A vancomicina é um antimicrobiano de escolha para o tratamento de microrganismos gram-positivos multirresistentes, como Staphylococcus aureus resistente à meticilina (MRSA). Apesar da importância ouso indiscriminado gera preocupação em relação à resistências bacteriana, além de ser considerado nefrotóxico e haver carência de dados em pediatria.Objetivo:Avaliar o perfil de utilização de vancomicina, a presença de eventos adversos, nefrotoxicidade e o desfecho clínico pacientes de uma Unidade de Terapia Intensiva. Método: Estudo transversal, retrospectivo, conduzido entre pacientes pediátricos internados em unidade de terapia intensiva que receberam vancomicina durante o período de abril de 2013 a junho de 2014. Os dados foram coletados de fontes secundárias como prontuário, prescrições e exames laboratoriais. Resultados: De 94 pacientes selecionados, 53,2% eram lactentes e o CID mais prevalente foi bronquiolite aguda devido a outros microrganismos especificados. A duração do tratamento com vancomicina teve uma média de 13±6,7 dias e uma dose média inicial de 51,2±14,7 mg/kg/dia. Entre os eventos adversos observados, edema foi o mais prevalente, seguido de toxicidade renal. Dos doze pacientes que tiveram MRSA identificado em hemocultura ou cultura de secreções, três tiveram infecção persistente e um foi a óbito em menos de 30 dias. Dos pacientes com nefrotoxicidade, 63,6% utilizaram vancomicina por mais de 14 dias e todos tiveram níveis séricos ≥15 μg/ml. Conclusão: As faixas de doses de vancomicina usadas estão dentro do que tem sido recomendado na literatura, contudo o aparecimento de toxicidade renal parece ter relação significativa não somente com o nível sérico de vancomicina, mas também com o tempo de tratamento. Sugere-se a realização de estudos que consigam confirmar essa tendência associativa entre tempo, concentração sérica de vancomicina e aparecimento de nefrotoxicidade. / Vancomycin is an antibiotic of choice for the treatment of multidrug-resistant grampositive microorganisms, as methicillin-resistant Staphylococcus aureus (MRSA). Despite the importance their indiscriminately use causes concern regarding the bacterial resistance, besides being nephrotoxic agent and there are few studies in pediatric patients. Objective: To evaluate the profile of use of vancomycin, the presence of adverse events, nephrotoxicity and clinic outcome in children admitted to the Intensive Care Unit. Method: Cross-sectional and retrospective study, with pediatric patients admitted to the intensive care unit who received vancomycin during the period from April 2013 to June 2014. Data were collected from secondary sources such as patient records, prescriptions and laboratory tests. Results: 94 selected patients, 53.2% were aged infants and the most prevalent CID was acute bronchiolitis due to other specified microorganism. The duration of vancomycin treatment averaged 13±6.7 days and an initial mean dose was 51.2±14.7 mg/kg/day. Among the adverse events observed edema was the most prevalent, followed by renal toxicity. Of the twelve patients who had MRSA identified in blood or secretions culture, three had persistent infection and one died in less than 30 days. Of patients with nephrotoxicity, 63.6% used vancomycin for more than 14 days and all had vancomycin serum levels ≥15 μg/mL. Conclusion: Vancomycin doses used are in agreement with recommended in literature, however the onset of renal toxicity seems to be related with serum level and the duration of vancomycin treatment. It is suggested to conduct studies to confirm this associative tendency among treatment duration, vancomycin serum levels and appearance of nephrotoxicity.

