A bactéria Lactococcus lactis subsp. lactis CECT-4434 foi empregada para investigar o efeito da composição do meio de cultivo na produção biotecnológica de biossurfactante e, adicionalmente bacteriocina. Utilizou-se resíduos agroindustriais, tais como soro de leite e vinhaça de uva, para formular meios de cultivos mais econômicos e naturais, suplementados sacarose e extrato de levedura. Um planejamento fatorial fracionado 24, com adição de três ensaios nos pontos centrais foi empregado para avaliar a influência destas variáveis. A produção de biossurfactante foi influenciada positivamente pela concentração soro de leite, onde 15 % deste demonstrou melhor resultado reduzindo a tensão superficial em cerca de 18,1 mN/m, alcançando produção máxima de biossurfactante equivalente em surfactina de 11,02 mg/L. Em relação à síntese de bacteriocina, a fonte de carbono adicional (sacarose) interferiu de forma antagonista, ou seja, quanto menor a concentração de sacarose, maior a síntese de bacteriocina (com aumento da zona de inibição em 14,2% contra Staphylococcus aureus CECT-239). Observou-se que o ensaio conduzido em biorreator, sob microaeração com 5% de oxigênio dissolvido, promoveu maior produção de biossurfactante (11,6 mg/L) quando comparados aos estudos conduzidos com maior concentração de oxigênio entre 30 a 100%, com produção em média de 2,3 mg/mL. Destaca-se que nenhum estudo da influência do oxigênio dissolvido, principalmente em microaerofilia, para a produção de biossurfactante por bactérias láticas já havia sido realizado. Ademais, o biossurfactante produzido se mostrou altamente estável frente a valores extremos de pH e temperatura, além de demonstrar notável propriedade antimicrobiana e antiadesiva, inibindo Listeria monocytogenes NADC 2045 e Salmonella entérica<i/> CECT-724 em mais de 90%. / Lactococcus lactis subsp. lactis CECT-4434 was used to investigate the effect of the composition of the culture media on the biotechnological production of biosurfactant and bacteriocin additionally. Agroindustrial residues, such as whey and grape vinasse, were used to formulate more economical and natural culture media, supplemented with sucrose and yeast extract. A fractional factorial design 24, with addition of three runs at the central points was used to evaluate the influence of these variables. The biosurfactant production was positively influenced by the concentration of whey, where 15% showed a better result reducing the surface tension by 18.1 mN/m, reaching a maximum production of biosurfactant equivalent in surfactin of 11.02 mg/L. In relation to bacteriocin synthesis, the sucrose interfered in an antagonistic way, that is, the lower the sucrose concentration, the greater the bacteriocin synthesis (with an increase in the zone of inhibition in 14.2% against Staphylococcus aureus CECT-239). It was observed that the bioreactor conducted under microaeration with 5% dissolved oxygen promoted a higher biosurfactant production (11.6 mg/L) when compared to studies conducted with a higher concentration of oxygen between 30 and 100%, with production on average 2.3 mg/mL. It is noteworthy that no study of the influence of dissolved oxygen, mainly in microaerophilic, for the biosurfactant production by lactic acid bacteria had already been carried out. In addition, the biosurfactant produced proved to be highly stable against extreme values of pH and temperature, and demonstrated remarkable antimicrobial and antiadhesive properties, inhibiting Listeria monocytogenes NADC 2045 and Salmonella entérica CECT-724 in more than 90%.

A bactéria Lactococcus lactis subsp. lactis CECT-4434 foi empregada para investigar o efeito da composição do meio de cultivo na produção biotecnológica de biossurfactante e, adicionalmente bacteriocina. Utilizou-se resíduos agroindustriais, tais como soro de leite e vinhaça de uva, para formular meios de cultivos mais econômicos e naturais, suplementados sacarose e extrato de levedura. Um planejamento fatorial fracionado 24, com adição de três ensaios nos pontos centrais foi empregado para avaliar a influência destas variáveis. A produção de biossurfactante foi influenciada positivamente pela concentração soro de leite, onde 15 % deste demonstrou melhor resultado reduzindo a tensão superficial em cerca de 18,1 mN/m, alcançando produção máxima de biossurfactante equivalente em surfactina de 11,02 mg/L. Em relação à síntese de bacteriocina, a fonte de carbono adicional (sacarose) interferiu de forma antagonista, ou seja, quanto menor a concentração de sacarose, maior a síntese de bacteriocina (com aumento da zona de inibição em 14,2% contra Staphylococcus aureus CECT-239). Observou-se que o ensaio conduzido em biorreator, sob microaeração com 5% de oxigênio dissolvido, promoveu maior produção de biossurfactante (11,6 mg/L) quando comparados aos estudos conduzidos com maior concentração de oxigênio entre 30 a 100%, com produção em média de 2,3 mg/mL. Destaca-se que nenhum estudo da influência do oxigênio dissolvido, principalmente em microaerofilia, para a produção de biossurfactante por bactérias láticas já havia sido realizado. Ademais, o biossurfactante produzido se mostrou altamente estável frente a valores extremos de pH e temperatura, além de demonstrar notável propriedade antimicrobiana e antiadesiva, inibindo Listeria monocytogenes NADC 2045 e Salmonella entérica<i/> CECT-724 em mais de 90%. / Lactococcus lactis subsp. lactis CECT-4434 was used to investigate the effect of the composition of the culture media on the biotechnological production of biosurfactant and bacteriocin additionally. Agroindustrial residues, such as whey and grape vinasse, were used to formulate more economical and natural culture media, supplemented with sucrose and yeast extract. A fractional factorial design 24, with addition of three runs at the central points was used to evaluate the influence of these variables. The biosurfactant production was positively influenced by the concentration of whey, where 15% showed a better result reducing the surface tension by 18.1 mN/m, reaching a maximum production of biosurfactant equivalent in surfactin of 11.02 mg/L. In relation to bacteriocin synthesis, the sucrose interfered in an antagonistic way, that is, the lower the sucrose concentration, the greater the bacteriocin synthesis (with an increase in the zone of inhibition in 14.2% against Staphylococcus aureus CECT-239). It was observed that the bioreactor conducted under microaeration with 5% dissolved oxygen promoted a higher biosurfactant production (11.6 mg/L) when compared to studies conducted with a higher concentration of oxygen between 30 and 100%, with production on average 2.3 mg/mL. It is noteworthy that no study of the influence of dissolved oxygen, mainly in microaerophilic, for the biosurfactant production by lactic acid bacteria had already been carried out. In addition, the biosurfactant produced proved to be highly stable against extreme values of pH and temperature, and demonstrated remarkable antimicrobial and antiadhesive properties, inhibiting Listeria monocytogenes NADC 2045 and Salmonella entérica CECT-724 in more than 90%.

