analyzing the role of the ATRs in knock-out mice with high dose Nme infections, revealed that the lack of C5aR2, like the lack of C5aR1, was beneficial for the outcome of meningococcal induced sepsis. In contrast, the lack of C3aR in knock-out mice was detrimental. The positive outcome associated with the C5aRs could be reproduced by using an antagonist against both C5aRs or an antagonist specifically against C5aR1 in WT mice. These findings are giving hope to future therapeutic applications. Next, a possible contribution of neutrophils to this positive outcome was analyzed. Absence of C5aR1 led to a decrease of degranulation by neutrophils in a murine whole blood model, while the other ATRs showed no effect. Neutrophil analysis in human whole blood, on the other hand, revealed a reduced oxidative burst and IL-8 secretion upon inhibition of all three ATRs. A functional difference between the C5aRs and the C3aR in neutrophils was observed in phagocytosis, which was reduced upon C3aR inhibition, but was unaltered with C5aR1 or C5aR2 inhibition. Possible underlying mechanisms in the phosphorylation of ERK1/2 were analyzed in bone marrow derived macrophages isolated from ATR knock-out mice. The later phosphorylation of ERK1/2 in macrophages without C5aR1 or C5aR2 expression might explain, why blocking the C5aRs is beneficial for the outcome of IMD in mice. In contrast to these findings, the colonization of the nasopharynx in huCEACAM 1 expressing mice by Nme did not seem to depend on the Complement system factors C3 and C5 nor the ATRs. Additionally, no difference in the colonization could be observed in this model using Nme mutants lacking different parts of the type 2 CRISPR/Cas system. Conclusively, this work highlights the importance of the complement system, the ATRs and the cellular components in IMD. Contrariwise, these factors did not play a role in the analyzed nasopharyngeal infection model. The beneficial effects of C5aR1 and C5aR2 lack/inhibition in IMD might have medicinal applications, which could support the standard therapies of IMD in the future. N2 - Das human spezifische pathogene Gram-negative Bakterium Neisseria meningitidis (Nme, Meningokokken) ist ein Kommensale angesiedelt im Nasopharynx. Bei invasiver Erkrankung können Nme Meningitis oder eine lebensbedrohliche Sepsis verursachen. Die wichtigste Verteidigung des Immunsystems in invasiver Meningokokken-Erkrankung (IMD) ist die Abtötung von Bakterien durch das Komplementsystem. Die Komplementkaskade wird durch verschiedene pathogenassoziierte Muster in Gang gesetzt und resultiert in dem Aufbau des Membranangriffskomplex, welcher die Bakterien schließlich lysiert. Darüber hinaus spielt das Komplementsystem auch eine wichtige Rolle in verschiedenen inflammatorischen Prozessen im Körper. Ein charakteristisches Merkmal von IMD ist eine übermäßige Reaktion des Immunsystems und dabei ist die Freisetzung der Anaphylatoxine C3a und C5a, durch das Komplementsystem, ein wichtiger Faktor. Es gibt drei Anaphylatoxin Rezeptoren (ATR), den C3aR, den C5aR1 und den C5aR2, welche die jeweiligen Anaphylatoxine erkennen. In murinen Modellen wurde bereits gezeigt, dass die Inhibition des C5aR1 einen positiven Einfluss auf den Verlauf von IMD hat. Im Kontrast dazu sind die Rollen der ATRs C3aR und C5aR2 in IMD weiter unklar. Diese Arbeit hat als Ziel, die Rolle der ATRs in Meningokokken induzierter Sepsis zu untersuchen und mögliche zugrundeliegende Mechanismen zu finden. Des Weiteren soll ein möglicher Einfluss des Komplementsystems, der ATRs und des Typ II CRISPR/Cas Systems auf die Kolonisation durch Nme im Nasopharynx untersuchen werden. In vivo Depletions-Versuche zeigten, dass ohne Neutrophile oder Monozyten/Makrophagen das Komplementsystem allein nicht in der Lage war eine Nme-Infektion mit einer niedrigen Infektionsdosis zu beseitigen. Dies zeigt die Wichtigkeit von Immunzellen neben dem Komplementsystem in IMD. Experimente mit hohen Nme-Dosen in ATR knock-out Mäusen zeigten, dass die fehlende Expression von C5aR2, wie die von C5aR1, sich positiv auf den Ausgang von IMD auswirkte. Im Gegensatz dazu, verschlimmerte das Fehlen des C3aR Rezeptors den Ausgang der IMD. Die positive Wirkung in den C5aR knock-out Mäusen, konnte auch mit der Gabe von einem gegen beide C5aRs oder einem spezifisch gegen C5aR1 gerichteten Antagonisten in WT Mäusen beobachtet werden. Diese Ergebnisse geben Hoffnung auf eine mögliche zukünftige therapeutische Applikation. Als nächstes wurde eine mögliche Beteiligung von Neutrophilen an dem positiven Ausgang von IMD in Abhängigkeit von den ATRs untersucht. Eine fehlende C5aR1 Expression führte zu einer verminderten Degranulation durch Neutrophile in dem verwendeten murinen Vollblutmodel, wohingegen die fehlende Expression der anderen ATRs keinen Effekt zeigte. Im Gegensatz dazu, zeigten Versuche mit humanem Vollblut einen verminderten Oxidativen Burst sowie eine verminderte Ausschüttung von IL-8 bei der Blockade von allen drei ATRs. Ein Unterschied zwischen den C5aRs und dem C3aR zeigte sich hingegen in der Phagozytose, welche mit C3aR Inhibierung reduziert war, aber unverändert nach der Inhibierung von C5aR1 oder C5aR2 blieb. Mögliche zugrundeliegende Mechanismen in der Phosphorylation von ERK1/2 wurden anschließend in Knochenmark-gereiften Makrophagen von ATR knock-out Mäusen untersucht. Ohne C5aR1 oder C5aR2 Expression wurde eine verzögerte Phosphorylierung von ERK1/2 in den Makrophagen beobachtet, was erklären könnte warum die Blockade von C5aRs den Ausgang von Meningokokken induzierter Sepsis in Mäusen positiv beeinflusst. Im Gegensatz zu diesen Ergebnissen wurde die Kolonisation des Nasopharynx durch Nme in huCEACAM-1 exprimierenden Mäusen, weder durch die Komplementfaktoren C3 und C5 noch durch die ATRs beeinflusst. Zusätzlich konnte auch kein Unterschied in der Besiedelung des Nasopharynx durch Nme-Mutanten, die verschiedene Mutationen des Typ 2 CRISPR/Cas Systems besaßen, beobachtet werden. Diese Arbeit zeigt die Wichtigkeit des Komplementsystems, der ATRs und der Immunzellen in IMD. Zusätzlich zeigt diese Arbeit, dass das Komplementsystem und die ATRs jedoch keine Auswirkungen auf die Kolonisation des Nasopharynx in Mäusen haben. Die äußerst positive Auswirkung auf IMD, wenn C5aR1 und C5aR2 nicht gebildet oder blockiert werden, könnte medizinisch von Bedeutung sein und eventuell in der Zukunft die Standarttherapie bei IMD unterstützen. KW - Anaphylatoxine KW - Komplement C3a KW - Komplement C5a KW - Neisseria meningitidis KW - Komplement KW - anaphylatoxin receptors KW - invasive meningococcal diseases KW - Anaphylatoxinrezeptoren Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-269759 ER - TY - JOUR A1 - Aldejohann, Alexander Maximilian A1 - Wiese-Posselt, Miriam A1 - Gastmeier, Petra A1 - Kurzai, Oliver T1 - Expert recommendations for prevention and management of Candida auris transmission JF - Mycoses N2 - Candida auris was first described as a yeast pathogen in 2009. Since then, the species has emerged worldwide. In contrast to most other Candida spp., C. auris frequently exhibits multi-drug resistance and is readily transmitted in hospital settings. While most detections so far are from colonised patients, C. auris does cause superficial and life-threatening invasive infections. During management of the first documented C. auris transmission in a German hospital, experts from the National Reference Centers for Invasive Fungal Infections (NRZMyk) and the National Reference Center for Surveillance of Nosocomial Infections screened available literature and integrated available knowledge on infection prevention and C. auris epidemiology and biology to enable optimal containment. Relevant recommendations developed during this process are summarised in this guidance document, intended to assist in management of C. auris transmission and potential outbreak situations. Rapid and effective measures to contain C. auris spread require a multi-disciplinary approach that includes clinical specialists of the affected unit, nursing staff, hospital hygiene, diagnostic microbiology, cleaning staff, hospital management and experts in diagnostic mycology / fungal infections. Action should be initiated in a step-wise process and relevant interventions differ between management of singular C. auris colonised / infected patients and detection of potential C. auris transmission or nosocomial outbreaks. KW - Candida auris KW - nosocomial transmission KW - infection prevention KW - expert recommendation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318570 VL - 65 IS - 6 SP - 590 EP - 598 ER - TY - THES A1 - Drayß, Maria T1 - Asymptomatisches Trägertum von Staphylococcus aureus und Haemophilus influenzae bei Senioren T1 - Asymptomatic carriage of Staphylococcus aureus and Haemophilus influenzae in elderly people N2 - Ältere Menschen sind gegenüber invasiven Infektionen und Sepsis besonders vulnerabel mit ungünstiger Prognose. Staphylococcus aureus und Haemophilus influenzae können beide invasive Infektionen verursachen. Oft geht eine asymptomatische Besiedelung einer Infektion voraus und ist ein Risikofaktor für eine invasive Infektion. Daher wurde eine bizentrische Querschnittstudie in den Regionen Aachen und Würzburg durchgeführt, um die Prävalenz von H. influenzae, S. aureus und MRSA (Methicillin resistenter S. aureus) bei asymptomatischen Senioren zu bestimmen, wie auch Risikofaktoren für eine Besiedelung. Von Oktober 2012 bis Mai 2013 wurden 677 Erwachsenen im Alter von 65 Jahren oder älter eingeschlossen, die zu Hause oder in Seniorenheimen lebten. Die Prävalenz von H. influenzae bei älteren Menschen war mit einer Trägerrate von nur 1,9% ([95% CI: 1,0 - 3,3%]; 13/677) sehr niedrig. Trägerisolate waren überwiegend nicht typisierbare H. influenzae, zeigten eine hohe clonale Diversität und waren alle Ampicillin-sensibel. Die Prävalenz von S. aureus war mit 28,5% ([95% CI: 25,1 - 32,1%]; 193/677) hoch, wie für die deutsche Allgemeinbevölkerung bekannt, während MRSA bei weniger als 1% der Teilnehmer gefunden wurde (0,7% [95% CI: 0,2 - 1,7%]; 5/677). Die Prävalenz von H. influenzae, S. aureus und MRSA unterschied sich nicht signifikant zwischen selbständig zu Hause lebenden Senioren und Pflegeheimbewohnern. Ältere, selbständig lebende Menschen mit höherem Bildungsniveau hatten signifikant höhere Kolonisierungsraten mit S. aureus (adjusted OR: 1,905 [95% CI: 1,248 - 2,908]; p = 0,003). Bei Pflegeheimbewohnern war eine Kolonisierung signifikant mit Verheiratet sein assoziiert (adjusted OR: 3,367 [95% CI: 1,502 - 7,546]; p = 0,003). Diese Ergebnisse unterstreichen die Bedeutung von sozio-demographischen Faktoren für eine Kolonisierung mit S. aureus und schließen eine Lücke bei epidemiologischen Daten zu H. influenzae. N2 - Elderly people are especially vulnerable to invasive infections and sepsis with often poor outcome. Staphyloccus aureus and Haemophilus influenzae both can cause invasive infections. Asymptomatic colonization often precedes infection and poses a risk for invasive infection. Therefore, a bi-centric cross-sectional carrier study was conducted in the regions of Aachen and Wuerzburg, Germany, to determine the prevalence of H. influenzae, S. aureus and MRSA (methicillin resistant S. aureus) in asymptomatic elderly people and to identify risk factors for colonization. From October 2012 to May 2013 677 adults aged 65 years and older were included, living at home or in nursing homes. In contrast to children and younger adults the prevalence of H. influenzae was very low among elderly people with a carriage rate of only 1.9% ([95% CI: 1.0 - 3.3%]; 13/677). Carrier isolates were predominantly non typeable H. influenzae, showed a high clonal diversity and were all susceptible to ampicillin. The prevalence of S. aureus was expectedly high as known for the German general population (28.5% [95% CI: 25.1 - 32.1%]; 193/677), while MRSA was found in less than 1% of the individuals (0.7% [95% CI: 0.2 - 1.7%]; 5/677). The prevalence of H. influenzae, S. aureus und MRSA did not differ significantly between community dwellers and nursing home residents. Elderly community-dwellers with higher education level had significantly higher colonization rates with S. aureus (adjusted OR: 1.905 [95% CI: 1.248 - 2.908]; p = 0.003). Among nursing home residents, colonization was significantly associated with being married (adjusted OR: 3.367 [1.502 - 7.546]; p = 0.003). These results underline the importance of socio-demographic factors for colonization with S. aureus and close a gap in epidemiological data on H. influenzae. KW - Staphylococcus aureus KW - Haemophilus influenzae KW - MRSA KW - Senioren KW - Besiedelung KW - Multilocus-Sequenz-Typisierung Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-272276 ER - TY - JOUR A1 - Rohde, Jörn A1 - Himmel, Wolfgang A1 - Hofinger, Clemens A1 - Lâm, Thiên-Trí A1 - Schrader, Hanna A1 - Wallstabe, Julia A1 - Kurzai, Oliver A1 - Gágyor, Ildikó T1 - Diagnostic accuracy and feasibility of a rapid SARS-CoV-2 antigen test in general practice - a prospective multicenter validation and implementation study JF - BMC Primary Care N2 - Background PCR testing is considered the gold standard for SARS-CoV-2 diagnosis but its results are earliest available hours to days after testing. Rapid antigen tests represent a diagnostic tool enabling testing at the point of care. Rapid antigen tests have mostly been validated by the manufacturer or in controlled laboratory settings only. External validation at the point of care, particularly in general practice where the test is frequently used, is needed. Furthermore, it is unclear how well point of care tests are accepted by the practice staff. Methods In this prospective multicenter validation study in primary care, general practitioners included adult individuals presenting with symptoms suggesting COVID-19. Each patient was tested by the general practitioner, first with a nasopharyngeal swab for the point of care test (Roche SARS-CoV-2 Rapid Antigen Test) and then with a second swab for PCR testing. Using the RT-PCR result as a reference, we calculated specificity, sensitivity, positive predictive value and negative predictive value, with their 95% confidence intervals. General practitioners and medical assistants completed a survey to assess feasibility and usefulness of the point of care tests. Results In 40 practices in Würzburg, Germany, 1518 patients were recruited between 12/2020 and 06/2021. The point of care test achieved a sensitivity of 78.3% and a specificity of 99.5% compared to RT-PCR. With a prevalence of 9.5%, the positive predictive value was 93.9% and the negative predictive value was 97.8%. General practitioners rated the point of care test as a helpful tool to support diagnostics in patients with signs and symptoms suggestive for infection, particularly in situations where decision on further care is needed at short notice. Conclusion The point of care test used in this study showed a sensitivity below the manufacturer’s specification (Sensitivity 96.25%) in the practice but high values for specificity and high positive predictive value and negative predictive value. Although widely accepted in the practice, measures for further patient management require a sensitive interpretation of the point of care test results. KW - COVID-19 testing KW - feasibility study KW - attitude of health personnel KW - sensitivity and specificity KW - general practice Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299659 VL - 23 IS - 1 ER - TY - THES A1 - Stieber, Hanna T1 - Auswirkungen des Sphingolipidsynthese-Inhibitors Myriocin auf Vitalität und Antimykotikaresistenz von \(Candida\) \(auris\) T1 - Impact of the sphingolipid synthesis inhibitor myriocin on viability and antifungal susceptibility of \(Candida\) \(auris\) N2 - Candida Spezies gehören als kommensale Organismen zur normalen menschlichen Mikroflora, können allerdings unter bestimmten Bedingungen Krankheitswert erlangen. Limitationen in der Behandlung durch immer mehr resistente Candida Spezies und die wachsende Zahl immunsupprimierter Patienten gelten als Hauptursachen für die steigende Häufigkeit invasiver Candidosen und systemischer Candidämien. Die 2009 entdeckte Spezies C. auris stellt durch ihre zahlreichen Resistenzen, das Potential zur Auslösung nosokomialer Ausbrüche in Krankenhäusern und die schnelle Verbreitung über mehrere Kontinente eine neue Herausforderung dar. Der Bedarf an neuen Antimykotika mit anderen Wirkmechanismen und neuen Zielstrukturen ist größer denn je. Die fungale Sphingolipid-Biosynthese wurde bereits mehrfach als potenzielles Ziel antimykotischer Therapie diskutiert, allerdings bezieht sich die meiste Forschung hierzu auf C. albicans]. In vorliegender Arbeit wurden die Auswirkungen der Inhibition der Sphingolipid Biosynthese durch Myriocin auf C. auris und sein Resistenzverhalten untersucht und mit denen auf andere Candida Spezies verglichen. Sowohl die Mikrodilution als auch die Plattentropftests zeigten, dass C. auris verglichen mit anderen Candida Spezies besonders sensitiv auf die Anwesenheit von Myriocin reagierte und stärker im Wachstum gehemmt wurde. Der Survival Assay ergab für alle drei Spezies ein Absenken der CFU durch Myriocin, die Abweichungen zwischen den Stämmen waren jedoch unwesentlich. Unterschiede konnten in Vitalität und Vermehrung der verschiedenen Spezies unter Myriocineinfluss festgestellt werden. Aus der Lebend/Tot-Färbung ging hervor, dass Myriocin bei allen Stämmen zum Absterben von Candida Zellen führte, C. albicans und C. glabrata allerdings signifikant niedrigere Überlebensraten im Vergleich zu den C. auris Isolaten aufwiesen. Im Gegensatz dazu konnte mithilfe der FITC-Mikroskopie gezeigt werden, dass Candida Zellen unter Zugabe von Myriocin weniger Tochterzellen ausbildeten, was auf eine erschwerte oder zumindest verlangsamte Zellvermehrung hindeutet. Dabei schien das Wachstum der C. auris Stämme durch Myriocin deutlich eingeschränkter zu sein als das von C. albicans und C. glabrata. Durch weitere Mikroskopie und die Kombination aus Lebend/Tot Färbung mittels PI und FITC Färbung, sollte die Verteilung der toten Zellen auf Mutter- und Tochterzellen evaluiert werden. Hier konnte ein Trend zu einem vermehrten Zellsterben der Tochterzellen, vor allem für C. auris, festgestellt werden. Abschließende E-Tests für Amphotericin B, Anidulafungin und Fluconazol ergaben eine signifikante Herabsetzung der MHK für alle C. auris Isolate durch Myriocin. Die hier vorgestellten Ergebnisse und die durch mehrere Studien festgestellten Differenzen in der Sphingolipidkomposition von C. auris verglichen mit anderen Candida Spezies geben Hinweis darauf, dass Sphingolipide für Vitalität, Zellteilung und vor allem für die Wirkung einiger Antimykotika auf C. auris eine besondere, wenn nicht übergestellte Bedeutung haben könnten. Zwar wurde die Sphingolipidsynthese bereits mehrfach als potenzieller Angriffspunkt für die antifungale Therapie diskutiert, allerdings lediglich am Beispiel anderer Candida Spezies. Der Sphingolipidstoffwechsel könnte somit ein vielversprechender Ansatz für die Behandlung des sonst so therapieresistenten und lebensbedrohlichen Pilzes C. auris sein. N2 - Candida species are commensal organisms belonging to the normal human microflora, but can become pathogenic under certain conditions. Limitations in treatment due to an increasing number of resistant Candida species and the growing number of immunosuppressed patients are considered to be the main reasons for the increasing frequency of invasive candidiasis and systemic candidemia. C. auris, a species discovered in 2009, shows potential to cause nosocomial outbreaks in hospitals, limited susceptibility to numerous antifungals and a rapid spread across several continents. This leads to a need for new antifungal agents with different mechanisms of action and new targets. Fungal sphingolipid biosynthesis has been discussed several times as a potential target of antifungal therapy, however most research on this relates to C. albicans. In the present work, the effects of inhibition of sphingolipid biosynthesis by myriocin on C. auris and its impact on fungal susceptibility were investigated and compared with those on other Candida species. Both microdilution and plate droplet assays showed that C. auris was more sensitive to myriocin compared with other Candida species and showed severe growth defects. The survival assay showed a lowering of CFU by myriocin for all three species, but the differences between the strains were insignificant. Live/dead staining showed that myriocin led to the death of Candida cells in all strains, but C. albicans and C. glabrata had significantly lower survival rates compared to the C. auris isolates. In contrast, FITC microscopy showed that Candida cells produced fewer daughter cells when myriocin was added, indicating that cell proliferation was impeded or at least slowed. In this regard, the growth of C. auris strains appeared to be significantly more restricted by myriocin than that of C. albicans and C. glabrata. Further microscopy and the combination of live/dead staining using PI and FITC staining, was performed to evaluate the distribution of dead cells between mother and daughter cells. Here, a trend towards increased cell death of daughter cells, especially for C. auris, was observed. Final E-tests for amphotericin B, anidulafungin, and fluconazole revealed a significant reduction in MIC for all C. auris isolates by myriocin. These results and the differences in sphingolipid composition of C. auris compared with other Candida species established by several studies provide evidence that sphingolipids may have a special, if not superimposed, importance for viability, cell division, and especially for the suscteptibility of C. auris to some antifungals. It is true that sphingolipid synthesis has been discussed several times as a potential target for antifungal therapy, but only using other Candida species as examples. Sphingolipid metabolism could thus be a promising approach for the treatment of the therapy-resistant and life-threatening fungus C. auris. KW - Candida KW - Sphingolipide KW - Sphingolipidstoffwechsel KW - Multiresistenz KW - pathogene Pilze KW - Candida auris KW - Antimykotikaresistenz KW - antifungal susceptibility KW - myriocin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-289121 ER - TY - JOUR A1 - Zoran, Tamara A1 - Seelbinder, Bastian A1 - White, Philip Lewis A1 - Price, Jessica Sarah A1 - Kraus, Sabrina A1 - Kurzai, Oliver A1 - Linde, Joerg A1 - Häder, Antje A1 - Loeffler, Claudia A1 - Grigoleit, Goetz Ulrich A1 - Einsele, Hermann A1 - Panagiotou, Gianni A1 - Loeffler, Juergen A1 - Schäuble, Sascha T1 - Molecular profiling reveals characteristic and decisive signatures in patients after allogeneic stem cell transplantation suffering from invasive pulmonary aspergillosis JF - Journal of Fungi N2 - Despite available diagnostic tests and recent advances, diagnosis of pulmonary invasive aspergillosis (IPA) remains challenging. We performed a longitudinal case-control pilot study to identify host-specific, novel, and immune-relevant molecular candidates indicating IPA in patients post allogeneic stem cell transplantation (alloSCT). Supported by differential gene expression analysis of six relevant in vitro studies, we conducted RNA sequencing of three alloSCT patients categorized as probable IPA cases and their matched controls without Aspergillus infection (66 samples in total). We additionally performed immunoassay analysis for all patient samples to gain a multi-omics perspective. Profiling analysis suggested LGALS2, MMP1, IL-8, and caspase-3 as potential host molecular candidates indicating IPA in investigated alloSCT patients. MMP1, IL-8, and caspase-3 were evaluated further in alloSCT patients for their potential to differentiate possible IPA cases and patients suffering from COVID-19-associated pulmonary aspergillosis (CAPA) and appropriate control patients. Possible IPA cases showed differences in IL-8 and caspase-3 serum levels compared with matched controls. Furthermore, we observed significant differences in IL-8 and caspase-3 levels among CAPA patients compared with control patients. With our conceptual work, we demonstrate the potential value of considering the human immune response during Aspergillus infection to identify immune-relevant molecular candidates indicating IPA in alloSCT patients. These human host candidates together with already established fungal biomarkers might improve the accuracy of IPA diagnostic tools. KW - host response KW - invasive pulmonary aspergillosis KW - alloSCT patients KW - galectin-2 KW - caspase-3 KW - matrix metallopeptidase-1 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262105 SN - 2309-608X VL - 8 IS - 2 ER - TY - JOUR A1 - Endres, Leo M. A1 - Jungblut, Marvin A1 - Divyapicigil, Mustafa A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Christodoulides, Myron A1 - Kim, Brandon J. A1 - Schubert-Unkmeir, Alexandra T1 - Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection JF - Fluids and Barriers of the CNS N2 - Background Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction. Methods We used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection. Results Both cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection. Conclusions Here, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting. KW - brain endothelial cells KW - bacterial meningitis KW - meningeal blood-csf barrier KW - induced pluripotent stem cells KW - neisseria meningitidis KW - leptomeningeal cells Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300208 VL - 19 IS - 1 ER - TY - JOUR A1 - Forster, Johannes A1 - Kohlmorgen, Britta A1 - Haas, Julian A1 - Weis, Philipp A1 - Breunig, Lukas A1 - Turnwald, Doris A1 - Mizaikoff, Boris A1 - Schoen, Christoph T1 - A streamlined method for the fast and cost-effective detection of bacterial pathogens from positive blood cultures for the BacT/ALERT blood culture system using the Vitek MS mass spectrometer JF - PLoS ONE N2 - Background and objective Prompt pathogen identification of blood stream infections is essential to provide appropriate antibiotic treatment. Therefore, the objective of this prospective single centre study was to establish an inexpensive, fast and accurate protocol for bacterial species identification with SDS protein-extraction directly from BacT/Alert® blood culture (BC) bottles by VitekMS®. Results Correct species identification was obtained for 198/266 (74.4%, 95%-CI = [68.8%, 79.6%]) of pathogens. The protocol was more successful in identifying 87/96 (91.4%, 95%-CI = [83.8%, 93.2%]) gram-negative bacteria than 110/167 (65.9%, 95%-CI = [58.1%, 73.0%]) gram-positive bacteria. The hands-on time for sample preparation and measurement was about 15 min for up to five samples. This is shorter than for most other protocols using a similar lysis-centrifugation approach for the combination of BacT/Alert® BC bottles and the Vitek® MS mass spectrometer. The estimated costs per sample were approx. 