Made available in DSpace on 2015-04-11T13:38:44Z (GMT). No. of bitstreams: 1 Dissertacao Mirna Miyamoto.pdf: 3227973 bytes, checksum: 7f5d27c4ca1e753ebe05cec66ce51343 (MD5) Previous issue date: 2010-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Staphylococcus aureus is a potential pathogen, accounting for 60% of infections in ICU`s and can be found in the nasopharyngeal region and also in the nasal cavity, especially in health care workers who become sources of dissemination of these microorganisms in hospital. However, the indiscriminate use of antibiotics to combat these pathogens, has caused the emergence of bacteria possessing resistance genes such as mecA. Of which are about 30 to 50% of the strains of S. aureus and more than 50% of coagulase-negative staphylococci. The CONS are increasingly becoming the target of concern in hospital environments, increasing the need for screening, prevention and control of resistant strains. Therefore, the objective of this work was the characterization of Staphylococcus sp., looking for the resistance gene and genetic mapping of S. aureus in clinical samples from the cities of Manaus and Porto Velho. The techniques used were biochemical tests, antibiogram, PCR (for 16 rRNA gene and mecA) and MLST. The four strains of CONS were found, three of Staphylococcus sciuri and S. epidermidis, characterized by phenotypic and genotypic tests. They were possessed of the methicillin resistance gene mecA. As for S. aureus studied, failed to detect the presence of the mecA gene, but gene mapping was performed from these samples with the technique of MLST, using five housekeeping genes. In this analysis we observed the formation of two large clusters between the cities of Manaus and Porto Velho, and two samples showed genetic divergence from other, thus demonstrating genetic variability between the cities of northern Brazil. The evidence related in the scientific literature, little research related to knowledge and epidemiological characterization of strains existing in the Amazon region, in this research, we discussed some questions of phenotypic analysis, characterization of new resistant strains and preparation of database for future development of biotechnological tools. / Staphylococcus aureus é um patógeno em potencial, sendo responsável por 60% das infecções nos CTIs, podendo ser encontrado na região da nasofaringe e também nas fossas nasais, principalmente em profissionais da saúde que se tornam fontes de disseminação desses micro-organismos no ambiente hospitalar. No entanto, o uso indiscriminado de antimicrobianos no combate desses patógenos, tem ocasionado o surgimento de bactérias possuidoras de genes de resistência, como o gene mecA, entre as quais, cerca de 30 a 50% das cepas de S. aureus e mais de 50% são de estafilococos coagulase-negativos são portadores deste gene. Os CONS estão cada vez mais se tornando alvo de preocupação nos ambientes hospitalares, aumentando a necessidade de rastreamento, prevenção e controle das cepas resistentes. Sendo assim, o objetivo deste trabalho foi realizar a caracterização de Staphylococcus sp., quanto a presença do gene de resistência e mapeamento genético de S. aureus em amostras clínicas das cidades de Manaus e Porto Velho. As técnicas aplicadas foram testes bioquímicos, antibiograma, PCR (para gene 16S rRNA e gene mecA) e MLST. Neste trabalho, encontramos quatro cepas de CONS, três de Staphylococcus sciuri e um S. epidermidis, caracterizados por testes fenotípicos e genotípicos, sendo estas possuidoras do gene de resistência a meticilina mecA. Quanto aos S. aureus estudados, não se detectou a presença do gene mecA, mas foi realizado o mapeamento gênico destas amostras com a técnica de MLST, utilizando cinco genes constitutivos. Nesta análise, foi observada a formação de dois grandes agrupamentos principais (clusters) entre as cidades de Manaus e Porto Velho, e duas amostras apresentaram divergência gênica das outras, demonstrando assim, variabilidade genética entre essas cidades da Região Norte do Brasil. Diante das evidências científicas na literatura, escassas pesquisas relacionadas ao conhecimento epidemiológico e caracterização das linhagens existentes na região Amazônica, nesta pesquisa, foram discutidas algumas questões de análise fenotípica, caracterizações de novas cepas resistentes e elaboração de banco de dados para futuro desenvolvimento de ferramentas biotecnológicas.

As doenças infecciosas estão entre as principais causas de morte no mundo. Nas infecções bacterianas, os componentes das paredes destes organismos são descritos como os principais antígenos. Os macrófagos estão diretamente envolvidos no reconhecimento e ataque de agentes patogênicos, além de atuarem como moduladores da resposta imunológica. Os macrófagos participam do combate às infecções bacterianas através da fagocitose dos agentes patogênicos e da produção de diversos mediadores inflamatórios como citocinas, metaloproteinases (MMP), espécies reativas de oxigênio (ROS) e óxido nítrico(NO). Estes mediadores são importantes na resposta inflamatória, contribuindo para o controle da infecção. Entretanto, a produção exacerbada destas moléculas contribui na patogênese das complicações associadas à inflamação, como a sepse e a falência múltipla de órgão. A maior parte dos estudos tem focado a resposta das células imunes a infecções por bactérias gram-negativas ou ao seu principal antígeno, o lipopolisacarídeo (LPS). No entanto, as infecções por bactérias gram-positivas são freqüentes e resultam em grande taxa de mortalidade. As doenças infecciosas estão entre as principais causas de morte no mundo. Nas infecções bacterianas, os componentes das paredes destes organismos são descritos como os principais antígenos. Os macrófagos estão diretamente envolvidos no reconhecimento e ataque de agentes patogênicos, além de atuarem como moduladores da resposta imunológica. Os macrófagos participam do combate às