Biofilms are characterized by surface attachment, structural heterogeneity; genetic diversity; complex community interactions and an extracellular matrix of polymeric substances (EPS). Biofilms deposit and adhere to all surfaces that are immersed in aqueous environments. EPS serves many functions including: facilitation of the initial attachment of bacterial cells to a surface; formation and maintenance of the micro colony; enables the bacteria to capture nutrients; causes biofouling; cell-cell communication and enhances bacterial resistance antimicrobial agents. EPS also function as a stabilizer of the biofilm structure and as a barrier against hostile environments. Extracelullar polymeric substances are composed of a wide variety of materials including polysaccharides, proteins, nucleic acid, uronic acid, DNA, lipid and even humid substances. EPS can be hydrophilic or hydrophobic depending on the structural components making up such EPS and the environmental conditions were the biofilms are developing. The exopolysachharides (EPS) synthesized by microbial cells vary greatly in their composition and in their chemical and physical properties within the bacterial strains. Due to variety in the structural components of the bacterial EPS, removal of biofilms by compounds that have no effects on the biofilm EPS would be difficult. Enzymes are proven to be effective in degrading biofilm EPS. The manner in which enzymes degrade the biofilm EPS is through binding and hydrolysis of the EPS components (proteins and carbohydrates) molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. The objectives of this study were to grow Pseudomonas fluorescens and mixed bacterial species biofilms in nutrient rich and nutrient limited medium conditions; to determine the EPS, protein and carbohydrate concentrations of the biofilm grown in rich and in limited nutrient conditions and to test the efficiency of protease and amylase enzymes for the degradation of the EPS and biofilm removal. In the results, there was a slight difference in the number of viable cells grown in biofilms that were fed than the cells of the unfed biofilms. As a result, the EPS, protein and carbohydrate concentrations were higher in the fed biofilms than the unfed biofilms. There are contradictory reports about the composition of EPS especially with the ratio of carbohydrate to protein. Some of these reports indicate that certain biofilms EPS have bigger proportion of proteins and some found polysaccharides to be the dominant composition of the EPS of the biofilms. Nonetheless, the quantity and the composition of the EPS produced by bacterial biofilms depend on a number of factors such as microbial species, growth phase and the type of limiting substrate. Enzymes were tested individually and in combination for the degradation of biofilm EPS. For efficient removal of biofilm, it is important that the structural components of the biofilm EPS should be known before application of the relevant enzymes. In this study, the test enzymes were effective for the degradation of the biofilm EPS except for the protease Polarzyme which had no activity. The reason for the inefficiency of Polarzyme may be due to its incompatibility with the specific protein structural components of the biofilm EPS tested in this study. The manner in which the enzymes degrade the biofilm EPS is through binding and hydrolysis of the protein and carbohydrate molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. In addition, the mode of enzymatic action will depend on the specific EPS components and this in turn will determine its efficacy. The protease enzymes tested individually and in combination were most effective for EPS degradation. The efficiency of the proteases may be due to their broad spectrum activity in degrading a variety of proteins acting partly as the multi structural components of Pseudomonas fluorescens and mixed bacterial species biofilm EPS. On the other hand, amylase enzymes tested individually and in combination was less effective for the EPS degradation. The structures of polysaccharides synthesized by microbial cells vary. Microbial exopolysaccharides are comprised of either homopolysachharides or heteoropolysaccharides. A number of lactic acid bacteria produce heteropolysaccharides and these molecules form from repeating units of monosaccharides including D- glucose, D- galactose, L- fructose, L- rhamnose, D- glucuronic acid, L- guluronic acid and D- mannuronic acid. The type of both linkages between monosaccharides units and the branching of the chain determines the physical properties of the microbial heteropolysaccharides. Due to a wide range of linkages and the complexity of polysaccharides structures, it would