1.80€ which is much cheaper than for commercial kits. Conclusion This optimized protocol allows for accurate identification of bacteria directly from blood culture bottles for laboratories equipped with BacT/Alert® blood culture bottles and VitekMS® mass spectrometer. KW - bacterial pathogens KW - blood stream infections KW - BacT/ALERT Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300213 VL - 17 IS - 4 ER - TY - JOUR A1 - Forster, Johannes A1 - Dichtl, Karl A1 - Wagener, Johannes T1 - Lower beta‐1,3‐D‐glucan testing cut‐offs increase sensitivity for non‐albicans Candida species bloodstream infections JF - Mycoses N2 - Purpose Fungal biomarkers support early diagnosis of invasive fungal infections. In this study, we evaluated the impact of a recent update to the manufacturer‐recommended cut‐off for beta‐1,3‐D‐glucan (BDG) testing (Fujifilm Wako BDG assay) on sensitivity and specificity for the detection of candidemia. Additionally, we compared the performance with tests for Candida antigen (Ag by Serion ELISA antigen Candida, Virion\Serion) and anti‐mannan antibodies (Ab by Hemkit Candida IHA, Ravo Diagnostika). Methods Sera of 82 patients with candidemia, which were sampled with a maximum distance of ±14 days from the date of sampling of the corresponding positive blood cultures, were retrospectively analysed for BDG, Ag and Ab. Results of BDG testing were compared with results from sera of 129 patients with candidemia from a different hospital. Results Sensitivity of BDG testing (47%) was higher than for Ag (17%) or Ab (20%). By combining Ag and Ab testing, sensitivity was raised to 32%. Lowering the cut‐off of BDG from 11 pg/ml to the newly recommended cut‐off of 7 pg/ml resulted in a significant increase in sensitivity (47% vs 58%, p = .01 and 63% vs 71% p < .01). At both centres, the increase was significant in NAC but not in C. albicans candidemia. No significant effects on specificity were observed. Conclusion BDG testing outperformed Ag and Ab testing and its combination. Lowering the BDG cut‐off had no significant impact on specificity. The increase in sensitivity can be mainly attributed to a gain in sensitivity for non‐albicans Candida species bloodstream infections. KW - antigen testing KW - BDG KW - beta‐d‐glucan KW - bloodstream infection KW - candidemia KW - mannan Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276515 VL - 65 IS - 5 SP - 500 EP - 507 ER - TY - THES A1 - Aldejohann, Alexander Maximilian T1 - Echinocandin-Resistenzen in \(Candida\) \(glabrata\) T1 - Echinocandin resistance in \(Candida\) \(glabrata\) N2 - Candida glabrata ist die zweithäufigste Ursache von Candidämien und invasiven Hefepilzinfektionen in Europa. Im Gegensatz zu C. albicans zeigt C. glabrata eine reduzierte Empfindlichkeit gegen bestimmte Antimykotika und kann unter Therapie rasch Resistenzen entwickeln. Diese Arbeit umfasst eine systematische geno- und phänotypische Resistenzanalyse einer der größten europäischen - durch das NRZMyk in 5 Jahren zusammengetragenen - C. glabrata Stammsammlungen bestehend aus 176 klinisch relevanter Isolate. 84 der Stämme wurden anhand Referenztestung nach EUCAST zunächst als Anidulafungin (AND) resistent eingestuft. 71 wiesen konkordante Mutationen in den für die Glucan-Synthetase kodierenden FKS-Genen auf (13 % in FKS1, 87 % in FKS2). Vor allem die Position Ser-663 (FKS2-HS1) imponierte mit signifikant erhöhten AND MHK-Werten. 11 FKS-Wildtyp-Isolate, die ursprünglich als AND resistent klassifiziert wurden, wiesen in multiplen Nachtestungen um den Breakpoint undulierende AND MHK-Werte auf. 2 FKS-Wildtyp Isolate zeigten durchgängig hohe AND MHK-Werte und mussten daher - trotz fehlender Zielgenmutationen - als resistent eingestuft werden. Diese extremen Phänotypen wurden durch einen verblindeten nationalen Ringversuch bestätigt. Über ein Drittel der Isolate war multiresistent. Stämme aus Blutstrominfektionen und Ser-663 Mutation waren mit einer erhöhten Mortalität assoziiert. Ein weiteres Kernelement war die Detektion von Azol-resistenten C. glabrata petite-Phänotypen in der Routinediagnostik. Hier wurden innerhalb von 8 Monaten 20 relevante Isolate identifiziert. Die Ergebnisse belegen das regelmäßige Auftreten single- / multidrug-resistenter C. glabrata Isolate in Deutschland. Phänotypische Resistenztestungen können zu Fehlklassifizierung von sensiblen Isolaten führen. FKS-Genotypisierungen hingegen sind ein nützliches Tool zur Identifizierung relevanter Resistenzen. In seltenen Fällen scheint jedoch eine Echinocandin-Resistenz ohne genotypisches Korrelat möglich zu sein. N2 - Candida glabrata is the second most common cause of candidaemia and invasive yeast infections in Europe. In contrast to C. albicans, C. glabrata shows reduced susceptibility to certain antifungal agents and can rapidly acquire resistance under therapy. This work comprises a systematic geno- and phenotypic resistance analysis of one of the largest European C. glabrata strain collections - compiled by NRZMyk in 5 years - consisting of 176 clinically relevant isolates. 84 of the strains were initially classified as anidulafungin (AND) resistant by reference testing according to EUCAST. 71 showed concordant mutations in FKS genes encrypting the glucan synthetase (13 % in FKS1, 87 % in FKS2). In particular, the position Ser-663 (FKS2-HS1) impressed with significantly increased AND MIC-values. 11 FKS wild-type isolates, originally classified as AND resistant, showed fluctuating AND MIC-values near the clinical breakpoint after retests with multiple assays. Two FKS wild-type isolates showed consistently high AND MIC values and therefore had to be classified as resistant - despite the absence of target gene mutations. These extreme phenotypes were confirmed in a blinded national ring trial. More than one third of echinocandin-resistant isolates showed concordant fluconazole resistance. Strains from bloodstream infections and Ser-663 mutation were associated with high mortality. Another core element was the detection of azole-resistant C. glabrata petite phenotypes in routine diagnostics. Here, 20 relevant isolates were identified within 8 months, which could be assigned to 8 patients. These results demonstrate the regular occurrence of single- / multidrug-resistant C. glabrata isolates in Germany. Phenotypic resistance testing can lead to misclassification of susceptible isolates. FKS genotyping, on the other hand, is a useful tool for identifying resistant strains. However, in rare cases, echinocandin resistance without a genotypic correlate seems to be possible. KW - Resistenzbestimmung KW - Candida KW - Multidrug-Resistenz KW - Anidulafungin KW - Micafungin KW - Invasive Mykosen KW - Invasive Fungal Infections KW - C. glabrata KW - Multidrug-Resistenzen KW - Antimykotika KW - Mikrodilution KW - Anidulafungin KW - MDR KW - Susceptibility Testing KW - FKS-genes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275840 ER - TY - JOUR A1 - Tappe, Beeke A1 - Lauruschkat, Chris D. A1 - Strobel, Lea A1 - Pantaleón García, Jezreel A1 - Kurzai, Oliver A1 - Rebhan, Silke A1 - Kraus, Sabrina A1 - Pfeuffer-Jovic, Elena A1 - Bussemer, Lydia A1 - Possler, Lotte A1 - Held, Matthias A1 - Hünniger, Kerstin A1 - Kniemeyer, Olaf A1 - Schäuble, Sascha A1 - Brakhage, Axel A. A1 - Panagiotou, Gianni A1 - White, P. Lewis A1 - Einsele, Hermann A1 - Löffler, Jürgen A1 - Wurster, Sebastian T1 - COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds JF - Frontiers in Immunology N2 - Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus- and R. arrhizus-induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients. KW - COVID-19 KW - immune impairment KW - T cells KW - granulocytes KW - Aspergillus KW - Rhizopus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-283558 SN - 1664-3224 VL - 13 ER - TY - JOUR A1 - Bauer, Hannah A1 - Concha Mendoza, Gustavo Andrés A1 - Kreienbrock, Lothar A1 - Hartmann, Maria A1 - Frickmann, Hagen A1 - Kann, Simone T1 - Prevalence of common diseases in Indigenous people in Colombia JF - Tropical Medicine and Infectious Disease N2 - The Indigenous tribe called the Wiwa lives retracted in the Sierra Nevada de Santa Marta, Colombia. Little is known about their health status and whether the health care system in place covers their needs. In 2017 and 2018, a permanent physician was in charge for the Wiwa. Diseases and complaints were registered, ranked, and classified with the ICD-10 coding. Datasets from the Indigenous health care provider Dusakawi, collected from local health points and health brigades travelling sporadically into the fields for short visits, were compared. Furthermore, a list of provided medication was evaluated regarding the recorded needs. The most common complaints found were respiratory, infectious and parasitic, and digestive diseases. The top ten diagnoses collected in the health points and in the health brigade datasets were similar, although with a different ranking. The available medication showed a basic coverage only, with a critical lack of treatment for many severe, chronic, and life-threatening diseases. Most of the detected diseases in the Indigenous population are avoidable by an improvement in health care access, an expansion of the provided medication, and an increase in knowledge, hygiene, and life standards. KW - Chagas disease KW - indigenous KW - public health KW - Colombia KW - Sierra Nevada KW - neglected groups Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-278953 SN - 2414-6366 VL - 7 IS - 6 ER - TY - JOUR A1 - Strobel, Katharina A1 - Sickenberger, Christina A1 - Schoen, Christoph A1 - Kneitz, Hermann A1 - Kolb-Mäurer, Annette A1 - Goebeler, Matthias T1 - Diagnosis and therapy of Mycobacterium marinum: a single-center 21-year retrospective analysis JF - Journal der Deutschen Dermatologischen Gesellschaft N2 - Background and Objectives In Europe, infections with Mycobacterium (M.) marinum are rare. We conducted a retrospective single-center study to assess the clinical spectrum of M. marinum infection and its diagnosis, treatment and outcome under real-world conditions. Patients and Methods Eighteen patients presenting with M. marinum infections between 1998 and 2018 were identified in the data warehouse of the University Hospital Würzburg and considered for detailed analysis. Results Twelve patients reported aquatic exposure. In 16/18 cases the upper extremities were affected. No invasive infections were detected. Mean time to diagnosis was 15 weeks. Histology revealed granulomatous inflammation in 14 patients while mycobacterial cultures were positive for M. marinum in 16 cases. Most patients received antibiotic monotherapy (14/18) while combination therapy was administered in four cases. Treatment (with a median duration of 10 weeks) was successful in 13 patients. Five patients were lost to follow-up. Conclusions Our retrospective analysis of M. marinum infections at a German tertiary referral center revealed a considerable diagnostic delay and the relevance of microbiological culture, PCR and histology for diagnosis. Monotherapy with clarithromycin (rather than doxycycline) appeared as a reasonable treatment option while immunosuppressed or -compromised patients and those with extended disease received combination therapy. KW - Mycobacterium marinum KW - diagnosis KW - therapy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318428 VL - 20 IS - 9 SP - 1211 EP - 1218 ER - TY - JOUR A1 - Nieuwenhuizen, Natalie E. A1 - Evans, Joanna C. T1 - Cellular and molecular mechanisms in mycobacterial infection JF - International Journal of Molecular Sciences N2 - No abstract available KW - mycobacterial infection Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284370 SN - 1422-0067 VL - 23 IS - 13 ER - TY - JOUR A1 - Streck, Laura Elisa A1 - Forster, Johannes A1 - von Hertzberg-Boelch, Sebastian Philipp A1 - Reichel, Thomas A1 - Rudert, Maximilian A1 - Rueckl, Kilian T1 - The role of synovial fluid aspiration in shoulder joint infections JF - BMC Musculoskeletal Disorders N2 - Background Joint aspiration with analysis of synovial fluid white blood cell count (WBC) and microbiological culture is a widely established aspect in the diagnosis of shoulder joint infections (SJI). In case of a two stage revision for SJI, joint aspiration before re−/implantation of a total shoulder arthroplasty (TSA) was used to rule out persistent infection for years but its value is under debate. Shoulder specific data on all aspects is rare. The current study aims to answer the following research questions: Does joint aspiration have an insufficient predictive value in the diagnosis of SJI in (1) initial workup and (2) before definite arthroplasty with polymethylmethacrylate (PMMA)-Spacer in place? Methods This retrospective evaluation investigates 35 patients that were treated for SJI with a two staged implantation of a TSA after debridement and implantation of an PMMA-Spacer. Joint aspirations were performed preoperatively (PA) and before re−/implantation of the prosthesis while spacer was in place (interstage aspiration, IA). Samples were taken for microbiological culture and analysis of WBC. Sensitivity and specificity were calculated with reference to intraoperative microbiological samples. Receiver Operating Characteristic (ROC), Area-Under-Curve analysis (AUC) and calculation of the Youden index were performed to find optimum cut-off for WBC. Results The sensitivity of microbiological cultures from PA was 58.3% and the specificity was 88.9%. The mean WBC was 27,800 leucocytes/mm3 (range 400-96,300). The maximum Youden index (0.857) was a cut-off of 2600 leucocytes/mm3 with a sensitivity of 85.7% and a specificity of 100.0%. The sensitivity and specificity of IA were 0.0% and 88.5%, respectively. Conclusions Preoperative aspiration is likely to miss Cutibacteria spp. and CoNS and cannot rule out infection for sure. However, we recommend it for its advantages of targeted antibiotic therapy in case of germ identification. Empiric antibiotic therapy should cover Cutibacteria and CoNS even if aspiration showed negative microbiological cultures. In contrast, the diagnostic value of interstage aspiration does not qualify for its routine use. KW - revision arthroplasty KW - periprosthetic joint infection KW - white blood cell count KW - septic KW - microbiological culture KW - interstage aspiration Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300795 VL - 23 ER - TY - JOUR A1 - Nyawale, Helmut A. A1 - Moremi, Nyambura A1 - Mohamed, Mohamed A1 - Njwalila, Johnson A1 - Silago, Vitus A1 - Krone, Manuel A1 - Konje, Eveline T. A1 - Mirambo, Mariam M. A1 - Mshana, Stephen E. T1 - High seroprevalence of SARS-CoV-2 in Mwanza, northwestern Tanzania: a population-based survey JF - International Journal of Environmental Research and Public Health N2 - The transmission of the SARS-CoV-2 virus, which causes COVID-19, has been documented worldwide. However, the evidence of the extent to which transmission has occurred in different countries is still to be established. Understanding the magnitude and distribution of SARS-CoV-2 through seroprevalence studies is important in designing control and preventive strategies in communities. This study investigated the seropositivity of the SARS-CoV-2 virus antibodies in the communities of three different districts in the Mwanza region, Tanzania. A household cross-sectional survey was conducted in September 2021 using the modified African Centre for Disease and Prevention (ACDC) survey protocol. A blood sample was obtained from one member of each of the selected households who consented to take part in the survey. Immunochromatographic rapid test kits were used to detect IgM and IgG SARS-CoV-2 antibodies, followed by descriptive data analysis. Overall, 805 participants were enrolled in the study with a median age of 35 (interquartile range (IQR):27–47) years. The overall SARS-CoV-2 seropositivity was 50.4% (95%CI: 46.9–53.8%). The IgG and IgM seropositivity of the SARS-CoV-2 antibodies was 49.3% and 7.2%, respectively, with 6.1% being both IgG and IgM seropositive. A history of runny nose (aOR: 1.84, 95%CI: 1.03–3.5, p = 0.036), loss of taste (aOR: 1.84, 95%CI: 1.12–4.48, p = 0.023), and living in Ukerewe (aOR: 3.55, 95%CI: 1.68–7.47, p = 0.001) and Magu (aOR: 2.89, 95%CI: 1.34–6.25, p= 0.007) were all independently associated with SARS-CoV-2 IgM seropositivity. Out of the studied factors, living in the Ukerewe district was independently associated with IgG seropositivity (aOR 1.29, CI 1.08–1.54, p = 0.004). Twenty months after the first case of COVID-19 in Tanzania, about half of the studied population in Mwanza was seropositive for SARS-CoV-2. KW - SARS-CoV-2 KW - COVID-19 KW - seroprevalence KW - antibodies KW - Mwanza KW - Tanzania Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288134 SN - 1660-4601 VL - 19 IS - 18 ER - TY - JOUR A1 - Straub, Anton A1 - Stapf, Maximilian A1 - Fischer, Markus A1 - Vollmer, Andreas A1 - Linz, Christian A1 - Lâm, Thiên-Trí A1 - Kübler, Alexander A1 - Brands, Roman C. A1 - Scherf-Clavel, Oliver A1 - Hartmann, Stefan T1 - Bone concentration of ampicillin/sulbactam: a pilot study in patients with osteonecrosis of the jaw JF - International Journal of Environmental Research and Public Health N2 - Osteonecrosis of the jaw (ONJ) occurs typically after irradiation of the head and neck area or after the intake of antiresorptive agents. Both interventions can lead to compromised bone perfusion and can ultimately result in infection and necrosis. Treatment usually consists of surgical necrosectomy and prolonged antibiotic therapy, usually through beta-lactams such as ampicillin/sulbactam. The poor blood supply in particular raises the question as to whether this form of antibiosis can achieve sufficient concentrations in the bone. Therefore, we investigated the antibiotic concentration in plasma and bone samples in a prospective study. Bone samples were collected from the necrosis core and in the vital surrounding bone. The measured concentrations in plasma for ampicillin and sulbactam were 126.3 ± 77.6 and 60.2 ± 35.0 µg/mL, respectively. In vital bone and necrotic bone samples, the ampicillin/sulbactam concentrations were 6.3 ± 7.8/1.8 ± 2.0 µg/g and 4.9 ± 7.0/1.7 ± 1.7 µg/g, respectively. These concentrations are substantially lower than described in the literature. However, the concentration seems sufficient to kill most bacteria, such as Streptococci and Staphylococci, which are mostly present in the biofilm of ONJ. We, therefore, conclude that intravenous administration of ampicillin/sulbactam remains a valuable treatment in the therapy of ONJ. Nevertheless, increasing resistance of Escherichia coli towards beta-lactam antibiotics have been reported and should be considered. KW - osteonecrosis of the jaw KW - ARONJ KW - MRONJ KW - ONJ KW - osteoradionecrosis KW - antibiotic bone concentration KW - jaw bone KW - beta-lactam KW - ampicillin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297413 SN - 1660-4601 VL - 19 IS - 22 ER - TY - JOUR A1 - Straub, Anton A1 - Vollmer, Andreas A1 - Lâm, Thiên-Trí A1 - Brands, Roman C. A1 - Stapf, Maximilian A1 - Scherf-Clavel, Oliver A1 - Bittrich, Max A1 - Fuchs, Andreas A1 - Kübler, Alexander C. A1 - Hartmann, Stefan T1 - Evaluation of advanced platelet-rich fibrin (PRF) as a bio-carrier for ampicillin/sulbactam JF - Clinical Oral Investigations N2 - Objectives Mechanisms of wound healing are often impaired in patients with osteonecrosis of the jaw (ONJ). According to the guidelines for the treatment of this disease, early surgical intervention is indicated. However, surgery often faces complications such as wound healing disorders. The application of platelet-rich fibrin (PRF) after necrosectomy between bone and mucosa may constitute a promising approach to improve surgical results. An aspect that was not investigated until now is that PRF acts as a “bio-carrier” for antibiotics previously applied intravenously. Materials and methods We investigated the antimicrobial properties of PRF in 24 patients presenting ONJ undergoing systemic antibiosis with ampicillin/sulbactam. We measured the concentration of ampicillin/sulbactam in plasma and PRF and performed agar diffusion tests. Ampicillin/sulbactam was applied intravenously to the patient 10 minutes for blood sampling for PRF. No further incorporation of patients’ blood or PRF product with antibiotic drugs was obtained. Four healthy patients served as controls. Results Our results revealed that PRF is highly enriched with ampicillin/sulbactam that is released to the environment. The antibiotic concentration in PRF was comparable to the plasma concentration of ampicillin/sulbactam. The inhibition zone (IZ) of PRF was comparable to the standard ampicillin/sulbactam discs used in sensitivity testing. Conclusions The results of our study demonstrated that PRF is a reliable bio-carrier for systemic applied antibiotics and exhibits a large antimicrobial effect. Clinical relevance We describe a clinically useful feature of PRF as a bio-carrier for antibiotics. Especially when applied to poorly perfused tissues and bone such as in ONJ, the local release of antibiotics can reduce wound healing disorders like infections. KW - osteonecrosis of the jaw KW - osteoradionecrosis KW - antiresorptive drug-related osteonecrosis of the jaw KW - ARONJ KW - oral microbiome KW - agar diffusion test Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324515 VL - 26 IS - 12 ER - TY - THES A1 - Herz, Michaela T1 - Genome wide expression profiling of Echinococcus multilocularis T1 - Genomweite Expressionsanalysen von Echinococcus multilocularis N2 - Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver. Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis. Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future. The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival. Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis. N2 - Alveoläre Echinokokkose wird durch das Metazestodenstadium des kleinen Fuchsbandwurms Echinococcus multilocularis verursacht und medizinisch als eine schwere Zoonose mit begrenzten Behandlungsmöglichkeiten betrachtet. Um ein besseres Verständnis für die Biologie der Zestoden zu erlangen, wurde das Genom von E. multilocularis, zusammen mit denen anderer Zestoden, bereits sequenziert. Bisher wurden nur wenige Studien zum Transkriptom von E. multilocularis durchgeführt und eine umfassende Analyse der Transkriptionsprofile über verschiedene Stadien des Lebenszyklus hinweg fehlt bislang. Diese Arbeit stellt die bisher umfassendste Untersuchung des Transkriptoms von E. multilocularis dar. Unterschiede in der Genexpression in verschiedenen Stadien des Lebenszyklus und unter experimentellen Bedingungen wurden qualitativ und quantitativ untersucht. Dazu wurden Daten aus RNA-Sequenzierungen in drei biologischen Replikaten verwendet. Die untersuchten Datensätze beruhen auf Proben von Metazestoden, die unter aeroben und anaeroben Bedingungen kultiviert, sowie von Metazestoden, die direkt aus Gerbilen isoliert wurden. Weitere Proben umfassen Stammzellkulturen zu drei verschiedenen Entwicklungszeitpunkten sowie nicht-aktivierte und aktivierte Protoskolizes, das Larvenstadium das sich zu Adulten entwickeln kann. Zusätzlich wurden zwei Datensätze von Metazestoden unter experimentellen Bedingungen, die zur Identifizierung stammzellspezifischer (keimzellspezifischer) Gene geeignet sind, sowie ein Datensatz von einem siRNA-Experiment untersucht. Die Analyse dieser Datensätze führte zu Genexpressionsprofilen für alle annotierten Gene, unter anderem für Gene, die im Tegument des Metazestoden exprimiert werden und eine Rolle spielen bei Wirt-Parasit-Interaktionen und der Modulierung der Immunantwort des Wirts. Genexpressionsprofile liefern zudem Informationen über Gene, die für das infiltrative Wachstum des Parasiten in der Leber verantwortlich sein könnten. Des Weiteren wurden keimzellspezifische Gene identifiziert. Keimzellen sind die einzigen proliferierenden Zellen in E. multilocularis und daher von essentieller Bedeutung für die Entwicklung und das Wachstum des Parasiten. Durch eine Kombination von Keimzelldepletierungs- und Keimzellanreicherungsverfahren wurden Gene mit keimzellspezifischer Expression identifiziert. Wie erwartet, sind viele dieser Gene in der Translation, der Zellzyklusregulation oder DNA-Replikation und –Reparatur involviert. Darüber hinaus wurden keimzellspezifisch exprimierte Transkriptionsfaktoren detektiert, von denen viele in der Festlegung des Zellschicksals eine Rolle spielen. Als Beispiel eines keimzellspezifischen Genes wurde das Gen, das für die reverse Transkriptase (TERT) kodiert, genauer untersucht. Die Expression von E. multilocularis tert in Keimzellen wurde experimentell bestätigt. Zellkulturexperimente weisen darauf hin, dass TERT für die Proliferation und die Entwicklung essentiell ist. TERT ist daher ein potentiell interessantes Wirkstofftarget für die chemotherapeutische Behandlung der alveolären Echinokokkose. Zu den keimzellspezifischen Genen in E. multilocularis gehören auch Gene densoviralen Ursprungs. Es wurden mehr als 20 Densovirusloci mit Informationen für nicht-strukturelle und strukturelle Densovirusproteine im E. multilocularis-Genom identifiziert. Densovirale Elemente wurden auch in vielen anderen Zestodengenomen detektiert. Die genomische Integration dieser Elemente deutet darauf hin, dass densovirus-basierte Vektoren zur genetischen Manipulation von Zestoden geeignet sein könnten. Interessanterweise sind nur drei von mehr als 20 Densovirusloci im E. multilocularis-Genom exprimiert. Da es in Zestoden keinen kanonischen piRNA-Signalweg gibt, stellt sich die Frage nach möglichen Genabschaltungsmechanismen. Die Analyse der RNA-Sequenzierdaten ergab Hinweise auf natürliche Antisense-Transkripte als einen möglichen Genregulationsmechanismus in E. multilocularis. Vorläufige Experimente und bisherige Studien deuten weiterhin darauf hin, dass DNA-Methylierung ein Mechanismus der Genregulation und -abschaltung in Zestoden sein könnte. Die Transkriptionsdaten enthalten auch Informationen zu Genen, die in differenzierten Zellen exprimiert werden, wie zum Beispiel das Serotonintransportergen, das in Nervenzellen exprimiert wird. Zellkulturversuche weisen darauf hin, dass Serotonin und Serotonintransport eine wichtige Rolle bei der Proliferation, der Entwicklung und dem überleben von E. multilocularis spielen. Insgesamt bietet diese Arbeit einen umfassenden Transkriptionsdatenatlas über die Stadien des Lebenszyklus von E. multilocularis. Die Identifizierung von keimzellspezifischen Genen und Genen, die für die Interaktion zwischen Wirt und Parasit wichtig sind, wird die zukünftige Forschung erheblich erleichtern. Ein globaler Überblick über die Genexpressionsprofile wird zudem hilfreich sein bei der Entdeckung geeigneter Wirkstofftargets und bei der Entwicklung neuer Chemotherapeutika gegen die alveoläre Echinokokkose. KW - Fuchsbandwurm KW - Serotonin KW - Telomerase KW - Stammzelle KW - Transkriptomanalyse KW - foxtapeworm KW - transcriptome data analysis KW - germinative cell Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203802 ER - TY - THES A1 - Stoll, Kristin T1 - Molekulare Charakterisierung von Mitogen-activated Protein Kinase (MAPK)- Komponenten aus \(Echinococcus\) \(multilocularis\) T1 - Molecular characterization of mitogen-activated protein kinase (MAPK) components from \(Echinococcus\) \(multilocularis\) N2 - Die alveoläre Echinokokkose (AE), verursacht durch das Metacestoden- Larvenstadium des Fuchsbandwurms Echinococcus multilocularis (E. multilocularis), ist eine lebensbedrohliche Zoonose der nördlichen Hemisphäre mit eingeschränkten therapeutischen Möglichkeiten. Bei der Suche nach neuen Therapeutika haben Mitogen-activated Proteinkinase (MAPK) -Kaskaden als pharmakologische Zielstrukturen aufgrund ihrer essentiellen Rolle bei der Zellproliferation und -differenzierung ein großes Potenzial. In der vorliegenden Arbeit wurden durch BLAST- und reziproke BLAST-Analysen elf potenzielle MAPK Kinase Kinasen (MAP3K), fünf potenzielle MAPK Kinasen (MAP2K) und sechs potenzielle MAPK im E. multilocularis-Genom identifiziert, die teils hoch konserviert sind und in nahezu allen Entwicklungsstadien des Parasiten exprimiert werden. Diese Erkenntnisse lassen auf ein komplexes MAPK-Signaltransduktions- system in E. multilocularis mit großer Bedeutung für den Parasiten schließen. Transkriptomdatenanalysen und Whole Mount in Situ Hybridisierung (WMISH) zeigten, dass emmkkk1 (EmuJ_000389600) als einzige MAP3K neben der Expression in postmitotischen Zellen in besonderem Maße in proliferativen Stammzellen des Parasiten exprimiert wird und somit eine wichtige Rolle bei der Differenzierung von Stammzellen spielen könnte. In Yeast-Two-Hybrid (Y2H) -Wechselwirkungsassays wurden Interaktionen von mehreren upstream- (EmGRB2) und downstream- wirkenden Signalkaskadekomponenten des JNK (EmMKK3, EmMPK3) und ERK (EmMKK3, EmMPK4) -Signalwegs gefunden. Daraus lässt sich schließen, dass EmMKKK1, analog zu seinem humanen Homolog HsM3K1, eine zentrale Rolle bei der Echinococcus-Wachstumsregulation durch Rezeptortyrosinkinasen und vielfältige weitere Funktionen im Parasiten besitzt. Anhand von Erkenntnissen an Platyhelminthes kann daher von einem großen Potenzial dieser neu charakterisierten Signalwege als chemotherapeutische Angriffspunkte ausgegangen werden, wenngleich erste RNA-Interferenz (RNAi)- und Inhibitorstudien an emmkkk1, emmpk1 und emmpk4 keine durchschlagenden Effekte auf das Überleben von Primärzellkulturen und die Bildung von Metacestodenvesikeln zeigten. Zusammenfassend konnte in der vorliegenden Arbeit mit EmMKKK1 und neuen ERK- und JNK-Signalwegen zentrale Komponenten der komplexen MAPK-Signalkaskaden in E. multilocularis identifiziert werden, die höchstwahrscheinlich einen großen Beitrag zur enormen Regenerationsfähigkeit der Echinococcus-Stammzellen leisten und vom Wirt abgeleitete Signale wie Insulin, Epidermaler Wachstumsfaktor (EGF) und Fibroblasten-Wachstumsfaktor (FGF) über EmGRB2 in Proliferationsnetzwerke des Parasiten integrieren. Arzneimittel-Screening-Assays, die auf diese Signalwege abzielen, könnten daher zu alternativen Arzneimitteln führen, die alleine oder in Kombination mit einer bestehenden Chemotherapie (Benzimidazol) die Prognose von für AE-Patienten verbessern könnten. N2 - Alveolar echinococcosis (AE), caused by the metacestode larval stage of the fox tapeworm Echinococcus multilocularis (E. multilocularis), is a life-threatening zoonosis of the Northern Hemisphere with limited therapeutic options. In searches for a new chemotherapy, mitogen-activated protein kinase (MAPK) cascades are of great potential as new drug targets due to their essential role in cell proliferation and differentiation. In this work eleven potential MAPK kinase kinases (MAP3K), five potential MAPK kinases (MAP2K) and six potential MAPK were identified in the E. multilocularis genome by BLAST and reciprocal BLAST analyses, of which some are highly conserved and expressed in almost all developmental stages of the parasite. These findings suggest a complex MAPK signal transduction system in E. multilocularis with major importance for the parasite. Transcriptome data analysis and Whole Mount in Situ Hybridization (WMISH) showed that emmkkk1 (EmuJ_000389600) is the only MAP3K being decisively expressed in the proliferative parasite in addition to expression in postmitotic cells, and thus could play an important role in the differentiation of stem cells. In Yeast-Two-Hybrid (Y2H) interaction assays, interactions between several upstream (EmGRB2) and downstream signaling cascade components of JNK (EmMKK3, EmMPK3) and ERK (EmMKK3, EmMPK4) signaling pathways were detected. Thus, EmMKKK1, like its human homologue HsM3K1, appears to play a central role in Echinococcus growth regulation by receptor tyrosine kinases and seems to have diverse other functions in the parasite. Based on findings on other platyhelminths it can be expected that these newly characterized signaling pathways are of great potential as drug target, although first RNA interference (RNAi) and inhibitor studies on emmkkk1, emmpk1 and emmpk4 revealed no substantive effects on the survival of primary cell cultures and the formation of metacestode vesicles. In conclusion, the present work identified with EmMKKK1 and new ERK and JNK signaling pathways central components of the complex MAPK signaling cascades in E. multilocularis, which most likely contribute to the enormous regenerative capacity of Echinococcus stem cells and integrate host derived signals such as insulin, epidermal growth factor (EGF), and fibroblast growth factor (FGF) via EmGBR2 into proliferative networks of the parasite. Targeting these signaling pathways in drug screening assays might thus lead to alternative drugs that alone, or in combination with existing chemotherapy (benzimidazole), could improve the prognose of AE patients. KW - Fuchsbandwurm KW - MAP-Kinase KW - Echinococcus multilocularis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-243180 ER - TY - JOUR A1 - Surat, Güzin A1 - Vogel, Ulrich A1 - Wiegering, Armin A1 - Germer, Christoph-Thomas A1 - Lock, Johan Friso T1 - Defining the scope of antimicrobial stewardship interventions on the prescription quality of antibiotics for surgical intra-abdominal infections JF - Antibiotics N2 - Background: The aim of this study was to assess the impact of antimicrobial stewardship interventions on surgical antibiotic prescription behavior in the management of non-elective surgical intra-abdominal infections, focusing on postoperative antibiotic use, including the appropriateness of indications. Methods: A single-center quality improvement study with retrospective evaluation of the impact of antimicrobial stewardship measures on optimizing antibacterial use in intra-abdominal infections requiring emergency surgery was performed. The study was conducted in a tertiary hospital in Germany from January 1, 2016, to January 30, 2020, three years after putting a set of antimicrobial stewardship standards into effect. Results: 767 patients were analyzed (n = 495 in 2016 and 2017, the baseline period; n = 272 in 2018, the antimicrobial stewardship period). The total days of therapy per 100 patient days declined from 47.0 to 42.2 days (p = 0.035). The rate of patients receiving postoperative therapy decreased from 56.8% to 45.2% (p = 0.002), comparing both periods. There was a significant decline in the rate of inappropriate indications (17.4% to 8.1 %, p = 0.015) as well as a significant change from broad-spectrum to narrow-spectrum antibiotic use (28.8% to 6.5%, p ≤ 0.001) for postoperative therapy. The significant decline in antibiotic use did not affect either clinical outcomes or the rate of postoperative wound complications. Conclusions: Postoperative antibiotic use for intra-abdominal infections could be significantly reduced by antimicrobial stewardship interventions. The identification of inappropriate indications remains a key target for antimicrobial stewardship programs. KW - antimicrobial stewardship KW - antibiotic prescription behavior KW - surgical intra-abdominal infections KW - post-operative antibiotic treatment Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223034 SN - 2079-6382 VL - 10 IS - 1 ER - TY - JOUR A1 - Marincola, Gabriella A1 - Liong, Olivia A1 - Schoen, Christoph A1 - Abouelfetouh, Alaa A1 - Hamdy, Aisha A1 - Wencker, Freya D. R. A1 - Marciniak, Tessa A1 - Becker, Karsten A1 - Köck, Robin A1 - Ziebuhr, Wilma T1 - Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany JF - Frontiers in Public Health N2 - Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates. KW - coagulase-negative staphylococci KW - antimicrobial resistance KW - One Health KW - community settings KW - Germany Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-240881 SN - 2296-2565 VL - 9 ER - TY - JOUR A1 - Walther, Grit A1 - Zimmermann, Anna A1 - Theuersbacher, Johanna A1 - Kaerger, Kerstin A1 - Lilienfeld-Toal, Marie von A1 - Roth, Mathias A1 - Kampik, Daniel A1 - Geerling, Gerd A1 - Kurzai, Oliver T1 - Eye infections caused by filamentous fungi: spectrum and antifungal susceptibility of the prevailing agents in Germany JF - Journal of Fungi N2 - Fungal eye infections can lead to loss of vision and blindness. The disease is most prevalent in the tropics, although case numbers in moderate climates are increasing as well. This study aimed to determine the dominating filamentous fungi causing eye infections in Germany and their antifungal susceptibility profiles in order to improve treatment, including cases with unidentified pathogenic fungi. As such, we studied all filamentous fungi isolated from the eye or associated materials that were sent to the NRZMyk between 2014 and 2020. All strains were molecularly identified and antifungal susceptibility testing according to the EUCAST protocol was performed for common species. In total, 242 strains of 66 species were received. Fusarium was the dominating genus, followed by Aspergillus, Purpureocillium, Alternaria, and Scedosporium. The most prevalent species in eye samples were Fusarium petroliphilum, F. keratoplasticum, and F. solani of the Fusarium solani species complex. The spectrum of species comprises less susceptible taxa for amphotericin B, natamycin, and azoles, including voriconazole. Natamycin is effective for most species but not for Aspergillus flavus or Purpureocillium spp. Some strains of F. solani show MICs higher than 16 mg/L. Our data underline the importance of species identification for correct treatment. KW - eye infection KW - fungal infection KW - keratitis KW - antifungal susceptibility KW - natamycin KW - Fusarium KW - Purpureocillium KW - Aspergillus KW - Alternaria KW - Scedosporium Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241810 SN - 2309-608X VL - 7 IS - 7 ER - TY - JOUR A1 - Kurotschka, Peter Konstantin A1 - Tiedemann, Elena A1 - Wolf, Dominik A1 - Thier, Nicola A1 - Forster, Johannes A1 - Liese, Johannes G. A1 - Gagyor, Ildiko T1 - Management of common infections in German primary care: a cross-sectional survey of knowledge and confidence among General Practitioners and outpatient pediatricians JF - Antibiotics N2 - Outpatient antibiotic use is closely related to antimicrobial resistance and in Germany, almost 70% of antibiotic prescriptions in human health are issued by primary care physicians (PCPs). The aim of this study was to explore PCPs, namely General Practitioners' (GPs) and outpatient pediatricians' (PDs) knowledge of guideline recommendations on rational antimicrobial treatment, the determinants of confidence in treatment decisions and the perceived need for training in this topic in a large sample of PCPs from southern Germany. Out of 3753 reachable PCPs, 1311 completed the survey (overall response rate = 34.9%). Knowledge of guideline recommendations and perceived confidence in making treatment decisions were high in both GPs and PDs. The two highest rated influencing factors on prescribing decisions were reported to be guideline recommendations and own clinical experiences, hence patients' demands and expectations were judged as not influencing treatment decisions. The majority of physicians declared to have attended at least one specific training course on antibiotic use, yet almost all the participating PCPs declared to need more training on this topic. More studies are needed to explore how consultation-related and context-specific factors could influence antibiotic prescriptions in general and pediatric primary care in Germany beyond knowledge. Moreover, efforts should be undertaken to explore the training needs of PCPs in Germany, as this would serve the development of evidence-based educational interventions targeted to the improvement of antibiotic prescribing decisions rather than being focused solely on knowledge of guidelines. KW - infectious diseases management KW - general practitioner KW - pediatrician KW - primary care KW - outpatient KW - antibiotic use KW - antimicrobial resistance KW - antimicrobial stewardship KW - survey KW - knowledge Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246272 SN - 2079-6382 VL - 10 IS - 9 ER - TY - JOUR A1 - Peters, Simon A1 - Fohmann, Ingo A1 - Rudel, Thomas A1 - Schubert-Unkmeir, Alexandra T1 - A Comprehensive Review on the Interplay between Neisseria spp. and Host Sphingolipid Metabolites JF - Cells N2 - Sphingolipids represent a class of structural related lipids involved in membrane biology and various cellular processes including cell growth, apoptosis, inflammation and migration. Over the past decade, sphingolipids have become the focus of intensive studies regarding their involvement in infectious diseases. Pathogens can manipulate the sphingolipid metabolism resulting in cell membrane reorganization and receptor recruitment to facilitate their entry. They may recruit specific host sphingolipid metabolites to establish a favorable niche for intracellular survival and proliferation. In contrast, some sphingolipid metabolites can also act as a first line defense against bacteria based on their antimicrobial activity. In this review, we will focus on the strategies employed by pathogenic Neisseria spp. to modulate the sphingolipid metabolism and hijack the sphingolipid balance in the host to promote cellular colonization, invasion and intracellular survival. Novel techniques and innovative approaches will be highlighted that allow imaging of sphingolipid derivatives in the host cell as well as in the pathogen. KW - sphingolipids KW - host–pathogen interaction KW - Neisseria meningitidis KW - Neisseria gonorrhoeae Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250203 SN - 2073-4409 VL - 10 IS - 11 ER - TY - JOUR A1 - Sattler, Janko A1 - Noster, Janina A1 - Brunke, Anne A1 - Plum, Georg A1 - Wiegel, Pia A1 - Kurzai, Oliver A1 - Meis, Jacques F. A1 - Hamprecht, Axel T1 - Comparison of two commercially available qPCR kits for the detection of Candida auris JF - Journal of Fungi N2 - Candida auris is an emerging pathogen with resistance to many commonly used antifungal agents. Infections with C. auris require rapid and reliable detection methods to initiate successful medical treatment and contain hospital outbreaks. Conventional identification methods are prone to errors and can lead to misidentifications. PCR-based assays, in turn, can provide reliable results with low turnaround times. However, only limited data are available on the performance of commercially available assays for C. auris detection. In the present study, the two commercially available PCR assays AurisID (OLM, Newcastle Upon Tyne, UK) and Fungiplex Candida Auris RUO Real-Time PCR (Bruker, Bremen, Germany) were challenged with 29 C. auris isolates from all five clades and eight other Candida species as controls. AurisID reliably detected C. auris with a limit of detection (LoD) of 1 genome copies/reaction. However, false positive results were obtained with high DNA amounts of the closely related species C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. The Fungiplex Candida Auris RUO Real-Time PCR kit detected C. auris with an LoD of 9 copies/reaction. No false positive results were obtained with this assay. In addition, C. auris could also be detected in human blood samples spiked with pure fungal cultures by both kits. In summary, both kits could detect C. auris-DNA at low DNA concentrations but differed slightly in their limits of detection and specificity. KW - qPCR KW - detection limits KW - sensitivity KW - strain specificity KW - commercial kits KW - Candida auris KW - Fungiplex Candida Auris KW - AurisID Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228879 SN - 2309-608X VL - 7 IS - 2 ER - TY - JOUR A1 - Streck, Laura Elisa A1 - Gaal, Chiara A1 - Forster, Johannes A1 - Konrads, Christian A1 - Hertzberg-Boelch, Sebastian Philipp von A1 - Rueckl, Kilian T1 - Defining a synovial fluid white blood cell count threshold to predict periprosthetic infection after shoulder arthroplasty JF - Journal of Clinical Medicine N2 - Background: The diagnosis of periprosthetic shoulder infection (PSI) requires a thorough diagnostic workup. Synovial fluid aspiration has been proven to be a reliable tool in the diagnosis of joint infections of the lower extremity, but shoulder specific data is limited. This study defines a threshold for synovial fluid white blood cell count (WBC) and assesses the reliability of microbiological cultures. Methods: Retrospective study of preoperative and intraoperative fluid aspiration of 31 patients who underwent a revision of a shoulder arthroplasty (15 with PSI defined by IDSA criteria, 16 without infection). The threshold for WBC was calculated by ROC/AUC analysis. Results: WBC was significantly higher in patients with PSI than in other patients. A threshold of 2800 leucocytes/mm\(^3\) showed a sensitivity of 87% and a specificity of 88% (AUROC 0.92). Microbiological cultures showed a sensitivity of 76% and a specificity of 100%. Conclusions: A threshold of 2800 leucocytes/mm\(^3\) in synovial fluid can be recommended to predict PSI. Microbiological culture has an excellent specificity and allows for targeted antibiotic therapy. Joint aspiration presents an important pillar to diagnose PSI. KW - upper extremity KW - joint infection KW - joint aspiration KW - leucocyte count KW - cutibacteria KW - ICM KW - MSIS KW - IDSA KW - WBC Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-252275 SN - 2077-0383 VL - 11 IS - 1 ER - TY - THES A1 - Peters, Simon T1 - The impact of sphingolipids on \(Neisseria\) \(meningitidis\) and their role in meningococcal pathogenicity T1 - Einfluss von Sphingolipiden auf \(Neisseria\) \(meningitidis\) und deren Bedeutung für die Pathogenität N2 - The obligate human pathogen Neisseria meningitidis is a major cause of sepsis and meningitis worldwide. It affects mainly toddlers and infants and is responsible for thousands of deaths each year. In this study, different aspects of the importance of sphingolipids in meningococcal pathogenicity were investigated. In a first step, the acid sphingomyelinase (ASM), which degrades membrane sphingomyelin to ceramide, was studied in the context of meningococcal infection. A requirement for ASM surface activity is its translocation from the lysosomal compartment to the cell surface, a process that is currently poorly understood. This study used various approaches, including classical invasion and adherence assays, flow cytometry, and classical and super resolution immunofluorescence microscopy (dSTORM). The results showed that the live, highly piliated N. meningitidis strain 8013/12 induced calcium-dependent ASM translocation in human brain microvascular endothelial cells (HBMEC). Furthermore, it promoted the formation of ceramide-rich platforms (CRPs). In addition, ASM translocation and CRP formation were observed after treating the cells with pili-enriched fractions derived from the same strain. The importance for N. meningitidis to utilize this pathway was shown by the inhibition of the calcium-dependent ASM translocation, which greatly decreased the number of invasive bacteria. I also investigated the importance of the glycosphingolipids GM1 and Gb3. The results showed that GM1, but not Gb3, plays an important role in the ability of N. meningitidis to invade HBMEC. By combining dSTORM imaging and microbiological approaches, we demonstrated that GM1 accumulated prolifically around bacteria during the infection, and that this interaction seemed essential for meningococcal invasion. Sphingolipids are not only known for their beneficial effect on pathogens. Sphingoid bases, including sphingosine, are known for their antimicrobial activity. In the last part of this study, a novel correlative light and electron microscopy approach was established in the combination with click chemistry to precisely localize azido-functionalized sphingolipids in N. meningitidis. The result showed a distinct concentration-dependent localization in either the outer membrane (low concentration) or accumulated in the cytosol (high concentration). This pattern was confirmed by mass spectrometry on separated membrane fractions. Our data provide a first insight into the underlying mechanism of antimicrobial sphingolipids. N2 - Der obligate Humanpathogen Neisseria meningitidis ist weltweit einer der Hauptursachen für Sepsis und Meningitis. Er befällt vor allem Kleinkinder und Säuglinge und ist jedes Jahr für Tausende von Todesfällen verantwortlich. In dieser Studie wurden verschiedene Aspekte der Bedeutung von Sphingolipiden bei der Pathogenität von Meningokokken untersucht. In einem ersten Schritt wurde die saure Sphingomyelinase (ASM), die Membran-Sphingomyelin zu Ceramid abbaut, im Zusammenhang mit einer Meningokokken-Infektion untersucht. Eine Voraussetzung für die Oberflächenaktivität der ASM ist ihre Translokation vom lysosomalen Kompartiment auf die Zelloberfläche, ein Prozess, der derzeit noch wenig verstanden wird. In dieser Studie wurden verschiedene Ansätze verwendet, darunter klassische Invasions- und Adhärenztests, Durchflusszytometrie sowie klassische und superauflösende Immunfluoreszenzmikroskopie (dSTORM). Die Ergebnisse zeigten, dass der lebende, hochpiliatisierte N. meningitidis Stamm 8013/12 eine kalziumabhängige ASM-Translokation in