Biofilms are bacterial communities that adhere to biotic and abiotc surfaces, and are embedded in a polymeric matrix composed mainly of polysaccharides and proteins. Not only are biofilms a public health problem, but they are also a hindrance in industrial practices. Due to their intractability by conventional cleaning agents, a number of alternative agents, including enzymes, have been investigated as potential biofilm detachment-promoting agents. Two major types of enzymes, i.e. proteases and polysaccharases, have been used for biofilm removal and their use is aimed at degrading or promoting the collapse of the biofilm matrix. Consequently, the aim of this investigation was primarily to assess the use of enzymes originating from a wastewater biofilm to remove biofilms from three Pseudomonas species, viz. P. aeruginosa PAO1, P. fluorescens and P. putida. To investigate, biofilms were sampled from an aerobic reactor at an industrial wastewater treatment plant. Dissolution of the biofilm, as evidenced by reductions in the soluble chemical oxygen demand (COD) and total suspended solids (TSS), coincided with detectable protease and carbohydrate-degrading enzyme activities. Crude extracellular enzyme extracts prepared from the wastewater biofilm were subsequently shown to remove P. aeruginosa PAO1 biofilms from a glass surface, suggesting that the wastewater biofilms expressed enzymes that may be used towards the removal of detrimental biofilms. Consequently, representative bacteria were isolated from the wastewater biofilm and, based on 16S rRNA gene sequencing and analyses, were found to represent four major phylogenetic divisions of bacteria, i.e. Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Screening of the bacterial isolates for different enzyme activities indicated that nine isolates produced proteases, while ten isolates produced polysaccharide-degrading enzymes that comprised amylase, xylanase, cellulase, á-glucosidase and â-glucosidase. The ability of these enzymes to degrade proteins and polysaccharides present in purified EPS from P. aeruginosa PAO1, P. putida and P. fluorescens was confirmed by SDS-polyacrylamide gel electrophoresis and an increase in the amount of reducing sugar, respectively, while their efficacy to remove single-and multi-species biofilms cultured in microtiter plates was evaluated using a quantitative spectrophotometric assay. Proteases produced by four of the strains were effective in degrading the EPS proteins of all three Pseudomonas spp., while all bacterial strains that produced polysaccharide-degrading enzymes were capable of degrading the EPS polysaccharides, albeit with different efficiencies. Efficient removal of P. aeruginosa PAO1 biofilms was only achieved when mixtures of enzyme extracts, containing protease and different types of polysaccharase activities, were used. Biofilms of P. putida and P. fluorescens were readily removed with single enzyme extracts prepared from B. subtilis and B. pumilus. Enzyme combinations showing high biofilm removal for all three Pseudomonas species were tested against a mixed species biofilm. These enzyme extracts yielded lower biofilm removal efficiencies than those obtained for mono-species pseudomonad biofilms, possibly due to the heterogenous nature of the EPS. Nevertheless, it may be possible that the enzymes identified in this study could be used in combination with other treatments to increase the biofilm removal effectiveness or in combination with other enzymes to degrade the mixture of proteins and polysaccharides present in the EPS of multi-species biofilms. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Biofilms are aggregations of bacteria that can thrive wherever there is a watersurface or water-interface. Sometimes they can be beneficial; for example, biofilms are used in water and waste-water treatment. The filter used to remove contaminants acts as a scaffold for microbial attachment and growth. However, biofilms could have bad effects, especially on a persons health. They can cause chronic diseases and serious infections. The importance of biofilms in industrial and medical settings, is the main reason of the mathematical studies performed up to now, concerning biofilms. Biofilms have been mathematical modelling targets over the last 30 years. The complex structure and growth of biofilms make them difficult to study. Biofilm formation is a multi-stage process and occurs in even the most unlikely of