therefore be difficult for the amylases to break down the bond linkages and the monomers making up polysaccharides which determine the physical and chemical structure of the EPS. It was therefore not surprising that the amylase enzymes tested for the degradation of Pseudomonas fluorescens and mixed bacterial species biofilms, were less effective than the proteases. Hence, when the amylase enzymes were tested in combination with the protease enzymes, efficiency improved. It was therefore concluded that the protease enzymes were the primary remedial compounds and the amylase enzymes were the secondary remedial compounds. Conclusion If a compound or compounds capable of destroying all the structural components of different EPS that are produced by different biofilms growing under different conditions is found then the “city of microbes” (biofilms) would be destroyed permanently. If only an enzyme or enzymatic mixture capable of shutting down or deactivating the quorum sensing systems of different biofilm EPS could be found, then there would not be any formation of biofilms. In this study, protease enzymes tested individually and in combination were the most effective in the degradation of biofilm EPS than the amylase enzymes resulting in the reduction of large population of the biofilm cells attached on the substratum. Recommendation Amylase enzymes tested individually and in combination were less efficient for the degradation of the biofilm EPS and biofilm removal. This may be due to the complex structure of the exopolysaccharides synthesized by different biofilms. Also, the bond linkages between monosaccharides units and the branching of the chain complex the structures and as a result confer in the physical properties of the microbial biofilms. Hence, when the amylase enzymes were tested in combination with the protease enzymes, activity improved. For efficient degradation of biofilm EPS, it is therefore recommended that, protease and amylase enzymes should be tested in combination. In addition, the structure of the biofilm EPS should be investigated so that relevant enzymatic mixtures are tested for biofilm removal. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Les travaux présentés traitent des cinétiques de décrochement des biofilms bactériens formés au sein des équipements communément utilisé dans les agro-industries. Deux espèces bactériennes Pseudomonas et Bacillus ayant une forte capacité à former des biofilms dont la présence quasi permanente est reportés dans les industries alimentaires (viande, lait, légumes frais…) ont servi de modèles pertinent pour ces travaux. Par ailleurs Bacillus, bactéries potentiellement pathogènes capable de former des spores dont les capacités à adhérer et résister aux environnements chimiques et thermiques défavorables (détergents, traitement de pasteurisation, stérilisation) sont reconnues pour présenter un risque sanitaire majeur pour l’agro-industrie du fait de la contamination des produits alimentaires par l’outil de fabrication lui-même. Deux type d’études ont été effectués sur le nettoyage d’équipements utilisés en industrie agroalimentaire.Une première approche cinétique a été mise en œuvre pour étudier l’élimination de ces biofilms des surfaces au cours du nettoyage. Nous avons cherchés à identifier les rôles respectifs de l’action mécanique via la contrainte pariétale générée par l’écoulement ou chimique via différentes concentrations en NaOH puis la combinaison des deux actions chimiques et mécaniques. Un modèle biphasique a été proposé pour décrire ces cinétiques de nettoyage en pertinence avec deux phases très distinctes que nous interprétons comme l’élimination de deux fractions différentes du biofilm. Un détachement plus ou moins rapide (1 à 5 min) correspondrait à l’élimination de structure tridimensionnelles composées ou non d’exopolymères suivi par un détachement lent des cellules directement en contact avec les surfaces.Dans le cas des biofilms à Bacillus, il existe une fraction du biofilm composée par des spores pouvant représenter près de 90% du biofilm. Cette fraction particulière semble s’éliminer plus rapidement dans la première phase et pas du dans la seconde phase. Les données préliminaires sur biofilms mixtes montrent une résistance accrue de l’ensemble qui se traduit par une plus grande résistance de la partie de biofilm constituée par des cellules à Pseudomonas.La seconde thématique développée traite de la possible récontamination par les sopres de Bacillus décrochées lors du processus de nettoyage.La réadhésion des bactéries à pu être prouvée par le positionnement des tubes dans le circuit de nettoyage, ainsi que par l’utilisation d’un matériel complexe (vanne bidirectionnelle) communément