"Bacterial infections persist as a public threat due to the ease by which bacteria adapt to commonly used antibiotics. In addition, bacteria on surfaces develop protective communities called biofilms that hinder the ability of antibiotics to completely eliminate the pathogens. The rapid development of bacterial resistance to antibiotics has made pharmaceutical companies reluctant to fund new antibiotics research. Hence, novel approaches to prevent and treat infections are needed. The development of infections can be divided into three steps: adhesion, invasion and multiplication. Antibiotics target at the latter two step and are prone to bacterial resistance as passive strategies. Bacterial adhesion to host cells/implanted medical devices is the first step leading to following invasion and multiplication. However, fundamental understanding of bacterial adhesion process is still lacking. The current studies are aimed to systematically investigate biological interactions between pathogenic bacteria and host cell, proteins and biomaterials with both macro and micro scale approaches. The macro scale methods include bacterial adhesion assay, viability studies, and thermodynamic modeling. The micro scale methods include direct adhesion force measurements, ultra surface visualization via atomic force microscopy (AFM) and surface structure modeling. Our work combines experiments and modeling aimed at understanding the initial steps of the bacterial adhesion process, focusing on two case studies: 1) Mechanisms by which cranberry can prevent urinary tract infections through interfering with bacterial adhesion; and 2) Design of anti-adhesive and antimicrobial coatings for biomaterials. We make direct adhesion force measurements between bacteria and substrates with an atomic force microscope (AFM), and combine such experiments with thermodynamic calculations, in order to develop a set of tools that allows for the prediction of whether bacteria will attach to a given surface. These fundamental investigations of the bacterial adhesion process help elucidate the underlying mechanisms behind bacterial adhesion, thus leading to improved clinical outcomes for a number of biomedical applications. "

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future. / Koagulase-negative Staphylokokken besiedeln die menschliche und tierische Haut, sowie die Schleimhäute. Durch Läsionen oder das Einbringen von medizinischen Instrumenten wie Kathetern gelangen sie in tiefere Hautschichten oder die Blutbahn und können dort schwerwiegende Infektionen auslösen, vor Allem bei Risikopersonen. Besonders Staphylococcus epidermidis hat sich als Verursacher von nosokomialen Infektionen, aber auch als Pathogen in der Tierhaltung etabliert. Die Bakterien bilden bei der Besiedlung sogenannte Biofilme aus (d.h. eine Akkumulation der Keime, die von einer extrazellulären Matrix umgeben sind). Diese Matrix besteht neben Proteinen und eDNA hauptsächlich aus einem Polysaccharid, dem interzellulären Adhäsin PIA (engl.: polysaccharide intercellular adhesin). Dieses wird durch die Ica-Proteine synthetisiert, die im icaADBC-Operon (engl.: intercellular adhesin operon) kodiert sind. Das Operon hat große Bedeutung in klinischen Stämmen und wurde daher innerhalb der letzten beiden Jahrzehnte eingehend untersucht, auch im Hinblick auf seine Regulation. In der unmittelbaren Umgebung des icaADBC-Operons, stromabwärts des icaR Gens, das für den Repressor des ica-Operons (IcaR) kodiert, wurde ein großes Transkript identifiziert, von dem vermutet wird, dass es möglicherweise an der Regulation der Biofilmbildung beteiligt ist (Eckart, 2006). Ziel dieser Arbeit war es, dieses Transkript zu charakterisieren und seine Funktion in S. epidermidis aufzudecken. Die nicht-kodierende RNA, genannt IcaZ, hat eine Länge von ~400 nt und ist spezifisch