mikrovaskulären Endothelzellen des menschlichen Gehirns (HBMEC) induzierte. Des Weiteren förderte er die Bildung Ceramid-reicher Plattformen (CRPs). Zusätzlich wurden ASM-Translokation und CRP-Bildung beobachtet, nachdem die Zellen mit pili-angereicherten Fraktionen desselben Stammes behandelt worden waren. Die Bedeutung für N. meningitidis in der Pathogenese zeigte sich durch die Hemmung der Calcium-abhängigen ASM-Translokation, wodurch die Zahl der invasiven Bakterien stark reduziert wurde. Ich untersuchte auch die Bedeutung der Glykosphingolipide GM1 und Gb3. Die Ergebnisse zeigten, dass GM1, aber nicht Gb3, eine wichtige Rolle bei der Fähigkeit von N. meningitidis spielt, in Gehirnendothelzellen einzudringen. Durch die Kombination von dSTORM-Bildgebung und mikrobiologischen Ansätzen konnten wir zeigen, dass sich GM1 während der Infektion vermehrt um die Bakterien herum anreicherte und dass diese Interaktion für die Invasion von Meningokokken essenziell ist. Sphingolipide sind nicht nur für ihre positive Wirkung auf Krankheitserreger bekannt. Sphingoidbasen, einschließlich Sphingosin, sind zusätzlich für ihre antimikrobielle Aktivität bekannt. Im letzten Teil dieser Studie wurde ein neuartiger korrelativer licht- und elektronenmikroskopischer Ansatz in der Kombination mit Click-Chemie etabliert, um azidofunktionalisierte Sphingolipide in N. meningitidis genau zu lokalisieren. Das Ergebnis zeigte eine deutliche konzentrationsabhängige Lokalisation entweder in der äußeren Membran (niedrige Konzentration) oder akkumuliert im Zytosol (hohe Konzentration). Dieses Muster konnte durch einen Massenspektrometrischen Ansatz bestätigt werden. Hierfür wurde eine Separation der inneren und äußeren Membran, nach Behandlung mit der niedrigen Konzentration, etabliert. Die verschiedenen Membranfraktionen wurden anschließend auf ihren Gehalt an funktionalisierten Sphingolipiden hin untersucht und bestätigten die lokalisierung in der äußeren Membran. Unsere Daten geben einen ersten Einblick in den zugrundeliegenden Mechanismus der antimikrobiellen Sphingolipide. KW - Neisseria meningitidis KW - Sphingolipide KW - Infektion KW - Pathogenität KW - host-pathogen interaction KW - antimicrobial Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226233 ER - TY - JOUR A1 - Peters, Simon A1 - Kaiser, Lena A1 - Fink, Julian A1 - Schumacher, Fabian A1 - Perschin, Veronika A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Kleuser, Burkhard A1 - Seibel, Juergen A1 - Schubert-Unkmeir, Alexandra T1 - Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria JF - Scientific Reports N2 - Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity. KW - antimicrobials KW - biological techniques KW - imaging KW - microbiology KW - microbiology techniques KW - microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259147 VL - 11 IS - 1 ER - TY - JOUR A1 - Page, Lukas A1 - Wallstabe, Julia A1 - Lother, Jasmin A1 - Bauser, Maximilian A1 - Kniemeyer, Olaf A1 - Strobel, Lea A1 - Voltersen, Vera A1 - Teutschbein, Janka A1 - Hortschansky, Peter A1 - Morton, Charles Oliver A1 - Brakhage, Axel A. A1 - Topp, Max A1 - Einsele, Hermann A1 - Wurster, Sebastian A1 - Loeffler, Juergen T1 - CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to Aspergillus fumigatus JF - Frontiers in Immunology N2 - Aspergillus fumigatus causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti-Aspergillus defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require ex vivo differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an A. fumigatus lysate and two A. fumigatus proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both A. fumigatus proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4\(^+\) and CD8\(^+\) T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with A. fumigatus germ tubes. Taken together, our in vitro data suggest that ex vivo CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA. KW - antigens KW - dendritic cells KW - cytokines KW - host defense KW - immunotherapy KW - Aspergillus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239493 SN - 1664-3224 VL - 12 ER - TY - JOUR A1 - Krüger, Sören A1 - Leskien, Miriam A1 - Schuller, Patricia A1 - Prifert, Christiane A1 - Weißbrich, Benedikt A1 - Vogel, Ulrich A1 - Krone, Manuel T1 - Performance and feasibility of universal PCR admission screening for SARS‐CoV‐2 in a German tertiary care hospital JF - Journal of Medical Virology N2 - Anamnestic screening of symptoms and contact history is applied to identify coronavirus disease 2019 (COVID‐19) patients on admission. However, asymptomatic and presymptomatic patients remain undetected although the viral load may be high. In this retrospective cohort study, all hospitalized patients who received polymerase chain reaction (PCR) admission testing from March 26th until May 24th, 2020 were included. Data on COVID‐19‐specific symptoms and contact history to COVID‐19 cases were retrospectively extracted from patient files and from contact tracing notes. The compliance to the universal testing protocol was high with 90%. Out of 6940 tested patients, 27 new severe acute respiratory syndrome coronavirus‐2 infections (0.4%) were detected. Seven of those COVID‐19 cases (26% of all new cases) were asymptomatic and had no positive contact history, but were identified through a positive PCR test. The number needed to identify an asymptomatic patient was 425 in the first wave of the epidemic, 1218 in the low incidence phase. The specificity of the method was above 99.9%. Universal PCR testing was highly accepted by staff as demonstrated by high compliance. The costs to detect one asymptomatic case in future studies need to be traded off against the costs and damage caused by potential outbreaks of COVID‐19. KW - admission screening KW - COVID‐19 KW - infection control KW - SARS‐CoV‐2 KW - testing strategy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238971 VL - 93 IS - 5 SP - 2890 EP - 2898 ER - TY - JOUR A1 - Springer, Jan A1 - Held, Jürgen A1 - Mengoli, Carlo A1 - Schlegel, Paul Gerhardt A1 - Gamon, Florian A1 - Träger, Johannes A1 - Kurzai, Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen A1 - Eyrich, Matthias T1 - Diagnostic performance of (1→3)-β-D-glucan alone and in combination with aspergillus PCR and galactomannan in serum of pediatric patients after allogeneic hematopoietic stem cell transplantation JF - Journal of Fungi N2 - Data on biomarker-assisted diagnosis of invasive aspergillosis (IA) in pediatric patients is scarce. Therefore, we conducted a cohort study over two years including 404 serum specimens of 26 pediatric patients after allogeneic hematopoietic stem cell transplantation (alloSCT). Sera were tested prospectively twice weekly for Aspergillus-specific DNA, galactomannan (GM), and retrospectively for (1→3)-β-D-glucan (BDG). Three probable IA and two possible invasive fungal disease (IFD) cases were identified using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSGERC) 2019 consensus definitions. Sensitivity and specificity for diagnosis of probable IA and possible IFD was 80% (95% confidential interval (CI): 28–99%) and 55% (95% CI: 32–77%) for BDG, 40% (95% CI: 5–85%) and 100% (95% CI: 83–100%) for GM, and 60% (95% CI: 15–95%) and 95% (95% CI: 75–100%) for Aspergillus-specific real-time PCR. However, sensitivities have to be interpreted with great caution due to the limited number of IA cases. Interestingly, the low specificity of BDG was largely caused by false-positive BDG results that clustered around the date of alloSCT. The following strategies were able to increase BDG specificity: two consecutive positive BDG tests for diagnosis (specificity 80% (95% CI: 56–94%)); using an optimized cutoff value of 306 pg/mL (specificity 90% (95% CI: 68–99%)) and testing BDG only after the acute posttransplant phase. In summary, BDG can help to diagnose IA in pediatric alloSCT recipients. However, due to the poor specificity either an increased cutoff value should be utilized or BDG results should be confirmed by an alternative Aspergillus assay. KW - beta-D-glucan KW - galactomannan KW - real-time PCR KW - Aspergillus KW - pediatric Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234179 SN - 2309-608X VL - 7 IS - 3 ER - TY - JOUR A1 - Mottola, Austin A1 - Ramírez-Zavala, Bernardo A1 - Hünninger, Kerstin A1 - Kurzai, Oliver A1 - Morschhäuser, Joachim T1 - The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans JF - Molecular Microbiology N2 - The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall. KW - cell wall KW - zinc cluster transcription factor KW - Candida albicans KW - protein kinases Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259583 VL - 116 IS - 2 ER - TY - JOUR A1 - Machata, Silke A1 - Sreekantapuram, Sravya A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Dunker, Christine A1 - Schubert, Katja A1 - Krüger, Wibke A1 - Schulze-Richter, Bianca A1 - Speth, Cornelia A1 - Rambach, Günter A1 - Jacobsen, Ilse D. T1 - Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood JF - Frontiers in Immunology N2 - Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts. KW - whole blood ex vivo model KW - host-pathogen interaction KW - neutrophils KW - mice Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222575 SN - 1664-3224 VL - 11 ER - TY - JOUR A1 - Beierle, Felix A1 - Schobel, Johannes A1 - Vogel, Carsten A1 - Allgaier, Johannes A1 - Mulansky, Lena A1 - Haug, Fabian A1 - Haug, Julian A1 - Schlee, Winfried A1 - Holfelder, Marc A1 - Stach, Michael A1 - Schickler, Marc A1 - Baumeister, Harald A1 - Cohrdes, Caroline A1 - Deckert, Jürgen A1 - Deserno, Lorenz A1 - Edler, Johanna-Sophie A1 - Eichner, Felizitas A. A1 - Greger, Helmut A1 - Hein, Grit A1 - Heuschmann, Peter A1 - John, Dennis A1 - Kestler, Hans A. A1 - Krefting, Dagmar A1 - Langguth, Berthold A1 - Meybohm, Patrick A1 - Probst, Thomas A1 - Reichert, Manfred A1 - Romanos, Marcel A1 - Störk, Stefan A1 - Terhorst, Yannik A1 - Weiß, Martin A1 - Pryss, Rüdiger T1 - Corona Health — A Study- and Sensor-Based Mobile App Platform Exploring Aspects of the COVID-19 Pandemic JF - International Journal of Environmental Research and Public Health N2 - Physical and mental well-being during the COVID-19 pandemic is typically assessed via surveys, which might make it difficult to conduct longitudinal studies and might lead to data suffering from recall bias. Ecological momentary assessment (EMA) driven smartphone apps can help alleviate such issues, allowing for in situ recordings. Implementing such an app is not trivial, necessitates strict regulatory and legal requirements, and requires short development cycles to appropriately react to abrupt changes in the pandemic. Based on an existing app framework, we developed Corona Health, an app that serves as a platform for deploying questionnaire-based studies in combination with recordings of mobile sensors. In this paper, we present the technical details of Corona Health and provide first insights into the collected data. Through collaborative efforts from experts from public health, medicine, psychology, and computer science, we released Corona Health publicly on Google Play and the Apple App Store (in July 2020) in eight languages and attracted 7290 installations so far. Currently, five studies related to physical and mental well-being are deployed and 17,241 questionnaires have been filled out. Corona Health proves to be a viable tool for conducting research related to the COVID-19 pandemic and can serve as a blueprint for future EMA-based studies. The data we collected will substantially improve our knowledge on mental and physical health states, traits and trajectories as well as its risk and protective factors over the course of the COVID-19 pandemic and its diverse prevention measures. KW - mobile health KW - ecological momentary assessment KW - digital phenotyping KW - longitudinal studies KW - mobile crowdsensing Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242658 SN - 1660-4601 VL - 18 IS - 14 ER - TY - JOUR A1 - Dichtl, Karl A1 - Koc, Özlem A1 - Forster, Johannes A1 - Scharf, Christina A1 - Suerbaum, Sebastian A1 - Andrassy, Joachim A1 - Wagener, Johannes A1 - Schroeder, Ines T1 - An invasive infection caused by the thermophilic mold Talaromyces thermophilus JF - Infection N2 - Background Increasing incidence of invasive infections caused by rare fungi was observed over the recent years. Case Here, we describe the first reported case of an infection caused by the thermophilic mold Talaromyces thermophilus. Cultivation and, hence, identification of this fastidious organism is challenging since standard incubation conditions are not sufficient. Retrospective analysis of patient samples and in vitro experiments demonstrated that testing for fungal antigens, i.e., the cell wall components galactomannan and β-1,3-D-glucan, is a promising tool. KW - Talaromyces KW - invasive fungal infection KW - thermophile KW - antigen testing KW - serology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-308970 SN - 0300-8126 SN - 1439-0973 VL - 49 IS - 6 ER - TY - THES A1 - Leitenberger, Karolin T1 - Vergleich der Bakterienlast in vivo und Wachstumskinetik in vitro hyperletaler Meningokokkentypen T1 - Comparison of bacterial load in vivo and growth characteristics in vitro of highly lethal meningococcal types N2 - Die invasive Meningokokkenerkrankung stellt weltweit mit einer Letalität von 5-10% trotz antibiotischer Therapie eine Herausforderung dar. Ein spezifisches Virulenzgen, welches die Schwere der Meningokokkenerkrankung bestimmt, konnte bisher nicht definiert werden. Vorangegangene Studien zeigen eine Korrelation der Letalität mit der Bakterienlast, Unterschiede bezüglich der Letalität je nach Serogruppe, eine erhöhte Letalität bei Infektionen mit sogenannten hyperletalen Feintypen (bisher nicht veröffentlichte Daten des NRZMHi) sowie einen Unterschied in der maximal in Flüssigkultur erreichten Konzentration der Bakterien zwischen invasiven Stämmen und Trägerstämmen. In dieser Arbeit wurden mögliche Gründe für die Hyperletalität bestimmter Meningokokkentypen experimentell untersucht. Insbesondere wird die Frage analysiert, ob die hyperletalen Meningokokkentypen mit einer höheren bakteriellen Last im Blut assoziiert sind und ob sie andere Wachstumscharakteristiken im Vergleich zu ihren Kontrollstämmen in vitro zeigen. Hierzu erfolgte mittels quantitativer Echtzeit-Polymerase-Kettenreaktion die Bestimmung der bakteriellen Last in 62 Blutproben von Patienten mit bestätigter invasiver Meningokokkenerkrankung über den Nachweis des ctrA-Gens. Darunter waren elf Proben des hyperletalen Feintyps B:P1.7-2,4:F1-5 und fünf Proben des hyperletalen Feintyps C:P1.5,2:F3-3. Die Wachstumsversuche wurden mit 30 zufällig gewählten Stämmen der hyperletalen Feintypen B:P1.7-2,4:F1-5, C:P1.5-1,10-8:F3-6 und C:P1.5,2:F3-3 mit ihren jeweiligen nach Alter und Geschlecht abgeglichenen nicht zu der Gruppe der hyperletalen Feintypen gehörenden Kontrollstämmen in dem Medium PPM+ durchgeführt. Die Wachstumsgeschwindigkeit μ sowie die Kapazität A (maximale Konzentrationszunahme als Logarithmus der gemessenen OD im Verhältnis zur Ausgangsdichte ODT0) wurden durch nicht-lineare Regression anhand der modifizierten Gompertz-Funktion ermittelt. Die Messung der optischen Dichte erfolgte alle 30 Minuten über 16 Stunden bei 620nm durch das Gerät TECAN Infinite 200 Pro (Tecan Group Ltd., Männedorf / Schweiz). Die Methode wurde anhand einer publizierten Studie zwischen Trägerstämmen und invasiven Stämmen (Schoen et al., 2014) validiert und bestätigte einen marginalen Unterschied in der optischen Dichte (p=0,057, Wilcoxon-Test) zwischen den Gruppen. Es zeigte sich kein Unterschied in der Wachstumsgeschwindigkeit. Aus den Ergebnissen dieser Arbeit können drei wesentliche Schlussfolgerungen gezogen werden: 1.) Die Bakterienlast in dieser Stichprobe ist, entgegen der Literatur, nicht abhängig von der Serogruppe und dem Feintyp, jedoch von der Krankheitsmanifestation. 2.) Die Kapazität A ist in der Gruppe der „hyperletalen“ Typen im Vergleich zu den Kontrollstämmen möglicherweise höher. 3.) Größere Stichproben (Nativmaterial, Stämme) sind erforderlich, um die Beobachtungen dieser Studie zu bestätigen. N2 - The invasive meningococcal disease still has a case fatality rate of 5 to 10 percent despite antibiotic treatment. A specific virulence gene, which determines disease severity could not be determined so far. Earlier studies have shown a correlation between lethality and bacterial load, differences in lethality between serogroups, a disproportionately high lethality of infections with distinct meningococcal finetypes (data not yet published by the NRZMHi) as well as a difference in the maximum concentration of bacteria in fluid culture between invasive and carrier strains. This dissertation analyses possible causes for the higher fatality rate of certain meningococcal finetypes. It tries to answer the question whether the so called highly lethal meningococcal finetypes are associated with a higher bacterial load in blood and whether they show different growth characteristics in comparison to their control strains in vitro. Bacterial loads in 62 blood samples from patients with confirmed invasive meningococcal disease were measured by real-time polymerase chain reaction targeting the ctrA gene. The samples contained eleven samples of the highly lethal meningococcal finetype B:P1.7-2,4:F1-5 and five samples of the highly lethal meningococcal finetype C:P1.5,2:F3-3. Growth characteristics of 30 randomly selected strains pertaining to the highly lethal finetypes B:P1.7-2,4:F1-5, C:P1.5-1,10-8:F3-6 and C:P1.5,2:F3-3 were compared to strains from age- and sex- matched controls in proteose-peptone-medium (PPM+). Growth rates (μ) and maximum densities (A) were estimated by non-linear regression on the basis of the modified Gompertz function. Optical density at 620nm was measured every 30 minutes over 16 hours using a heatable plate reader (Tecan, Maennedorf, Switzerland). The method was validated on a published sample of invasive and carrier strains (Schoen et al., 2014) confirming marginally significant differences in maximal densities (p=0,057, Wilcoxon test), yet not growth rates, between groups. In summary we conclude: 1. The bacterial load in this sampling is not dependent on serogroup and finetype, but on the clinical picture. This finding contrasts the current literature. 2. The maximum density A is possibly higher in the group of highly lethal finetypes in comparison to the control strains. 3. Larger samples of both patient material and bacterial strains are required to confirm the observations of this study. KW - Letalität KW - Virulenzfaktor KW - Gompertz-Funktion KW - Wachstumsrate KW - Optische Dichte KW - Inasive Meningokokkenerkrankung KW - Hyperletale Feintypen KW - Bakterienlast KW - Kapazität KW - ctrA Gen KW - in vivo KW - in vitro KW - Quantitative Echtzeit-Polymerase-Kettenreaktion Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203133 ER - TY - THES A1 - Eibicht, Sebastian Johannes T1 - Die Kontamination von Flächen mit MRSA in Krankentransport- und Rettungswagen bei Kurzzeittransporten von MRSA-Patienten T1 - Meticillin-resistant Staphylococcus aureus (MRSA) contamination of ambulance cars after short term transport of MRSA-colonised patients N2 - Die Innenausbauflächen von Krankentransport- bzw. Rettungswagen unterliegen dem Risiko einer Verunreinigung durch Methicillin-resistenten S. aureus (MRSA). In der vorliegenden Arbeit wurden Krankentransport- bzw. Rettungswagen unmittelbar nach dem Transport von MRSA-kolonisierten oder -infizierten Patienten auf MRSA untersucht. Zu diesem Zweck wurden an 2 Stellen der Trage und an 3 Stellen der Innenausbauflächen Proben entnommen. 89 von 100 untersuchten Transporten, welche das Einschlusskriterium einer Transportzeit von weniger als 20 Minuten erfüllten, wurden weitergehend analysiert. 8 der untersuchten Kranken- bzw. Rettungswagen (7,1%) wiesen eine Kontamination auf (90% Konfidenzintervall: 4-14 %), wobei die Transportzeit keinen Einfluss auf die Kontamination hatte. MRSA wurde nur an der Trage nachgewiesen und zwar ausschließlich am Kopfteil der Trage und an den Tragegriffen. Die beprobten Stellen der Innenausbauflächen waren nicht kontaminiert. Bei Kurzzeittransporten von MRSA-positiven Patienten sollte daher der Fokus der Desinfektion auf die Oberflächen in unmittelbarer Patientennähe gelegt werden. Eine weitere Untersuchung von 60 Transporten MRSA-negativer Patienten blieb ohne MRSA-Befund. In 12 dieser Krankentransportwagen wurde jedoch Methicillin-sensibler S. aureus nachgewiesen, der sich ebenfalls vorwiegend an Kopfteil und Handgriffen der Trage fand. Auch dieses Ergebnis unterstreicht die Bedeutung der Desinfektion patientennaher Flächen unabhängig vom MRSA-Status der transportierten Patienten. N2 - Patients carrying MRSA provide a risk of surface contamination with MRSA in ambulance cars. This study analysed whether surfaces of ambulance cars, in which MRSA-colonised or -infected patients were transported, were contaminated with MRSA and where the contamination occurred. 89 transportation events lasting less than 20 minutes were included in the analysis. 8 ambulance cars (7,1%) were found to be contaminated with MRSA following transport (90% confidence interval: 4-14%). The transport time was not relevant for the contamination rate. MRSA was exclusively found on the stretcher, i.e. the headrest and the handles. This finding sug¬gests that disinfection measures after transport should be focused on the patients' contact surfaces. Consecutive investigation included 60 transport events in the absence of MRSA-notification and demonstrated that MSSA, but not MRSA, could be detected in 12 cars, again mostly at handles and headrests. This study shows the importance of disinfection of surfaces in the vicinity of patients transported in an ambulance irrespective of the patients' MRSA-status KW - MRSA KW - Methicillin-resistenter S. aureus KW - Krankentransportwagen KW - ambulance car KW - MRSA-Kontamination von Oberflächen KW - Cabin surfaces contaminated with MRSA KW - MRSA-kolonisierten Patienten KW - MRSA-colonised patients KW - Kurzzeittransporten KW - Shorttime transport KW - Rettungsdienst KW - rescue service Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-207130 ER - TY - THES A1 - Fuß, Antje T1 - Evaluierung des Nachweises von Schistosoma mansoni DNA mittels Real-Time PCR in verschiedenen humanen Proben sowie den Zwischenwirtschnecken in einer Hochprävalenzregion am Viktoriasee in Tansania T1 - Evaluation of the detection of Schistosoma mansoni DNA by real-time PCR in different human samples and the intermediate host snails in a high prevalence region at Lake Victoria in Tanzania N2 - Die Schistosomiasis ist nach wie vor eine der häufigsten parasitären Erkrankungen der Welt und verursacht erhebliche gesundheitliche und wirtschaftliche Folgen, insbesondere in ärmeren, ländlichen Regionen. Durch Immunreaktionen auf die im Wirt abgelegten Eier des Parasiten können sich chronische Verlaufsformen manifestieren. Dabei kann es zu irreversiblen Schäden kommen. Um dies zu verhindern sind eine frühe und sichere Diagnose sowie eine Behandlung mit Praziquantel (PZQ) unabdingbar. Zudem spielt der zuverlässige Nachweis der Schistosomiasis eine Schlüsselrolle bei der Überwachung, Prävention und Kontrolle der Erkrankung. In epidemiologischen Studien findet am häufigsten die mikroskopische Kato-Katz (KK)-Methode zum Nachweis von Schistosoma mansoni Eiern im Stuhl Anwendung. Dieses Verfahren ist äußerst spezifisch und bietet die Möglichkeit der Quantifizierung, wodurch die Intensität der vorliegenden Infektion bestimmt werden kann. Die Sensitivität der Testmethode ist jedoch nur moderat, insbesondere bei einer niedrigen Infektionsintensität. Zudem kann eine Infektion erst nach der Präpatenzzeit nachgewiesen werden. Der ebenfalls häufig eingesetzte urinbasierte Point-of-Care Circulating Cathodic Antigen (POC-CCA)-Test weist zwar eine höhere Sensitivität aber geringere Spezifität als das KK-Verfahren auf. Als hochsensitive und sehr spezifische Methode zur Diagnose der Schistosomiasis hat sich der Nachweis von Schistosoma-spezifischer DNA mittels Real-Time PCR herausgestellt. Allerdings wird für die Durchführung dieser Technik ein gut ausgestattetes Labor benötigt, das sich in der Regel nicht in unmittelbarer Nähe zum Patienten im Feld befindet. Daher ist es besonders wichtig, über praktikable und schnelle Konservierungsmethoden zu verfügen, die bevor die Extraktion und Amplifikation der DNA stattfindet, einen einfachen Transport und eine einfache Lagerung des Probenmaterials ermöglichen. Das Ziel des ersten Teils der vorliegenden Arbeit war, die Sensitivität und Spezifität der klassischerweise verwendeten KK-Methode und des POC-CCA-Tests mit der Real-Time PCR- Methode unter Verwendung von Stuhlproben, Urinproben, Serumproben sowie auf Filterpapier getrocknete Blutproben (dried blood spots – DBSs) zu vergleichen. Zudem wurde die Anwendbarkeit der Real-Time PCR aus Serum- und Urinproben zur Therapiekontrolle überprüft. Die dazu notwendigen Studien wurden alle in der Region Mwanza in Tansania durchgeführt, welche als hochendemisch für S. mansoni gilt. Für die Untersuchungen zur stuhlbasierten Real-Time PCR wurden als Studienteilnehmer Schulkinder gewählt. Aufgrund der erforderlichen Blutabnahme wurden die anderen Teilstudien nur mit erwachsenen Probanden durchgeführt. Unter Verwendung