Einleitung: Atemwegsinfekte gehören zu den häufigsten Krankheitsbildern in der Allgemeinarztpraxis. Ziel der Erhebung war es, Daten zur Diagnostik und Therapie von Atemwegsinfekten in der hausärztlichen Praxis zu erheben. Methoden: Während einer jeweils eintägigen Hospitation bei 30 Fachärzten für Allgemeinmedizin wurde deren Vorgehensweise bei allen Patienten mit einem akuten Atemwegsinfekt dokumentiert, die im Zeitraum der Beobachtung den Arzt konsultierten. Es erfolgte eine Unterscheidung zwischen Erst- und Folgekontakten. Ergebnisse: Diagnostisch wurden am häufigsten die Auskultation der Lunge und die Inspektion des Mund-Rachen-Raumes durchgeführt. 98,4% der Patienten mit Erstkonsultationen und 62,5% der Patienten mit Folgekonsultationen erhielten eine medikamentöse Verordnung. Im Durchschnitt erhielten erstkonsultierende Patienten 2,1 (+-1,0), Patienten im Folgekontakt 1,3 (+-1,1) Medikamente. Am häufigsten wurden Medikamente aus der Gruppe der Husten- und Erkältungspräparate verordnet (87,1% der Erstkontakte und 52,9% der Folgekontakte). 43,5% der Erstkontakte und 29,9% der Folgekontakte erhielten ein Antibiotikum (37,5% Makrolide, 21,5% Penicilline, 20,8% Doxycyclin als Monosubstanz oder in Kombination mit Expektorantien). Schlussfolgerung: Nahezu alle Patienten erhielten ein Rezept über mindestens ein Medikament. Die erhobenen Daten lassen vermuten, dass sich bei einer höheren Gewichtung der so genannten "Hausmittel" ein deutliches Einsparungspotential böte. Angesichts des hohen Anteils der Antibiotikaverordnungen sollte die entsprechende Indikationsstellung kritisch überdacht werden.

Die Mehrzahl der jährlich 400.000 bis 600.000 Krankenhausinfektionen in Deutschland wird von Erregern der sog. ESCAPE-Gruppe (Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa und verschiedene Enterobacteriaceae, u.a. Klebsiella pneumoniae) verursacht. Besondere Sorge bereitet dabei die Ausbreitung von K. pneumoniae-Stämmen mit enzymvermittelter Resistenz gegenüber Carbapenem-Antibiotika (K. pneumoniae-Carbapenemase, KPC) und die Zunahme von C. difficile-Infektionen (CDI) durch hypervirulente Epidemiestämme (z.B. Ribotyp 027). Die spezifischen Erfahrungen eines prolongierten Ausbruchsgeschehens durch einen KPC-bildenden K. pneumoniae-Stamm (KPC-KP) am Leipziger Universitätsklinikum machen deutlich, dass bei diesem Erregertyp ein hohes Transmissionspotential bei enormer Tenazität (Umweltresistenz) zu berücksichtigen ist, ein Versagen von Standardhygienemaßnahmen in Betracht zu ziehen ist, und Infektionsketten oftmals unklar bleiben. Die Anwendung von Antibiotika ist bei KPC-KP-Infektionen auf einzelne Substanzen (Colistin, Tigecyclin, Gentamicin) beschränkt und vor allem bei immunsupprimierten Patienten (z.B. Lebertransplantierte) mit einem relevanten Risiko des Therapieversagens behaftet. Die Therapie von CDI wird gerade bei Immunsupprimierten durch eine steigende Zahl an Rezidiven erschwert, die teilweise antibiotisch (Vancomycin, Fidaxomicin) nicht beherrschbar sind, so dass alternative Therapieverfahren wie die fäkale Bakterientherapie („Stuhltransplantation“) zur Anwendung kommen. CDI-Rezidive, aber auch eine dauerhafte intestinale Besiedelung mit multiresistenten Enterobakterien wie KPC-KP, scheinen neben wirtsspezifischen Faktoren der Immunantwort durch eine Dysregulation der physiologischen intestinalen Standortflora mit Störung der Kolonisationsresistenz bedingt zu sein. Der Versuch einer Eradikationsbehandlung von Patienten mit persistierender intestinaler Besiedelung durch KPC-KP mittels oraler Applikation der nicht resorbierbaren Antibiotika Colistin und Gentamicin ist mit einem relevanten Risiko der Entstehung von Sekundärresistenzen behaftet. Die Zulassung neuer, besser wirksamer Antibiotika ist für die nächsten Jahre nicht in Sicht, so dass der Infektionsprävention überragende Bedeutung zukommt. Die Erfahrungen der KPC-Ausbruchsbewältigung am Leipziger Universitätsklinikum zeigen, dass nahezu lückenlose Compliance bei der Händedesinfektion, rigoros praktizierte und kontrollierte Barriere- und Isolationsmaßnahmen, Optimierung des Gebrauchs von Breitspektrum-Antibiotika (sog. „Antibiotic Stewardship“) und systematisches mikrobiologisches Erregerscreening dabei unabdingbar sind. Nachhaltige Verbesserungen hinsichtlich der globalen Ausbreitung von multiresistenten Krankenhausbakterien werden sich nur durch grundlegende Umgestaltungen in Umwelt, Landwirtschaft, Tierzucht und Gesundheitswesen mit sparsamer und möglichst gezielter Anwendung von Antibiotika erzielen lassen. Um Risikopopulationen hospitalisierter Patienten vor potentiell lebensbedrohlichen Erregertransmissionen effektiv schützen zu können, sind erweiterte Surveillance und konsequent umgesetzte krankenhaushygienische Maßnahmen erforderlich.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