environmental conditions. Models of biofilms vary from the discrete to the continuous; accounting for one-species to multi-species and from one-scale to multi-scale models. A model may even have both discrete and continuous parts. The implication of these differences is that the tools used to model biofilms differ; we present and review some of these models. The aim in this thesis is to model the early initiation of biofilm formation. This stage involves bacterial movement towards a surface and the attachment to the boundary which seeds a biofilm. We use a diffusion equation to describe a bacterial random walk and appropriate boundary conditions to model surface attachment. An analytical solution is obtained which gives the bacterial density as a function of position and time. The model is also analysed for stability. Independent of this model, we also give a reaction diffusion equation for the distribution of sensing molecules, accounting for production by the bacteria and natural degradation. The last model we present is of Keller-Segel type, which couples the dynamics of bacterial movement to that of the sensing molecules. In this case, bacteria perform a biased random walk towards the sensing molecules. The most important part of this chapter is the derivation of the boundary conditions. The adhesion of bacteria to a surface is presented by zero-Dirichlet boundary conditions, while the equation describing sensing molecules at the interface needed particular conditions to be set. Bacteria at the boundary also produce sensing molecules, which may then diffuse and degrade. In order to obtain an equation that includes all these features we assumed that mass is conserved. We conclude with a numerical simulation. / AFRIKAANSE OPSOMMING: Biofilms is die samedromming van bakterieë wat kan floreer waar daar ’n wateroppervlakte of watertussenvlak is. Soms kan hulle voordelig wees, soos byvoorbeeld, biofilms word gebruik in water en afvalwater behandeling. Die filter wat gebruik word om smetstowwe te verwyder, dien as ’n steier vir mikrobiese verbinding en groei. Biofilms kan ook egter slegte gevolge he, veral op ’n persoon se gesondheid. Hulle kan slepende siektes en ernstige infeksies veroorsaak. Die belangrikheid van biofilms in industriële en mediese omgewings, is die hoof rede vir die wiskundige studies wat tot dusver uitgevoer is met betrekking tot biofilms. Biofilms is oor die afgelope 30 jaar al ’n teiken vir wiskundige modellering. Die komplekse struktuur en groei van biofilms maak dit moeilik om hul te bestudeer. Biofilm formasie is ’n multi-fase proses, en gebeur selfs in die mees onwaarskynlikste omgewings. Modelle wat biofilms beskryf wissel van die diskreet tot die kontinu, inkorporeer een of meer spesies, en strek van eentot multi-skaal modelle. ’n Model kan ook oor beide diskreet en kontinue komponente besit. Dit beteken dat die tegnieke wat gebruik word om biofilms te modelleer ook verskil. In hierdie proefskrif verskaf ons ’n oorsig van sommige van hierdie modelle. Die doel in hierdie proefskrif is om die vroeë aanvang van biofilm ontwikkeling te modeleer. Hierdie fase behels ’n bakteriële beweging na ’n oppervlak toe en die aanvanklike aanhegsel wat sal ontkiem in ’n biofilm. Ons gebruik ’n diffusievergelyking om ’n bakteriële kanslopie te beskryf, met geskikte randvoorwaardes. ’n Analities oplossing is verkry wat die bakteriële bevolkingsdigtheid beskryf as ’n funksie van tyd en posisie. Die model is ook onleed om te toets vir stabiliteit. Onafhanklik van die model, gee ons ook ’n reaksiediffusievergelyking vir die beweging van waarnemings-molekules, wat insluit produksie deur die bakterieë en natuurlike afbreking. Die laaste model wat ten toon gestel word is ’n Keller-Segel tipe model, wat die bakteriese en waarnemings-molekule dinamika koppel. In hierdie geval, neem die bakterieë ’n sydige kanslopie agter die waarnemings molekules aan. Die belangrikste deel van hierdie hoofstuk is die afleiding van die randvoorwaardes. Die klewerigheid van die bakterieë tot die oppervlak word vvorgestel deur nul-Dirichlet randvoorwaardes, terwyl die vergelyking wat waarnemingsmolekule gedrag by die koppelvlak beskryf bepaalde voorwaardes nodig het. Bakterieë op die grensvlak produseer ook waarnemings-molekules wat diffundeer en afbreek. Om te verseker dat al hierdie eienskappe omvat is in ’n vergelyking is die aanname gemaak dat massa behoud bly. Ter afsluiting is numeriese simulasie van die model gedoen.