für ica-positive S. epidermidis. Sie wird in der frühen bis mittleren exponentiellen Phase temperaturabhängig exprimiert. Stromaufwärts überlappt das icaZ-Gen und dessen Promotor mit der 3' UTR vom icaR-Gen. Stromabwärts wird das icaZ-Gen vom einem Transkriptionsterminator begrenzt, der auch für das tRNAThr-4-Gen benutzt wird, das auf dem gegenüberliegenden Strang in Richtung des icaZ-Gens lokalisiert ist. Die Deletion der RNA führte zu einem makroskopisch sichtbaren Biofilm-negativen Phänotyp mit deutlich verminderter PIA Bildung. Die Biofilmzusammensetzung wurde in vitro mittels eines klassischen Kristallviolett-Assays gemessen und die Biofilmbildung in vivo in Echtzeit mittels konfokaler Mikroskopie (CLSM) betrachtet. Dabei wurde mit einer peristaltischen Pumpe ein Mediumfluss appliziert. Die Mutante zeigte klare Defekte in der initialen Adhärenz und in der Zell-Zell Adhäsion. Sie bildete im Gegensatz zum Wildtyp keinen strukturierten Biofilm aus. Zur Komplementierung des Biofilms wurde die IcaZ von einem Plasmid exprimiert und die Biofilmzusammensetzung nach 18-20 Stunden Wachstum gemessen. Die Ergebnisse dieser Untersuchungen in den verschiedenen Mutanten waren nicht eindeutig. Um die Funktion von IcaZ aufzudecken, wurden Transkriptom- und Proteomvergleiche zwischen Wildtyp und Mutante gemacht. Diese lieferten einige Hinweise, aber da der metabolische Unterschied eines Biofilmbildners zu einem Nicht-Biofilmbildner zu groß war, wurde eine direktere Methode angewandt, die induzierte Expression (Pulsexpression). Zudem wurden potentielle Interaktionspartner der IcaZ mittels computer-basierter Bindungsvorhersagen analysiert. Die icaR mRNA kristallisierte sich dabei als Target heraus und die Interaktion zwischen IcaZ und icaR mRNA wurde mit Gelshift-Assays (EMSA) untersucht. Eine Bandenverschiebung wurde mit icaR 3' UTR und mit dem icaR-5'-3' UTR-Fusionsprodukt detektiert, wohingegen keine Interaktion zwischen IcaZ und icaA mRNA stattfand. Aufgrund dieser Assays wurde vermutet, dass IcaZ die Translation von icaR in S. epidermidis reguliert. In S. aureus fehlt die nicht-kodierende RNA IcaZ und für icaR mRNA wurde eine Autoregulation gezeigt, bei der die icaR 5' UTR mit der icaR 3' UTR intramolekular oder intermolekular durch Basenpaarung interagiert, wodurch die Shine-Dalgarno Sequenz blockiert wird und es aufgrund dessen zu einer Hemmung der Translation kommt. Die Umweltfaktoren, die dazu führen sind bisher unbekannt. Der Komplex wird durch eine Endoribonuklease, RNase III, abgebaut (Ruiz de los Mozos et al., 2013). In S. epidermidis wurde eine solche Interaktion theoretisch ausgeschlossen. Experimentelle Analysen dieser Arbeit haben gezeigt, dass diese Autoregulation in S. epidermidis nicht stattfinden kann und es wird angenommen, dass IcaZ diese Regulation übernimmt. Um die Interaktion zu visualisieren wurden GFP-Reporter Plasmide generiert, die aber für weitere Experimente noch zu verbessern sind. Zusammenfassend lässt sich sagen, dass IcaZ mit der icaR mRNA interagiert, was höchstwahrscheinlich zu einer Hemmung der Translation des Repressors IcaR führt und damit letztlich PIA-Synthese und Biofilmbildung positiv reguliert. Zusätzlich wurde gefunden, dass Ethanol die Expression der IcaZ-RNA induziert, während NaCl nur schwache Effekte zeigte und Glucose keinen Einfluss auf die Expression von icaZ hatte. Ethanol ist ein Bestandteil von Desinfektionsmitteln, die in Krankenhäusern verwendet werden und ist bekannt dafür Biofilmbildung auszulösen. Da die Bildung von Biofilmen auf medizinischen Geräten kritisch ist und diese die Behandlung von S. epidermidis Infektionen erschweren, tragen die Ergebnisse dieser Arbeit nicht nur zu einem besseren Verständnis des komplexen Netzwerks der Biofilmregulation bei, sondern haben möglicherweise auch praktischen Nutzen in der Zukunft.