The aim of this work was to gain new knowledge about mechanisms of autoimmune uveitis and to test new therapeutic possibilities that have not yet been studied in uveitis or whose effect is questionable. The main emphasis was placed on the role of microorganisms in the process of uveitis. A mouse model of experimental autoimmune uveitis including a germ-free model was used to achieve the aims and samples of patients' intraocular fluids were analyzed. In the experimental model, the intensity of inflammation was evaluated in vivo clinically and post mortem histologically. The effect of immunomodulatory treatment was evaluated. The intensity of inflammation was compared between groups of germ-free and conventional mice. The therapeutic effect of antibiotics administered to affect microbiome was investigated in conventional mice. In intraocular fluid samples of patients with autoimmune uveitis signs of infection were monitored and levels of cytokines and other factors were evaluated. Evaluation of the effect of immunomodulatory therapy has demonstrated the efficacy of mycophenolate mofetil, which supports its wider use in the treatment of autoimmune posterior uveitis in human medicine. The decrease in bacterial load has led to a decrease in the intensity of inflammation, thereby confirming the importance of...

Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.

Infectious diseases are a very hot topic nowadays in society. In recent years, there were several epidemics, infectious diseases (hepatitis A, Avian influenza, pandemic influenza A) and nobody knows when will the next "new" infection. At the outbreak of epidemics, there is great interest in the company to obtain information about the disease, but often also to unnecessary panic, because the media often publish incorrect information. It would be preferable, in order to improve public awareness and prevent the unnecessary spread of disease. Great emphasis should be given to prevent the disease - primarily on immunization, hygiene rules and principles of safe sex. Quality information should be mainly from teachers in teaching their pupils, as is clear from research books, infectious diseases are only mentioned in textbooks and the number of substantive information in them is missing. The fact that pupils of primary schools and grammar schools with basic information on infectious diseases do not meet in the classroom, evidenced by the results of a survey carried out in the 6th classes and first year at selected elementary schools and grammar schools in Prague. To raise awareness of the pupils in school was to create a methodical manual for teachers, which summarizes information about bacterial and...