Os microrganismos podem apresentar-se tanto em forma de vida livre como aderidos a uma superfície ou interface ar-líquido, formando comunidades complexas e dinâmicas conhecidas como biofilmes. Nos últimos anos, com o avanço das pesquisas em nível molecular, foi identificado que a maioria das bactérias utilizam guanosina monofosfato (3´-5´)-cíclica dimérica (c-di-GMP) como um segundo mensageiro. De forma geral, essa molécula controla a sinalização celular, virulência, comunicação entre células e a expressão de proteínas relacionadas com o fenótipo de biofilmes, em resposta à sua concentração intracelular. Sua síntese e degradação são controladas respectivamente por diguanilto ciclases (DGCs) contendo domínio GGDEF e fosfodiesterases (PDEs) que possuem os domínios EAL ou HD-GYP. Em Pseudomonas aeruginosa (PA14) foi identificada uma nova classe de receptor específico para c-di-GMP, a proteína transmembranar PelD, cuja porção citoplasmática contém os domínios GAF e GGDEF degenerado. Sua modulação através desse dinucleotídeo controla a produção de exopolissacarídeos pelos componentes do conservado operon pel e influencia diretamente na capacidade de formação de biofilmes. Devido à escassez de dados a respeito dos eventos moleculares do mecanismo de sinalização mediado por c-di-GMP, este trabalho teve como objetivo principal a caracterização biofísica/estrutural da proteína PelD, bem como o reconhecimento de interação entre este ligante e a porção citoplasmática da proteína. Diversas construções solúveis de PelD foram clonadas e expressas, sendo que a construção compreendendo os resíduos 176-455 (PelD176-455) foi cristalizada com sucesso e teve sua estrutura determinada por iodo-SAD. O modelo final apresentou os dois domínios com enovelamentos característicos das famílias GAF e GGDEF, sendo a interface inter-domínios composta majoritariamente por resíduos hidrofóbicos. Visando uma compreensão das bases moleculares de reconhecimento e ativação de PelD por c-di-GMP, uma estrutura em complexo com o ligante foi resolvida. Como esperado, o dinucleotídeo foi encontrado no sítio inibitório do domínio GGDEF, onde o motivo R367xxD370 e o resíduo R402 são responsáveis pela maior parte das interações com c-di-GMP. No entanto, nenhuma grande mudança estrutural foi observada entre as formas apo e holo de PelD, ao contrário de outros sistemas efetores tal como LapD e domínios PilZ. Curiosamente, apenas uma molécula de c-di-GMP foi encontrada no sítio, contrastando com a forma dimérica intercalada normalmente ligada aos sítios inibitórios de domínios GGDEF, tais como em PleD e WspR. Estudos de ITC confirmaram a estequiometria 1:1 em solução. Isso mostra a versatilidade dos diversos receptores já identificados até o momento, frente à ligação desse dinucleotídeo. Estudos de bioinformática identificaram uma potencial região de coiled-coil na hélice juxtamembrana de PelD, resíduos 115-160, provavelmente responsável pela homodimerização. Visando uma comprovação experimental dessa hipótese, uma construção contendo toda a porção citoplasmática, PelD111-455, foi expressa e purificada. Estudos comparativos de dicroísmo circular e ultracentrifugação analítica entre as construções PelD176-455 e PelD111-455 realmente demonstraram que os resíduos extras presentes em PelD111-455 formam uma hélice-α e são responsáveis pela dimerização da porção citoplasmática da proteína. De modo geral, os resultados aqui apresentados não só contribuirão para o entendimento dos mecanismos de regulação das vias