Staphylococcus epidermidis has emerged in recent years as an important nosocomial pathogen, especially in infections associated with implanted foreign body materials (e.g., prosthetic joints and heart valves) and in individuals with a compromised immune system (e.g., cancer patients and neonates). Although rare, implant infections are long lasting and cause severe suffering for the patient that includes pain and disability and even increased mortality. One aim of the present thesis was to develop and evaluate a genetic method for species identification and simultaneous detection of rifampicin resistance in staphylococci. A second aim was to examine S. epidermidis isolated from prosthetic joint infections (PJIs) and from wrists and nares of healthy individuals regarding their antibiotic susceptibility, biofilm production, virulence factors, and epidemiology. Comparison with phenotypic diagnostics revealed that 8 (16%) of 49 isolates differed in their species identification in favour of the genetic method. In addition, mutations associated with rifampicin resistance, including two not previously reported, were possible to detect in all isolates resistant to rifampicin. Antibiotic susceptibility testing of 61 PJI isolates showed multi-drug resistance in 91%. Furthermore, the results of the synergy testing revealed that no antibiotic combination was significantly better than the others. Hence, the effects that were possible to detect were isolate dependent. To find a method for discriminating between invasive (n=61) and commensal (n=24) isolates of S. epidermidis genotypic and phenotypic characterisations of biofilm production (including the ica and aap genes), antibiotic susceptibility, virulence-related genes (such as agr and ACME) and epidemiology were performed (using multilocus sequence typing [MLST], typing of the staphylococcal chromosome cassette mec [SCCmec] and PhenePlate). Significant differences were found in antibiotic susceptibility, i.e. there was more resistance among invasive isolates. MLST sequence types (ST) ST2 and ST215 dominated the invasive isolates.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Bacterial contamination of platelet concentrates (PCs) represents the highest post-transfusion infectious risk. The skin flora bacterium Staphylococcus epidermidis has been reported to be the predominant aerobic contaminant of PCs. The Ramirez' group has shown that S. epidermidis can form surface-attached bacterial aggregates known as biofilms, and can outcompete other coagulase-negative staphylococci, such as Staphylococcus capitis, in PCs. The ability of S. epidermidis to form biofilms has been linked to increased pathogenicity and missed detection during PC screening with an automated culture system (BacT/ALERT). This thesis aimed at investigating the proliferative advantage and resistance mechanisms displayed by S. epidermidis in the PC milieu. Furthermore, in an effort to enhance PC safety for transfusion patients, I studied the anti-biofilm properties of essential oils and antimicrobial peptides (AMPs). My studies aimed at improving PC safety by focussing on both the point of introduction of bacterial contaminants (blood collection), and the stage at which bacterial contaminants can form biofilms and proliferate (PC storage). S. epidermidis can be found in the skin of blood donors as biofilms, which are resistant to the blood donor skin disinfectant currently used by Canadian Blood Services, chlorhexidine-gluconate and isopropyl alcohol (CHG-IPA). Here, several plant-extracted essential oils were evaluated for their ability to enhance the anti-biofilm activity of CHG-IPA. Data revealed that the Lavandula multifida oil and its main component (linalool) greatly enhanced the activity of CHG-IPA against S. epidermidis biofilms. Furthermore, the ability of a combination of three synthetic AMPs to inhibit S. epidermidis biofilm formation during PC storage was assessed These results showed that the combination of AMPs could inhibit biofilm formation but was ineffective against pre-formed S. epidermidis biofilms. The accumulation associated protein (Aap) encoded by the aap gene, found in most S. epidermidis strains and absent in S. capitis, plays a role in biofilm formation. When S. epidermidis aap is transformed into S. capitis, this bacterium displayed increased biofilm formation and proliferated to higher concentrations compared to untransformed S. capitis and to a S. epidermidis aap deletion mutant. Based on these results, aap appears to play a role in providing S. epidermidis a proliferative advantage in PCs by enhancing biofilm formation. Lastly, the GraRS system and SepA were studied for their role in S. epidermidis resistance to platelet-derived AMPs using the synthetic AMP PD4 as a model molecule. Results indicate that the GraS mechanism is involved in resistance towards PD4. The work presented in my thesis provides further insights into why S. epidermidis has a proliferative advantage in the PC storage environment and allows for the proposal of alternative methods to enhance PC safety for transfusion patients.