utilisé dans les agro-industries. Ces travaux ont permis d’identifier les conditions présidant à cette récontamination. La conception hygiénique des outils de transformation prend tout son sens ici. / This work deals with removal kinetics of bacterial biofilms potentially formed in commonly used equipment in agro-industries. Two bacterial species Pseudomonas and Bacillus with a strong ability to form biofilms in which the almost permanent presence is reported in the food industry (meat, milk, vegetables…) were chosen as relevant models for the study. Moreover Bacillus, potentially pathogenic bacteria can form spores which are known to strongly adhere to solid surfaces and for their ability to withstand adverse environments as thermal and chemical detrimental conditions (detergents, pasteurization or sterilization temperatures). Bacillus species are known to pose a major health risk for agro-industry is contamination of food by the technical equipment itself.Two main areas were considered in the field of food processing equipment cleaning.At first a kinetic approach was implemented to study the removal of biofilms from surfaces during cleaning operations. Thus respective roles of the mechanical action (wall shear stress under turbulent flow regime), the chemical action (different NaOH concentrations) and the combination of both chemical and mechanical actions were studied. A biphasique model was proposed to describe cleaning kinetics according to the observation of two very different phases which we interpreted as the elimination of two different fractions of the biofilm. A more or less fast detachment in 1 to 5 min would correspond to the three-dimensional elimination of structure with more or less exopolymeric materials followed by a slow detachment of bacteria cells directly in contact with solid surfaces. In the case of Bacillus, can represent about 90% of the total biofilm. Spores removal appeared to be a two phases phenomenon as the whole biofilm. However, the removal was essentially observed during the short first phase of the kinetics. Preliminary data on mixed biofilms with both species Bacillus and Pseudomonas had shown an increase in their resistance to cleaning comparing to the corresponded monospecies biofilm counterpart. This is really significant for Pseudomonas. The second developed theme considered as a consequence of the above one deals with possible equipment re-contamination by Bacillus spores removed during cleaning operations. Post contamination of food processing equipment during cleaning was thus observed In pipes located at different places in the cleaning loop, as well as in more complex materials as a mixproof valve commonly encountered in Dairies. This work allowed us to identify all the conditions explaining such phenomenon. Equipment hygienic design issues appeared to be here.

This diploma thesis focuses on an optimalization of simple laboratory model systems which serve as an innovative tool for an experimental study on the adsorption of bacterial cells on solid surfaces. In the description of living biological systems, an adsorption is labelled as an adhesion. Designed model systems were validated with a physical-chemical analysis. Various techniques were used to determine bacteria properties, more specifically Burkholderia cepacia and Bacillus megaterium. The solid surfaces after sorption of bacterial cells of Bacillus megaterium were subjected to a structural and visual analysis. Applying the theoretical approach (e.g. using different physical-chemical models) to study the adhesion of microorganisms to a particular surface allows a prediction of the conditions for a successful adhesion. The results will give us a better understanding of a formation and development of a biofilm.

No description available.

No description available.

No description available.

O biofilme bacteriano é o agente etiológico primário no desenvolvimento da resposta inflamatória observada na doença periodontal. Os receptores do tipo NOD (NLRs) são proteínas citosólicas que reconhecem componentes microbianos presentes no citoplasma liberados por bactérias invasoras. Sabendo que bactérias periodontopatogênicas têm a capacidade de invadir e colonizar diversas células do tecido periodontal, o presente projeto tem o objetivo de estudar a participação dos receptores do tipo NOD (NOD1 e NOD2) no reconhecimento das bactérias Porphyromonas gingivalis, na modulação da resposta imune e no processo de reabsorção óssea. Camundongos isogênicos machos da linhagem C57BL/6 (WT) e camundongos deficientes para o receptor NOD1 (NOD1-/-) e receptor NOD2 (NOD2-/-) foram infectados com Phorphyromonas gingivalis utilizando um modelo experimental de doença periodontal. O