de sinalização mediadas por c-di-GMP como, em longo prazo, poderão levar ao desenvolvimento de agentes contra infecções bacterianas. / Microorganisms may be presented either in planktonic free-swimming life-style or adhered to surfaces, forming a complex and dynamic community known as biofilm. In recent years, with the progress of research at the molecular level, it was identified that the majority of bacteria use guanosine monophosphate (3\'-5 \')-cyclic dimeric (c-di-GMP) as a second messenger. Generally, this molecule controls the cell signaling, virulence, communication between cells and expression of proteins related to the phenotype of biofilms in response to its intracellular concentration. Its synthesis and degradation are controlled respectively by diguanylate cyclases (DGC) containing the GGDEF domain and phosphodiesterases (PDE) with the domains EAL or HD-GYP. In Pseudomonas aeruginosa (PA14) a novel class of receptor specific for c-di-GMP has been identified, the transmembrane protein PelD, which contains a GAF and degenerate GGDEF domains in the cytoplasmic portion. Its modulation through this dinucleotide controls the production of exopolysaccharides by the components of the conserved operon pel and directly influences the ability of biofilm formation. Due to the paucity of data about the molecular events of the signaling mechanism mediated by c-di-GMP, this study aimed to characterize biophysically and structurally the protein PelD. Various soluble constructions of PelD were cloned and expressed, and the construction comprising the residues 176-455 (PelD176-455) was successfully crystallized and its structure was determined by iodine-SAD. The final model showed the two characteristic domains of families GAF and GGDEF, and the inter-domain interface composed primarily of hydrophobic residues. Seeking an understanding of the molecular basis of recognition and activation of PelD by c-di-GMP, a structure in complex with the ligand was solved. As expected, the dinucleotide was found at the inhibitory site of the GGDEF domain, where the motif R367xxD370 and the residue R402 are responsible for most of the interactions with c-di-GMP. However, no major structural change was observed between the apo and holo forms of PelD, unlike other effector systems such as LapD and domains PilZ. Interestingly, only one molecule of c-di-GMP was present on the site, in contrast to the dimeric intercalated form normally found at I-sites GGDEF domains, such as PleD and in WspR. ITC studies confirmed the 1:1 stoichiometry in solution. This shows the versatility of the various receptors identified so far, compared to the binding of dinucleotide. Bioinformatics studies have identified a potential coiled-coil region in the juxtamembrane helix of PelD, residues 115-160, probably responsible for homodimerization. Aiming at an experimental confirmation of this hypothesis, a construct containing the full cytoplasmic portion, PelD111-455 was expressed and purified. Comparative studies of circular dichroism and analytical ultracentrifugation between constructs PelD176-455 and PelD111-455 indeed demonstrated that the extra residues present in PelD111-455 form an α-helix and are responsible for dimerization of the cytoplasmic portion of the protein. Overall, the results presented here not only contribute to the understanding of the mechanisms of regulation of signaling pathways mediated by c-di-GMP as in the long run, may lead to the development of agents against bacterial infections.