Made available in DSpace on 2014-07-28T18:09:54Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 103.pdf: 652449 bytes, checksum: ea164ab2e7463c3fab8f472fadef32d6 (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:35Z (GMT). No. of bitstreams: 2 103.pdf: 652449 bytes, checksum: ea164ab2e7463c3fab8f472fadef32d6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Staphylococus aureus e Staphylococcus Coagulase negative(CNS) são reconhecidos como causadores de infecções hospitalares e comunitárias em todas as regiões do mundo. Nós estudamos a produção de biofilme em 43 cepas de Staphylococus aureus meticilina resistente (MRSA) e em 21 isolados de Staphylococus epidermidis meticilina resistente (MRSE) e a susceptibilidade a antibióticos destas cepas que foram obtidas de pacientes infectados do Hospital Universitário Antônio Pedro (HUAP), localizado na cidade de Niterói no estado do Rio de Janeiro. Foi realizado também a Eletroforese em campo pulsado (PFGE) de fragmentos de macrorestrição de SmaI do DNA genômico, bem como a tipificação do cassete cromossômico estafilocócico mec (SCCmec).Os isolados que carreaiam mecA foram usados para estudar a distribuição do tipo clonal entre 43 cepas de MRSA colhidas no HUAP de 2003 a 2006. Nossos resultados demonstraram que a maioria das cepas de MRSA (83.7 por cento) e MRSE (52,4 por cento) produziram biofilme,e altas taxas de resistência de MRSA e MRSE à eritromicina, à clindamicina e ao ciprofloxacino foram observadas. Dois tipos clonais predominantes (A e B) foram identificados por PFGE, tendo ambos compreendendo a maioria das cepas (32,6 por cento cada um). De acordo com a tipificação de SCCmec, o SCCmec tipo IV foi o que prevaleceu (51,2 por cento). Porém, o mais interessante em nosso estudo foi a mudança observada no background genético dos MRSA isolados do HUAP, pois o Clone Epidêmico Brasileiro(CEB) que era o clone predominante neste hospital, está sendo substituído pelo USA-400, um clone que a princípio foi denominado de clone comunitário Staphylococcus aureus meticilina resistente (CA-MRSA). / Staphylococus aureus and coagulase-negative staphylococci (CNS) are recognized as causing nosocomial and community-acquired infection in every region of the world. We study biofilm production in 43 isolates of methicillin-resistant Staphylococcus aureus (MRSA) and 21 isolates methicillin-resistant Staphylococcus epidermidis (MRSE) and the antibiotics susceptibilities of these strains that were obtained from infected patients at Antônio Pedro University Hospital (HUAP) located in Niterói city, Rio de Janeiro. In addition, Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA as well as staphylococcal cassette chromosome mec (SCCmec) typing for mecA-carrying isolates were used to study the distribution of clonal types among 43 MRSA recovered in HUAP between 2003 and 2006. Our results demonstrated that the majority strains of MRSA (83.7%) and MRSE (52,4%) produced biofilm and high resistance of MRSA and MRSE to erytromicin, clindamicin and ciprofloxacin were observed. The two major clones types (A and B) were identified by PFGE, with both them comprising the majority of strains (32,6% each). According to SCCmec typing, SCCmec type IV were the most prevalent type, showing 51,2% . But the most interesting in our study is a changed observed in the genetic background of MRSA isolates from HUAP, because of Brazilian clone, which was the major clone in this hospital, was replaced by the USA-400, one type of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA).

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.

