This document deals with devices which are used for the automatic Gram staining. The document starts by a research with the aim to discover existing devices and to compare them to the concept of the device developed by company Biovendor. There is theoretically and then practically dealed with the methods of the rotor imbalance detection and measurement of water-level. The Practical part of this document describes the development of the control unit with the microcontroller ARM Cortex-M3 which will be used for the prototype development of Biovendor’s device. The Last part describes measurement of imbalance of three different rotors with the aid of the control unit.

Der generalisierte Einsatz von Antibiotika im medizinischen Sektor erzeugt einen hohen Selektionsdruck auf Bakterien. Daher weisen bakterielle Resistenzen gegenüber antimikrobiellen Wirkstoffen eine zunehmende Inzidenz auf. Gegen Antibiotika multiresistente Erreger (MRE), insbesondere Multiresistente Gram-Negative Erreger (MRGN), stellen in Krankenhäusern, aber auch in Tierkliniken ein ernstzunehmendes Problem dar. Das Therapieversagen bei der Behandlung von Infektionen mit 3MRGN oder 4MRGN gefährdet die Gesundheitsversorgung. Die Bedeutung effektiver Kontrollmaßnahmen in der Unterbrechung nosokomialer Infektionsketten rückt in den Fokus. Infektionsprophylaxe wird durch eine Umweltdekontamination in Form von Reinigung und Desinfektion durchgeführt. Zertifizierte Biozide werden routinemäßig eingesetzt. Jedoch weisen einigen Studien auf eine Toleranz von Bakterien gegenüber Desinfektionsmitteln hin. Spezifische Dekontaminationsmaßnahmen könnten daher für die Bekämpfung von MRGN notwendig sein. Ziel der Untersuchung war es zu evaluieren, ob klinische 3MRGN und 4MRGN im Vergleich zu nicht resistenten Referenzstämmen unempfindlicher gegenüber Desinfektionsmitteln reagieren und Desinfektionsprotokolle gegebenenfalls angepasst werden müssen. Dazu wurden sechzehn klinische Stämme der Gattungen Acinetobacter, Pseudomonas und Klebsiella wurden mit fünf korrespondierenden Referenzstämmen in vitro auf deren Empfindlichkeit gegenüber den gängigen Desinfektionsmitteln Natriumhypochlorit, Ethanol, Peressigsäure und Benzalkoniumchlorid untersucht. Die Effizienz wurde anhand von vier aufeinander aufbauenden Testverfahren gemäß den Richtlinien des Verbunds für angewandte Hygiene (VAH, 2015) beurteilt. Diese beinhalten die Bestimmung der minimalen Hemmkonzentration (MHK) und die Durchführung von Suspensionstests und Keimträgerversuchen. Es wurden wirksame Konzentrations-Zeit-Relationen für jeden Teststamm und jede aktive Substanz determiniert. Die Ergebnisse der Suspensionstests über- beziehungsweise unterschreiten die Werte der MHK-Bestimmung um ein Vielfaches. P. aeruginosa tolerierte die höchsten Benzalkoniumchlorid-Konzentrationen, während Acinetobacter-Stämme am empfindlichsten reagierten. Im Vergleich zu Suspensionstests wurden in den praxisnahen Keimträgerversuchen signifikant höhere Konzentrationen an Peressigsäure und Benzalkoniumchlorid ermittelt. Die geringere Empfindlichkeit von P. aeruginosa gegenüber Benzalkoniumchlorid wurde bestätigt. Signifikante Unterschiede zwischen klinischen 3MRGN, 4MRGN und den jeweiligen Referenzstämmen in ihrer Sensitivität gegenüber Bioziden wurden - unabhängig von der angewandten Testmethodik - nicht festgestellt. Im Vergleich der Bakterienspezies wiesen Pseudomonaden eine geringere Sensibilität gegenüber Benzalkoniumchlorid auf, jedoch lagen die bakteriziden Konzentrationen in jedem Fall unterhalb der gelisteten Anwendungskonzentrationen. Der Einfluss organischer Belastung auf die Wirksamkeit von Natriumhypochlorit war signifikant. Die Expositionsdauer führte zu keiner signifikanten Absenkung der Wirkkonzentrationen. Die Annahme, dass antibiotikaresistente Bakterien zwangsläufig biozidresistent sind, konnte anhand unserer Ergebnisse nicht bestätigt werden. Spezielle Desinfektionsprotokolle sind nicht notwendig, solange eine konsequente Einhaltung der durch den Hersteller vorgeschriebenen Konzentrations-Zeit-Relationen gegeben ist. Die Höhe der Desinfektionsmittelkonzentration ist für den Desinfektionserfolg ausschlaggebend, während der Einfluss der Einwirkzeit vernachlässigbar ist. Die MHK-Bestimmung eignet sich im Gegensatz zu praxisnahen Keimträgerversuchen nicht zur Ableitung von Anwendungs-konzentrationen von Desinfektionsmitteln.:1 Einleitung 2 Literatur 2.1 Desinfektion 2.1.1 Allgemeines 2.2 Wirksamkeitsprüfung chemischer Desinfektionsmittel 2.2.1 Allgemeines 2.2.2 VAH-Richtlinie 2.2.3 Minimale Hemmkonzentration 2.2.4 Qualitativer Suspensionstest 2.2.5 Quantitativer Suspensionstest 2.2.6 Quantitativer Keimträgertest 2.3 Bakterielle Resistenzen gegenüber Antibiotika 2.3.1 Allgemeines 2.3.2 Nosokomiale Infektionen 2.3.3 Acinetobacter spp. 2.3.4 Klebsiella spp. 2.3.5 Pseudomonas aeruginosa 2.4 Bakterielle Resistenzen gegenüber Bioziden 2.4.1 Allgemeines 3 Veröffentlichungen 3.1 Veröffentlichung 1 3.2 Veröffentlichung 2 4 Diskussion 5 Zusammenfassung 6 Summary 7 Referenzen 7.1 Literaturverzeichnis 7.2 Tabellenverzeichnis 8 Danksagung