der KK-Methode als Goldstandard erzielten die Real-Time PCR aus Stuhlproben und der POC-CCA-Test sehr hohe Sensitivitäten von 99,5% bzw. 89,7%, jedoch nur geringe Spezifitäten von 29,55% und 22,73%. Die KK-Methode weist bekanntermaßen nur eine geringe bis moderate Sensitivität auf und ist daher nicht gut als Referenz geeignet. Deshalb wurde zusätzlich eine latente Klassenanalyse angewandt, um die tatsächlich Erkrankten zu ermitteln und anhand dieser die diagnostische Güte der verwendeten Tests zu bestimmen. Hier zeigte der POC-CCA-Test die höchste Sensitivität (99,5%) sowie eine Spezifität von 63,4%. Der Real-Time PCR-Test hatte eine Sensitivität von 98,7% und die höchste Spezifität (81,2%). Die Spezifität der KK-Technik betrug 72,8%, die Sensitivität war signifikant niedriger (89,7%) als bei den anderen beiden Methoden. Diese Ergebnisse verdeutlichen, dass der POC-CCA-Schnelltest empfindlicher ist als die KK-Methode und zum Screening von S. mansoni-Infektionen eingesetzt werden kann. Die Stuhl-PCR war zwar ebenfalls hochsensitiv und zeigte unter den drei getesteten Diagnoseverfahren die höchste Spezifität, aber aufgrund der höheren Kosten und der komplizierten Anwendung sollte für epidemiologische Untersuchungen in Hochprävalenzregionen der POC-CCA-Test bevorzugt werden. Bei unklaren Diagnosen kann die Real-Time PCR-Methode als Bestätigungstest Anwendung finden. In der Teilstudie zur serum- und urinbasierten Real-Time PCR in einer endemischen Region vor und nach der Behandlung mit PZQ wurden folgende Ergebnisse erzielt: Unter Verwendung einer kombinierten Referenz aus den Ergebnissen des parasitologischen KK-Tests und / oder der serumbasierten PCR konnte zu Studienbeginn eine Prävalenz von S. mansoni von 77,1% ermittelt werden. In Bezug auf die Sensitivität zeigte der DNA-Nachweis aus Serum (96,3%) und der POC-CCA-Assay (77,8%) die höchsten Ergebnisse. Die urinbasierte Real-Time PCR zeigte die geringste Empfindlichkeit (33,3%). Durch die Behandlung mit Praziquantel wurde eine signifikante Reduktion der S. mansoni Prävalenz erreicht. Zwanzig Wochen nach Therapie konnte durch die KK-Methode keine, mit dem POC-CCA-Test 33,3% und mit der serumbasierten Real-Time PCR 58,3% Infektionen festgestellt werden. Die Analyse der mittels der serumbasierten PCR bestimmten mittleren Ct-Werte im zeitlichen Verlauf zeigte, dass dieser eine Woche nach der Behandlung signifikant abnahm (von 30,3 auf 28) und 20 Wochen später über den Basiswert (34,9) anstieg. Der Ct-Wert ist umgekehrt proportional zur DNA-Ausgangsmenge, die in die PCR eingesetzt wurde. Dies deutet darauf hin, dass kurz nach der Therapie ein DNA-Anstieg zu verzeichnen war und 20 Wochen später weniger DNA als zu Beginn der Studie nachweisbar war. Dieser DNA-Verlauf lässt verschiedene Interpretationsmöglichkeiten zu. Die Daten zeigen jedoch, dass die serumbasierte Real-Time PCR eine ausgezeichnete diagnostische Genauigkeit aufweist. Da die nachgewiesene DNA jedoch keine Rückschlüsse auf das Parasitenstadium zulässt und es sich hierbei auch um DNA aus im Gewebe verbliebenen Eiern oder Reinfektionen handeln könnte, ist diese Methode in Hochprävalenz- Regionen nicht zur Therapiekontrolle geeignet. Die Verwendung von Urin zum DNA-Nachweis erzielte keine vielversprechenden Ergebnisse. Die Sensitivität der Real-Time PCR aus DBSs war ebenfalls sehr gering (45,4%) und kann ohne weitere ausführliche Testung hinsichtlich Lagertemperatur, Lagerdauer, verschiedener Filterpapierarten und Extraktionsmethoden nicht empfohlen werden. Zusammenfassend zeigten die Ergebnisse dieser Studien, dass sowohl die stuhl- als auch die serumbasierte Real-Time PCR bei der Erkennung und Bewertung der Infektionsprävalenz, einem wichtigen Aspekt epidemiologischer Studien, deutlich empfindlicher ist als das mikroskopische KK-Verfahren. Aufgrund des hohen Kosten- und Personalaufwandes und der Notwendigkeit eines gut ausgestatteten Labors wird sich diese Methode aber nicht zum Screening in hochendemischen Ländern durchsetzen. Sie kann jedoch einen Mehrwert bei der Diagnose der Schistosomiasis bieten, vor allem bei frühen oder leichten Infektionen. Zudem kann diese hochsensitive und spezifische Methode als Bestätigungstest bei unklaren Diagnosen herangezogen werden. Im zweiten Teil dieser Arbeit wurden malakologische Untersuchungen zur Identifizierung potenzieller Übertragungsorte für die Schistosomiasis rund um die im Viktoriasee gelegene Insel Ijinga durchgeführt. Diese Analysen fanden innerhalb eines Pilotprojektes zur Eliminierung der Erkrankung auf der Insel Ijinga statt, wobei ein intensiviertes Behandlungsprotokoll, welches die gesamte Inselbevölkerung einschloss, Anwendung fand. Die Kontrolle der Praziquanteleffektivität nach mehreren Behandlungsrunden bringt eine Reihe diagnostischer Herausforderungen mit sich. Hier könnte die Beurteilung der Schistosoma-Infektion in den Zwischenwirtschnecken vor und nach der Therapie als Indikator für den Erfolg der Maßnahme dienen. Zu diesem Zweck erfolgte zunächst eine Baseline-Untersuchung, bei der Schnecken an Uferregionen gesammelt wurden, an denen die Inselbewohner häufigen Wasserkontakt hatten. Die Schnecken wurden anhand morphologischer Merkmale identifiziert und mithilfe der Real-Time PCR-Methode auf Infektionen mit S. mansoni untersucht. Insgesamt wurden 35,4% (279/788) S. mansoni- positive Zwischenwirtschnecken (Biomphalaria) detektiert. Dies verdeutlicht, dass an den meisten Wasserkontaktstellen um die Insel Ijinga ein potentielles Risiko für die Übertragung der Schistosomiasis besteht. Die mithilfe der KK-Methode ermittelte Gesamtprävalenz von S. mansoni in der humanen Bevölkerung betrug 68,9%. Nachdem die Bewohner der Insel viermal mit PZQ behandelt wurden, zeigte sich in der kontinuierlich überwachten Sentinelgruppe eine Reduktion der Prävalenz auf 28,7%. Zu diesem Zeitpunkt wurde ebenfalls die Analyse der Schnecken wiederholt und es konnten 16,8% (57/350) Schnecken mit einer S. mansoni Infektion nachgewiesen werden. Die Reduktion der Infektionshäufigkeit in den Schnecken vor und nach der viermaligen Behandlung der Bevölkerung war signifikant (χ² = 74.335, p < 0,001). Dies deutet darauf hin, dass die intermediären Wirtsschnecken zur Überwachung von Kontrollmaßnahmen verwendet werden können. N2 - Schistosomiasis remains one of the most common parasitic infections in the world, causing significant health and economic consequences, particularly in rural areas. Immune reactions to the parasite's eggs deposited in the host can lead to chronic progression. This may result in to irreversible damage. In order to prevent this, an early and reliable diagnosis and a concomitant therapy will be indispensable. In addition, the reliable detection of schistosomiasis plays a key role in monitoring, prevention and control of the disease. In epidemiological studies, the microscopic Kato-Katz (KK) method is most frequently used to detect eggs in stool. This method is highly specific and offers the possibility of quantification, which allows to determine the intensity of the existing infection. However, especially at low infection intensities, the sensitivity of the test method is rather moderate. Furthermore, infections can only be detected after the prepatent period. The also frequently used urine-based Point-of-Care Circulating Cathodic Antigen (POC-CCA) test shows a higher sensitivity but lower specificity than the KK method. The detection of schistosome-specific DNA through Real-Time PCR has proven to be a highly sensitive and very specific method for the diagnosis of schistosomiasis. However, this assay requires a well-equipped laboratory, which is usually not located in the immediate vicinity of the patient in the field. Therefore, it is particularly important to have practical and rapid preservation methods that allow easy transport and storage of the sample material before DNA extraction and amplification takes place. The aim of the first part of this dissertation was to compare the sensitivity and specificity of the classical KK method and the POC-CCA test with the Real-Time PCR method using stool samples, urine samples, serum samples and blood samples dried on filter paper (dried blood spots – DBS). In addition, tests were conducted regarding the applicability of Real-Time PCR from serum and urine samples for therapy control. The necessary studies were all carried out in the Mwanza region of Tanzania, which is considered to be highly endemic for S. mansoni. Schoolchildren were selected as study participants for the studies on stool-based Real-Time PCR. Due to the required blood collection, the other partial studies were conducted with adults only. Using the KK method as gold standard, the Real-Time PCR from stool samples and the POC-CCA test achieved very high sensitivities of 99.5% and 89.7%, respectively, but only low specificities of 29.55% and 22.73%. The KK method is known to have low to moderate sensitivity and is therefore not well suited as a reference. For this reason, a latent class analysis was carried out to determine the true patients and the diagnostic quality of the tests used. The POC-CCA test showed the highest sensitivity (99.5%) and a specificity of 63.4%. The Real-Time PCR test had a sensitivity of 98.7% and the highest specificity (81.2%). The specificity of the KK technique was 72.8%, the sensitivity was significantly lower (89.7%) than with the other two methods. These results show that the POC-CCA rapid test is more sensitive than the KK method and can be used to screen for S. mansoni infections. Although stool PCR is also highly sensitive and shows the highest specificity of the three diagnostic methods tested, the POC-CCA test should be preferred for epidemiological investigations in high prevalence regions on account of the higher costs and complicated application. If the diagnosis is unclear, the Real-Time PCR method can be used as a confirmatory test. The following results were obtained in the partial study regarding the use of serum and urine-based Real-Time PCR in an endemic region before and after treatment with Praziquantel (PZQ): Using a combined reference of the results of the parasitological KK test and / or the serum-based PCR, a prevalence of S. mansoni of 77.1% could be determined at the beginning of the study. In terms of sensitivity, DNA detection from serum (96.3%) and the POC-CCA assay (77.8%) showed the highest results. Urine-based Real-Time PCR showed the lowest sensitivity (33.3%). Treatment with Praziquantel significantly reduced the prevalence of S. mansoni. Twenty weeks after therapy, no infections could be detected with the KK method, 33.3% with the POC-CCA test and 58.3% with the serum-based Real-Time PCR. Analysis of mean Ct-values determined by serum-based Real-Time PCR over time showed that it decreased significantly (from 30.3 to 28) one week after treatment and increased above the baseline value (34.9) 20 weeks later. The Ct-value is inversely proportional to the initial amount of DNA added to the PCR. This suggests that shortly after therapy there was an increase in DNA and 20 weeks later less DNA was detectable compared to the beginning of the study. This DNA progression offers various interpretation possibilities. However, the data show that serum-based Real-Time PCR has excellent diagnostic accuracy. Yet, since the detected DNA does not allow conclusions to be drawn about the parasite stage and this could also be DNA from eggs remaining in the tissue or reinfections, this method is not suitable for therapy control in high prevalence regions. The use of urine for DNA detection did not yield promising results. The sensitivity of the Real-Time PCR from DBSs was also very low (45.4%) and cannot be recommended without further extensive testing with regard to storage temperature, storage time, different filter paper types and extraction methods. In summary, the results of these studies showed that Real-Time PCR is significantly more sensitive than microscopy in the detection and evaluation of infection prevalence, an important aspect of epidemiological studies. However, due to the high costs and personnel involved and the need for a well-equipped laboratory, this method will not be accepted for screening in high-endemic countries. Yet, it can provide added value in the diagnosis of schistosomiasis, especially in early or light infections. In addition, this highly sensitive and specific method can be used as a confirmatory test for unclear diagnoses. The second part of this dissertation describes malacological investigations to identify potential transmission sites for schistosomiasis around Ijinga Island located on Lake Victoria. These analyses took place as part of a pilot project to eliminate the disease on this island, using an intensified treatment protocol that included the entire island population. The control of Praziquantel effectiveness after several rounds of treatment poses a number of diagnostic challenges. Here, the assessment of the schistosoma infection in the intermediate host snails before and after the therapies could serve as an indicator for the success of the measure. For this purpose, a baseline study was carried out, in which snails were collected from shore regions where the islanders had frequent contact with water. The snails were identified on the basis of morphological characteristics and examined for infections with S. mansoni using the Real-Time PCR method. A total of 35.4% (279/788) S. mansoni positive intermediate host snails (Biomphalaria) were detected. This shows that there is a potential risk of schistosomiasis transmission at most water contact points around Ijinga. The total prevalence of S. mansoni in the human population determined by the KK method was 68.9%. After treating the community with Praziquantel four times, the prevalence of S. mansoni in the continuously monitored sentinel group was reduced to 28.7%. At this time, the analysis of the snails was repeated and 16.8% (57/350) snails with a S. mansoni infection were detected. The reduction in the frequency of infection in the snails before and after the fourfold treatment of the population was significant (χ² = 74,335, p < 0.001). This suggests that the intermediate host snails can be used to monitor control measures. KW - Schistosomiasis KW - Bilharziose KW - Real-time PCR KW - Tansania KW - Trematoden KW - Vernachlässigte Tropenkrankheiten Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215061 ER - TY - THES A1 - Kaiser, Lena Franziska T1 - Wirkmechanismus von Sphingolipiden und Sphingosin gegen mikrobielle Erreger T1 - Mechanism of the bactericidal effect of sphingolipids and sphingosine against microbial pathogens N2 - Die zunehmende Antibiotikaresistenz vieler Krankheitserreger ist ein weltweites Problem, welches zu einem klinischen Bedarf an neuen antimikrobiellen Substanzen führt. Sphingolipide einschließlich Ceramide stellen eine vielfältige Gruppe strukturverwandter Lipide dar und bestehen aus einem Sphingosin-Grundgerüst, welches mit einer Fettsäure verbunden ist. Sowohl das Sphingosin-Grundgerüst allein als auch Sphingolipide zeigen eine antibakterielle Wirkung gegenüber einer Vielzahl pathogener Mikroorganismen. Die Intensität der Hemmung hängt von der Sphingolipidstruktur und dem Mikroorganismus ab. Neuere Studien konnten zeigen, dass Sphingosin, Ceramide und Ceramid-Analoga in N. meningitidis aufgenommen werden und eine bakteriostatische oder bakterizide Wirkung zeigen. Jedoch ist die antibakterielle Wirkungsweise noch nicht genau bekannt. Um mehr über den Wirkmechanismus zu erfahren haben wir die ultrastrukturellen Veränderungen von N. meningitidis nach Inkubation mit azido-funktionalisierten Sphingolipiden mit elektronenmikroskopischen Verfahren (transmissionselektronenmikroskopische und rasterelektronenmikroskopische Aufnahmen) untersucht. Mittels korrelativer Licht- und Elektronenmikroskopie (CLEM) konnten wir die azido-funktionalisierten Sphingolipide nach Aufnahme in N. meningitidis lokalisieren. Zum Anfärben der funktionalisierten Sphingolipide wurde die kupferfreie Azid-Alkin-Cyccloaddition verwendet. N2 - The increasing antibiotic resistance of many pathogens is a worldwide problem that leads to a clinical need of new anti-microbial compounds. Sphingolipids, including ceramides, represent a diverse group of structurally related lipids and are composed of a sphingosine backbone coupled to a fatty acid. Solely the sphingosine backbone as well as the sphingolipids show antibacterial activity against a wide range of pathogenic microorganisms. The rate of inhibition depends on the sphingolipid structure and the microbial strain. Recent studies revealed the uptake of sphingosine, ceramides and ceramide analogues by N. meningitidis and a bacteriostatic or bactericidal effect. However, the mechanism of the bactericidal effect is still unknown. To elucidate the antibacterial mechanism, we studied the ultrastructural changes after incubation of N. meningitidis with azido-modified sphingolipids by using electron microscopy techniques (transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Due to correlative light-electron microscopy (CLEM) we were able to localize the azido-modified sphingolipids after incorporation in N. meningitidis. Copper-free azide-alkyne cycloaddition was used to stain the azido-modified sphingolipids. KW - Neisseria meningitidis KW - Sphingolipide KW - Sphingosin KW - Antimikrobieller Wirkstoff KW - Mikroskopie KW - sphingolipids Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-218970 ER - TY - JOUR A1 - Lorson, Thomas A1 - Ruopp, Matthias A1 - Nadernezhad, Ali A1 - Eiber, Julia A1 - Vogel, Ulrich A1 - Jungst, Tomasz A1 - Lühmann, Tessa T1 - Sterilization Methods and Their Influence on Physicochemical Properties and Bioprinting of Alginate as a Bioink Component JF - ACS Omega N2 - Bioprinting has emerged as a valuable threedimensional (3D) biomanufacturing method to fabricate complex hierarchical cell-containing constructs. Spanning from basic research to clinical translation, sterile starting materials are crucial. In this study, we present pharmacopeia compendial sterilization methods for the commonly used bioink component alginate. Autoclaving (sterilization in saturated steam) and sterile filtration followed by lyophilization as well as the pharmacopeia non-compendial method, ultraviolet (UV)-irradiation for disinfection, were assessed. The impact of the sterilization methods and their effects on physicochemical and rheological properties, bioprinting outcome, and sterilization efficiency of alginate were detailed. Only sterile filtration followed by lyophilization as the sterilization method retained alginate's physicochemical properties and bioprinting behavior while resulting in a sterile outcome. This set of methods provides a blueprint for the analysis of sterilization effects on the rheological and physicochemical pattern of bioink components and is easily adjustable for other polymers used in the field of biofabrication in the future. KW - hydrogels Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229460 N1 - Lizenz: https://pubs.acs.org/page/policy/authorchoice_termsofuse.html VL - 5 IS - 12 ER - TY - JOUR A1 - Apsemidou, Athanasia A1 - Füller, Miriam Antonie A1 - Idelevich, Evgeny A. A1 - Kurzai, Oliver A1 - Tragiannidis, Athanasios A1 - Groll, Andreas H. T1 - Candida lusitaniae breakthrough fungemia in an immuno-compromised adolescent: case report and review of the literature JF - Journal of Fungi N2 - Candida lusitaniae is a rare cause of candidemia that is known for its unique capability to rapidly acquire resistance to amphotericin B. We report the case of an adolescent with grade IV graft-vs.-host disease after hematopoietic cell transplantation who developed catheter-associated C. lusitaniae candidemia while on therapeutic doses of liposomal amphotericin B. We review the epidemiology of C. lusitaniae bloodstream infections in adult and pediatric patients, the development of resistance, and its role in breakthrough candidemia. Appropriate species identification, in vitro susceptibility testing, and source control are pivotal to optimal management of C. lusitaniae candidemia. Initial antifungal therapy may consist of an echinocandin and be guided by in vitro susceptibility and clinical response. KW - Candida lusitaniae KW - candidemia KW - resistance KW - breakthrough KW - infection KW - transplantation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-220125 SN - 2309-608X VL - 6 IS - 4 ER - TY - JOUR A1 - Dichtl, Karl A1 - Forster, Johannes A1 - Ormanns, Steffen A1 - Horns, Heidi A1 - Suerbaum, Sebastian A1 - Seybold, Ulrich A1 - Wagener, Johannes T1 - Comparison of β-D-Glucan and galactomannan in serum for detection of invasive aspergillosis: retrospective analysis with focus on early diagnosis JF - Journal of Fungi N2 - The early diagnosis of invasive aspergillosis (IA) relies mainly on computed tomography imaging and testing for fungal biomarkers such as galactomannan (GM). We compared an established ELISA for the detection of GM with a turbidimetric assay for detection of the panfungal biomarker β-D-glucan (BDG) for early diagnosis of IA. A total of 226 serum specimens from 47 proven and seven probable IA cases were analysed. Sensitivity was calculated for samples obtained closest to the day of IA-diagnosis (d0). Additional analyses were performed by including samples obtained during the presumed course of disease. Most IA cases involved the respiratory system (63%), and Aspergillus fumigatus was the most frequently isolated species (59%). For proven cases, sensitivity of BDG/GM analysis was 57%/40%. Including all samples dating from –6 to +1 weeks from d0 increased sensitivities to 74%/51%. Sensitivity of BDG testing was as high as or higher than GM testing for all subgroups and time intervals analysed. BDG testing was less specific (90–93%) than GM testing (99–100%). Combining BDG and GM testing resulted in sensitivity/specificity of 70%/91%. Often, BDG testing was positive before GM testing. Our study backs the use of BDG for diagnosis of suspected IA. We suggest combining BDG and GM to improve the overall sensitivity. KW - BDG KW - beta-D-glucan KW - GM KW - galactomannan KW - IA KW - invasive aspergillosis KW - biomarker KW - fungal antigens KW - serology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216298 SN - 2309-608X VL - 6 IS - 4 ER - TY - JOUR A1 - Bauriedl, Saskia A1 - Gerovac, Milan A1 - Heidrich, Nadja A1 - Bischler, Thorsten A1 - Barquist, Lars A1 - Vogel, Jörg A1 - Schoen, Christoph T1 - The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition JF - Nature Communications N2 - FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition. FinO-domain proteins are bacterial RNA-binding proteins with a wide range of target specificities. Here, the authors employ UV CLIP-seq and show that minimal ProQ protein of Neisseria meningitidis binds to various small non-coding RNAs and mRNAs involved in virulence. KW - Neisseria meningitidis KW - natural transformation KW - dual function KW - FinO family KW - HFQ KW - chaperone KW - transcriptome KW - regulator KW - sequence KW - in vivo Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230040 VL - 11 ER - TY - THES A1 - Bauriedl, Saskia Corinna T1 - The influence of riboregulation on fitness and virulence in Neisseria meningitidis T1 - Der Einfluss der Riboregulation auf Fitness und Virulenz von Neisseria meningitidis N2 - Neisseria meningitidis (N. meningitidis) is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experi-mental evidence that the expression of small non-coding RNAs (sRNAs) as well as the RNA chaperone Hfq affect meningococcal physiology, the impact of RNA-based regula-tion (riboregulation) on fitness and virulence in N. meningitidis is only poorly understood. Therefore, this study addressed these issues using a combination of high-throughput tech-nologies. A differential RNA-sequencing (dRNA-seq) approach was applied to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8013. The dRNA-seq analysis predicted 1,625 transcriptional start sites including 65 putative sRNAs, of which 20 were further validated by northern blot analysis. By Hfq RNA im-munopreci-pitation sequencing a large Hfq-centered post-transcriptional regulatory net-work comprising 23 sRNAs and 401 potential mRNA targets was identified. Rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on its associat-ed sRNAs. Based on these data, the interactions of two paralogous sRNAs and their cog-nate target mRNA prpB were validated in vivo as well as in vitro. Both sRNAs directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx. Besides the well-described RNA chaperone Hfq, FinO-domain proteins have recently been recognized as a widespread family of RNA-binding proteins (RBPs) with regulatory roles in diverse bacteria. They display an intriguing bandwidth of target sites, ranging from a single RNA pair as recognized by plasmid-encoded FinO to the global RNA regu-lons of enterobacterial ProQ proteins. To better understand the intrinsic targeting mode of this RBP family, in vivo targets of the minimal ProQ protein of N. meningitidis were de-termined. In vivo UV crosslinking with RNA deep sequencing (UV-CLIP) identified as-sociations of ProQ with 16 sRNAs and 166 mRNAs encoding a variety of biological functions and thus revealed ProQ as another global RBP in meningococci. It could be shown that meningococcal ProQ predominantly binds to highly structured RNA regions including DNA uptake