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 - Stijnis, Kees A1 - Dijkmans, Anneke C. A1 - Bart, Aldert A1 - Brosens, Lodewijk A. A. A1 - Muntau, Birgit A1 - Schoen, Christoph A1 - Barth, Thomas F. A1 - van Gulik, Thomas A1 - van Gool, Tom A1 - Grobusch, Martin P. A1 - Tappe, Dennis T1 - Echinococcus vogeli in Immigrant from Suriname to the Netherlands JF - Emerging Infectious Diseases KW - alveolar echinococcosis KW - monoclonal antibody KW - multilocularis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143953 VL - 21 IS - 3 ER - TY - JOUR A1 - Tappe, Dennis A1 - Meyer, Michael A1 - Oesterlein, Anett A1 - Jaye, Assan A1 - Frosch, Matthias A1 - Schoen, Christoph A1 - Pantchev, Nikola T1 - Transmission of Armillifer armillatus Ova at Snake Farm, The Gambia, West Africa JF - Emerging Infectious Diseases N2 - Visceral pentastomiasis caused by Armillifer armillatus larvae was diagnosed in 2 dogs in The Gambia. Parasites were subjected to PCR; phylogenetic analysis confirmed relatedness with branchiurans/crustaceans. Our investigation highlights transmission of infective A. armillatus ova to dogs and, by serologic evidence, also to 1 human, demonstrating a public health concern. KW - Pentastomiasis Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142804 N1 - All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. VL - 17 IS - 2 ER - TY - JOUR A1 - Heisig, Martin A1 - Frentzen, Alexa A1 - Bergmann, Birgit A1 - Gentschev, Katharina Ivaylo A1 - Hotz, Christian A1 - Schoen, Christoph A1 - Stritzker, Jochen A1 - Fensterle, Joachim A1 - Rapp, Ulf R. A1 - Goebel, Werner T1 - Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines N2 - Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization. KW - Listeria monocytogenes Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68705 ER - TY - JOUR A1 - Biju, Joseph A1 - Schwarz, Roland A1 - Linke, Burkhard A1 - Blom, Jochen A1 - Becker, Anke A1 - Claus, Heike A1 - Goesmann, Alexander A1 - Frosch, Matthias A1 - Müller, Tobias A1 - Vogel, Ulrich A1 - Schoen, Christoph T1 - Virulence Evolution of the Human Pathogen Neisseria meningitidis by Recombination in the Core and Accessory Genome JF - PLoS One N2 - Background Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences. Principal Findings We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins. Conclusions Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence. KW - population genetics KW - DNA recombination KW - meningococcal disease KW - recombinant proteins KW - genomic databases KW - comparative genomics KW - neisseria meningitidis KW - homologous recombination Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137960 VL - 6 IS - 4 ER - TY - JOUR A1 - Schoen, Christoph A1 - Kischkies, Laura A1 - Elias, Johannes A1 - Ampattu, Biju Joseph T1 - Metabolism and virulence in Neisseria meningitidis N2 - A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions. KW - Neisseria meningitidis KW - virulence KW - pathometabolism KW - oxidative stress KW - glutathione KW - γ-glutamyl cycle KW - glutamate dehydrogenase KW - nitrite respiration Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113118 ER - TY - THES A1 - Schoen, Christoph T1 - Entwicklung neuartiger bakterieller Vektoren zur Übertragung von zellassoziierten Antigenen, DNA und RNA auf der Basis virulenzattenuierter L. monocytogenes T1 - Development of novel bacterial carrier strains for the delivery of cell wall associated antigens, DNA and RNA on the basis of virulence attenuated Listeria monocytogenes N2 - Listeria monocytogenes ist ein fakultativ humanpathogenes Bakterium und aufgrund seiner Fähigkeit, in Zellen des Wirtes einzudringen und sich im Zytoplasma der befallenen Wirtszelle zu vermehren, ein attraktiver Träger, um heterolog exprimierte Antigene in den MHC-I- und -II-Präsentationsweg antigenpräsentierender Zellen (APC) einzuschleusen und so eine effektive zelluläre Immunreaktion zu erzeugen. Dabei hat die Art und Weise der Antigenexpression einen wesentlichen Einfluss auf die Erzeugung der antigenspezifischen Immunität. So konnte unter Verwendung extrazellulärer Trägerbakterien gezeigt werden, dass insbesondere die Verankerung von Antigenen auf der Zelloberfläche der Bakterien zu einer effektiven Induktion einer humoralen Immunantwort führt. Mit dem Ziel, mit einem derartigen Ansatz auch eine zelluläre Immunität zu erzeugen, wurde ein Plasmidsystem für die Expression von heterologen Proteinen in der Zellwand von L. monocytogenes entwickelt. Dabei gelang die Verankerung zahlreicher Proteine eukaryontischer wie auch prokaryontischer Herkunft über das LPXTG-Ankermotiv von Internalin A. Die so erzielte starke Expression in der Zellwand setzte aber sowohl die Fitness der Bakterien als auch deren Invasivität in vitro deutlich herab und verhinderte damit eine effektive MHC-I-Präsentation des verwendeten Modellantigens. Alternativ wurde L. monocytogenes bereits erfolgreich zur Übertragung von DNA-Vakzinen eingesetzt, um so auch die Synthese gegebenenfalls