infecções bacterianas através da fagocitose dos agentes patogênicos e da produção de diversos mediadores inflamatórios como citocinas, metaloproteinases (MMP), espécies reativas de oxigênio (ROS) e óxido nítrico(NO). Estes mediadores são importantes na resposta inflamatória, contribuindo para o controle da infecção. Entretanto, a produção exacerbada destas moléculas contribui na patogênese das complicações associadas à inflamação, como a sepse e a falência múltipla de órgão. A maior parte dos estudos tem focado a resposta das células imunes a infecções por bactérias gram-negativas ou ao seu principal antígeno, o lipopolisacarídeo (LPS). No entanto, as infecções por bactérias gram-positivas são freqüentes e resultam em grande taxa de mortalidade. Os receptores de adenosina, em macrófagos, possuem função antiinflamatória em modelos de infecções por bactérias gram-negativas, no entanto, pouco se investigou a ação destes receptores em infecções por bactérias gram-positivas. O diabetes é uma doença metabólica comum que apresenta diversas complicações secundárias, muitas delas associadas aos níveis elevados de glicose. Os pacientes diabéticos apresentam uma ocorrência aumentada de infecções bacterianas, além de o diabetes tipo 2 estar associado com uma inflamação crônica, caracterizada por níveis plasmáticos aumentados de citocinas pró-inflamatórias. Adicionalmente, estes pacientes possuem maior risco de desenvolver doenças cardiovasculares e aterosclerose, o que tem sido relacionado à hiperglicemia e à inflamação crônica. A regulação, pela glicose, da resposta inflamatória em macrófagos expostos a bactérias gram-negativas ou ao seu antígeno principal, o LPS, é descrita, entretanto, a ação da glicose em modelos de infecções por bactérias gram-positivas não é conhecida. O tratamento dos macrófagos RAW 264.7 com concentrações elevadas de glicose aumentaram a resposta inflamatória ao LTA, com o aumento da produção de NO, TNF-a e MMP-9. Estes resultados mostram que tanto os receptores de adenosina como concentrações elevadas de glicose modulam a resposta inflamatória a S. aureus. Os receptores de adenosina parecem atuar num mecanismo autócrino de modulação da resposta inflamatória, apontando um possível alvo terapêutico. Já a modulação da resposta inflamatória dos macrófagos por concentrações aumentadas de glicose pode contribuir para complicações associadas ao diabetes, como a aterosclerose e a inflamação crônica. / Infectious diseases are among the majors causes of mortality around the world. In bacterial infections, the bacterial cell wall components are described as the main antigens. The macrophages are key mediators of inflammatory response, acting in the attack to the pathogens and in the regulation of immune response. These cells act in the phagocytosis of pathogenic agents and in the production of inflammatory mediators, like cytokines, matrix-metalloproteinases (MMP), reactive oxygen species (ROS) and nitric oxide (NO). These mediators participate of inflammatory response, acting in infection control, notwithstanding, exacerbated production of these molecules could contribute to inflammatory complications, like sepsis and multiple organ failure. Several studies has addressed the inflammatory response to gram-negative bacterial infections or lipopolysaccharide (LPS), the main antigen of these microorganisms, however grampositive bacterial infections are prevalent and associated to high mortality. The adenosine receptors are described to possess anti-inflammatory properties in macrophages in gram-negative bacterial infections models but the role of these receptors in gram-positive bacterial infections is unknown. Diabetes is a prevalent metabolic disorder which presents several associated complications; much of them associated to increased glucose levels. Diabetic patients shown increased occurrence of bacterial infections and type 2 diabetes is associated to a chronic inflammatory state, with increased circulatory levels of pro-inflammatory cytokines. Type 2 diabetic patients possess increased risk of cardiovascular diseases and atherogenesis, which has been associated to hyperglycemia and chronic inflammation. The regulation of macrophages inflammatory response to gram-negative bacterial infections or to its antigen LPS by increased levels of glucose is described, however, the role of increased glucose in gram-positive bacterial infections is unknown. For this reasons, we studied the role of adenosine receptors and increased glucose in macrophages activation by Staphylococcus aureus antigens, considering that this bacteria is the major organism in hospital infections. The treatment of RAW 264.7 macrophages with S. aureus antigens lipoteichoic acid (LTA) and peptidoglycan (PEG) resulted in the production of proinflammatory mediators. Diabetes is a prevalent metabolic disorder which presents several associated complications; much of them associated to increased glucose levels. Diabetic patients shown increased occurrence of bacterial infections and type 2 diabetes is associated to a chronic inflammatory state, with increased circulatory levels of pro-inflammatory cytokines. Type 2 diabetic patients possess increased risk of cardiovascular diseases and atherogenesis, which has been associated to hyperglycemia and chronic inflammation. The regulation of macrophages inflammatory response to gram-negative bacterial infections or to its antigen LPS by increased levels of glucose is described, however, the role of increased glucose in gram-positive bacterial infections is unknown. For this reasons, we studied the role of adenosine receptors and increased glucose in macrophages activation by Staphylococcus aureus antigens, considering that this bacteria is the major organism in hospital infections. The treatment of RAW 264.7 macrophages with S. aureus antigens lipoteichoic acid (LTA) and peptidoglycan (PEG) resulted in the production of proinflammatory mediators.