grau de reabsorção óssea foi avaliado por análise morfométrica macroscópica e histológica da maxila, além da quantificação do número de osteoclastos na crista óssea alveolar. O grau de inflamação local foi realizado por contagem do número total de bactérias orais, quantificação de neutrófilos no tecido gengival (MPO) e avaliação dos mediadores inflamatórios por ELISA. Foi também avaliada a expressão de marcadores osteogênicos e osteoclastogênicos no tecido gengival pela técnica de qPCR. Animais NOD1-/- e NOD2-/- apresentaram menor delta de reabsorção óssea quando comparados aos animais WT. Animais NOD2-/- infectados apresentaram debilitado controle bacteriano quando comparados aos animais WT infectados. Animais NOD1-/- e NOD2-/- infectados apresentaram baixa expressão de Cxcl1 e MPO quando comparados aos animais WT infectados. Além disso, foi observado que animais NOD2-/- infectados apresentaram reduzida produção de TNF-α e elevada produção de IL-10 quando comparados aos animais WT infectados. Não foi detectado diferença estatística na expressão de fatores osteogênicos, Runx2 e osteocalcina, entre animais WT, NOD1-/- e NOD2-/- infectados. Embora não houve diferença no número de células TRAP positivas presentes na crista óssea alveolar entre os grupos no tempo avaliado, animais WT infectados apresentaram elevada expressão gênica de RANKL/OPG quando comparados aos animais NOD2-/- infectados. Além disso, a expressão de marcadores de atividades dos osteoclastos, catepsina k e matrix metaloproteinase-9, foi significantemente baixa nos animais NOD1-/- e NOD2-/- infectados quando comparados aos animais WT infectados. Esses resultados permitem sugerir que independente das variáveis observadas, verificamos que a ausência dos dois receptores impede a rápida progressão da reabsorção óssea alveolar observada na periodontite, portanto os receptores NOD1 e NOD2 contribuem para progressão da reabsorção óssea no modelo experimental de periodontite. / The bacterial biofilm has been identified as an etiological agent in the pathogenesis of periodontal disease. The NOD-like receptors (NLRs) are cytoplasmic proteins that sense microbial by products released by invasive bacteria. Since periodontopathogenic bacteria are able to invade and colonize some periodontal tissue cells, the purpose of this study is to determine the role of NOD1 and NOD2 receptors in the recognition of invasive periodontopathogenic bacteria such as Porphyromonas gingivalis, modulating the immune response and bone resorption. Isogenic strain C57BL/6 males (WT), NOD1 (NOD1-/-) and NOD2 (NOD2-/-) knockout mice were infected with Porphyromonas gingivalis in an experimental model of periodontal disease. Alveolar bone resorption was evaluated by macroscopic and histological morphometric analysis, quantification of osteoclasts numbers in bone crest alveolar. Imune inflammatory response was evaluated by, bacterial load, neutrophils quantification and inflammatory mediators levels by ELISA. We also evaluated the osteogenic and osteoclastogenic markers expression in gingival tissue by Real time PCR techniques. NOD1-/- and NOD2-/- animals showed lower bone resorption when compared to WT animals. NOD2-/- infected animals expressed higher bacterial load compared to WT infected ones. NOD1-/- and NOD2-/- infected animals presented lower Cxcl1 and MPO levels compared to WT infected animals. In addition, NOD2-/- infected animals presented lower level of TNF-α and higher level of IL-10 when compared to WT infected animals. There was no significant difference in the osteogenic factors expression, Runx2 and osteocalcin, when compared NOD1-/- and NOD2-/- infected animals to WT infected ones. Although there was no difference in TRAP-positive cells number evaluated in the alveolar bone crest among the studied groups, WT infected animals showed elevated ratio RANKL/OPG when compared to NOD2-/- infected animals. Moreover, the expression of osteoclasts activity markers, cathepsin k and matrix metalloproteinase-9, was significantly lower in NOD1-/- and NOD2-/- infected animals compared to WT infected ones. These results suggest that NOD1 and NOD2 receptors contribute to progression of bone resorption in experimental model of periodontitis, since the lack of NOD like receptors impair the bone resorption.