<p><b>Part I: Design of a Photolabile Backbone Amide Linker for the Synthesis of C-terminally Modified Peptides</b></p> <p>A new photolabile backbone amide linker has been developed for the on-resin synthesis of cyclic and C-terminally modified peptides. The linker (Hcnb) is stable to strongly acidic conditions and instead releases the completed peptide through photolytic cleavage at 365 nm. Hcnb possesses four degrees of orthogonality and is amenable to the preparation of cyclic peptides, C-terminally modified peptides, and fully protected peptides due to its photolabile backbone amide linkage. The Hcnb precursor can be conveniently synthesized in 4 steps from commercially available 4-methyl-3,5-dinitrobenzoic acid. The C-terminal amino acid residue is loaded via reductive amination of the precursor followed by an O→N transacylation for the addition of the second residue in quantitative yields, even when employing sterically bulky residues. Standard Fmoc- or Boc-based synthesis can then be utilized to complete the desired peptide. Hcnb has been used to demonstrate the linear synthesis and subsequent on-resin cyclization of various cyclic peptides of interest, as well as synthesis of C-terminal thioesters on-resin. </p> <p><b>Part II: Development of II-HMG CoA Reductase Inhibitors for use as Gram-Positive Selective Antimicrobials. </b></p> <p>Bacterial resistance to antibiotic drugs is an issue that humans have faced since the first use of sulfa drugs in the 1930s. In recent years, the rate of production of new antimicrobial drugs has diminished, as they are no longer financially beneficial to pharmaceutical companies due to short term use and rapid resistance development. This places the burden of the development of new antimicrobial drug on the academic research field. In the work presented here, progress has been made toward the development of a novel class of antimicrobial compounds. These small molecule inhibitors target II-HMG CoA Reductase, a key enzyme involved in cell wall synthesis in gram-positive bacteria. Based on analysis of co-crystal structures obtained from first- and second- generation inhibitors, structural alterations were made to design a new generation of compounds. Efforts have also been made toward identification of a potential secondary target of these inhibitors. </p>

Abstract Time-consuming diagnosis of bacterial blood stream infections and inappropriate antibiotic therapy have critical implications for patient outcome – with mortality figures rising for every hour of delayed treatment. The development of diagnostic methods that are capable of selective and rapid bacteria detection, and do not rely on preliminary blood culturing and Gram-staining procedures, is imperative in providing effective therapy and preventing multi-resistance. The aim of this dissertation was to develop a quantum dot based and Gram-specific bacteria labelling protocol. Focused on the detection of Gram-negative species, a two-step conjugation protocol was produced to functionalise quantum dots with anti-lipid A antibodies. Ionic adsorption and EDC chemistry were used to obtain oriented and covalent conjugation of antibodies to the quantum dot surface. In order to reduce non-specific binding of unreacted carboxylic groups on the conjugates to the bacterial membrane, and optimise the accuracy of detection, blocking experiments were conducted with molecules that could provide a neutral surface charge and sterically block open sites. To access lipid A on E. coli cells, three different antigen retrieval methods were tested. As a result, the developed quantum dot-anti lipid A conjugates were able to detect and specifically label Gram-negative E. coli cells after treatment with 0.6mM EDTA or acetic acid pH 3.58 at 42.5°C. 1% BSA reduced non-specific binding to untreated E. coli cells. Furthermore, in comparison to experiments performed with Tris as a blocking agent, the protein reduced non-specific binding to Gram-positive cells. The results obtained in this project are a step further in the development of a new method to rapidly detect bacteria Gram-specifically.