O Staphylococcus aureus está freqüentemente envolvido em surtos de intoxicação alimentar. Geralmente esses surtos têm início abrupto, causando náusea e vômito nos acometidos. Na elucidação deste tipo de surtos é necessário que seja investigada a presença da enterotoxina no alimento e/ou a capacidade enterotoxigênica da bactéria isolada. A partir disso, o objetivo do presente estudo foi implantar uma técnica de detecção de enterotoxinas e avaliar a capacidade enterotoxigênica de linhagens de Staphylococcus aureus isolados de surtos. Trinta linhagens isoladas, no período de 2002 a 2003, de alimentos envolvidos em surtos no Rio Grande do Sul foram identificadas e avaliadas quanto à capacidade de produzir enterotoxinas SEA, SEB, SEC e SED, através da técnica de sensibilidade ótima em placa-OSP.Ao lado disso, os dados epidemiológicos relativos aos surtos de origem foram coletados. Os resultados obtidos demonstraram que todas as linhagens foram positivas no teste da catalase, coagulase em tubo, prova de termonuclease, teste de hemólise e fermentaram a maltose e o manitol, sendo confirmadas como Staphylococcus aureus. Todas as linhagens produziram enterotoxina SEA, 24 produziram SEB, 12 SEC e 6 SED, isoladamente ou em associação.A maioria das linhagens foi isolada de alimentos submetidos à grande manipulação durante seu preparo e apresentaram contagens superiores a 106 UFC/g de Staphylococcus aureus, demonstrando que condições favoráveis à produção de enterotoxinas devem ter ocorrido durante o seu preparo e armazenamento. / Staphylococcus aureus often occurs in food poisoning outbreaks. These outbreaks usually start abruptly, causing nausea and vomit on affected people. In order to investigate these outbreaks, it is necessary to check the presence of enterotoxin in food and/or enterotoxigenic capacity of isolate. So the purpose of this study was to propose a method to the enterotoxin detection and evaluate the enterotoxigenic capacity of coagulase positive Staphylococci strains which were isolated from foods involved with outbreaks. On the period of 2002 to 2003, thirty strains detected in foods poisoning outbreaks in RS were identified and evaluated on their SEA, SEB, SEC and SED enterotoxins production capacity checking their sensitivity on OSP - plates. Moreover, epidemiological information related to the origin outbreaks was collected. The results showed that all the strains were positive according to following tests: catalase, coagulase in vitro/tube, termonuclease and hemolysis. Besides the strains have fermented maltose and manitol as to confirm as being Staphylococcus aureus. All the strains produced SEA enterotoxin; 24 of them produced SEB, 12 SEC and 6 SED, isolated or in association. Most strains were isolated in food that had been under intense manipulation during their preparation and which had presented levels higher than 106 CFU/g to Staphylococcus aureus, indicating that favorable conditions to the production of enterotoxins should have occured during their preparation and storage.

El uso de plantas para el alivio de afecciones de manera exitosa nos lleva a investigar su actividad y hacer formulaciones aprovechando los principios activos de estas. Es así; que en el presente trabajo se determinó el efecto antimicrobiano del gel elaborado con extracto etanólico de hojas de Senecio rhizomatus Rusby; planta seleccionada en base a la etnobotánica y etnofarmacia, se recolectó en la zona de Chavín de Huantar, distrito de Huari, Departamento de Ancash, su nombre común es “Llancahuasha”; con la cual se hizo un extracto etanólico de hojas para la determinación de metabolitos secundarios utilizando reacciones en tubo y Cromatografía en Capa Fina para la determinación de Alcaloides. Con el extracto etanólico se elaboró un gel antimicrobiano a concentraciones de 12,5 y 25 mg/mL; se evaluó su efecto antimicrobiano mediante el método de difusión en agar, utilizando para ello cepas de Staphylococcus aureus aisladas de muestra clínica hospitalaria y de la comunidad. Se encontró alcaloides, flavonoides y saponinas esteroidales y mediante la lectura e interpretación de zonas claras de inhibición del crecimiento bacteriano se comprobó el efecto antibacterial significativo del gel a concentración de 25 mg/mL frente a cepa de la comunidad de S. aureus, el halo de inhibición formado fue de 20 mm y con la cepa hospitalaria el halo de inhibición formado fue de 18 mm. Se concluye entonces que el gel elaborado con extracto etanólico de hojas de Senecio rhizomatus Rusby tiene efecto antibacteriano in vitro. Palabras clave: Senecio rhizomatus, gel antimicrobiano, difusión en agar, Staphylococcus aureus, inhibición. / --- The use of plants to relieve infections successfully leads us to investigate their activities and make formulations taking advantage of the active ingredients present. Is thus; in this study the antimicrobial effect of the gel made from ethanol extract of leaves of Senecio rhizomatus Rusby, selected based on ethnopharmacy and ethnobotany. It was collected in the area of Chavin de Huantar, Huari district, Department of Ancash, the common name of this species