Dans le cadre de ce travail, l’objectif était d’élaborer des surfaces PVC antibactériennes. Deux types de surfaces ont été élaborés, des surfaces bactéricides visant à tuer les bactéries au contact et des surfaces antiadhésives (bactériophobes) empêchant ou limitant l’adhésion bactérienne.Pour ce faire, des copolymères porteurs de groupements cationiques bactéricides ainsi que des copolymères renfermant des structures à effets antiadhésifs de type polysaccharides ou PEG ont été synthétisés et caractérisés. Ces composés ont été ensuite greffés chimiquement par click addition sur des surfaces PVC porteuses de fonctions azoture (PVC-N3). Les propriétés physico-chimiques des nouvelles surfaces obtenues ont été caractérisées par diverses techniques (IRTF, XPS, angle de contact, AFM, microscopie confocale à fluorescence, ATG etDSC). Les propriétés bactéricides ou antiadhésives ont été évaluées à l’égard de Escherichia coli et Staphylococcus epidermidis. La microscopie confocale à fluorescence a mis en évidence le caractère hautement bactéricide des surfaces PVC cationiques via le test Live and Dead.Cette technique a aussi permis de démontrer l’effet antiadhésif de la méthyl cellulose, del’hydroxyéthyl cellulose et du PEG greffés en surface, diminuant d’au moins 10⁴ fois le nombre de bactéries adhérées par rapport aux surfaces témoins. / This work aimed to elaborate antibacterial PVC surfaces. Two kinds of surface were prepared, i.e. non leaching bactericidal surfaces killing bacteria by contact and antiadhesive (bacteriarepellent) surfaces preventing or limiting bacteria attachment. Thus, copolymers bearing cationic bactericidal groups as well as copolymers bearing polysaccharide or PEG antiadhesive structures were synthesized and characterized. These polymers were then successfully grafted by click chemistry onto PVC surfaces bearing azide groups (PVC-N3).The physico-chemical properties of the obtained surfaces were assessed by several techniques(FTIR, XPS, contact angle measurements, AFM, confocal fluorescence microscopy, TGA and DSC). The bactericidal and antiadhesive properties were assessed against Escherichia coli and Staphylococcus epidermidis. The fluorescence microscopy evidenced the high bactericidal effect of the cationic surfaces, using the Live and Dead test. This microscopytechnique has also shown high bacteria repellent effect of the surfaces based on methyl cellulose, hydroxyethyl cellulose and PEG, i.e. decrease of bacteria attachment by more than 10⁴ fold compared to the ungrafted surface.

This thesis describes studies directed towards development of novel antiadhesive agents, with particular emphasis on compounds that prevent attachment of bacteria to a host-cell. Three different proteins involved in the assembly or function of adhesive pili in uropathogenic Escherichia coli have been targeted either by rational structure based design or statistical molecular methods. A library of substituted galabiose (Galα1-4Gal) derivatives was screened for binding to the E. coli adhesin PapG in an assay based on surface plasmon resonance, and for inhibition of Streptococcus suis adhesins PN and PO in a hemagglutination assay. The results were used to generate QSAR models which had good predictive powers and provided further insight in the structural requirements needed for high affinity binding. 2-pyridones and amino acid derivatives were modelled into the binding site of chaperones involved in pilus assembly in E. coli and a heuristic method, VALIDATE, was used for affinity prediction. The affinity of the compounds for the chaperones PapD and FimC were assessed in assays based on surface plasmon resonance and relaxation-edited NMR spectroscopy. Their ability to disrupt chaperone/subunit complexes was investigated in vitro through a FPLC assay and their capacity to inhibit pilus formation in vivo was determined via hemagglutination and confirmed with atomic force microscopy. Statistical molecular design was used to design a diverse peptide library targeting pili subunits, and an ELISA was developed to investigate the ability of the peptides to inhibit chaperone/subunit complexation. The resulting QSAR model provided extensive information regarding binding of the peptides to the subunits. Because the peptides were suggested to bind in an extended β-strand formation, β-strand mimetics consisting of oligomeric enaminones were designed. Finally, new methods to synthesize enaminone building blocks were developed using microwave assisted chemistry. The projects described have generated compounds that besides their value as leads for developing novel antibacterial agents, also constitute new chemical tools to study the mechanisms underlying bacterial virulence.