Biofilmes são comunidades bacterianas tridimensionais complexas, que vivem organizadas e aderidas a uma superfície, biótica ou abiótica, embebidas em uma matriz exopolimérica. Cerca de 80% das bactérias vivem organizadas na forma de biofilmes, pois dentro destas estruturas são menos sensíveis aos antibióticos e à resposta imune do hospedeiro. Dentre as principais bactérias formadoras de biofilmes têm-se Staphylococcus spp., Pseudomonas aeruginosa e enterobacteriaceas. Estas bactérias formadoras de biofilmes são importantes colonizadoras da superfície de dispositivos médicos e implantes, aumentando a morbidade e mortalidade dos pacientes que apresentam este tipo de infecção. A investigação de novas estratégias para prevenção e tratamento de infecções por biofilmes é urgentemente necessária. Dentre estas estratégias estão a pesquisa de diferentes mecanismos ou substâncias capazes de provocar a inibição da formação ou a erradicação do biofilme formado. Neste contexto, os venenos animais representam uma fonte ainda inexplorada de uma vasta quantidade de moléculas bioativas, candidatas ao desenvolvimento de novas terapias, inclusive antibiofilme. O principal objetivo deste estudo é avaliar diferentes venenos de serpentes e análogos sintéticos como fonte de moléculas contra biofilmes bacterianos patogênicos. O capítulo 1 revisa estudos que relatam a atividade antimicrobiana (contra bactérias, vírus, protozoários e fungos) de 170 peptídeos isolados de venenos de oito diferentes animais. Peptídeos antimicrobianos vêm ganhando destaque em pesquisas para o tratamento de infecções e peptídeos com atividade antibiofilme são uma nova e promissora abordagem, para o tratamento de infecções relacionadas. O capítulo 2 mostra as atividades antimicrobiana, antibiofilme e de erradicação de biofilmes pré-estabelicidos de 18 análogos de peptídeos de oriundos de venenos de serpentes. Inicialmente foram analisadas e alinhadas 170 sequências peptídeos oriundos de venenos animais. O pepptídeos 16 apresentou considerável atividade antimicrobiana contra cepas bacterianas Gram-positivas, sensíveis e resistentes. Para S. epidermidis os peptídeos 1, 2, 3, 4, 12, 13 e 16 apresentaram menos de 50% de formação de biofilme e os peptídeos 2, 3 e 16 reduziram o biofilme pré-formad. A citotoxicidade e a actividade hemolítica foram testadas e os peptídeos ativos 2 e 16 apresentaram citotoxicidade e hemólise significativas em comparação com os controles. A posição dos aminoácidos pode contribuir para as atividades, sendo que mais testes são necessários para entender a relação das posições de aminoácidos na ação. O capítulo 3 mostra as atividades antiformação e erradicação de biofilmes pré-estabelecidos de dezessete diferentes venenos de serpentes, duas de escorpiões e três de anêmonas marinhas, bem como as secreções de pele de três espécies de sapos. Consideráveis atividades antiformação e de erradicação foram verificadas contra as cepas de S. aureus, S. epidermidis e E. cloaceae por todos os venenos de serpentes testados, em diferentes concentrações. Além disso, um fracionamento inicial foi realizado e as melhores condições foram selecionadas para um novo fracionamento, onde foram testadas 27 frações de veneno de B. diporus. As frações 8, 14 e 23 apresentaram atividades com menos de 50% de formação de biofilme e menos de 80% do biofilme remanescente. Os resultados indicam a capacidade dos venenos, especialmente da serpente de B. diporus, de serem potenciais fontes de moléculas como estratégia para combater os biofilmes patogênicos bacterianos. O presente estudo aborda o potencial de venenos animais, principalmente venenos de serpentes, como fonte de moléculas, que podem apresentar inúmeras atividades farmacológicas inéditas, incluindo àquelas relacionadas com a prevenção da formação e de erradicação de biofilmes bacterianos patogênicos. Atualmente, existem seis medicamentos aprovados pelo Food and Drug Administration (FDA), oriundos de venenos, como o Captopril (Capoten®). Este estudo mostra a capacidade de venenos como fonte de novas moléculas ativas contra biofilmes patogênicos. / Biofilms are complex three-dimensional bacterial communities living organized and attached on a surface, embedded in exopolimeric matrix. About 80% of live bacteria are organized in the form of biofilms because in these structures are less sensitive to antibiotics and host immune response. Staphylococcus spp., Pseudomonas aeruginosa and enterobacteriaceas are the main biofilm forming bacteria. These forming biofilms bacteria are important colonizing surface of medical devices and implants, increasing the morbidity and mortality of patients with this kind of infection. The investigation of new strategies for prevention and treatment of infections caused by biofilms is urgently needed. Among these strategies, there are the research of different mechanisms or substances capable of inhibit the formation or to eradicate the formed biofilm. In this context, animal venoms represent an untapped source of vast amounts of bioactive molecules, candidates for the development of new therapies, including antibiofilm. The main objective of this study is to evaluate different venoms of snakes and synthetic analogs as source of molecules against