is "Llancahuasha", which it was made an ethanolic extract of leaves for determination of secondary metabolites using tube reactions and Thin Layer Chromatography for determination of alkaloids. With ethanolic extract, it is elaborated an antimicrobial gel at concentrations of 12,5 and 25 mg/mL ; Its antimicrobial effect was evaluated by the agar well diffusion method, using strains of Staphylococcus aureus isolated from hospital clinic and community samples. It was found alkaloids, flavonoids and steroidal saponins by reading and interpretating of clear zones of inhibition of bacterial growth, it was found significant antibacterial effect of the gel to a concentration of 25 mg/mL versus community strain of S. aureus, halo inhibition formed was 20 mm and hospital strain, with the inhibition halo formed was 18 mm. It is concluded that the gel made from ethanol extract of leaves of Senecio rhizomatus Rusby has antibacteril effect in vitro. Keywords: Senecio rhizomatus, antimicrobial gel, agar well diffusion, Staphylococcus aureus, inhibition / Tesis

A semi-synthetic medium for use in determining adherent growth with Staphylococcus epidermidis and Staphylococcus saprophyticus was developed. Production of an adherent biofilm was dependent upon the presence of hematin in the growth medium. Clinical strains of Staphylococcus epidermidis were tested for production of an adherent biofilm in trypticase soy broth, the semi-synthetic medium and the hyperalimentary nutrient solution used in the neonatal hospital unit. An adherent biofilm was obtained when Staphylococcus epidermidis was cultured m hematin supplemented hyperalimentary solution. Growth in the hyperalimentary nutrient solution diluted with fetal calf serum showed the same growth rate as when the nutrient solution was diluted with water. The final growth yield was always higher in serum diluted nutrients. There was no effect of hematin on the growth rate of the organisms.

Staphylococcus aureus encodes two extracellular nuclease enzymes, Nuc and Nuc2. Nuc is a secreted enzyme that is cut by signal peptidase (SpsB) at the cell membrane and is further processed into two active forms, NucA and NucB, by an unknown protease. Nuc2 is predicted to be a second extracellular nuclease based on sequence homology to the staphylococcal nuclease (SNase) and is tethered to the membrane with a N-terminal anchor. At the beginning of these studies, little was understood about the biological and physiological roles of Nuc and Nuc2 in S. aureus. The goal of this dissertation was to characterize the extracellular nuclease activity of S. aureus in order to better understand the contributions of Nuc and Nuc2 to the S. aureus life cycle. The studies presented in Chapter II focus on the role of Nuc in regulating S. aureus biofilm growth. The secreted forms of Nuc, called NucA and NucB, were first identified as anti-biofilm agents present in spent media from a S. aureus alternative sigma factor B (sigB) mutant. Regulation studies identified the major repressors and activators of nuc expression and showed that nuc is repressed under biofilm-forming conditions. By bypassing the native regulatory mechanisms using a nuc inducible plasmid, biofilm growth could be inhibited in a dose-dependent manner. Biofilm testing of nuc mutant strains across genetic backgrounds led to the observation that biofilm thickness increased two-fold in the absence of Nuc. More high molecular weight extracellular DNA (eDNA) accumulated in the nuc mutant compared to wild-type cells, indicating a direct link between Nuc and the availability of eDNA to contribute to the biofilm matrix. These studies showed that nuc expression is tightly regulated in S. aureus biofilms, and Nuc activity can greatly impact biofilm formation and maturation. In Chapter III, studies were performed to determine whether Nuc2 is an active nuclease in S. aureus and where the protein is localized in the cell. Upon initial comparison to Nuc, Nuc2 has 42% amino acid identity in the proposed SNase domain, and 7 of 9 residues known to be required for Nuc activity are conserved in Nuc2. Fluorescence microscopy of a Nuc2-sGFP translational fusion demonstrated the protein is localized to the cell membrane, and alkaline phosphatase fusion studies showed that the C-terminus of Nuc2 faces out of the cell. Fluorescence resonance energy transfer (FRET) assays facilitated the detection of low levels of Nuc2 activity on the S. aureus cell surface, demonstrating for the first time the enzyme is a functional nuclease, and mutations in the nuc2 gene eliminated this activity. Purification of recombinant Nuc2 also showed that enzyme has DNase activity that is calcium-dependent. Through the construction of Nuc/Nuc2 chimeric proteins, it was determined that localization to the cell membrane does not impair nuclease activity, and the low levels measured for Nuc2 on S. aureus cells is likely due instead to weak expression. The knowledge that Nuc2 is an active nuclease, localized to the cell surface, provides insight into the potential roles Nuc2 may play in a biofilm environment and during S. aureus infection.