Made available in DSpace on 2016-06-02T19:02:42Z (GMT). No. of bitstreams: 1 5562.pdf: 2010923 bytes, checksum: 99b0ace4e740c99eb2fc0708b19a70fd (MD5) Previous issue date: 2013-05-10 / Financiadora de Estudos e Projetos / The staphylococci belong a diverse group of bacteria that cause diseases ranging from minor skin infections to death-threatening bacteraemia. The two major opportunistic pathogens of this genus, Staphylococcus epidermidis and S. aureus are the most frequent causes of nosocomial infections and infections associated with the use of medical devices. Staphylococci, is the leading cause of infections associated with biofilm formation. Biofilmrelated infections are challenging to treat with conventional antimicrobial agents, limiting the efficacy of antibiotic therapy and becoming a crucial problem for treatment of chronic infections. Therefore, the main aim of this thesis was to study the susceptibility of staphylococcal biofilm cells against antimicrobial agents. For that, it was evaluated in vitro activities of N-acetylcysteine (NAC), rifampicin, linezolid, daptomycin and vancomycin alone and in combination on biofilm cells of Staphylococcus epidermidis and S. aureus. The activities of antimicrobial agents were evaluated in concentrations CIM, 10xCIM and peak serum. The biofilms susceptibility to agents studied was assessed through, colony-forming units (CFU/ml), staining with crystal violet (CV) that is the measure total biofilm biomass and cellular activity using XTT reduction assay. The results of viable cells (expressed as log10 CFU/ml) to N-acetylcysteine alone, in the concentration 10xCIM on both the biofilms of staphylococcal evaluated showed a greater effect compared with other antimicrobial agents evaluated, with reductions of approximately 4-5 log10. The combination NAC (10xCIM) - vancomycin (independent of concentration evaluated) showed a greater reduction (p<0.05) on viable cells of S. epidermidis and S. aureus, compared with other combinations evaluated. This combination presented a reduction about of 5-6 log10 CFU/ml. The results of CV showed loss the total biofilm biomass and were observed decrease in the metabolic activity measured by the XTT, these results are in very good agreement with those obtained in terms of cell viability. In conclusion, the results obtained in the studies of this thesis constitutes a promising therapeutic strategy in the treatment of infections associated with biofilm formation by S. epidermidis and S. aureus. In addition, the use of antimicrobial agents in combinations may be an alternative for monotherapy, thus avoiding the development of resistance. / Os estafilococos pertencem a um grupo diversificado de bactérias que causam doenças que vão desde infecções de pele, a risco de morte como bacteriemia. Os dois principais patógenos oportunistas deste gênero, Staphylococcus epidermidis e S. aureus são as causas mais frequentes de infecções nosocomiais e infecções associadas ao uso de dispositivos médicos. Estafilococos é a principal causa de infecções associadas com a formação de biofilme. As infecções relacionadas à formação de biofilme são difíceis de tratar com agentes antimicrobianos convencionais, limitando a eficácia da terapia com antibióticos e tornando um problema crucial para o tratamento de infecções crônicas. Portanto, o objetivo principal desta tese foi estudar a susceptibilidade das células de estafilococos em biofilme contra agentes antimicrobianos. Para isso, foram avaliadas as atividades in vitro de N-acetilcisteína (NAC), rifampicina, linezolida, daptomicina e vancomicina isoladamente e em combinação contra as células em biofilme de Staphylococcus epidermidis e S. aureus. As atividades dos agentes antimicrobianos foram avaliadas nas concentrações CIM, 10xCIM e a concentração máxima no soro. A susceptibilidade dos biofilmes para os agentes estudados foi avaliada através de unidades formadoras de colônia (UFC/ml), a coloração com cristal violeta (CV), que é a medida da biomassa total do biofilme e, a atividade celular usando ensaio de redução de XTT. Os resultados das células viáveis (expressos em log10 UFC/ml) para N-acetilcisteína sozinho, na concentração 10xCIM para ambos os biofilmes de estafilococos avaliados mostraram um maior efeito em comparação com outros agentes antimicrobianos avaliados, com redução de cerca de 4-5 log10. A combinação de NAC (10xCIM) - vancomicina (independente da concentração avaliada) mostrou uma maior redução (p<0,05) em células viáveis de S. epidermidis e S. aureus, em comparação com as outras combinações avaliadas. Esta combinação apresentou uma redução de cerca de 5-6 log10 CFU/ml. Os resultados de CV mostraram perda da biomassa total do biofilme e, foram observados diminuição da atividade metabólica, medida pelo ensaio de XTT, este resultados estão em boa concordância com os resultados obtidos em termos de viabilidade celular. Em conclusão, os resultados obtidos nos estudos desta tese constituem uma estratégia terapêutica promissora para o tratamento de infecções associadas a formação de biofilme por S. epidermidis e S. aureus. Além disso, a utilização de agentes antimicrobianos em combinação pode ser uma alternativa para a monoterapia, evitando assim o desenvolvimento de resistência.

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.