pathogenic bacterial biofilms. The chapter 1 reviewed numerous studies reporting the antimicrobial activity (against bacteria, viruses, protozoa and fungi) of 170 peptides isolated from venoms of eight different animals. Antimicrobial peptides have been gaining attention in research for the treatment of infections and peptides with antibiofilm activity are a new and promising approach for the treatment of infections related. The chapter 2 shows the antimicrobial, antibiofilm and eradication of established biofilms activities of 18 analogs of peptides derived from snake venoms. Initially, 170 peptide sequences from animal venoms were analyzed and aligned. The peptide 16 showed considerable antimicrobial activity against Gram-positive bacterial strains, sensitive and resistant. For S. epidermidis, the peptides 1, 2, 3, 4, 12, 13 and 16 showed less than 50% of biofilm formation and peptides 2, 3 and 16 reduced preformed biofilm. Cytotoxicity and hemolytic activity were tested and active peptides 2 and 16 showed significant cytotoxicity and hemolysis compared with the controls. The position of the amino acids can contribute to the activities and more tests are needed to understand the relationship of the amino acid positions in the action. The chapter 3 shows the antiformation and eradication of established biofilms activities of seventeen different venoms of snakes, two of scorpions and three of marine anemones, as well as the skin secretions of three species of frogs. Significant activities were observed against strains of S. aureus, S. epidermidis and E. cloaceae for all venom of snakes tested at different concentrations. In addition, an initial fractionation was performed and the best conditions were selected for a new fractionation, where 27 fractions of B. diporus enom were tested. Fractions 8, 14 and 23 presented activities with less than 50% of biofilm formation and less than 80% of the remaining biofilm. The results indicate the ability of venoms, especially the snake B. diporus, to be potential sources of molecules as a strategy to combat bacterial pathogenic biofilms. This study addresses the potential of animal venoms, especially snake venoms, as source of molecules, which can present numerous unpublished pharmacological activities, including those related to the prevention of the formation and eradication of pathogenic bacterial biofilms. Currently, there are six drugs approved by Food and Drug Administration (FDA), derived from venoms, such as Captopril®. This study shows the ability of venoms as source of new bioactive molecules against pathogenic biofilms.

Abstract: The role of bacterial biofilms in patients with persistent CRS is of growing concern. The limited efficacy of some medical and surgical treatments for CRS illustrates the need for further progress in this area. The current treatments of chronic rhinosinusitis are concentrated on medical +/- surgical therapy. In this thesis, we consider two methods performed in chronic rhinosinusitis, endoscopic sinus surgery and long term low-dose macrolide therapy, and consider how they can affect bacterial biofilms. The effect of endoscopic sinus surgery on bacterial biofilms and the clinical impact of this condition on CRS patients may be far more profound than we can currently understand. To understand the impact of ESS on bacterial biofilms, we have performed the first prospective study to evaluate the effect of ESS on bacterial biofilms in patients with CRS and patients’ clinical outcomes after 3 months follow-up. We have shown that ESS results in a statistically significant improvement in QoL, subjective and objective outcome measures. In terms of bacterial biofilms, the mean OD630nm of isolates was significantly reduced after 3 months follow-up (p=0.043). No correlations between the reduction of bacterial biofilms with any of the objective, subjective and QoL outcomes were seen in our study. Macrolides have demonstrated their anti-inflamatory effects in the treatment of diffuse panbronchiolitis, asthma, cystis fibrosis and chronic rhinosinusitis. In recent years, there are a number of in vitro studies supporting the anti-biofilm effects of macrolide antibiotics, especially at sub-MICs level. These have shown that macrolides alter the outer membrane, lipopolysaccharide of biomass and inhibit the expression of other bacterial virulence factors which may disrupt the adherence of bacteria to form biofilms. Long term low dose macrolide therapy, therefore, may transform bacterial biofilms from the protected organized form into the plantonic form. In this thesis, we also report the first in vivo efficacy of long term low dose macrolides on bacterial biofilms in patients with CRS. Patients receiving oral macrolide showed significant improvements in subjective, objective and QoL scores following a 12 week course. Nasal swabs were taken from CRS patients at the first visit and 3 months after macrolide therapy. Using the microtiter biofilm assay, these swabs showed a reduction in the mean OD630nm of isolates in 8/12 patients. While it is well-known that bacterial biofilms are established in CRS patients, the relationship between the improvement of clinical symptoms and the severity of bacterial biofilm is less clear.