Background: Multidrug-resistant Gram-negative bacteria (MDRGN) are found with rising prevalence in non-hemodialysis risk populations as well as hemodialysis (HD) cohorts in Asia, Europe and North America. At the same time, colonization and consecutive infections with such pathogens may increase mortality and morbidity of affected individuals. We aimed to monitor intestinal MDRGN colonization in a yet not investigated German HD population. Methods: We performed cross-sectional point-prevalence testing with 12 months follow-up and selected testing of relatives in an out-patient HD cohort of n = 77 patients by using microbiological cultures from fresh stool samples, combined with Matrix Assisted Laser Desorption Ionization—Time of Flight Mass Spectrometry (MALDI-TOF-MS) and antimicrobial susceptibility testing. Results: We detected MDRGN in 8 out of 77 patients (10.4%) and 1 out of 22 relatives (4.5%), indicating only colonization and no infections. At follow-up, 2 patients showed phenotypic persistence of MDRGN colonization, and in 6 other patients de-novo MDRGN colonization could be demonstrated. Pathogens included Escherichia coli and Klebsiella pneumoniae (with extended-spectrum beta-lactamase [ESBL]-production as well as fluoroquinolone resistance), Stenotrophomonas maltophilia and Enterobacter cloacae. Conclusions: In a single-center study, MDRGN colonization rates were below those found in non-HD high-risk populations and HD units in the US, respectively. Reasons for this could be high hygiene standards and a strict antibiotic stewardship policy with evidence of low consumption of fluoroquinolones and carbapenems in our HD unit and the affiliated hospital.

The work in the Brown laboratory has two long-range objectives. Both are derived from an interest in the replication of the genome of Gram-positive eubacteria. One objective is to gain a deeper understanding of the structure and function of DNA polymerase III, the unique species of DNA polymerase which is essential for chromosome replication. The second objective, the one from which this thesis is derived, is to determine whether a selective inhibitor of this DNA polymerase can serve as a basis for producing a new generation of clinically useful Gram-positive-selective antimicrobial agents. The polymerase III-specific inhibitor prototypes investigated in this work are members of a family of simple 6-substituted uracils. The following members of this family, TMAU and EMAU, were used as platforms for the manipulation of the N3 atom (arrow), the only ring component which could be substituted without severe reduction of inhibitory activity. The N3 position was substituted with a series of alkyl groups of increasing size. The resulting structure-activity relationships at the level of the polymerase was consistent with the presence of an N3-specific subdomain within the inhibitor binding site which could accommodate a wide variety of substituents. Although specific alkyl substituents at N3 also significantly enhanced the antibacterial potency of TMAU and EMAU, the respective compounds were found to have insufficient aqueous solubility for successful application in in vivo infection. To increase aqueous solubility, the N3 atom of the EMAU platform was substituted with selected hydroxy- and methoxyalkyl groups. The latter agents retained both anti-polymerase and antibacterial activity, and, as expected, they displayed a combination of lipid and aqueous solubility favorable to efficacy in in vivo infection. Two of the agents, N3-hydroxypropyl- and N3-methoxypropyl-EMAU were examined for their ability to protect mice from lethal staphylococcal infection. Both were found to be active in this model. In sum, the results of this work demonstrated, for the first time, that: (1) the eubacterial replication-specific DNA polymerase III is a valid target for antibiotic development, and (2) the N3-substituted 6-anilinouracil platform has strong potential as a basis for novel antibiotics useful against Gram-positive bacterial infection.

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Several Gram-negative bacteria modify their outer most surface structure, lipopolysaccharide (LPS), to evade immune surveillance and survive within the host. Many of these changes occur within the lipid A domain, a region that is recognized by the innate immune system via Toll-like receptor-4 (TLR4). One such pathogen, Porphyromonas gingivalis, orchestrates chronic inflammatory disease by disrupting immune homeostasis. P. gingivalis initially synthesizes a penta-acylated lipid A that functions as a weak TLR4 agonist but displays tetra-acylated forms that are either immunologically silent or TLR4 antagonists. The impact of lipid A modifications on downstream signaling and antigen-specific immunity are unclear. TLR4 signals from the plasma membrane through a MyD88-dependent pathway and intracellularly through a TRIF-dependent pathway. Here we show that expression of immunological silent or antagonistic lipid A enables P. gingivalis to evade TRIF-dependent signaling in dendritic cells (DCs). Evasion of TRIF signaling accelerated antigen degradation and impaired priming of pathogen-specific T cells. In contrast, a P. gingivalis strain expressing agonist lipid A potently activated TRIF signaling and delayed antigen degradation, thereby preserving peptides for optimal T cell activation. We propose that lipid A modifications control the endocytic activity of DCs and the efficiency at which microbe-specific T cells are primed. We next investigated the impact of purified P. gingivalis LPS on innate signaling and antigen presentation. All P. gingivalis LPS species induced a program of DC maturation that allowed for constitutive antigen uptake and cross-presentation. This was independent of TLR4 agonist activity and required CD14, a protein that transports TLR4 to endosomes where TRIF signaling can occur. Agonist LPS induced signaling through both MyD88 and TRIF and elicited T cell priming. Antagonistic LPS potently accelerated CD14 endocytosis and induced TRIF-biased signaling leading to comparable degree of cross-priming. Immunologically silent LPS promoted CD14 endocytosis but failed to activate signaling and induced T cell tolerance. Collectively, our results demonstrate that modification of lipid A structure enables Gram-negative bacteria to direct the host immune system towards tolerance or immunity. We propose that these findings can be harnessed for therapeutic modulation of the immune system to treat a variety of immune-mediated diseases. / 2021-06-14T00:00:00Z

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