O biofilme bacteriano é o agente etiológico primário no desenvolvimento da resposta inflamatória observada na doença periodontal. Os receptores do tipo NOD (NLRs) são proteínas citosólicas que reconhecem componentes microbianos presentes no citoplasma liberados por bactérias invasoras. Sabendo que bactérias periodontopatogênicas têm a capacidade de invadir e colonizar diversas células do tecido periodontal, o presente projeto tem o objetivo de estudar a participação dos receptores do tipo NOD (NOD1 e NOD2) no reconhecimento das bactérias Porphyromonas gingivalis, na modulação da resposta imune e no processo de reabsorção óssea. Camundongos isogênicos machos da linhagem C57BL/6 (WT) e camundongos deficientes para o receptor NOD1 (NOD1-/-) e receptor NOD2 (NOD2-/-) foram infectados com Phorphyromonas gingivalis utilizando um modelo experimental de doença periodontal. O grau de reabsorção óssea foi avaliado por análise morfométrica macroscópica e histológica da maxila, além da quantificação do número de osteoclastos na crista óssea alveolar. O grau de inflamação local foi realizado por contagem do número total de bactérias orais, quantificação de neutrófilos no tecido gengival (MPO) e avaliação dos mediadores inflamatórios por ELISA. Foi também avaliada a expressão de marcadores osteogênicos e osteoclastogênicos no tecido gengival pela técnica de qPCR. Animais NOD1-/- e NOD2-/- apresentaram menor delta de reabsorção óssea quando comparados aos animais WT. Animais NOD2-/- infectados apresentaram debilitado controle bacteriano quando comparados aos animais WT infectados. Animais NOD1-/- e NOD2-/- infectados apresentaram baixa expressão de Cxcl1 e MPO quando comparados aos animais WT infectados. Além disso, foi observado que animais NOD2-/- infectados apresentaram reduzida produção de TNF-α e elevada produção de IL-10 quando comparados aos animais WT infectados. Não foi detectado diferença estatística na expressão de fatores osteogênicos, Runx2 e osteocalcina, entre animais WT, NOD1-/- e NOD2-/- infectados. Embora não houve diferença no número de células TRAP positivas presentes na crista óssea alveolar entre os grupos no tempo avaliado, animais WT infectados apresentaram elevada expressão gênica de RANKL/OPG quando comparados aos animais NOD2-/- infectados. Além disso, a expressão de marcadores de atividades dos osteoclastos, catepsina k e matrix metaloproteinase-9, foi significantemente baixa nos animais NOD1-/- e NOD2-/- infectados quando comparados aos animais WT infectados. Esses resultados permitem sugerir que independente das variáveis observadas, verificamos que a ausência dos dois receptores impede a rápida progressão da reabsorção óssea alveolar observada na periodontite, portanto os receptores NOD1 e NOD2 contribuem para progressão da reabsorção óssea no modelo experimental de periodontite. / The bacterial biofilm has been identified as an etiological agent in the pathogenesis of periodontal disease. The NOD-like receptors (NLRs) are cytoplasmic proteins that sense microbial by products released by invasive bacteria. Since periodontopathogenic bacteria are able to invade and colonize some periodontal tissue cells, the purpose of this study is to determine the role of NOD1 and NOD2 receptors in the recognition of invasive periodontopathogenic bacteria such as Porphyromonas gingivalis, modulating the immune response and bone resorption. Isogenic strain C57BL/6 males (WT), NOD1 (NOD1-/-) and NOD2 (NOD2-/-) knockout mice were infected with Porphyromonas gingivalis in an experimental model of periodontal disease. Alveolar bone resorption was evaluated by macroscopic and histological morphometric analysis, quantification of osteoclasts numbers in bone crest alveolar. Imune inflammatory response was evaluated by, bacterial load, neutrophils quantification and inflammatory mediators levels by ELISA. We also evaluated the osteogenic and osteoclastogenic markers expression in gingival tissue by Real time PCR techniques. NOD1-/- and NOD2-/- animals showed lower bone resorption when compared to WT animals. NOD2-/- infected animals expressed higher bacterial load compared to WT infected ones. NOD1-/- and NOD2-/- infected animals presented lower Cxcl1 and MPO levels compared to WT infected animals. In addition, NOD2-/- infected animals presented lower level of TNF-α and higher level of IL-10 when compared to WT infected animals. There was no significant difference in the osteogenic factors expression, Runx2 and osteocalcin, when compared NOD1-/- and NOD2-/- infected animals to WT infected ones. Although there was no difference in TRAP-positive cells number evaluated in the alveolar bone crest among the studied groups, WT infected animals showed elevated ratio RANKL/OPG when compared to NOD2-/- infected animals. Moreover, the expression of osteoclasts activity markers, cathepsin k and matrix metalloproteinase-9, was significantly lower in NOD1-/- and NOD2-/- infected animals compared to WT infected ones. These results suggest that NOD1 and NOD2 receptors contribute to progression of bone resorption in experimental model of periodontitis, since the lack of NOD like receptors impair the bone resorption.