Die vorliegende Arbeit, die im Rahmen des SFB 630 „Erkennung, Gewinnung und funktiona-le Analyse von Wirkstoffen gegen Infektionskrankheiten“ erstellt worden ist, beschäftigt sich mit der Entwicklung und Synthese der bisquartären Bisnaphthalimide und deren antimikro-biellen Eigenschaften, speziell gegen Erreger tropischer Infektionskrankheiten, wie Plasmo-dien und Trypanosomen aber auch Bakterien wie Staphylococcus aureus. Erste Testungen einer kleinen Bibliothek verschiedener mono- und bisquartärer Phthal- und Naphthalimide im SFB 630 offenbarten deren antimikrobielles Potenzial. Daher war es das Hauptziel dieser Arbeit, durch systematische Variation der verschiedenen Strukturbestandteile diese Bibliothek zu erweitern. Dazu mussten zuerst die entsprechenden Naphthalin-1,8-dicarbonsäure-Anhydride hergestellt werden. Im nächsten Schritt wurden diese mit einem N,N-Dimethylaminopropylamin-Derivat zum Imid kondensiert und abschließend mit einem Alkyl-Linker zur bisquartären Verbindung alkyliert. So konnte die Bibliothek um 25 Verbin-dungen erweitert werden. Dabei umfassten die Variationen die Alkylkettenlänge zwischen den quartären Stickstoffen mit 3–14 Methylen-Einheiten, das aromatische Substitutionsmuster mit Amino- bzw. Nitrogruppen und symmetrische, wie asymmetrische Bisnaphthalimide. Durch anschließende antimikrobielle Testung und qualitativen Vergleich der ermittelten IC50-Werte konnten verschiedene strukturelle Merkmale der Bisnaphthalimide identifiziert werden, die einen positiven Einfluss auf die Aktivität gegen den untersuchten Mikroorganismus haben. / The present work focuses on the design and synthesis of bisquaternary bisnaphthalimides and their antimicrobial properties, esp. against causative agents of tropical infectious diseases like Plasmodia or Trypanosoma but also bacteria like Staphylococcus aureus. The work was sup-ported by the SFB 630 “Recognition, Preparation and functional Analysis of Agents against Infectious Diseases”. First tests of a small compound library, consisting of several mono- and bisquaternary phthal- and naphthalimides, revealed their antimicrobial potential. Hence, it was the primary purpose of this work to broaden this library through systematical structural variations. Therefor the corresponding naphthalic anhydrides had to be synthesized. In the next step the anhydrides were condensed to the corresponding imide by reaction with an N,N-dimethylaminopropyl-amine derivative. The obtained naphthaimides were finally alkylated by means of α,ω-dihaloalkane to give the bisquaternary compound. The so achieved variations included the length of the alkyl-chain linker (C3–C14), the aromatic substitution pattern (nitro- and amino groups) and, symmetrical as well as unsymmetrical bisnaphthalimides. Subsequently, these compounds were tested against the aforementioned microorganisms. By comparing of the determined IC50-values several structural characteristics could be identified which are important to the antimicrobial activity.

Das grampositive Bakterium Staphylococcus epidermidis ist ein wesentlicher Bestandteil der kommensalen Flora der Haut und der Schleimhäute des Menschen. Jedoch stellen diese Bakterien eine häufige Ursache nosokomialer Katheter-assozierter Infektionen bei immunsupprimierten Patienten dar. Dies liegt zum einen an der Fähigkeit von S. epidermidis, Biofilm zu bilden. Diese physikalische Barriere schützt die Bakterien vor dem Immunsystem sowie vor Antibiotika. Dabei zählen sie zu den häufigsten Erregern von Infektionen an implantierten Fremdkörpern mit Plastikoberflächen, wie z. B. Venenkathetern, künstlichen Herzklappen oder Gefäßprothesen. Zum anderen stellt die Antibiotikaresistenzentwicklung unter S. epidermidis ein zunehmendes Problem dar. Vor allem die late-onset Sepsis, die durch S. epidermidis als Erreger verursacht werden kann, stellt für Frühgeborene eine Gefahr dar. Ziel der vorliegenden Arbeit war, für S. epidermidis als häufigsten und klinisch bedeutsamen KoNS zu eruieren, ob die zunehmende Dauer des stationären Krankenhausaufenthaltes von sehr kleinen Frühgeborenen mit einer höheren Rate an ica-Präsenz, Biofilmbildung und Antibiotikaresistenz assoziiert ist, sowie die Verbreitungswege und das Reservoir für diese S. epidermidis-Stämme zu identifizieren. Hierzu wurden sequenzielle Isolate von S. epidermidis bei Müttern, Kindern und vom Krankenhauspersonal gewonnen und mittels MLST (Multilocus-Sequence-Typing) klonal typisiert. Sie wurden auf Antibiotikaresistenzen, Biofilmbildung und Präsenz des icaA-Gens, das eine Rolle bei der Biofilmbildung spielt, sowie des mecA-Gens untersucht und mit Isolaten, die aus Blutkulturen oder Venenkatheter des Kindes isoliert wurden, verglichen. Es fiel auf, dass die Isolate der sehr kleinen Frühgeborenen deutlich mehr Virulenzfaktoren, wie z.B. Biofilmbildung, hohe Antibiotikaresistenzraten sowie die Präsenz des mecA- und des icaA- Gens, als die maternalen Stämme besaßen. Im Vergleich mit den Ergebnissen der untersuchten Personalstämme