sequences (DUS) and rho-independent transcription terminators and stabilizes many of its RNA targets as proved by rifampicin stability experiments. As expected from the large suite of ProQ-bound RNAs, proQ deletion globally affects both gene and protein expression in N. meningitidis, changing the expression levels of at least 244 mRNAs and 80 proteins. Phenotypic analyses suggested that ProQ promotes oxida-tive stress tolerance and UV damage repair capacity, both of which are required for full virulence of N. meningitidis. Together, this work uncovers the co-existence of two major post-transcriptional regulons, one governed by ProQ, the other by Hfq, in N. meningitidis. It further highlights the role of these distinct RBPs and its associated sRNAs to bacterial virulence and indicates that riboregulation is likely to contribute to the way how meningococci adapt to different host niches. N2 - Neisseria meningitidis (N. meningitidis) ist ein kommensal lebendes Bakterium, welches unter nicht vollständig geklärten Bedingungen auch lebensbedrohliche Infektionen im Menschen wie bakterielle Meningitis und Sepsis verursachen kann. Obwohl experimentell nachgewiesen wurde, dass die Expression kleiner, nicht kodierender RNAs (sRNAs) so-wie des RNA-Chaperons Hfq in Meningokokken physiologisch relevant ist, blieb der Ein-fluss der RNA-basierten Genregulation (Riboregulation) auf die Fitness und Virulenz von N. meningitidis bisher unvollständig verstanden. Daher befasste sich diese Studie durch Kombination verschiedener Hochdurchsatz-Technologien mit dieser Fragestellung. Es wurde differentielle RNA-Sequenzierung (dRNA-seq) angewendet, um das primäre Transkriptom des N. meningitidis Stamms 8013 möglichst genau zu kartieren. Die durch-geführte dRNA-seq-Analyse detektierte 1.625 Transkriptionsstartstellen (TSS) einschließ-lich 65 potentieller sRNAs. Durch Anwendung von Northern-Blot-Analysen konnten an-schließend 20 sRNAs experimentell validiert werden. Darüber hinaus wurde durch Ko-Immunopräzipitation mit Hfq (RIP-seq) ein großes, Hfq-zentriertes, post- transkripti-onelles regulatorisches Netzwerk identifiziert, welches 23 sRNAs und 401 mRNAs um-fasst. Rifampicin-Stabilitätsversuche zeigten, dass durch Hfq-Bindung die Stabilität die-ser sRNAs erhöht wird. Basierend auf diesen Daten konnte die Interaktion zwischen zweier Hfq-gebundener paraloger sRNAs und der prpB mRNA sowohl in vivo als auch in vitro bestätigt werden. Beide sRNAs reprimieren die Translation des PrpB-Genes, wel-ches für eine Methylisocitratlyase kodiert und wahrscheinlich die Kolonisation des menschlichen Nasopharynxs durch Meningokokken begünstigt. Neben dem ausführlich charakterisierten RNA-Chaperon Hfq wurden Proteine mit FinO-Domäne kürzlich als eine neue Familie von RNA-bindenden Proteinen (RBPs) mit regula-torischen Funktionen in verschiedenen Bakterien identifiziert. Sie weisen eine große Bandbreite regulierter Gene auf: Während das Plasmid-kodierte FinO-Protein nur ein ein-zelnes RNA-Paar bindet, stellt das enterobakterielle ProQ-Protein ein globales RBP dar. Um die Wirkungsweise dieser RBP-Familie besser zu verstehen, wurde in vivo untersucht, wie viele RNAs mit dem minimalen ProQ-Protein in N. meningitidis assoziiert sind. Durch Kombination von UV-Crosslinken mit RNA-Sequenzierung (UV-CLIP) konnte die Bin-dung von 16 sRNAs und 166 biologisch diverser mRNAs mit ProQ identifiziert werden, welches daher ebenfalls ein globales RBP in Meningokokken darstellt. Es konnte gezeigt werden, dass ProQ vorwiegend RNA-Regionen mit ausgeprägter Sekundärstruktur bin-det, darunter DNA-Aufnahmesequenzen (DUS) und Rho-unabhängige Transkriptions-terminatoren. Die ProQ-Bindung führt dabei häufig zur Stabilisation der RNAs, was durch Rifampicin-Stabilitätsexperimente nachgewiesen wurde. Wie aufgrund der großen Zahl ProQ-gebundener RNAs zu erwarten, beeinflusste die Deletion des ProQ Proteins die zelluläre Expression von mindestens 244 mRNAs und 80 Proteinen. Phänotypische Analysen deuten darauf hin, dass ProQ sowohl die Toleranz gegenüber oxidativem Stress als auch die Reparatur von DNA-Schäden reguliert, die beide für die vollständige Viru-lenz von N. meningitidis von Bedeutung sind. Zusammenfassend beschreibt diese Arbeit die Koexistenz von zwei großen posttranskrip-tionellen Regulons in N. meningitidis, von denen eines von ProQ und das andere von Hfq kontrolliert wird. Im Rahmen dieser Arbeit wurde die Rolle beider RBPs und ihrer assozi-ierten sRNAs für die bakterielle Virulenz verdeutlicht und hervorgehoben, dass Riboregu-lation sehr wahrscheinlich dazu beiträgt, wie sich Meningokokken an verschiedene Wirts-nischen anpassen. KW - Neisseria meningitidis KW - Small non-messenger RNS KW - Hfq KW - ProQ KW - Non-coding RNA KW - High throughput screening Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192978 ER - TY - JOUR A1 - Wurmb, Thomas A1 - Scholtes, Katja A1 - Kolibay, Felix A1 - Schorscher, Nora A1 - Ertl, Georg A1 - Ernestus, Ralf-Ingo A1 - Vogel, Ulrich A1 - Franke, Axel A1 - Kowalzik, Barbara T1 - Hospital preparedness for mass critical care during SARS-CoV-2 pandemic JF - Critical Care N2 - Mass critical care caused by the severe acute respiratory syndrome corona virus 2 pandemic poses an extreme challenge to hospitals. The primary goal of hospital disaster preparedness and response is to maintain conventional or contingency care for as long as possible. Crisis care must be delayed as long as possible by appropriate measures. Increasing the intensive care unit (ICU) capacities is essential. In order to adjust surge capacity, the reduction of planned, elective patient care is an adequate response. However, this involves numerous problems that must be solved with a sense of proportion. This paper summarises preparedness and response measures recommended to acute care hospitals. KW - Mass critical care KW - Disaster response KW - SARS-CoV-2 KW - Hospital emergency plan Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230201 VL - 24 ER - TY - JOUR A1 - Götz, Ralph A1 - Panzer, Sabine A1 - Trinks, Nora A1 - Eilts, Janna A1 - Wagener, Johannes A1 - Turrà, David A1 - Di Pietro, Antonio A1 - Sauer, Markus A1 - Terpitz, Ulrich T1 - Expansion Microscopy for Cell Biology Analysis in Fungi JF - Frontiers in Microbiology N2 - Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes. KW - Expansion microscopy KW - fluorescence microscopy KW - fungi KW - sporidia KW - hyphae Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202569 SN - 1664-302X VL - 11 ER - TY - GEN A1 - Tort, Jose F. A1 - Mitreva, Makedonka A1 - Brehm, Klaus R. A1 - Rinaldi, Gabriel T1 - Editorial: Novel Frontiers in Helminth Genomics T2 - Frontiers in Genetics N2 - No abstract available. KW - flatworm KW - nematodes KW - genomics KW - helminths KW - neglected diseases Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-210209 SN - 1664-8021 VL - 11 IS - 791 ER - TY - JOUR A1 - Sturm, Laura A1 - Geißel, Bernadette A1 - Martin, Ronny A1 - Wagener, Johannes T1 - Differentially Regulated Transcription Factors and ABC Transporters in a Mitochondrial Dynamics Mutant Can Alter Azole Susceptibility of Aspergillus fumigatus JF - Frontiers in Microbiology N2 - Azole resistance of the fungal pathogen Aspergillus fumigatus is an emerging problem. To identify novel mechanisms that could mediate azole resistance in A. fumigatus, we analyzed the transcriptome of a mitochondrial fission/fusion mutant that exhibits increased azole tolerance. Approximately 12% of the annotated genes are differentially regulated in this strain. This comprises upregulation of Cyp51A, the azole target structure, upregulation of ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) transporters and differential regulation of transcription factors. To study their impact on azole tolerance, conditional mutants were constructed of seven ABC transporters and 17 transcription factors. Under repressed conditions, growth rates and azole susceptibility of the mutants were similar to wild type. Under induced conditions, several transcription factor mutants showed growth phenotypes. In addition, four ABC transporter mutants and seven transcription factor mutants exhibited altered azole susceptibility. However, deletion of individual identified ABC transporters and transcription factors did not affect the increased azole tolerance of the fission/fusion mutant. Our results revealed the ability of multiple ABC transporters and transcription factors to modulate the azole susceptibility of A. fumigatus and support a model where mitochondrial dysfunctions trigger a drug resistance network that mediates azole tolerance of this mold. KW - mitochondrial dynamics KW - azole resistance KW - efflux pumps KW - transcription factors Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204874 SN - 1664-302X VL - 11 ER - TY - JOUR A1 - Schlesinger, Tobias A1 - Weißbrich, Benedikt A1 - Wedekink, Florian A1 - Notz, Quirin A1 - Herrmann, Johannes A1 - Krone, Manuel A1 - Sitter, Magdalena A1 - Schmid, Benedikt A1 - Kredel, Markus A1 - Stumpner, Jan A1 - Dölken, Lars A1 - Wischhusen, Jörg A1 - Kranke, Peter A1 - Meybohm, Patrick A1 - Lotz, Christpher T1 - Biodistribution and serologic response in SARS-CoV-2 induced ARDS: A cohort study JF - PLoS One N2 - Background The viral load and tissue distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain important questions. The current study investigated SARS-CoV-2 viral load, biodistribution and anti-SARS-CoV-2 antibody formation in patients suffering from severe corona virus disease 2019 (COVID-19) induced acute respiratory distress syndrome (ARDS). Methods This is a retrospective single-center study in 23 patients with COVID-19-induced ARDS. Data were collected within routine intensive care. SARS-CoV-2 viral load was assessed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Overall, 478 virology samples were taken. Anti-SARS-CoV-2-Spike-receptor binding domain (RBD) antibody detection of blood samples was performed with an enzyme-linked immunosorbent assay. Results Most patients (91%) suffered from severe ARDS during ICU treatment with a 30-day mortality of 30%. None of the patients received antiviral treatment. Tracheal aspirates tested positive for SARS-CoV-2 in 100% of the cases, oropharyngeal swabs only in 77%. Blood samples were positive in 26% of the patients. No difference of viral load was found in tracheal or blood samples with regard to 30-day survival or disease severity. SARS-CoV-2 was never found in dialysate. Serologic testing revealed significantly lower concentrations of SARS-CoV-2 neutralizing IgM and IgA antibodies in survivors compared to non-survivors (p = 0.009). Conclusions COVID-19 induced ARDS is accompanied by a high viral load of SARS-CoV-2 in tracheal aspirates, which remained detectable in the majority throughout intensive care treatment. Remarkably, SARS-CoV-2 RNA was never detected in dialysate even in patients with RNAemia. Viral load or the buildup of neutralizing antibodies was not associated with 30-day survival or disease severity. KW - viral load Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231348 VL - 15, 2020 IS - 11 ER - TY - JOUR A1 - Flemming, S. A1 - Hankir, M. A1 - Ernestus, R.-I. A1 - Seyfried, F. A1 - Germer, C.-T. A1 - Meybohm, P. A1 - Wurmb, T. A1 - Vogel, U. A1 - Wiegering, A. T1 - Surgery in times of COVID-19 — recommendations for hospital and patient management JF - Langenbeck's Archives of Surgery N2 - Background The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), has escalated rapidly to a global pandemic stretching healthcare systems worldwide to their limits. Surgeonshave had to immediately react to this unprecedented clinical challenge by systematically repurposing surgical wards. Purpose To provide a detailed set of guidelines developed in a surgical ward at University Hospital Wuerzburg to safelyaccommodate the exponentially rising cases of SARS-CoV-2 infected patients without compromising the care of emergencysurgery and oncological patients or jeopardizing the well-being of hospital staff. Conclusions The dynamic prioritization of SARS-CoV-2 infected and surgical patient groups is key to preserving life whilemaintaining high surgical standards. Strictly segregating patient groups in emergency rooms, non-intensive care wards andoperating areas prevents viral spread while adequately training and carefully selecting hospital staff allow them to confidentlyand successfully undertake their respective clinical duties. KW - SARS-CoV-2 KW - COVID-19 KW - Surgery Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231766 SN - 1435-2443 VL - 405 ER - TY - JOUR A1 - Weiss, Esther A1 - Schlegel, Jan A1 - Terpitz, Ulrich A1 - Weber, Michael A1 - Linde, Jörg A1 - Schmitt, Anna-Lena A1 - Hünniger, Kerstin A1 - Marischen, Lothar A1 - Gamon, Florian A1 - Bauer, Joachim A1 - Löffler, Claudia A1 - Kurzai, Oliver A1 - Morton, Charles Oliver A1 - Sauer, Markus A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus JF - Frontiers in Immunology N2 - Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56\(^{bright}\)CD16\(^{dim}\) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility. KW - natural killer cell KW - stem cell transplantation KW - corticosteroids KW - CCL3 KW - CCL4 KW - CCL5 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212581 SN - 1664-3224 VL - 11 ER - TY - THES A1 - Hauer [geb. Lein], Nina T1 - Genetische Variabilität und Expression der ADP-Ribosyltransferase NarE T1 - Genetic variability and expression of ADP-ribosyltransferase NarE N2 - Neisseria meningitidis ist Auslöser invasiver Infektionen, die Sepsis und Meningitis hervorrufen. Bakterielle ADP-Ribosyltransferasen wurden als Toxine zahlreicher Bakterien wie E.coli, V. cholerae und B. pertussis beschrieben, die postranslationale Modifikationen bei eukaryotischen Proteinen mit pathologischer Wirkung für den Menschen hervorrufen. Die ADP-Ribosyltransferase NarE von Neisserien ist auf der Basis von Sequenzhomologien identifiziert worden. Die enzymatische Aktivität des Proteins wurde bereits in Studien gezeigt. Ziel dieser Arbeit war, NarE aus epidemiologischem und populationsbiologischem Blickwinkel zu betrachten. Insgesamt wurden 576 Meningokokkenisolate (109 Isolate aus der Stammsammlung des Instituts für Hygiene und Mikrobiologie Würzburg und 467 Isolate der Meningococcus Genome Library der Meningitis Research Foundation) auf das Vorhandensein von narE sowie auf Sequenzvariationen untersucht. Das Ergebnis zeigte den Besitz des Gens bei insgesamt 247 Stämmen. Bis auf zwei Punktmutationen waren alle untersuchten narE-Sequenzen identisch. Die narE-positiven Isolate konnten neun klonalen Komplexen zugeordnet werden. Zusätzlich wurde veranschaulicht, dass das Gen in Komplexen vorkommt, die verwandtschaftlich nicht eng miteinander verbunden sind. Mittels Western Blot konnte bei allen narE-positiven Meningokokken die Proteinexpression bestätigt werden, wobei ein signifikanter Unterschied zwischen Stämmen des cc32 und cc41/44 festzustellen war. Auf Transkriptionsebene konnte mittels qRT-PCR kein Unterschied zwischen diesen Komplexen ermittelt werden, so dass der Expressionsunterschied auf einem posttranskriptionellen Mechanismus beruhen muss. Neisseria gonorrhoeae ist ebenfalls im Besitz des Gens wie von Masignani et al. (2003) am Beispiel weniger Isolate beschrieben. In dieser Arbeit konnte für alle 29 getesteten Gonokokken die Insertion von vier Basenpaaren bestätigt werden, die zu einer Verschiebung im Leseraster führt, so dass NarE nicht exprimiert wird. Auch ein Neisseria sicca Stamm beinhaltet und exprimiert das narE-Gen. N2 - Bacterial ADP-ribosyltransferases have been described as toxins of numerous bacteria such as E. coli, V. cholerae and B. pertussis that cause posttranslational modifications of eukaryotic proteins with pathological effects in humans. The ADP-ribosyltransferase NarE from Neisseria has been identified on the basis of sequence homologies. The enzymatic activity of the protein has already been shown in studies. The aim of this work was to look at NarE from an epidemiological and population biological point of view. A total of 576 meningococcal isolates were investigated for the presence of narE and for sequence variations. The result showed the possession of the gene in a total of 247 strains. With the exception of two point mutations, all investigated narE sequences were identical. The narE-positive isolates could be assigned to nine clonal complexes. In addition, it was demonstrated that the gene occurs in complexes that are not closely related. Western blot confirmed protein expression in all narE-positive meningococci with a significant difference between cc32 and cc41/44 strains. At the transcriptional level, qRT-PCR could not detect any difference between these complexes, so that the expression difference must be based on a post-transcriptional mechanism. Neisseria gonorrhoeae is also in possession of the gene as described by Masignani et al. (2003) using the example of a few isolates. In this work, the insertion of four base pairs was confirmed for all 29 gonococci tested, which leads to a shift in the reading frame so that NarE is not expressed. A Neisseria sicca strain also contains and expresses the narE gene. KW - Neisseria meningitidis KW - ADP-Ribosyltransferasen KW - NarE KW - Virulenzfaktor KW - Meningokokken Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187803 ER - TY - THES A1 - Benke, Dominik T1 - Charakterisierung der T2-Ribonuklease des Fuchsbandwurms \(Echinococcus\) \(multilocularis\) T1 - Charcterisation of the T2-Ribonuclease of \(Echinococcus\) \(multilocularis\) N2 - Die alveoläre Echinokokkose ist eine lebensbedrohliche Erkrankung, die durch tumorartig in der Leber wachsende Larven (Metazestoden) des Fuchsbandwurms ausgelöst wird. Während Th1-dominierte Immunantworten zur Expulsion des Parasiten führen können, sind Th2-Antworten mit chronischer Infektion assoziiert. Über seine exkretorisch-sekretorischen Produkte (ESPs) nimmt Echinococcus multilocularis Einfluss auf die Polarisierung der Immunantwort. Allerdings ist bislang nur wenig über die zugrundeliegenden Mechanismen und aktiven Komponenten der ESPs bekannt. Die Immunmodulation durch Eier des Pärchenegels Schistosoma mansoni, der wie E. multilocularis zu den Plattwürmern gehört, ist dagegen schon besser charakterisiert. Hier hat omega-1, eine Ribonuklease der T2-Familie, Aufmerksamkeit als starker Induktor von Th2-Antworten und als Hepatotoxin erregt. Die Fragestellung dieser Arbeit war nun, ob die T2-RNase des Fuchsbandwurms (EmRNASET2) hinsichtlich ihrer Wirkungen auf Zellen des Immunsystems und der Leber Ähnlichkeiten mit omega-1 besitzt. Es konnte gezeigt werden, dass EmRNASET2 von allen Larvenstadien und auch vom adulten Wurm exprimiert wird. Der Einsatz polyklonaler Antikörper gegen rekombinant in Escherichia coli exprimierte recEmRNASET2 ermöglichte den Nachweis des Proteins in den ESPs von Primärzellen, die das frühe Stadium sich entwickelnder Metazestoden darstellen, und, wenngleich geringer ausgeprägt, in ESPs reifer Metazestoden. Zur Untersuchung einer möglichen immunmodulatorischen Wirkung wurden dendritische Zellen (DCs) aus murinem Knochenmark generiert und mit Überständen recEmRNASET2-produzierender HEK-Zellen exponiert. Diese zeigten im Vergleich zu Überständen von mit leerem Transfektionsvektor behandelten HEK-Zellen keine signifikante Inhibition der LPS-induzierten Reifung und Interleukin-12-Produktion von DCs, wie sie für omega-1 beschrieben ist. Auch ein Pilotexperiment mit der Leberzelllinie Hep3B lieferte keinen Anhalt für eine hepatotoxische Wirkung von EmRNASET2. Somit sprechen die Ergebnisse dieser Arbeit gegen eine funktionelle Verwandtschaft von EmRNASET2 und omega-1. Unterstützt wird diese Beobachtung durch eine orientierende phylogenetische Untersuchung, in der sich EmRNASET2 näher verwandt zu einer zweiten T2-RNase von S. mansoni zeigte. Omega-1 könnte also das Resultat einer Genduplikation mit anschließender Akquirierung immunmodulatorischer Funktionen sein. N2 - Alveolar Echinococcosis is a life-threatening disease caused by larvae (metacestodes) of the fox tapeworm, growing infiltratively in the liver. While Th1-dominated immune responses can lead to parasite expulsion, Th2-responses are associated with chronic infection. Through its excretory-secretory products (ESPs), Echinoccoccus multilocularis can influence polarisation of the immune response. However, little is known about the underlying mechanisms and active components of the ESPs. In contrast, immunomodulation by eggs of the blood fluke Schistosoma mansoni, a flat worm like E. multilocularis, has already been characterised more intensively and omega-1, a ribonuclease of the T2-family, has been identified as a major inductor of Th2-responses and as hepatotoxin. The aim of this study was to determine if the T2-RNase of E. multilocularis (EmRNASET2) has effects on cells of the immune system and the liver that are similar to those of omega-1. It could be shown that EmRNASET2 is expressed by all larval stages and by the adult worm. The use of polyclonal antibodies raised against the recombinantly expressed recEmRNASET2 allowed the detection of the protein in ESPs of primary cells, representing an early stage of developing metacestodes, and, however at a lower level, in ESPs of mature metacestodes. In order to address a potential immunomodulatory role, dendritic cells (DCs) were generated from murine bone marrow and exposed to supernatants of recEmRNASET2-producing HEK-cells. Compared to supernatants of HEK-cells transfected with the empty vector, conditioned medium did not show a significant inhibition of the LPS-induced maturation and interleukin-12 production of DCs, as described for omega-1. Moreover, a pilot experiment using the liver cell line Hep3B did not show evidence for a hepatotoxic effect of EmRNASET2. The results of this work therefore do not speak for a functional similarity of EmRNASET2 and omega-1. These observations are underlined by phylogenetic analyses, showing that EmRNASET2 clusters with a second T2-RNase from S. mansoni. Omega-1 could therefore be the result of a gene duplication event, with subsequent acquisition of its immunomodulatory functions. KW - Parasitologie KW - Immunologie KW - Alveoläre Echinokokkose KW - Ribonucleasen KW - Dendritische Zelle KW - Immunoparasitologie Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181064 ER - TY - JOUR A1 - Schlegel, Jan A1 - Peters, Simon A1 - Doose, Sören A1 - Schubert-Unkmeir, Alexandra A1 - Sauer, Markus T1 - Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites JF - Frontiers in Cell and Developmental Biology N2 - Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection. KW - Neisseria meningitidis KW - sphingolipids KW - gangliosides and lipid rafts KW - super-resolution microscopy KW - single-molecule tracking Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201639 VL - 7 IS - 194 ER - TY - JOUR A1 - Silwedel, Christine A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Speer, Christian P. A1 - Glaser, Kirsten T1 - More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells JF - Journal of Neuroinflammation N2 - Background Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death. Methods Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties. Results Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05). Conclusions By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp. KW - Ureaplasma urealyticum KW - Ureaplasma parvum KW - Neuroinflammation KW - Meningitis KW - Caspase KW - Apoptosis KW - HBMEC Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200711 VL - 16 ER - TY - JOUR A1 - Walther, Grit A1 - Wagner, Lysett A1 - Kurzai, Oliver T1 - Updates on the taxonomy of Mucorales with an emphasis on clinically important taxa JF - Journal of Fungi N2 - Fungi of the order Mucorales colonize all kinds of wet, organic materials and represent a permanent part of the human environment. They are economically important as fermenting agents of soybean products and producers of enzymes, but also as plant parasites and spoilage organisms. Several taxa cause life-threatening infections, predominantly in patients with impaired immunity. The order Mucorales has now been assigned to the phylum Mucoromycota and is comprised of 261 species in 55 genera. Of these accepted species, 38 have been reported to cause infections in humans, as a clinical entity known as mucormycosis. Due to molecular phylogenetic studies, the taxonomy of the order has changed widely during the last years. Characteristics such as homothallism, the shape of the suspensors, or the formation of sporangiola are shown to be not taxonomically relevant. Several genera including Absidia, Backusella, Circinella, Mucor, and Rhizomucor have been amended and their revisions are summarized in this review. Medically important species that have been affected by recent changes include Lichtheimia corymbifera, Mucor circinelloides, and Rhizopus microsporus. The species concept of Rhizopus arrhizus (syn. R. oryzae) is still a matter of debate. Currently, species identification of the Mucorales is best performed by sequencing of the internal transcribed spacer (ITS) region. Ecologically, the Mucorales represent a diverse group but for the majority of taxa, the ecological role and the geographic distribution remain unknown. Understanding the biology of these opportunistic fungal pathogens is a prerequisite for the prevention of infections, and, consequently, studies