Many studies show the ability of gut microbes to modulate the anti-tumour immune response by direct triggering the immune cells or by bacterial metabolites. Interestingly bacteria may even migrate to the tumour tissue and orchestrate the immune response on site. These anti-tumour effects can be improved by the administration of immune checkpoint inhibitors (ICI). Notably, some microbial effects occur only in the presence of ICI. On the contrary, microbiota may also promote tumour growth and negatively impact the effects of ICI therapy. We have disrupted the gut microbiota homeostasis by antibiotics (ATB) to study the effects of gut microbiota on the ICI. This disturbance led surprisingly to reduced tumour growth and enhanced pro-inflammatory immune response not only in the gut but also within the tumour tissue, where especially IFN-γ orchestrated the anti-tumour immune response. Importantly the anti-tumour immune response could be transferred through colonisation of germ-free mice by ATB-changed gut microbiota if concomitantly anti- programmed cell death protein 1 (αPD-1) monoclonal antibody was administrated. These mice had elevated levels of segmented filamentous bacteria (SFB), which induced systemic immune response with increased expression of IL-17 and elevated amounts of Th 17 cells,...

This diploma thesis deals with the determination of macrolide antibiotics in wastewater, especially with erythromycin, clarithromycin and roxithromycin. In this time are these pharmaceuticals prescribed quite frequently. Solid phase extraction (SPE) was used for the isolation and the purification of selected analytes from an aqueous matrix; as the suitable procedure was found the using Oasis HLB cartridges. High performance liquid chromatography with mass spectrometry detection (HPLC-MS) was optimized for its analysis of selected pharmaceuticals. The optimized method was used for the determination of pharmaceuticals in real water samples, which was taken at the inflow and the outflow of the urban wastewater treatment plant in Brno-Modřice.

Adsorption and advanced oxidation processes, especially photocatalysis, are amongst the most common water treatment methodologies. Unfortunately, using each of these techniques independently does not fully eliminate the pollutants of diverse nature, which are present in wastewater. Here, an avenue for multifunctional materials for water treatment is opened by reporting for the first time the preparation, characterization, and study of the properties of a novel multifunctional nanocomposite with both adsorption and visible-light-driven photocatalysis abilities. These multifunctional nanocomposites, namely iron (II, III) oxide/poly(N-isopropylacrylamide-co-methacrylic acid)/silver-titanium dioxide (Fe3O4/P(NIPAM-co-MAA)/Ag-TiO2), are prepared by combining magnetic polymeric microspheres (Fe3O4/P(NIPAM-co-MAA)) with silver-decorated titanium dioxide nanoparticles (Ag-TiO2 NPs). Cationic dyes, such as basic fuchsin (BF), can be adsorbed by the nanocomposites thanks to the carboxylic groups of Fe3O4/P(NIPAM-co-MAA) microspheres. Concomitantly, the presence of Ag-TiO2 NPs endows the system with the visible-light-driven photocatalytic degradation ability toward antibiotics such as ciprofloxacin (CIP) and norfloxacin (NFX). Furthermore, the proposed nanocomposites show antibacterial activity toward Escherichia coli (E. coli), thanks to the presence of silver nanoparticles (Ag NPs). Due to the superparamagnetic properties of iron (II, III) oxide nanoparticles (Fe3O4 NPs), the nanocomposites can be also recycled and reused, after the cleaning process, by using an external magnetic field.

Solar-driven photocatalysis is of great interest in terms of a sustainable use of energy and its application in wastewater treatment. The UV light-driven photogeneration of H2O2 by solar irradiation is an advanced strategy for the treatment of bacteria and recalcitrant pollutants in wastewater, but suffers from low efficiencies. In this work, a solar-driven multifunctional nanocomposite consisting of Tm3+ upconverting nanoparticles, poly(vinyl alcohol), poly(acrylic acid) and hydroxylated sulfonated poly(ether ether ketone) was prepared. The components were crosslinked via a heating treatment at 170 °C, resulting in a non-leaching porous material. This nanocomposite exhibited excellent adsorption ability (89 % in 150 min) toward a 100 mg/L ciprofloxacin aqueous solution and proved to photodegrade it (50 %) upon 4 h artificial solar irradiation, exploiting photon upconversion processes. Moreover, an 80 % bactericidal effect against E. coli was registered upon sunlight irradiation. Altogether, these results suggest the feasibility of a solar-driven wastewater treatment based on upconverting nanoparticles.