liegt der Verdacht nahe, dass oftmals auch das Personal als Transmitter, vor allem von Klonen mit mehreren Virulenzfaktoren, dient. Das Krankenhaus-Milieu scheint dabei ein ideales Reservoir für die Ausbreitung solcher gefährlichen S. epidermidis-Stämme zu sein. / Staphylococcus epidermidis is usually a commensal inhabitant of the human skin and mucosa. However, they are a common cause of nosocomial infections, especially in context with medical devices and catheters in immunocompromised patients. This is due to the ability of Staphylococcus epidermidis to form biofilms on inert surfaces of medical devices, like intravenous catheters or artificial arthroplastics. Furthermore the development of antibiotic resistances among Staphylococcus epidermidis is another problem. Especially the late-onset-sepsis is a danger for very low birth weight premature infants. The aim of the study was to identify for S. epidermidis isolates in very preterm infants, if a longer stay in the hospital is associated with a higher carriage of the ica-operon, a higher antibiotic resistance and the ability to form biofilms. The reservoir and the means of distribution of these nosocomial isolates should be idendified. Therefore sequential isolates were taken from mothers, their children and from the hospital staff. They were clonally typified by MLST (Multilocus-Sequence-Typing) and tested for antibiotic resistances, biofilm formation and the presence of the icaA- and mecA gene. Then they were compared with isolates from blood cultures or venous catheters from the preterm infants. Herby it was remarkable that the isolates of the very premature infants had more virulence factors, e.g. formation of biofilm, antibiotic resistances and the presence of the icaA- and mecA gene, as the maternal isolates. Compared to the results of the isolates from the medical staff it could be suggested that the staff transmits these opportunistic pathogens. The hospital environment seems to be an ideal reservoir for these dangerous S. epidermidis isolates.

Staphylococcus aureus (SA) causes nosocomial infections including life threatening sepsis by multi-resistant strains (MRSA). It has the ability to form biofilms to protect it from the host immune system and from anti staphylococcal drugs. Biofilm and planctonic life style is regulated by a complex Quorum-Sensing (QS) system with agr as a central regulator. To study biofilm formation and QS mechanisms in SA a Boolean network was build (94 nodes, 184 edges) including two different component systems such as agr, sae and arl. Important proteins such as Sar, Rot and SigB were included as further nodes in the model. System analysis showed there are only two stable states biofilm forming versus planctonic with clearly different subnetworks turned on. Validation according to gene expression data confirmed this. Network consistency was tested first according to previous knowledge and literature. Furthermore, the predicted node activity of different in silico knock-out strains agreed well with corresponding micro array experiments and data sets. Additional validation included the expression of further nodes (Northern blots) and biofilm production compared in different knock-out strains in biofilm adherence assays. The model faithfully reproduces the behaviour of QS signalling mutants. The integrated model allows also prediction of various other network mutations and is supported by experimental data from different strains. Furthermore, the well connected hub proteins elucidate how integration of different inputs is achieved by the QS network. For in silico as well as in vitro experiments it was found that the sae-locus is also a central modulator of biofilm production. Sae knock-out strains showed stronger biofilms. Wild type phenotype was rescued by sae complementation. To elucidate the way in which sae takes influence on biofilm formation the network was used and Venn-diagrams were made, revealing nodes regulated by sae and changed in biofilms. In these Venn-diagrams nucleases and extracellular proteins were found to be promising nodes. The network revealed DNAse to be of great importance. Therefore qualitatively the DNAse amount, produced by different SA mutants was measured, it was tried to dissolve biofilms with according amounts of DNAse and the concentration of nucleic acids, proteins and polysaccharides were measured in biofilms of different SA mutants. With its thorough validation the network model provides a powerful tool to study QS and biofilm formation in SA, including successful predictions for different knock-out mutant behaviour, QS signalling and biofilm formation. This includes implications for the behaviour of MRSA strains and mutants. Key regulatory mutation combinations (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) were directly tested in the model but also in experiments. High connectivity was a good guide to identify master regulators, whose detailed behaviour was studied both in vitro and