on the ecology of the Mucorales are urgently needed. KW - Mucorales KW - taxonomy KW - pathogens KW - identification KW - ecology KW - Circinella KW - Lichtheimia KW - Mucor KW - Rhizomucor KW - Rhizopus Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193081 SN - 2309-608X VL - 5 IS - 4 ER - TY - THES A1 - von Papen, Hans Michael T1 - Untersuchungen zum Einfluss der Meningokokkeninfektion auf den Zellzyklus von Epithelzellen T1 - Disease and carrier isolates of Neisseria meningitidis cause G1 cell cycle arrest in human epithelial cells N2 - Zahlreiche humanpathogene bakterielle Erreger können ihre Fähigkeit zur Kolonisation epithelialer Barrieren optimieren, indem sie mit dem Zellzyklus der infizierten Wirtszelle in Wechselwirkung treten und so die Abschilferung und Erneuerung des Epithels verzögern. Die hierbei wirksamen bakteriellen Effektoren sind als „Cyclomoduline“ bekannt und gelten als neue Klasse bakterieller Pathogenitätsfaktoren. Ziel der vorliegenden Promotionsarbeit war es zu untersuchen, ob durch die Infektion menschlicher pharyngealer Epithelzellen mit N. meningitidis der Zellzyklus der Wirtszelle beeinflusst wird. Mit zwei verschiedenen Untersuchungsmethoden konnte übereinstimmend gezeigt werden, dass die Infektion der Epithelzelllinie Detroit 562 mit verschiedenen Meningokokkenisolaten zu einer signifikanten Akkumulation von Epithelzellen in der G1-Phase führte. Dieser Effekt wurde sowohl von pathogenen Meningokokkenstämmen als auch von Trägerstämmen ausgelöst, jedoch nur durch Isolate, die fähig zur Adhärenz und zur Invasion in die Epithelzelle waren. Durch Hitzebehandlung der Bakterien konnte der Zellzyklusarrest vollständig aufgehoben werden. Ebenso konnte der Effekt durch Inkubation der Epithelzellen mit bakteriellen Kulturüberständen und durch Infektion der Zellen mit E. coli-Stämmen, welche die Meningokokkenadhäsine Opa und Opc überexprimieren, nicht ausgelöst werden. Es konnte weiterhin nachgewiesen werden, dass die Infektion mit N. meningitidis in der Zielzelle zu einer signifikant gesteigerten Expression des CDK-Inhibitors p21WAF1/Cip1 führte, begleitet von einer vermehrten Lokalisation im Zellkern. Auch zeigte sich eine veränderte Proteinexpression der für die G1-Phase relevanten Cycline D und E. Diese scheint sich erst posttranslational zu ereignen, da die unterschiedliche Expression auf mRNA-Ebene nicht festgestellt werden konnte. Zusammenfassend konnte dargestellt werden, dass die Infektion von Pharynxepithelzellen mit lebenden, zur Adhärenz und Invasion fähigen Meningokokkenstämmen in der menschlichen Zielzelle einen Zellzyklusarrest in der G1-Phase verursacht, vermutlich durch veränderte Expression der Zellzyklusregulatoren p21WAF1/Cip1, Cyclin D und Cyclin E. Möglicherweise stellt die Induktion dieses Zellzyklusarrestes einen wichtigen Schritt in der Pathogenese der bakteriellen Kolonisation des oberen Atemwegsepithels durch N. meningitidis dar. N2 - Several microbial pathogens have developed mechanisms to modulate host cell cycle progression in order to improve bacterial colonization of epithelial barriers. The required bacterial effectors were summarized as “cyclomodulins” and have been proposed to be a new class of virulence factors. The objective of this doctoral research study was to analyze the capability of N. meningitidis to interfere with the cell cycle progression in human pharyngeal epithelial cells. Using two different methods for cell cycle analysis, we show that infection of the human pharyngeal epithelial cell line Detroit 562 with different meningococcal isolates induces an arrest of epithelial host cells in the G1 phase. This effect was caused by infection with both pathogenic isolates and carriage isolates, but only by strains able to adhere to and to invade into the host cells. Heat-inactivation of the bacteria prior to infection completely prevented the cell cycle arrest. Moreover treatment of epithelial cells with bacterial supernatants, as well as infection with E. coli strains expressing neisserial adhesins Opa and Opc did not induce the cell cycle arrest. We further demonstrate that infection of Detroit 562 cells with N. meningitidis leads to a significantly increased expression of the CDK-inhibitor p21WAF1/Cip1 in the host cell, as well as its increased nuclear localization. The protein expression of cyclin D and E, which are relevant for progression through the G1 phase, were altered by bacterial infection, too. This effect is most likely induced by posttranslational modification, since bacterial infection did not affect Cyclin D and E mRNA levels. In conclusion, we demonstrate that infection of human pharyngeal epithelial cells with different isolates of N. meningitidis arrests the host cells at the G1 phase, most likely by affecting the expression of the cell cycle regulators p21WAF1/Cip1, cyclin D and Cyclin E. Potentially, induction this cell cycle arrest is an important step in the pathogenesis of meningococcal colonization and further infection. KW - Neisseria meningitidis KW - Zellzyklus KW - Epithel KW - cell cycle Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192862 ER - TY - JOUR A1 - Gomes, Sara F. Martins A1 - Westermann, Alexander J. A1 - Sauerwein, Till A1 - Hertlein, Tobias A1 - Förstner, Konrad U. A1 - Ohlsen, Knut A1 - Metzger, Marco A1 - Shusta, Eric V. A1 - Kim, Brandon J. A1 - Appelt-Menzel, Antje A1 - Schubert-Unkmeir, Alexandra T1 - Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection JF - Frontiers in Microbiology N2 - Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs. KW - Neisseria meningitidis KW - meningococcus KW - bacteria KW - stem cells KW - blood-cerebrospinal fluid barrier KW - blood-brain barrier KW - brain endothelial cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201562 VL - 10 IS - 1181 ER - TY - JOUR A1 - Muenstermann, Marcel A1 - Strobel, Lea A1 - Klos, Andreas A1 - Wetsel, Rick A. A1 - Woodruff, Trent M. A1 - Köhl, Jörg A1 - Johswich, Kay O. T1 - Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections JF - Virulence N2 - The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar\(^{-/-}\) mice, whereas C5ar1\(^{-/-}\) and C5ar2\(^{-/-}\) mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1\(^{-/-}\) mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8\(^{Δ71−73}\)) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a “super-agonist” peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis. KW - inflammation KW - C3a KW - C5a KW - C3aR KW - C5aR1 KW - C5aR2 KW - meningococcal disease KW - sepsis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200496 VL - 10 IS - 1 ER - TY - JOUR A1 - Herz, Michaela A1 - Brehm, Klaus T1 - Evidence for densovirus integrations into tapeworm genomes JF - Parasites & Vectors N2 - Background Tapeworms lack a canonical piRNA-pathway, raising the question of how they can silence existing mobile genetic elements (MGE). Investigation towards the underlying mechanisms requires information on tapeworm transposons which is, however, presently scarce. Methods The presence of densovirus-related sequences in tapeworm genomes was studied by bioinformatic approaches. Available RNA-Seq datasets were mapped against the Echinococcus multilocularis genome to calculate expression levels of densovirus-related genes. Transcription of densovirus loci was further analyzed by sequencing and RT-qPCR. Results We herein provide evidence for the presence of densovirus-related elements in a variety of tapeworm genomes. In the high-quality genome of E. multilocularis we identified more than 20 individual densovirus integration loci which contain the information for non-structural and structural virus proteins. The majority of densovirus loci are present as head-to-tail concatemers in isolated repeat containing regions of the genome. In some cases, unique densovirus loci have integrated close to histone gene clusters. We show that some of the densovirus loci of E. multilocularis are actively transcribed, whereas the majority are transcriptionally silent. RT-qPCR data further indicate that densovirus expression mainly occurs in the E. multilocularis stem cell population, which probably forms the germline of this organism. Sequences similar to the non-structural densovirus genes present in E. multilocularis were also identified in the genomes of E. canadensis, E. granulosus, Hydatigera taeniaeformis, Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana, Taenia asiatica, Taenia multiceps, Taenia saginata and Taenia solium. Conclusions Our data indicate that densovirus integration has occurred in many tapeworm species. This is the first report on widespread integration of DNA viruses into cestode genomes. Since only few densovirus integration sites were transcriptionally active in E. multilocularis, our data are relevant for future studies into gene silencing mechanisms in tapeworms. Furthermore, they indicate that densovirus-based vectors might be suitable tools for genetic manipulation of cestodes. KW - Echinococcus KW - Echinococcosis KW - Densovirus KW - Parvovirus KW - Mobile genetic element KW - Gene silencing KW - Stem cell KW - Epigenetic Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202478 VL - 12 ER - TY - JOUR A1 - Kim, Brandon J. A1 - McDonagh, Maura A. A1 - Deng, Liwen A1 - Gastfriend, Benjamin D. A1 - Schubert-Unkmeir, Alexandra A1 - Doran, Kelly S. A1 - Shusta, Eric V. T1 - Streptococcus agalactiae disrupts P-glycoprotein function in brain endothelial cells JF - Fluids and Barriers of the CNS N2 - Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood–borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood–brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS. KW - Group B Streptococcus KW - Streptococcus agalactiae KW - Brain endothelial cells KW - P-glycoprotein KW - Efflux transport KW - Meningitis KW - Stem cells KW - P-gp Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201895 VL - 16 ER - TY - JOUR A1 - Kim, Brandon J. A1 - Shusta, Eric V. A1 - Doran, Kelly S. T1 - Past and current perspectives in modeling bacteria and blood–brain barrier interactions JF - Frontiers in Microbiology N2 - The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future. KW - bacteria KW - blood–brain barrier KW - meningitis KW - stem cells KW - brain endothelial cell Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201766 VL - 10 IS - 1336 ER - TY - JOUR A1 - Claus, Heike A1 - Hubert, Kerstin A1 - Becher, Dörte A1 - Otto, Andreas A1 - Pawlik, Marie-Christin A1 - Lappann, Ines A1 - Strobel, Lea A1 - Vogel, Ulrich A1 - Johswich, Kay T1 - A homopolymeric adenosine tract in the promoter region of nspA influences factor H-mediated serum resistance in Neisseria meningitidis JF - Scientific Reports N2 - Although usually asymptomatically colonizing the human nasopharynx, the Gram-negative bacterium Neisseria meningitidis (meningococcus) can spread to the blood stream and cause invasive disease. For survival in blood, N. meningitidis evades the complement system by expression of a polysaccharide capsule and surface proteins sequestering the complement regulator factor H (fH). Meningococcal strains belonging to the sequence type (ST-) 41/44 clonal complex (cc41/44) cause a major proportion of serogroup B meningococcal disease worldwide, but they are also common in asymptomatic carriers. Proteome analysis comparing cc41/44 isolates from invasive disease versus carriage revealed differential expression levels of the outer membrane protein NspA, which binds fH. Deletion of nspA reduced serum resistance and NspA expression correlated with fH sequestration. Expression levels of NspA depended on the length of a homopolymeric tract in the nspA promoter: A 5-adenosine tract dictated low NspA expression, whereas a 6-adenosine motif guided high NspA expression. Screening German cc41/44 strain collections revealed the 6-adenosine motif in 39% of disease isolates, but only in 3.4% of carriage isolates. Thus, high NspA expression is associated with disease, but not strictly required. The 6-adenosine nspA promoter is most common to the cc41/44, but is also found in other hypervirulent clonal complexes. KW - Meningitis KW - Pathogens Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200956 VL - 9 ER - TY - JOUR A1 - Silwedel, Christine A1 - Speer, Christian P. A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Schlegel, Nicolas A1 - Glaser, Kirsten T1 - Ureaplasma species modulate cytokine and chemokine responses in human brain microvascular endothelial cells JF - International Journal of Molecular Science N2 - Ureaplasma species are common colonizers of the adult genitourinary tract and often considered as low-virulence commensals. Intraamniotic Ureaplasma infections, however, facilitate chorioamnionitis and preterm birth, and cases of Ureaplasma-induced neonatal sepsis, pneumonia, and meningitis raise a growing awareness of their clinical relevance. In vitro studies are scarce but demonstrate distinct Ureaplasma-driven impacts on immune mechanisms. The current study addressed cytokine and chemokine responses upon exposure of native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) to Ureaplasma urealyticum or U. parvum, using qRT-PCR, RNA sequencing, multi-analyte immunoassay, and flow cytometry. Ureaplasma exposure in native HBMEC reduced monocyte chemoattractant protein (MCP)-3 mRNA expression (p < 0.01, vs. broth). In co-stimulated HBMEC, Ureaplasma spp. attenuated LPS-evoked mRNA responses for C-X-C chemokine ligand 5, MCP-1, and MCP-3 (p < 0.05, vs. LPS) and mitigated LPS-driven interleukin (IL)-1α protein secretion, as well as IL-8 mRNA and protein responses (p < 0.05). Furthermore, Ureaplasma isolates increased C-X-C chemokine receptor 4 mRNA levels in native and LPS co-stimulated HBMEC (p < 0.05). The presented results may imply immunomodulatory capacities of Ureaplasma spp. which may ultimately promote chronic colonization and long-term neuroinflammation. KW - Ureaplasma urealyticum KW - Ureaplasma parvum KW - neuroinflammation KW - meningitis KW - blood–brain barrier KW - HBMEC Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201848 SN - 1422-0067 VL - 20 IS - 14 ER - TY - JOUR A1 - Springer, Jan A1 - Walther, Grit A1 - Rickerts, Volker A1 - Hamprecht, Axel A1 - Willinger, Birgit A1 - Teschner, Daniel A1 - Einsele, Hermann A1 - Kurzai, Oliver A1 - Loeffler, Juergen T1 - Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR JF - Journal of Fungi N2 - The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens. KW - probe-based real-time PCR KW - Fusarium KW - bronchoalveolar lavage fluid KW - fungal molecular diagnostics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193111 SN - 2309-608X VL - 5 IS - 4 ER - TY - JOUR A1 - Drayß, Maria A1 - Claus, Heike A1 - Hubert, Kerstin A1 - Thiel, Katrin A1 - Berger, Anja A1 - Sing, Andreas A1 - van der Linden, Mark A1 - Vogel, Ulrich A1 - Lâm, Thiên-Trí T1 - Asymptomatic carriage of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Group A Streptococcus and Staphylococcus aureus among adults aged 65 years and older JF - PLoS ONE N2 - Objective The aim of this study was to determine the prevalence of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, group A Streptococcus (GAS), and Staphylococcus aureus in asymptomatic elderly people and to unravel risk factors leading to colonization. Methods A multi-centre cross-sectional study was conducted including 677 asymptomatic adults aged 65 years or more, living at home or in nursing homes. Study areas were Greater Aachen (North-Rhine-Westphalia) and Wuerzburg (Bavaria), both regions with medium to high population density. Nasal and oropharyngeal swabs as well as questionnaires were collected from October 2012 to May 2013. Statistical analysis included multiple logistic regression models. Results The carriage rate was 1.9% ([95%CI: 1.0–3.3%]; 13/677) for H. influenzae, 0.3% ([95%CI: 0–1.1%]; 2/677) for N. meningitidis and 0% ([95% CI: 0–0.5%]; 0/677) for S. pneumoniae and GAS. Staphylococcus aureus was harboured by 28.5% of the individuals ([95% CI: 25.1–32.1%]; 193/677) and 0.7% ([95% CI: 0.2–1.7%]; 5/677) were positive for methicillin-resistant S. aureus. Among elderly community-dwellers colonization with S. aureus was significantly associated with higher educational level (adjusted OR: 1.905 [95% CI: 1.248–2.908]; p = 0.003). Among nursing home residents colonization was associated with being married (adjusted OR: 3.367 [1.502–7.546]; p = 0.003). Conclusion The prevalence of N. meningitidis, H. influenzae, S. pneumoniae and GAS was low among older people in Germany. The S. aureus rate was expectedly high, while MRSA was found in less than 1% of the individuals. KW - Geriatric care KW - Geriatrics KW - Elderly KW - Staphylococcus aureus KW - Nursing homes KW - Haemophilus influenzae KW - Neisseria meningitidis KW - Methicillin-resistant Staphylococcus aureus Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201042 VL - 14 IS - 2 ER - TY - JOUR A1 - Luber, Verena A1 - Lutz, Mathias A1 - Abele-Horn, Marianne A1 - Einsele, Hermann A1 - Grigoleit, Götz Ulrich A1 - Mielke, Stephan T1 - Excretion of Ascaris lumbricoides following reduced‐intensity allogeneic hematopoietic stem cell transplantation and consecutive treatment with mebendazole JF - Transplant Infectious Disease N2 - Here, we present the unique case of a 51‐year‐old German patient with multiple myeloma excreting Ascaris lumbricoides in his stool five weeks after allogeneic hematopoietic stem cell transplantation. Stool analysis remained negative for the presence of eggs, and there was no eosinophilia in the peripheral blood at any time around stem cell transplantation. The patient was commenced on a three‐day treatment with mebendazole, which was well tolerated. No serious interactions with the concomitant post‐transplant medication or negative effects on the hematopoiesis were observed, and the myeloma still is in complete remission. To our knowledge, this is the first report on excretion of A lumbricoides in the context of allogeneic stem cell transplantation. The case is remarkable with view to the fact that the parasite has supposedly survived all courses of myeloma treatment including autologous and allogeneic conditioning. Parasitosis with A lumbricoides has a worldwide prevalence of about a billion and is extremely rare in northern Europe. Possibly the patient got infected during a trip to Egypt years before multiple myeloma was diagnosed. KW - sirolimus KW - mycophenolic acid KW - multiple myeloma KW - mebendazole KW - hematopoietic stem cell transplantation KW - Ascaris lumbricoides Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219608 SN - 1399-3062 VL - 22 IS - 1 ER - TY - JOUR A1 - Abimannan, Nagarajan A1 - Sumathi, G. A1 - Krishnarajasekhar, O. R. A1 - Sinha, Bhanu A1 - Krishnan, Padma T1 - Clonal Clusters and Virulence Factors of Methicillin-Resistant \(Staphylococcus\) \(Aureus\): Evidence for Community-Acquired Methicillin-Resistant \(Staphylococcus\) \(Aureus\) Infiltration into Hospital Settings in Chennai, South India JF - Indian Journal of Medical Microbiology N2 - Background and Objective: Staphylococcus aureus is one of the major pathogens of nosocomial infections as wells as community-acquired (CA) infections worldwide. So far, large-scale comprehensive molecular and epidemiological characterisation of S. aureus from very diverse settings has not been carried out in India. The objective of this study is to evaluate the molecular, epidemiological and virulence characteristics of S. aureus in both community and hospital settings in Chennai, southern India. Methods: S. aureus isolates were obtained from four different groups (a) healthy individuals from closed community settings, (b) inpatients from hospitals, (c) outpatients from hospitals, representing isolates of hospital-community interface and (d) HIV-infected patients to define isolates associated with the immunocompromised. Antibiotic susceptibility testing, multiplex polymerase chain reactions for detection of virulence and resistance determinants, molecular typing including Staphylococcal cassette chromosome mec (SCCmec) and agr typing, were carried out. Sequencing-based typing was done using spa and multilocus sequence typing (MLST) methods. Clonal complexes (CC) of hospital and CA methicillin-resistant S. aureus (MRSA) were identified and compared for virulence and resistance. Results and Conclusion: A total of 769 isolates of S. aureus isolates were studied. The prevalence of MRSA was found to be 7.17%, 81.67%, 58.33% and 22.85% for groups a, b, c and d, respectively. Of the four SCCmec types (I, III, IV and V) detected, SCCmec V was found to be predominant. Panton-Valentine leucocidin toxin genes were detected among MRSA isolates harbouring SCCmec IV and V. A total of 78 spa types were detected, t657 being the most prevalent. 13 MLST types belonging to 9 CC were detected. CC1 (ST-772, ST-1) and CC8 (ST238, ST368 and ST1208) were found to be predominant among MRSA. CA-MRSA isolates with SCCmec IV and V were isolated from all study groups including hospitalised patients and were found to be similar by molecular tools. This shows that CA MRSA has probably infiltrated into the hospital settings. KW - Community-acquired methicillin-resistant Staphylococcus aureus KW - HIV KW - hospital-acquired methicillin-resistant Staphylococcus aureus KW - innate immune evasions KW - MLST KW - microbial surface component recognising adhesive matrix molecules KW - spa typing KW - ST 772 KW - Inducible Clindamycin Resistance KW - Valentine Leukocidin Genes KW - Multiplex PCR KW - Nasal Carriage KW - Colonization KW - Prevalence KW - Emergence KW - Skin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226963 VL - 37 IS - 3 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Mshana, Stephen E. T1 - The role of patients and healthcare workers Staphylococcus aureus nasal colonization in occurrence of surgical site infection among patients admitted in two centers in Tanzania JF - Antimicrobial Resistance & Infection Control N2 - Background Colonization with Staphylococcus aureus has been identified as a risk for subsequent occurrence of infection. This study investigated the relationship between S. aureus colonization of patients and healthcare workers (HCWs), and subsequent surgical site infections (SSI). Methods Between December 2014 and September 2015, a total of 930 patients and 143 HCWs were enrolled from the Bugando Medical Centre and Sekou Toure hospital in Mwanza, Tanzania. On admission and discharge nasal swabs, with an additional of wound swab for those who developed SSI were collected from patients whereas HCWs were swabbed once. Identification and antimicrobial susceptibility testing were done by VITEK-MS and VITEK-2, respectively. Detection of Panton Valentine leukocidin (PVL) and mecA genes was done by PCR. S. aureus isolates were further characterized by spa typing and Multi-Locus Sequence Typing (MLST). Results Among 930 patients screened for S. aureus on admission, 129 (13.9%) were positive of which 5.4% (7/129) were methicillin-resistant S. aureus (MRSA). Amongst 363 patients rescreened on discharge, 301 patients had been tested negative on admission of whom 29 (9.6%) turned positive after their hospital stay. Three (10.3%) of the 29 acquired S. aureus were MRSA. Inducible Clindamycin resistance occurred more often among acquired S. aureus isolates than among isolates from admission [34.5% (10/29) vs. 17.1% (22/129), P = 0.018]. S. aureus contributed to 21.1% (n = 12) of the 57 cases of investigated SSIs among 536 patients followed. Seven out of eight S. aureus carriage/infection pairs had the same spa and sequence types. The previously reported dominant PVL-positive ST88 MRSA strain with spa type t690 was detected in patients and HCW. Conclusion A significant proportion of patients acquired S. aureus during hospitalization. The finding of more than 90% of S. aureus SSI to be of endogenous source underscores the need of improving infection prevention and control measures including screening and decolonization of high risk patients. KW - S. aureus KW - colonization KW - surgical site infection KW - Tanzania Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224185 VL - 8 ER - TY - JOUR A1 - Al-Zaben, Naim A1 - Medyukhina, Anna A1 - Dietrich, Stefanie A1 - Marolda, Alessandra A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Figge, Marc Thilo T1 - Automated tracking of label-free cells with enhanced recognition of whole tracks JF - Scientific Reports N2 - Migration and interactions of immune cells are routinely studied by time-lapse microscopy of in vitro migration and confrontation assays. To objectively quantify the dynamic behavior of cells, software tools for automated cell tracking can be applied. However, many existing tracking algorithms recognize only rather short fragments of a whole cell track and rely on cell staining to enhance cell segmentation. While our previously developed segmentation approach enables tracking of label-free cells, it still suffers from frequently recognizing only short track fragments. In this study, we identify sources of track fragmentation and provide solutions to obtain longer cell tracks. This is achieved by improving the detection of low-contrast cells and by optimizing the value of the gap size parameter, which defines the number of missing cell positions between track fragments that is accepted for still connecting them into one track. We find that the enhanced track recognition increases the average length of cell tracks up to 2.2-fold. Recognizing cell tracks as a whole will enable studying and quantifying more complex patterns of cell behavior, e.g. switches in migration mode or dependence of the phagocytosis efficiency on the number and type of preceding interactions. Such quantitative analyses will improve our understanding of how immune cells interact and function in health and disease. KW - image processing KW - software Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221093 VL - 9 ER - TY - THES A1 - Aumann, Ralf T1 - Vorkommen und Expression des opcA Gens in Meningokokkenstämmen von Erkrankten und asymptomatischen Trägern T1 - Prevalence and expression of the opcA gene in meningococci from invasive and carrier strains N2 - Das Opc-Protein ist ein Außenmembranprotein von Meningokokken, das über extrazelluläre Matrixproteine mit Integrinen der Wirtszelle interagiert. Opc ist in Menschen immunogen und induziert bakterizide Antikörper. Das Opc-Protein wurde daher als aussichtsreicher Impfstoff-Kandidat angesehen, da es außerdem relativ gut konserviert ist. Allerdings wird das Opc-Protein nicht von allen Meningokokkenstämmen exprimiert. Einerseits fehlt das opc-Gen in einigen klonalen Komplexen (z.B. ST-8, ST-11, ST-53), andererseits ist die Opc-Expression nicht konstitutiv wegen einer phasenvariablen Transkription, die auf einem Poly-Cytidin-Bereich im Promotor des opc-Gens beruht. In dieser Arbeit wurde die Präsenz des opc-Gens und die Opc-Expression in zwei großen Sammlungen deutscher Meningokokkenisolate von invasiven Erkrankungen (n=1141) und gesunden Trägern (n=792) untersucht. Das opc-Gen war bei 71% der invasiven und 77% der Trägerstämme nachweisbar. Der größte Teil der opc-Gen negativen Stämme gehörte zu den klonalen Komplexen ST-8, ST-11, ST-213, ST-231, ST-334 und ST-53. Der Anteil opc-positiver Stämme, die Opc in vitro exprimieren, war bei den invasiven Stämmen kleiner als bei den Trägerstämmen (13% vs. 29%, p<0,001, Chi-square-Test). Der größere Anteil Opc-exprimierender Trägerstämme ist u.a. am ehesten mit der Überrepräsentation von wenig pathogenen klonalen Komplexen (ST-23, ST-35, ST-198) mit einer hohen Opc-Expressionsrate zu erklären. 