Analyse der molekularen Struktur und der Verbreitung clyA-homologer Zytolysingene in bakteriellen Krankheitserregern aus der Familie der Enterobacteriaceae

Zytolysin A (ClyA) von Escherichia coli ist der Prototyp einer neuartigen Familie von bakteriellen porenbildenden Zytolysinen. Es handelt sich bei diesem Toxin um ein Protein von 34 kDa, das hämolytische und zytotoxische Aktivität aufweist und das in Zellmembranen stabile Poren bildet, indem es sich zu ringförmigen ClyA-Oligomeren zusammenlagert. Das Strukturgen des ClyA-Proteins, clyA, wurde ursprünglich im Chromosom des E. coli-Laborstammes K-12 identifiziert, später wurde es aber auch im Genom von vielen E. coli-Stämmen gefunden, die intestinale Infektionen beim Menschen verursachen (insbesondere in enteroinvasiven, Shigatoxin-produzierenden, enteroaggregativen und enterotoxischen E. coli-Stämmen). Die Expression des clyA-Gens unterliegt einer komplexen Regulation; unter normalen in vitro-Kulturbedingungen ist das clyA-Gen in E. coli stark reprimiert. In mehreren Salmonella enterica Serovar Typhi-Stämmen und in einem S. enterica Serovar Paratyphi A-Stamm (ATCC 9150) wurden kürzlich funktionale (intakte) clyA-homologe Gene gefunden, deren Proteinprodukte 90-91% Aminosäuresequenzidentität zu ClyA von E. coli aufweisen. Im Genom eines aviären (vogelpathogenen) E. coli-Stammes wurde ein intaktes clyA-homologes Hämolysingen identifiziert, dessen Proteinprodukt ca. 75% Identität zu ClyA von E. coli K-12 und anderen E. coli-Stämmen zeigt. Auch in mehreren Shigella flexneri-Stämmen sowie in einem Shigella sonnei- und einem Shigella dysenteriae-Stamm wurden clyA-homologe DNA-Sequenzen nachgewiesen. Alle bisher untersuchten Shigella-Stämme enthalten jedoch nur ein defektes clyA-Gen: in den untersuchten Sh. flexneri-Stämmen weist das clyA-Gen eine identische 11 bp-Deletion auf, welche aufgrund der entsprechenden Leserasterverschiebung zu einem vorzeitigen Abbruch der kodierenden Sequenz führt; in dem untersuchten Sh. sonnei-Stamm ST3112/01 ist das clyA-Gen durch ein Insertionselement (IS1 von Sh. sonnei) unterbrochen; und in dem untersuchten Sh. dysenteriae-Stamm ist ein großer Teil des clyA-Gens deletiert und durch ein IS-Element (iso-IS1 von Sh. dysenteriae) ersetzt. In der vorliegenden Doktorarbeit wurden zwei weitere Sh. sonnei-Stämme, ein weiterer S. enterica Serovar Paratyphi A-Stamm sowie verschiedene andere Bakterien aus der Familie der Enterobacteriaceae hinsichtlich des Vorhandenseins von clyA bzw. von clyA-homologen DNA-Sequenzen untersucht. Aus den beiden Sh. sonnei-Stämmen ST2757/01 und ST3135/01 und aus dem S. enterica Serovar Paratyphi A-Stamm FR1/99 konnten durch PCR clyA-spezifische DNA-Fragmente amplifiziert werden. Die Sequenzierung des clyA-spezifischen PCR-Produktes von S. enterica Serovar Paratyphi A FR1/99 zeigte, dass dieser Stamm ein intaktes clyA-Gen enthält, welches einschließlich der flankierenden DNA-Sequenzen 100% Nukleotidsequenzidentität zum clyA-Gen des Serovar Paratyphi A-Stammes ATCC 9150 aufweist. Um die Promotorregion dieses clyA-Gens (clyAS. enterica Serovar ParatyphiA) einzugrenzen, wurde es mit unterschiedlich langen 5'-flankierenden DNA-Sequenzen amplifiziert und in den Plasmidvektor pUC18 kloniert. Plasmidkonstrukte, die stromaufwärts vom clyAS. enterica Serovar ParatyphiA-Gen die ersten 102 bzw. 128 5'-flankierenden Basenpaare enthielten, führten nach Transformation in einen E. coli-Laborstamm (E. coli K-12-Derivat DH5a) zu einem ähnlich starken hämolytischen Phänotyp, während ein isogenes Plasmidkonstrukt mit 471 bp 5'-flankierender DNA-Sequenz deutlich schwächere hämolytische Aktivität vermittelte. Innerhalb der ersten 102 bp stromaufwärts vom clyAS.enterica Serovar ParatyphiA -Startcodon scheinen demnach regulatorische Sequenzen zu liegen, die die Expression dieses Gens gestatten, während DNA-Sequenzen, die mehr als 128 bp vor dem ATG-Startcodon liegen, die Expression anscheinend eher behindern. Die von Sh. sonnei ST2757/01 und Sh. sonnei ST3135/01 erhaltenen clyA-spezifischen PCRProdukte zeigten identische Größen; es wurde deshalb nur mit einem der beiden Stämme (ST2757/01) weitergearbeitet. Eine DNA-Sequenzanalyse ergab, dass Sh. sonnei ST2757/01 ein clyA-Gen enthält, welches an zwei Stellen (zwischen Nukleotidposition 80 und 81 und zwischen Nukleotidposition 354 und 355 von clyA) durch IS1 von Sh. sonnei unterbrochen ist. Die hintere der beiden IS1-Insertionen war dabei identisch mit der IS1-Insertion, die im clyA-Gen des bereits früher untersuchten Sh. sonnei-Stammes (ST3112/01) nachgewiesen worden war. Aus Stämmen der Species Citrobacter freundii, Citrobacter koseri, Citrobacter amalonaticus, Citrobacter gillenii, Citrobacter murliniae, Citrobacter braakii, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Hafnia alvei, Yersinia enterocolitica, Proteus mirabilis und Morganella morganii konnten durch PCR keine clyA-homologen