in the model. Together, both lines of evidence support in particular a refined regulatory role for sae and agr with involvement in biofilm repression and/or SA dissemination. With examination of the composition of different mutant biofilms as well as with the examination of the reaction cascade that connects sae to the biofilm forming ability of SA and also by postulating that nucleases might play an important role in that, first steps were taken in proving and explaining regulatory links leading from sae to biofilms. Furthermore differences in biofilms of different mutant SA strains were found leading us in perspective towards a new understanding of biofilms including knowledge how to better regulate, fight and use its different properties. / Staphylococcus aureus (SA) ist Auslöser nosocomialer Infektionen, darunter auch die, durch multiresistente Stämme (MRSA) verursachte, lebensbedrohliche Sepsis. Er hat die Fähigkeit Biofilme zu bilden, um sich vor dem Immunsystem des Wirtes und vor Antibiotika zu schützen. Biofilm und planktonische Lebensweise werden durch ein komplexes Quorum-Sensing (QS) System mit agr als zentralem Regulator gesteuert. Um die Biofilm Bildung und QS Mechanismen in SA zu untersuchen, wurde ein Boole´sches Netzwerk erstellt (94 Knoten, 184 Kanten) das verschiedene Zwei-Komponenten-Systeme wie agr, sae und arl mit einschließt. Wichtige Proteine wie Sar, Rot und SigB wurden als weitere Knoten im Modell eingefügt. Die Systemanalyse zeigte, dass es nur zwei stabile Zustände gibt, Biofilm bildend versus planktonisch, in denen deutlich unterschiedliche Subnetzwerke angeschaltet sind. Überprüfungen anhand von Gen-Expressions-Daten bestätigten dies. Die Netzwerkstabilität wurde zuerst an Hand von bestehendem Wissen und Literatur getestet. Zudem stimmte die vorhergesagte Aktivität der Knoten in verschiedenen in silico Knock-out Stämmen sehr gut mit den zugehörigen Micro-array Experimenten und Daten überein. Zusätzliche Validierungen schlossen die Expression weiterer Knoten (Northern Blots) und die Biofilm Produktion, verglichen durch Biofilm adherence assays, in verschiedenen Knock-out Stämmen mit ein. Das Modell spiegelt zuverlässig das Verhalten von QS-Signal Mutanten wieder. Das integrierte Modell erlaubt auch Vorhersagen von diversen anderen Netzwerk Mutationen und wird durch experimentelle Daten unterschiedlicher Stämme gestützt. Außerdem zeigen die gut vernetzten Hubproteine im Detail auf, wie die Verarbeitung unterschiedlicher Eingangssignale durch das QS-Netzwerk erreicht wird. Sowohl für in silico als auch für in vitro Experimente konnte gezeigt werden, dass der sae-Locus auch einen zentralen Modulator der Biofilm Produktion darstellt, sae Knock-out Stämme zeigten stärkere Biofilme. Der Wildtyp Phänotyp wurde durch sae Komplementierung wiederhergestellt. Um die Art und Weise, mit der sae Einfluss auf die Biofilm Bildung nimmt, aufzuklären wurde das Netzwerk genutzt und Venn-Diagramme angefertigt, welche Knoten aufzeigten, die durch sae reguliert- und in Biofilmen verändert sind. In den Venn-Diagrammen wurden Nucleasen und extrazelluläre Proteine als vielversprechende Knoten gefunden. Das Netzwerk zeigte, dass DNAse von großer Bedeutung ist. Deswegen wurde qualitativ die, durch unterschiedliche SA Mutanten produzierte, DNAse-Menge gemessen, es wurde versucht den Biofilm mit vergleichbaren DNAse-Mengen aufzulösen und die Konzentration von Nukleinsäuren, Proteinen und Polysacchariden wurde in Biofilmen unterschiedlicher SA Mutanten gemessen. Aufgrund seiner sorgfältigen Überprüfung stellt das Netzwerk-Modell ein mächtiges Werkzeug zur Untersuchung von QS und Biofilm Bildung in SA dar, erfolgreiche Vorhersagen über das Verhalten unterschiedlicher Knock-out Mutanten, QS Signale und Biofilm Bildung eingeschlossen. Dies beinhaltet Prognosen für das Verhalten von MRSA Stämmen und Mutanten. Zentrale regulatorische Mutationskombinationen (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) wurden direkt im Model aber auch in Experimenten getestet. Hohe Konektivität war ein guter Anhaltspunkt, um Hauptregulatoren zu identifizieren, deren Verhalten in vitro und im Modell untersucht wurde. Zusammen unterstützen beide Beweisführungen im Besonderen eine präzise regulatorische Rolle von sae und agr in Bezug auf Biofilm Unterdrückung und/oder SA Ausbreitung. Mit der Untersuchung der Zusammensetzung von Biofilmen unterschiedlicher Mutanten, ebenso wie mit der Untersuchung der Reaktionskaskade die sae mit der Biofilm Bildungsfähigkeit von SA verbindet und auch dem Überprüfen der Annahme, dass Nukleasen eine bedeutende Rolle hierin spielen könnten, wurden erste Schritte unternommen, um regulatoische Interaktionen zwischen sae und Biofilmen zu belegen und zu untersuchen. Des Weiteren wurden Unterschiede in Biofilmen verschiedener mutierter SA Stämme gefunden, die uns voraussichtlich zu einem neuem Verständnis von Biofilmen und damit zu Wissen führen, wie ihre Eigenschaften reguliert, bekämpft und genutzt werden können.