24 von den 176 invasiven Stämmen mit einer Anzahl von 11 - 14 Cs in der Promotor-Region, die die Opc-Expression begünstigt, zeigten weder im ELISA noch im Westernblot eine Opc-Expression. Bei 14 dieser 24 Stämme wurde als Ursache ein phasenvariabler, intragenischer Poly-Adenin-Bereich identifiziert, der zu einer Leserasterverschiebung führte. Die Vermutung mehrerer Autoren, dass die Opc-Expression mit dem klinischen Bild der Meningitis verknüpft ist, konnte mit der hier genutzten großen Stammsammlung nicht bestätigt werden. Invasive Stämme, die das Opc-Protein exprimierten, wurden genauso häufig von Patienten mit dem klinischen Bild der Meningitis isoliert wie Stämme, die das Opc-Protein nicht exprimierten (46% vs. 47%, Chi-square-Test: p<0,9). Allerdings gibt es eine starke Assoziation der Gegenwart des opc-Gens mit dem klinischen Merkmal Meningitis. Dieser Befund gibt Anlass zu der Hypothese, dass in vitro und in vivo Expression von Opc sich unterscheiden. Zusammenfassend lässt sich festhalten, dass das Opc-Protein nur in 19,8% aller Isolate (invasive und Trägerstämme zusammengenommen) exprimiert wurde. Es zeigte sich eine Tendenz zu häufigerer Opc-Expression in apathogenen Trägerisolaten. Das Vorhandensein des opc-Gens, nicht aber die in vitro Expression konnten mit dem klinischen Merkmal Meningitis assoziiert werden. Zusätzlich wurde ein weiterer Mechanismus der intragenischen Phasenvariation beschrieben. N2 - Presence of opc was associated with meningitis, mostly because ST-11/ST-8 cc meningococci with low meningitis rates were consistently opc negative. On the other hand, lack of opc did not exclude meningitis. Opc was expressed in only 13% of all invasive isolates. In vitro Opc expression was not associated with meningitis. Limitation: Definite conclusion about expression in vivo is not possible with cultured isolates. Evidence for intragenic opc phase-variation was provided. KW - Neisseria meningitidis KW - opc KW - Medizin KW - Mikrobiologie KW - Meningitis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157278 ER - TY - JOUR A1 - Herrmann, Johannes A1 - Muenstermann, Marcel A1 - Strobel, Lea A1 - Schubert-Unkmeir, Alexandra A1 - Woodruff, Trent M. A1 - Gray-Owen, Scott D. A1 - Klos, Andreas A1 - Johswich, Kay O. T1 - Complement C5a receptor 1 exacerbates the pathophysiology of N. meningitidis sepsis and is a potential target for disease treatment JF - mBio N2 - Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1\(^{-/-}\) mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1\(^{-/-}\) mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy. Importance: The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease. KW - C5aR1 KW - whole-blood model KW - Neisseria meningitidis KW - anaphylatoxins KW - complement system KW - inflammation KW - invasive disease KW - mouse model KW - neutrophils KW - sepsis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175792 VL - 9 IS - 1 ER - TY - JOUR A1 - Silwedel, Christine A1 - Speer, Christian P. A1 - Haarmann, Axel A1 - Fehrholz, Markus A1 - Claus, Heike A1 - Buttmann, Mathias A1 - Glaser, Kirsten T1 - Novel insights into neuroinflammation: bacterial lipopolysaccharide, tumor necrosis factor α, and Ureaplasma species differentially modulate atypical chemokine receptor 3 responses in human brain microvascular endothelial cells JF - Journal of Neuroinflammation N2 - Background: Atypical chemokine receptor 3 (ACKR3, synonym CXCR7) is increasingly considered relevant in neuroinflammatory conditions, in which its upregulation contributes to compromised endothelial barrier function and may ultimately allow inflammatory brain injury. While an impact of ACKR3 has been recognized in several neurological autoimmune diseases, neuroinflammation may also result from infectious agents, including Ureaplasma species (spp.). Although commonly regarded as commensals of the adult urogenital tract, Ureaplasma spp. may cause invasive infections in immunocompromised adults as well as in neonates and appear to be relevant pathogens in neonatal meningitis. Nonetheless, clinical and in vitro data on Ureaplasma-induced inflammation are scarce. Methods: We established a cell culture model of Ureaplasma meningitis, aiming to analyze ACKR3 variances as a possible pathomechanism in Ureaplasma-associated neuroinflammation. Non-immortalized human brain microvascular endothelial cells (HBMEC) were exposed to bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), and native as well as LPS-primed HBMEC were cultured with Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). ACKR3 responses were assessed via qRT-PCR, RNA sequencing, flow cytometry, and immunocytochemistry. Results: LPS, TNF-α, and Ureaplasma spp. influenced ACKR3 expression in HBMEC. LPS and TNF-α significantly induced ACKR3 mRNA expression (p < 0.001, vs. control), whereas Ureaplasma spp. enhanced ACKR3 protein expression in HBMEC (p < 0.01, vs. broth control). Co-stimulation with LPS and either Ureaplasma isolate intensified ACKR3 responses (p < 0.05, vs. LPS). Furthermore, stimulation wielded a differential influence on the receptor’s ligands. Conclusions: We introduce an in vitro model of Ureaplasma meningitis. We are able to demonstrate a pro-inflammatory capacity of Ureaplasma spp. in native and, even more so, in LPS-primed HBMEC, underlining their clinical relevance particularly in a setting of co-infection. Furthermore, our data may indicate a novel role for ACKR3, with an impact not limited to auto-inflammatory diseases, but extending to infection-related neuroinflammation as well. AKCR3-induced blood-brain barrier breakdown might constitute a potential common pathomechanism. KW - atypical chemokine receptor 3 KW - human brain microvascular endothelial cells KW - meningitis KW - neuroinflammation KW - Ureaplasma species Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175952 VL - 15 IS - 156 ER - TY - JOUR A1 - Strobel, Lea A1 - Johswich, Kay O. T1 - Anticoagulants impact on innate immune responses and bacterial survival in whole blood models of Neisseria meningitidis infection JF - Scientific Reports N2 - Neisseria meningitidis (meningococcus) causes invasive diseases such as meningitis or septicaemia. Ex vivo infection of human whole blood is a valuable tool to study meningococcal virulence factors and the host innate immune responses. In order to consider effects of cellular mediators, the coagulation cascade must be inhibited to avoid clotting. There is considerable variation in the anticoagulants used among studies of N. meningitidis whole blood infections, featuring citrate, heparin or derivatives of hirudin, a polypeptide from leech saliva. Here, we compare the influence of these three different anticoagulants, and additionally Mg/EGTA, on host innate immune responses as well as on viability of N. meningitidis strains isolated from healthy carriers and disease cases, reflecting different sequence types and capsule phenotypes. We found that the anticoagulants significantly impact on cellular responses and, strain-dependently, also on bacterial survival. Hirudin does not inhibit complement and is therefore superior over the other anticoagulants; indeed hirudin-plasma most closely reflects the characteristics of serum during N. meningitidis infection. We further demonstrate the impact of heparin on complement activation on N. meningitidis and its consequences on meningococcal survival in immune sera, which appears to be independent of the heparin binding antigens Opc and NHBA. KW - infection KW - pathogens KW - Neisseria meningitidis KW - anticoagulants Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176226 VL - 8 IS - 10225 ER - TY - JOUR A1 - Ruf, Dominik A1 - Brantl, Victor A1 - Wagener, Johannes T1 - Mitochondrial Fragmentation in \(Aspergillus\) \(fumigatus\) as Early Marker of Granulocyte Killing Activity JF - Frontiers in Cellular and Infection Microbiology N2 - The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. KW - Aspergillus fumigatus KW - killing KW - assay KW - PMNs KW - granulocytes KW - mitochondria KW - mitochondrial morphology KW - fungicidal activity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227133 VL - 8 IS - 128 ER - TY - JOUR A1 - Prauße, Maria T. E. A1 - Lehnert, Teresa A1 - Timme, Sandra A1 - Hünniger, Kerstin A1 - Leonhardt, Ines A1 - Kurzai, Oliver A1 - Figge, Marc Thilo T1 - Predictive Virtual Infection Modeling of Fungal Immune Evasion in Human Whole Blood JF - Frontiers in Immunology N2 - Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata. However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism. KW - immune evasion KW - state-based model KW - innate immune response KW - polymorphonuclear neutrophils KW - whole-blood infection assay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197493 SN - 1664-3224 VL - 9 IS - 560 ER - TY - JOUR A1 - Kohlmorgen, Britta A1 - Elias, Johannes A1 - Schoen, Christoph T1 - Improved performance of the artus Mycobacterium tuberculosis RG PCR kit in a low incidence setting: a retrospective monocentric study JF - Scientific Reports N2 - Tuberculosis (TB) and the spread of Mycobacterium tuberculosis complex (MTBC) strains resistant against rifampin (RIF) and isoniazid (INH) pose a serious threat to global health. However, rapid and reliable MTBC detection along with RIF/INH susceptibility testing are challenging in low prevalence countries due to the higher rate of false positives. Here, we provide the first performance data for the artus MTBC PCR assay in a low prevalence setting. We analyze 1323 respiratory and 311 non-respiratory samples with the artus MTBC PCR assay as well as by mycobacterial culture and microscopy. We propose retesting of specimens in duplicate and consideration of a determined cycle-threshold value cut-off greater than 34, as this significantly increases accuracy, specificity, and negative predictive value without affecting sensitivity. Furthermore, we tested fourteen MTBC positive samples with the GenoType MTBDRplus test and demonstrate that using an identical DNA extraction protocol for both assays does not impair downstream genotypic testing for RIF and INH susceptibility. In conclusion, our procedure optimizes the use of the artus MTB assay with workload efficient methods in a low incidence setting. Combining the modified artus MTB with the GenoType MTBDRplus assays allows rapid and accurate detection of MTBC and RIF/INH resistance. KW - laboratory techniques and procedures KW - Tuberculosis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159248 VL - 7 IS - 14127 ER - TY - JOUR A1 - Ampattu, Biju Joseph A1 - Hagmann, Laura A1 - Liang, Chunguang A1 - Dittrich, Marcus A1 - Schlüter, Andreas A1 - Blom, Jochen A1 - Krol, Elizaveta A1 - Goesmann, Alexander A1 - Becker, Anke A1 - Dandekar, Thomas A1 - Müller, Tobias A1 - Schoen, Christoph T1 - Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence JF - BMC Genomics N2 - Background: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. Results: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. Conclusions: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis. KW - neisseria meningitidis KW - MITE KW - virulenceregulatory evolution KW - systems biology KW - metabolism KW - cryptic KW - genetic variation KW - stringent response KW - relA Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157534 VL - 18 IS - 282 ER - TY - JOUR A1 - Klughammer, Johanna A1 - Dittrich, Marcus A1 - Blom, Jochen A1 - Mitesser, Vera A1 - Vogel, Ulrich A1 - Frosch, Matthias A1 - Goesmann, Alexander A1 - Müller, Tobias A1 - Schoen, Christoph T1 - Comparative genome sequencing reveals within-host genetic changes in Neisseria meningitidis during invasive disease JF - PLoS ONE N2 - Some members of the physiological human microbiome occasionally cause life-threatening disease even in immunocompetent individuals. A prime example of such a commensal pathogen is Neisseria meningitidis, which normally resides in the human nasopharynx but is also a leading cause of sepsis and epidemic meningitis. Using N. meningitidis as model organism, we tested the hypothesis that virulence of commensal pathogens is a consequence of within host evolution and selection of invasive variants due to mutations at contingency genes, a mechanism called phase variation. In line with the hypothesis that phase variation evolved as an adaptation to colonize diverse hosts, computational comparisons of all 27 to date completely sequenced and annotated meningococcal genomes retrieved from public databases showed that contingency genes are indeed enriched for genes involved in host interactions. To assess within-host genetic changes in meningococci, we further used ultra-deep whole-genome sequencing of throat-blood strain pairs isolated from four patients suffering from invasive meningococcal disease. We detected up to three mutations per strain pair, affecting predominantly contingency genes involved in type IV pilus biogenesis. However, there was not a single (set) of mutation(s) that could invariably be found in all four pairs of strains. Phenotypic assays further showed that these genetic changes were generally not associated with increased serum resistance, higher fitness in human blood ex vivo or differences in the interaction with human epithelial and endothelial cells in vitro. In conclusion, we hypothesize that virulence of meningococci results from accidental emergence of invasive variants during carriage and without within host evolution of invasive phenotypes during disease progression in vivo. KW - blood KW - comparative genomics KW - throat KW - genetic loci KW - Neisseria meningitidis KW - genomic libraries KW - genome sequencing KW - sequence assembly tools Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159547 VL - 12 IS - 1 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Mshana, Stephen E. T1 - Surveillance of surgical site infections by Pseudomonas aeruginosa and strain characterization in Tanzanian hospitals does not provide proof for a role of hospital water plumbing systems in transmission JF - Antimicrobial Resistance and Infection Control N2 - Background The role of hospital water systems in the development of Pseudomonas aeruginosa (P. aeruginosa) surgical site infections (SSIs) in low-income countries is barely studied. This study characterized P. aeruginosa isolates from patients and water in order to establish possible epidemiological links. Methods: Between December 2014 and September 2015, rectal and wound swabs, and water samples were collected in the frame of active surveillance for SSIs in the two Tanzanian hospitals. Typing of P. aeruginosa was done by multi-locus sequence typing. Results: Of 930 enrolled patients, 536 were followed up, of whom 78 (14.6%, 95% CI; 11.6–17.5) developed SSIs. P. aeruginosa was found in eight (14%) of 57 investigated wounds. Of the 43 water sampling points, 29 were positive for P. aeruginosa. However, epidemiological links to wound infections were not confirmed. The P. aeruginosa carriage rate on admission was 0.9% (8/930). Of the 363 patients re-screened upon discharge, four (1.1%) possibly acquired P. aeruginosa during hospitalization. Wound infections of the three of the eight P. aeruginosa SSIs were caused by a strain of the same sequence type (ST) as the one from intestinal carriage. Isolates from patients were more resistant to antibiotics than water isolates. Conclusions: The P. aeruginosa SSI rate was low. There was no evidence for transmission from tap water. Not all P. aeruginosa SSI were proven to be endogenous, pointing to other routes of transmission. KW - Tanzania KW - P. aeruginosa KW - surgical site infection KW - water microbiology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158168 VL - 6 IS - 56 ER - TY - THES A1 - Schlag, Stephanie T1 - Mikrobiologie, Klinik und Antibiotika-Therapie invasiver bakterieller Infektionen an der Würzburger Universitäts-Kinderklinik zwischen 2006 und 2012 T1 - Microbiology, clinical appearance and antibiotic therapy of invasive bacterial infections in children at the University Children's Hospital Würzburg (2006 - 2012) N2 - In einem Zeitraum von sieben Jahren untersuchten wir invasive bakterielle Infektionen bei Kindern durch die wichtigsten Erreger von Blutstrombahninfektionen an der Würzburger Universitäts-Kinderklinik. N2 - We analysed invasive bacterial infections in children caused by the most common and important pathogens of bloodstream infections in children over a 7-year-period. KW - Antibiotikum KW - Bakterielle Infektion KW - Kinderheilkunde KW - Kind KW - Würzburger Universitäts-Kinderklinik Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-156236 ER - TY - JOUR A1 - Dick, Julia A1 - Krauß, Patrizia A1 - Hillenkamp, Jost A1 - Kohlmorgen, Britta A1 - Schoen, Christoph T1 - Postoperative Tropheryma whipplei endophthalmitis – a case report highlighting the additive value of molecular testing JF - JMM Case Reports N2 - Introduction. Tropheryma whipplei is the causative agent of Whipple’s disease. Gastrointestinal and lymphatic tissues are affected in the majority of cases, resulting in diarrhoea, malabsorption and fever. Here, we report a rare case of ocular manifestation in a patient lacking the typical Whipple symptoms. Case presentation. A 74-year-old Caucasian female presented with blurred vision in the right eye over a period of 1–2 months, accompanied by stinging pain and conjunctival hyperaemia for the last 2 days. Upon admission, visual acuity was hand motion in the affected eye. Ophthalmological examination showed typical signs of intraocular inflammation. Diagnostic and therapeutic pars plana vitrectomy including vitreous biopsy and intravitreal instillation of vancomycin and amikacin was performed within hours of initial presentation. Both microscopic analysis and microbial cultures of the vitreous biopsy remained negative for bacteria and fungi. The postoperative antibiotic regime included intravenous administration of ceftriaxone in combination with topical tobramycin and ofloxacin. Due to the empirical therapy the inflammation ceased and the patient was discharged after 5 days with cefpodoxime orally and local antibiotic and steroidal therapy. Meanwhile, the vitreous body had undergone testing by PCR for the eubacterial 16S rRNA gene, which was found to be positive. Analysis of the PCR product revealed a specific sequence of T. whipplei. Conclusion. In our patient, endophthalmitis was the first and only symptom of Morbus Whipple, while most patients with Whipple’s disease suffer from severe gastrointestinal symptoms. 16S rDNA PCR should be considered for any intraocular infection when microscopy and standard culture methods remain negative. KW - intravitreal vancomycin and amikacin KW - intravenous ceftriaxone KW - topic ofloxacin KW - Whipple's disease KW - endophthalmitis KW - Tropheryma whipplei KW - ocular infection KW - vitrectomy KW - oral cefpodoxime KW - oral doxycycline Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158823 VL - 4 ER - TY - JOUR A1 - Moremi, Nyambura A1 - Claus, Heike A1 - Vogel, Ulrich A1 - Mshana, Stephen E. T1 - Faecal carriage of CTX-M extended-spectrum beta-lactamase-producing Enterobacteriaceae among street children dwelling in Mwanza city, Tanzania JF - PLoS ONE N2 - Background Data on ESBL carriage of healthy people including children are scarce especially in developing countries. We analyzed the prevalence and genotypes of ESBL-producing Enterobacteriaceae (EPE) in Tanzanian street children with rare contact to healthcare facilities but significant interactions with the environment, animals and other people. Methodology/ Principle findings Between April and July 2015, stool samples of 107 street children, who live in urban Mwanza were analyzed for EPE. Intestinal carriage of EPE was found in 34 (31.8%, 95% CI; 22.7–40.3) children. Of the 36 isolates from 34 children, 30 (83.3%) were Escherichia coli (E. coli) and six Klebsiella pneumoniae (K. pneumoniae). Out of 36 isolates, 36 (100%), 35 (97%), 25 (69%) and 16 (44%) were resistant to tetracycline, trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin, respectively. Beta-lactamase genes and the multilocus sequence types of E. coli and K. pneumoniae were characterized. ESBL gene bla\(_{CTX-M-15}\) was detected in 75% (27/36) of ESBL isolates. Sequence types (STs) 131, 10, 448 and 617 were the most prevalent in E. coli. Use of local herbs (OR: 3.5, 95% CI: 1.51–8.08, P = 0.003) and spending day and night on streets (OR: 3.6, 95% CI: 1.44–8.97, P = 0.005) were independent predictors of ESBL carriage. Conclusions/ Significance We observed a high prevalence of bla\(_{CTX-M-15}\) in EPE collected from street children in Tanzania. Detection of E. coli STs 131, 10, 38 and 648, which have been observed worldwide in animals and people, highlights the need for multidisciplinary approaches to understand the epidemiology and drivers of antimicrobial resistance in low-income countries. KW - Tanzania KW - children KW - Enterobacteriaceae KW - ESBL Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170331 VL - 12 IS - 9 ER - TY - JOUR A1 - Becam, Jérôme A1 - Walter, Tim A1 - Burgert, Anne A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Seibel, Jürgen A1 - Schubert-Unkmeir, Alexandra T1 - Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria JF - Scientific Reports N2 - Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C\(_{6}\) and long-chain C\(_{16}\)-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C\(_{6}\)-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω–azido-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C\(_{6}\)-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae. KW - ceramide analogs KW - Neisseria KW - ceramide Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159367 VL - 7 ER - TY - JOUR A1 - Wagener, Johannes A1 - Loiko, Veronika T1 - Recent insights into the paradoxical effect of echinocandins JF - Journal of Fungi N2 - Echinocandin antifungals represent one of the most important drug classes for the treatment of invasive fungal infections. The mode of action of the echinocandins relies on inhibition of the β-1,3-glucan synthase, an enzyme essentially required for the synthesis of the major fungal cell wall carbohydrate β-1,3-glucan. Depending on the species, echinocandins may exert fungicidal or fungistatic activity. Apparently independent of this differential activity, a surprising in vitro phenomenon called the “paradoxical effect” can be observed. The paradoxical effect is characterized by the ability of certain fungal isolates to reconstitute growth in the presence of higher echinocandin concentrations, while being fully susceptible at lower concentrations. The nature of the paradoxical effect is not fully understood and has been the focus of multiple studies in the last two decades. Here we concisely review the current literature and propose an updated model for the paradoxical effect, taking into account recent advances in the field. KW - echinocandin KW - caspofungin KW - micafungin KW - anidulafungin KW - paradoxical effect KW - paradoxical growth KW - glucan synthase KW - Fks1 KW - antifungals KW - echinocandins Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197960 SN - 2309-608X VL - 4 IS - 1 ER -