Biofilmes são comunidades bacterianas tridimensionais complexas, que vivem organizadas e aderidas a uma superfície, biótica ou abiótica, embebidas em uma matriz exopolimérica. Cerca de 80% das bactérias vivem organizadas na forma de biofilmes, pois dentro destas estruturas são menos sensíveis aos antibióticos e à resposta imune do hospedeiro. Dentre as principais bactérias formadoras de biofilmes têm-se Staphylococcus spp., Pseudomonas aeruginosa e enterobacteriaceas. Estas bactérias formadoras de biofilmes são importantes colonizadoras da superfície de dispositivos médicos e implantes, aumentando a morbidade e mortalidade dos pacientes que apresentam este tipo de infecção. A investigação de novas estratégias para prevenção e tratamento de infecções por biofilmes é urgentemente necessária. Dentre estas estratégias estão a pesquisa de diferentes mecanismos ou substâncias capazes de provocar a inibição da formação ou a erradicação do biofilme formado. Neste contexto, os venenos animais representam uma fonte ainda inexplorada de uma vasta quantidade de moléculas bioativas, candidatas ao desenvolvimento de novas terapias, inclusive antibiofilme. O principal objetivo deste estudo é avaliar diferentes venenos de serpentes e análogos sintéticos como fonte de moléculas contra biofilmes bacterianos patogênicos. O capítulo 1 revisa estudos que relatam a atividade antimicrobiana (contra bactérias, vírus, protozoários e fungos) de 170 peptídeos isolados de venenos de oito diferentes animais. Peptídeos antimicrobianos vêm ganhando destaque em pesquisas para o tratamento de infecções e peptídeos com atividade antibiofilme são uma nova e promissora abordagem, para o tratamento de infecções relacionadas. O capítulo 2 mostra as atividades antimicrobiana, antibiofilme e de erradicação de biofilmes pré-estabelicidos de 18 análogos de peptídeos de oriundos de venenos de serpentes. Inicialmente foram analisadas e alinhadas 170 sequências peptídeos oriundos de venenos animais. O pepptídeos 16 apresentou considerável atividade antimicrobiana contra cepas bacterianas Gram-positivas, sensíveis e resistentes. Para S. epidermidis os peptídeos 1, 2, 3, 4, 12, 13 e 16 apresentaram menos de 50% de formação de biofilme e os peptídeos 2, 3 e 16 reduziram o biofilme pré-formad. A citotoxicidade e a actividade hemolítica foram testadas e os peptídeos ativos 2 e 16 apresentaram citotoxicidade e hemólise significativas em comparação com os controles. A posição dos aminoácidos pode contribuir para as atividades, sendo que mais testes são necessários para entender a relação das posições de aminoácidos na ação. O capítulo 3 mostra as atividades antiformação e erradicação de biofilmes pré-estabelecidos de dezessete diferentes venenos de serpentes, duas de escorpiões e três de anêmonas marinhas, bem como as secreções de pele de três espécies de sapos. Consideráveis atividades antiformação e de erradicação foram verificadas contra as cepas de S. aureus, S. epidermidis e E. cloaceae por todos os venenos de serpentes testados, em diferentes concentrações. Além disso, um fracionamento inicial foi realizado e as melhores condições foram selecionadas para um novo fracionamento, onde foram testadas 27 frações de veneno de B. diporus. As frações 8, 14 e 23 apresentaram atividades com menos de 50% de formação de biofilme e menos de 80% do biofilme remanescente. Os resultados indicam a capacidade dos venenos, especialmente da serpente de B. diporus, de serem potenciais fontes de moléculas como estratégia para combater os biofilmes patogênicos bacterianos. O presente estudo aborda o potencial de venenos animais, principalmente venenos de serpentes, como fonte de moléculas, que podem apresentar inúmeras atividades farmacológicas inéditas, incluindo àquelas relacionadas com a prevenção da formação e de erradicação de biofilmes bacterianos patogênicos. Atualmente, existem seis medicamentos aprovados pelo Food and Drug