posttranslationell modifizierter Antigene in ihrer korrekten Konformation durch die infizierte Wirtszelle zu erzielen. Allerdings erfolgte die Expression des als Reporterprotein verwendeten EGFP insbesondere in APC sehr langsam und war von geringer Effizienz. Dabei konnte in der vorliegenden Arbeit erstmals gezeigt werden, dass nach bakterieller Übertragung von DNA-Vakzinen der Import der Plasmidmoleküle in den Kern insbesondere sich nicht teilender Zellen einen der wichtigsten Engpässe für eine möglichst frühzeitige und effektive Reportergenexpression darstellt. Einen Ausweg bietet die bakterielle Übertragung codierender mRNA, die unmittelbar nach der Freisetzung aus der Bakterienzelle im Zytoplasma der infizierten Wirtszelle zur Translation zu Verfügung steht. Dazu wurde das 5’-Ende der EGFP-codierenden Sequenz mit dem IRES-Element des Encephalomyocarditisvirus genetisch fusioniert. Um eine möglichst hohe Syntheserate zu erzielen und damit dem Abbau der mRNA in der Bakterienzelle entgegenzuwirken, erfolgte die Synthese der mRNA in L. monocytogenes mit Hilfe eines T7-RNA-Polymerase-basierten Transkriptionssystems. Im Gegensatz zur bakteriellen Übertragung von Plasmid-DNA konnte so bereits 4 h nach Infektion sowohl in epithelialen als auch in APC wie Makrophagen und humanen dendritischen Zellen eine deutliche EGFP-Expression nachgewiesen werden sowie bei Verwendung von Ovalbumin als Reporterprotein eine effektive MHC-I-Präsentation in vitro. Damit stellt die bakterielle Übertragung von mRNA einen vielversprechenden neuartigen Ansatz dar zur Erzeugung einer zellulären und gegebenenfalls auch humoralen Immunantwort gegen posttranslational modifizierte Antigene. N2 - Listeria monocytogenes is a facultative intracellular bacterium that enters the cell by phagocytosis after which it colonizes the cytosol of the host cell. It is thus a potent vaccine vector for the presentation of passenger antigens to the major histocompatibility complex class II and class I pathways. The mode of antigen expression has a major impact on the generation of a specific immune response. Compared to the expression of antigens in a secreted form or localized inside the bacterial cell, the display of heterologous antigens on the surface of bacteria has shown to be a potent strategy for the elicitation of efficient immune responses. Therefore, a plasmid system for the expression of heterologous proteins covalently linked to the cell wall of L. monocytogenes via the LPXTG cell wall anchor of Internalin A was developed. Although efficient expression of a number of proteins could be achieved by this approach the resulting high expression levels in turn reduced the invasiveness and overall fitness of the carrier Listeria and thus impaired the delivery efficiency of the encoded antigen to the antigen processing machinery of the infected host cell. As an alternative approach to the delivery of protein antigens synthetisized by the prokaryotic cell Listeria might also be exploited for the delivery of DNA vaccines enabling the correct expression also of posttranslationally modified passenger antigens by the eukaryotic cell. However, it is demonstrated that the uptake of the plasmid DNA into the nucleus is a major limiting step for achieving early and efficient gene expression in non-dividing and therefore also in antigen presenting cells. Comparable to other non-viral transfection techniques like lipofection, the limited access to the nuclear compartment may thus constitute a major barrier also after bacteria-mediated expression plasmid DNA delivery. To overcome this bottleneck, a self-destructing L. monocytogenes strain was employed for the release of translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. EGFP-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5´- end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro. In conclusion, this approach might also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response also against posttranslationally modified antigens such as viral envelope proteins and thus might lead to the development of novel vaccines. KW - Listeria monocytogenes KW - Impfstoff KW - DNS KW - Listeria monocytogenes KW - Impfstoffe KW - RNA delivery KW - DNA delivery KW - Listeria monocytogenes KW - recombinant vaccines KW - RNA delivery KW - DNA delivery KW - antigen delivery Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-16560 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 - Heidrich, Nadja A1 - Bauriedl, Saskia A1 - Barquist, Lars A1 - Li, Lei A1 - Schoen, Christoph A1 - Vogel, Jörg T1 - The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq JF - Nucleic Acids Research N2 - Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of −35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx. KW - RNA KW - Neisseria meningitidis KW - dRNA-seq KW - transcriptome KW - RNA chaperone Hfq Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170828 VL - 45 IS - 10 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 -