DNA-Sequenzen amplifiziert werden. Da auch in Southern Blot-Analysen mit einer clyA-spezifischen, 712 bp großen Gensonde keiner dieser Stämme eine signifikant positive Reaktion zeigte, wurde gefolgert, dass diese Stämme kein clyA-Gen und keine clyA-ähnlichen Sequenzen besitzen. Es spricht somit einiges dafür, dass das Zytolysingen clyA innerhalb der Familie der Enterobacteriaceae nur in den Gattungen Escherichia, Salmonella und Shigella vorkommt.Cytolysin A (ClyA) of Escherichia coli is a novel cytolysin with little apparent resemblance to previously characterized cytolysins. It is the prototype of a new family of pore-forming bacterial toxins. ClyA is a hemolytic and cytotoxic 34 kDa protein which forms stable pores in cell membranes by assembling to ring-shaped oligomers. The gene encoding the ClyA protein, clyA, was first identified in E. coli K-12, a laboratory strain. Later, it was also found in many E. coli strains causing intestinal infections in humans (particularly in enteroinvasive, Shiga toxin-producing, enteroaggregative and enterotoxic E. coli strains). The expression of clyA is controlled by a complex regulation system; under normal laboratory conditions the gene is strongly repressed. Recently, clyA homologues were identified in several Salmonella enterica serovar Typhi strains and in an S. enterica serovar Paratyphi A strain (strain ATCC 9150). The ClyA proteins encoded by these clyA homologues show 90-91% identity in amino acid sequence to ClyA of E. coli. In the genome of an avian E. coli strain, an intact clyA gene was found whose protein product is 75% identical in amino acid sequence to E. coli ClyA. Furthermore, clyAhomologous genes were identified in Shigella strains. However, in all Shigella strains analyzed so far, clyA is obviously nonfunctional: in several tested S. flexneri strains, clyA shows an 11-bp deletion leading to a frameshift with premature truncation of the gene sequence; in a tested Sh. sonnei strain, clyA is interrupted by an insertion sequence (IS1 of Sh. sonnei); and in a tested Sh. dysenteriae strain, a large part of clyA is deleted and replaced by an insertion sequence (iso-IS1 of Sh. dysenteriae). In this work, two additional Sh. sonnei strains, another S. enterica serovar Paratyphi A strain and several other bacteria belonging to the Enterobacteriaceae were examined with regard to the presence and integrity of the clyA-gene or of clyA-homologous DNA-sequences. From the Sh. sonnei strains (ST2757/01 and ST3135/01) and from S. enterica serovar Paratyphi A strain FR1/99 clyA-homologous sequences could be amplified by PCR. Sequencing of the PCR product obtained from S. enterica serovar Paratyphi A FR1/99 showed that this strain harbors an intact clyA gene which is 100% identical in nucleotide sequence to clyA of S. enterica serovar Paratyphi A ATCC 9150. To identify the promoter region of this clyA gene, it was amplified with different long fragments of the 5'-flanking DNA-sequence and cloned into the plasmid vector pUC18. Plasmid constructs containing the first 102 and 128 basepairs, respectively, upstream from clyA of S. enterica serovar Paratyphi A caused a similar hemolytic phenotype when transformed into the E. coli laboratory strain DH5a, while an isogenic plasmid construct containing 471 bp of the 5'-flanking DNA sequence caused only weaker hemolytic activity. Thus, regulatory sequences permitting gene expression appear to be present within the first 102 bp upstream from clyA of S.enterica serovar Paratyphi A, while DNA sequences located more than 128 bp upstream from the clyA startcodon obviously inhibit gene expression. The clyA-specific PCR products obtained from Sh. sonnei ST2757/01 and Sh. sonnei ST3135/01 were identical in size. Therefore, only one of these strains (ST2757/01) was further studied. DNA sequence analyses showed that Sh. sonnei ST2757/01 harbors a clyA gene which is interrupted twice by IS1 of Sh. sonnei (between nucleotide position 80 and 81 and between position 354 and 355 of clyA). The IS1 insertion at nucleotide position 354-355 proved to be identical to the single IS1 insertion that was previously found in the clyA gene of another Sh. sonnei (strain ST3112/01). From strains of the species Citrobacter freundii, Citrobacter koseri, Citrobacter amalonaticus, Citrobacter gillenii, Citrobacter murliniae, Citrobacter braakii, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Hafnia alvei, Yersinia enterocolitica, Proteus mirabilis and Morganella morganii, no clyA-homologous DNA sequences could be amplified by PCR. Furthermore, in Southern blot analyses conducted with a 712-bp clyA gene probe, none of these strains showed a significant positive reactivity. It can therefore be concluded that these strains do not harbor clyA-related DNA sequences. Thus, one may speculate that the cytolysin gene clyA is only found in Escherichia, Salmonella and Shigella species