Made available in DSpace on 2015-02-24T17:42:50Z (GMT). No. of bitstreams: 1 MagdaRAF_DISSERT.pdf: 2639129 bytes, checksum: 3c6efd5066d343bdfde86e4cf41e1a70 (MD5) Previous issue date: 2012-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The increase in the incidence of fungal infections due to the drug-resistance or to the number of patients with immune alterations such as AIDS, chemotherapy or organ transplantation, has done the research necesseray for new antifungal drugs. The species from Northeastern Brazil may become an important source of innovative natural molecules. To evaluate the antifungal activity of 10 medicinal plants from Northeastern Brazil, traditionally used as antimicrobial agents, 30 crude extracts (CE) were tested in vitro against four standard species of Candida spp. The CE most promising of these plants were evaluated against yeasts of the oral cavity of kidney transplant patients and through a bioassay-guided fractionation. The extracts form leaves of E. uniflora, the stem bark of L. ferrea and leaves of P. guajava showed significant activity against all yeasts evaluated, with MIC values between 15.62 and 62.5 μg/mL. E. uniflora also showed fungicidal properties against all yeasts, especially against Candida dubliniensis. In patients with immune systems compromised, such as transplanted, oral candidiasis manifests mainly due to immunosuppressive therapy, and resistance to conventional antifungals. The CE of E. uniflora presented range of MIC values between 1.95 to 1000 μg/mL, and lower MIC50 and MIC90 values were observed against C. non-albicans. Due the better results, the CE of E. uniflora was elected to performe the bioassay-guided fractionation. Thus it was possible to obtain enriched fractions, which showed good inhibitory ability against ATCC strains of Candida spp. It was also possible to perform experiments to verify the production of biofilm in two strains of C. dubliniensis and action of extracts and fractions on the same. With this, we observed a behavior between the yeast ATCC and clinical isolate. In addition, CE, fractions and subfractions of E. uniflora inhibit planktonic cells to preventing the growth of biofilm. The preliminary chemical characterization of the fractions obtained revealed the presence of polyphenols (especially flavonoids and tannins). Finally, the results suggests that among the plant species studied, E. uniflora showed a pattern very promising as regards the antifungal, requiring further study of purification and structural elucidation of compounds in order to verify that the antifungal effect found can be attributed to a specific compound or some mechanism depends on synergistic the mixture of polyphenols / O aumento na incid?ncia de infec??es por fungos, devido ? resist?ncia ?s drogas ou ao n?mero de pacientes com altera??es imunol?gicas, tais como SIDA, quimioterapia ou transplante de ?rg?os; tem feito a investiga??o de novas drogas antif?ngicas necess?ria. As esp?cies vegetais da regi?o Nordeste do Brasil podem se tornar uma importante fonte de mol?culas naturais inovadoras. Para avaliar a atividade antif?ngica de 10 plantas medicinais da regi?o Nordeste do Brasil, tradicionalmente usadas como antimicrobianas, 30 extratos brutos (EB) foram submetidos ? teste in vitro contra quatro cepas padr?o ATCC de Candida spp. Extratos das folhas de E. uniflora, cascas do caule de L. ferrea, e folhas de P. guajava mostraram atividade significativa contra as leveduras avaliadas, com valores MIC entre 15,62 e 62,5 μg/mL. E. uniflora tamb?m mostrou propriedade fungicida contra todas as leveduras, principalmente contra C. dubliniensis. O EB de E. uniflora apresentou intervalo de valores CIM entre 1,95-1000 μg/mL, e menores valores de MIC50 e MIC90 foram observados contra C. n?o albicans. Devido ao melhor desempenho, o EB de E. uniflora foi eleito para realiza??o do fracionamento biomonitorado. Assim, foi poss?vel obter fra??es enriquecidas, as quais apresentaram boa capacidade inibit?ria, na faixa entre 0,48 a 500 μg/mL, frente ?s cepas ATCC de Candida spp. Tamb?m foi poss?vel realizar experimentos para verificar a produ??o de biofilme de duas cepas de C. dubliniensis e a??o dos extratos e fra??es sobre o mesmo. Com isso, observou-se um comportamento diferente entre a levedura ATCC e o isolado cl?nico. Al?m disso, extrato bruto, fra??es e subfra??es de E. uniflora inibiram as c?lulas planct?nicas impedindo de se agragarem ao biofilme. A caracteriza??o qu?mica preliminar das fra??es obtidas revelou a presen?a de polifen?is (principalmente, flavon?ides e taninos). Por fim, os resultados permitiram afirmar que entre as esp?cies vegetais estudadas, E. uniflora apresentou comportamento bastante promissor no que diz respeito a a??o antif?ngica, sendo necess?rio a continua??o do estudo de purifica??o e elucida??o estrutural dos compostos presentes, a fim de verificar se a a??o antif?ngica encontrada poder? ser atribu?da a um composto espec?fico ou depende de algum mecanismo sin?rgico da mistura de polifen?is

This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.