Administration (FDA), oriundos de venenos, como o Captopril (Capoten®). Este estudo mostra a capacidade de venenos como fonte de novas moléculas ativas contra biofilmes patogênicos. / Biofilms are complex three-dimensional bacterial communities living organized and attached on a surface, embedded in exopolimeric matrix. About 80% of live bacteria are organized in the form of biofilms because in these structures are less sensitive to antibiotics and host immune response. Staphylococcus spp., Pseudomonas aeruginosa and enterobacteriaceas are the main biofilm forming bacteria. These forming biofilms bacteria are important colonizing surface of medical devices and implants, increasing the morbidity and mortality of patients with this kind of infection. The investigation of new strategies for prevention and treatment of infections caused by biofilms is urgently needed. Among these strategies, there are the research of different mechanisms or substances capable of inhibit the formation or to eradicate the formed biofilm. In this context, animal venoms represent an untapped source of vast amounts of bioactive molecules, candidates for the development of new therapies, including antibiofilm. The main objective of this study is to evaluate different venoms of snakes and synthetic analogs as source of molecules against pathogenic bacterial biofilms. The chapter 1 reviewed numerous studies reporting the antimicrobial activity (against bacteria, viruses, protozoa and fungi) of 170 peptides isolated from venoms of eight different animals. Antimicrobial peptides have been gaining attention in research for the treatment of infections and peptides with antibiofilm activity are a new and promising approach for the treatment of infections related. The chapter 2 shows the antimicrobial, antibiofilm and eradication of established biofilms activities of 18 analogs of peptides derived from snake venoms. Initially, 170 peptide sequences from animal venoms were analyzed and aligned. The peptide 16 showed considerable antimicrobial activity against Gram-positive bacterial strains, sensitive and resistant. For S. epidermidis, the peptides 1, 2, 3, 4, 12, 13 and 16 showed less than 50% of biofilm formation and peptides 2, 3 and 16 reduced preformed biofilm. Cytotoxicity and hemolytic activity were tested and active peptides 2 and 16 showed significant cytotoxicity and hemolysis compared with the controls. The position of the amino acids can contribute to the activities and more tests are needed to understand the relationship of the amino acid positions in the action. The chapter 3 shows the antiformation and eradication of established biofilms activities of seventeen different venoms of snakes, two of scorpions and three of marine anemones, as well as the skin secretions of three species of frogs. Significant activities were observed against strains of S. aureus, S. epidermidis and E. cloaceae for all venom of snakes tested at different concentrations. In addition, an initial fractionation was performed and the best conditions were selected for a new fractionation, where 27 fractions of B. diporus enom were tested. Fractions 8, 14 and 23 presented activities with less than 50% of biofilm formation and less than 80% of the remaining biofilm. The results indicate the ability of venoms, especially the snake B. diporus, to be potential sources of molecules as a strategy to combat bacterial pathogenic biofilms. This study addresses the potential of animal venoms, especially snake venoms, as source of molecules, which can present numerous unpublished pharmacological activities, including those related to the prevention of the formation and eradication of pathogenic bacterial biofilms. Currently, there are six drugs approved by Food and Drug Administration (FDA), derived from venoms, such as Captopril®. This study shows the ability of venoms as source of new bioactive molecules against pathogenic biofilms.