Novel outpatient treatment strategy for cranial infections – a single-center experience [Abstract]

Oral e-Poster Presentations - Booth 1: Trends & Innovation A, September 26, 2023, 1:00 PM - 2:30 PM Background: Antibiotic therapy of cranial infections is a resource-intensive process. On the background of the recommendation for longer-term antibiotic administration for cranial infections, we established an outpatient intravenous antibiotic administration in our hospital. Methods: The aim of this study was to evaluate the usefulness of outpatient antibiotic therapy in cranial neurosurgery. For this purpose, we included all patients who received a peripherally inserted central catheter (PICC line) for intravenous antibiotic therapy for cranial infections between 01/20 and 9/22. We evaluated the available patient data with regard to the infectiological and neurosurgical issues. All patients received intravenous antibiotics for at least 6 weeks (inpatient and outpatient). Results: In total, we were able to include 30 patients. The median age was 58.12 years (SD +/- 13.39 years). The proportion of female patients was 43%. The mean hospital stay was 18.4 days (SD +/- 4.97 days) for total inpatient treatment. Subsequent mean outpatient antibiotic therapy was admitted for 71.7 days (SD +/- 23.18 days). Outpatient mean IV therapy accounted for 53.88 days (SD +/- 18.56 days). The most common pathogens were Staphylococcus epidermis and cutibacteria. In 9%, microbiological samples were. In all patients, neither radiographic nor laboratory evidence of inflammation was found in the final control. During outpatient intravenous therapy, 12% of patients experienced a difficult patency of the PICC line due to the prolonged administration of antibiotics. This could be corrected radiologically in each case. In addition, one patient, independent of therapy, showed structural epilepsy after the abscess healing. Conclusions: Outpatient IV antibiotic therapy via a PICC line catheter is a safe and feasible method for long-term antibiotic treatment of cranial infections

Novel low energy hydrogen–deuterium isotope breakthrough separation using a trapdoor zeolite

AbstractCs-chabazite, a type of zeolite with caesium counter-cations, possesses interesting gas separation properties due to a highly selective molecular “trapdoor” effect. Herein the use of this material for H2/D2 isotope separation is demonstrated. Isotope separation was achieved using breakthrough separation with a single pass through a packed bed at moderate temperatures (293K) and pressures (0.17MPa) when one species was in a sufficiently low concentration. The breakthrough separation curves were successfully modelled using the Thomas kinetic breakthrough model and the Yoon and Nelson kinetic breakthrough model, where working transferable kinetic rate constants were developed. Use of this material for hydrogen isotope separation would significantly lower the total energy demand compared with current hydrogen isotope separation techniques such as cryogenic distillation and is applicable to separating out low concentrations of D2 (0.0156%) present in standard grade H2

Mesoporous tertiary oxides via a novel amphiphilic approach

We report a facile biomimetic sol-gel synthesis using the sponge phase formed by the lipid monoolein as a structure-directing template, resulting in high phase purity, mesoporous dysprosium- and gadolinium titanates. The stability of monoolein in a 1,4-butanediol and water mixture complements the use of a simple sol-gel metal oxide synthesis route. By judicious control of the lipid/solvent concentration, the sponge phase of monoolein can be directly realised in the pyrochlore material, leading to a porous metal oxide network with an average pore diameter of 10 nm

Synthesis of porous high-temperature superconductors via a melamine formaldehyde sacrificial template

Nanostructured high-temperature superconductors YBa(2)Cu(3)O(6+δ) and Bi(2)Sr(2)CaCu(2)O(8+δ) were synthesised using a melamine formaldehyde sponge as a sacrificial template, via three solution-based approaches. In the case of YBa(2)Cu(3)O(6+δ), a modified Pechini method produced a material with a superconducting transition at 92 K and a specific surface area of 4.22 m(2) g(−1). Further analysis with Hg porosimetry determined that the sponge exhibited a porosity of 82%. In the case of Bi(2)Sr(2)CaCu(2)O(8+δ), this method produced a material that exhibited superconductivity at 86 K with a specific surface area of 9.62 m(2) g(−1). Hg-porosimetry determined that the BSCCO sponge exhibited a porosity of 78%

Visible light promoted photocatalytic water oxidation:effect of metal oxide catalyst composition and light intensity

Simply prepared low-cost nanoparticulate transition metal oxides were used as catalysts in visible light promoted water oxidations. The activity using daylight equivalent light intensities was assessed.</p

Mixed-linker approach in designing porous zirconium-based metal–organic frameworks with high hydrogen storage capacity

YesThree highly porous Zr(IV)-based metal–organic frameworks, UBMOF-8, UBMOF-9, and UBMOF-31, were synthesized by using 2,2′-diamino-4,4′-stilbenedicarboxylic acid, 4,4′-stilbenedicarboxylic acid, and combination of both linkers, respectively. The mixed-linker UBMOF-31 showed excellent hydrogen uptake of 4.9 wt% and high selectivity for adsorption of CO2 over N2 with high thermal stability and moderate water stability with permanent porosity and surface area of 2552 m2 g−1.University of Bath; Royal Society of Chemistry; Engineering and Physical Sciences Research Counci

NFDI4Culture - Consortium for research data on material and immaterial cultural heritage

Digital data on tangible and intangible cultural assets is an essential part of daily life, communication and experience. It has a lasting influence on the perception of cultural identity as well as on the interactions between research, the cultural economy and society. Throughout the last three decades, many cultural heritage institutions have contributed a wealth of digital representations of cultural assets (2D digital reproductions of paintings, sheet music, 3D digital models of sculptures, monuments, rooms, buildings), audio-visual data (music, film, stage performances), and procedural research data such as encoding and annotation formats. The long-term preservation and FAIR availability of research data from the cultural heritage domain is fundamentally important, not only for future academic success in the humanities but also for the cultural identity of individuals and society as a whole. Up to now, no coordinated effort for professional research data management on a national level exists in Germany. NFDI4Culture aims to fill this gap and create a usercentered, research-driven infrastructure that will cover a broad range of research domains from musicology, art history and architecture to performance, theatre, film, and media studies. The research landscape addressed by the consortium is characterized by strong institutional differentiation. Research units in the consortium's community of interest comprise university institutes, art colleges, academies, galleries, libraries, archives and museums. This diverse landscape is also characterized by an abundance of research objects, methodologies and a great potential for data-driven research. In a unique effort carried out by the applicant and co-applicants of this proposal and ten academic societies, this community is interconnected for the first time through a federated approach that is ideally suited to the needs of the participating researchers. To promote collaboration within the NFDI, to share knowledge and technology and to provide extensive support for its users have been the guiding principles of the consortium from the beginning and will be at the heart of all workflows and decision-making processes. Thanks to these principles, NFDI4Culture has gathered strong support ranging from individual researchers to highlevel cultural heritage organizations such as the UNESCO, the International Council of Museums, the Open Knowledge Foundation and Wikimedia. On this basis, NFDI4Culture will take innovative measures that promote a cultural change towards a more reflective and sustainable handling of research data and at the same time boost qualification and professionalization in data-driven research in the domain of cultural heritage. This will create a long-lasting impact on science, cultural economy and society as a whole