O objetivo desse trabalho foi construir o fago recombinante D29::gfp e testar a sua utilização como um agente revelador da viabilidade bacilar na determinação da concentração inibitória mínima (GIM) aos principais fármacos administrados no tratamento da tuberculose. O fago recombinante contém o promotor hsp70 e o gene da proteína verde fluorescente (gfp) e foi construído através da restrição pela Spe I em uma região intergênica próxima a extremidade coesiva direita no genoma do fago D29. O promotor hsp70 e gfp clonados no pYL GFP foram amplificados pela PCR utilizando iniciadores com sítios para Spe I. O DNA do fago D29 digerido pela Spe I foi ligado com o fragmento hsp 70- gfp empregando a T 4 DNA ligase e os produtos da reação de ligação foram transformados de acordo com o protocolo de encapsulamento. A infecção do M.smegmatis com esse fago recombinante induziu a expressão da proteína verde fluorescente (GFP). Para avaliar o uso do fago recombinante em teste de sensibilidade aos fármacos anti-tuberculose, 100 isolados clínicos foram testados quanto ao perfil de sensibilidade a isoniazida (H), rifampicina (R), estreptomicina (S) e etambutol (E), utilizando o método das proporções em Lowenstein-Jensen (L-J), técnica em microplaca com a resazurina (REMA) e técnica em microplaca com D29::gfp. Os resultados do REMA demonstraram que 30 isolados clínicos foram sensíveis à H e 58 (66 %) isolados clínicos foram resistentes, dentre os quais a CIM foi 1 µg/mL ou maior para 41 (71 %). A CIM da R para 49 (56%) dos isolados clínicos resistentes foi de 0,5 µg/mL para 17 (35%). A CIM da S para 33 (37%) dos isolados clínicos resistentes foi de 2 µg/mL para 13 (40%) e CIM do E para 34 (39%) dos isolados clínicos resistentes foi de 16 µg/mL ou maior para 19 (56%). A caracterização molecular pela PCR IS6110 identificou 88 isolados clínicos como M.tuberculosis e pelo PRA hsp65, sete isolados clínicos foram M.kansasii, quatro foram M.abscessus e um M.szulgai. Após empregar o fago recombinante como um agente indicador da viabilidade bacilar para testar a atividade dos fármacos anti-tuberculose conclui-se que a expressão da proteína verde fluorescente foi inespecífica e não reprodutiva, não justificando o seu uso para determinar a CIM para os principais fármacos administrados no tratamento da tuberculose. / The objective of this work was to construct the recombinant phage D29::gfp and to use this phage as an indicator agent of cell viability in a minimal inhibitory concentration (MIC) assay for the mains drugs used for tuberculosis treatment. The recombinant phage contains the mycobacteria-specific hsp70 promoter controlling the green fluorescent protein gene (gfp) and was constructed by Spe I restriction in the intergenic region next to the right cohesive termini of the D29 phage genome. An hsp 70 promoter and gfp previously cloned in p YL GFP was amplified by PCR using primers with Spe I sites. The Spe I-restricted D29 phage DNA was ligated with the hsp 70-gfp fragment using T4 DNA ligase and ligated product was transformed using the packing protocol. Infection of M.smegmatis with this recombinant phage indicated the expression of green f1uorescent protein (GFP). To use the recombinant phage for assaying the activity of anti-TB drugs, 100 clinical isolates was tested for susceptibility to isoniazid (H), rifampicin (R), streptomycin (S), and ethambutol (E) using both the proportion method on Lowenstein-Jensen (L-J) medium, resazurin microtiter assay plate (REMA), as well as a microplate assay using D29::gfp. The REMA plate method showed that 30 clinical isolates were susceptible to H and 58 (66%) clinical isolates were resistant, where the MICs were 1 µg/mL or higher for 41 (71%). The R MICs for 49 (56%) resistant clinical isolates were 0,5 µg/mL for 17 (35%). The S MICs for 33 (37%) resistant clinical isolates were 2 µg/mL for 13 (40%) and E MICs for 34 (39%) resistant clinical isolates were 16 µg/mL or higher for 19 (56%). Molecular characterization by PCR IS6110 showed that 88 clinical isolates were M.tuberculosis and by PRA hsp65 were seven clinical isolates were M.kansasii and four was M.abscessus, and one M.zulgai. After using the recombinant phage as an indicator agent of cell viability for assaying the activitity of anti-TB drugs we can conclude that the expression of green fluorescent protein was non-specific and not reproducible, rendering it not useful for the determination of the MIC of the principal drugs used for the treatment of tb.

La biocontamination des surfaces et les risques associés sont des enjeux majeurs économiques et de santé publique. Afin de limiter ou empêcher l’adhésion bactérienne, une des solutions possible consiste en la modification des propriétés des surfaces, afin de leur conférer les fonctions voulues. Dans ce contexte, l’objectif de cette étude est de modifier des surfaces de types métallique (acier) ou polymère : poly(téréphtalate d’éthylène) par des glycomonomères ou des monomères bioactifs. La stratégie de modification des surfaces s’effectue en plusieurs étapes.La première étape permet d’incorporer des fonctions réactives sur les surfaces par traitement acide puis réaction avec la dopamine, ou par traitement plasma ammoniaque. Des fonctions hydroxyle et amine sont introduites. Par la suite un amorceur de polymérisation par transfert d’atome est greffé sur les surfaces. Les monomères sont synthétisés et leur polymérisation étudiée en solution, dans un premier temps dans les conditions de polymérisation radicalaire classique, puis par polymérisation par transfert d’atome. Les conditions optimales sont déterminées, puis les polymérisations sur surfaces effectuées. La dernière étape concerne l’étude des propriétés microbiologiques des surfaces synthétisées.Les glycopolymères protégés et déprotégés de galactose ainsi que les polymères de méthacrylate de gaïacyle et de méthacrylate de thymyle ont été synthétisés. Les monomères ont été polymérisés par polymérisation par transfert d’atome à partir de la surface, sur les surfaces d’acier et de poly(téréphtalate d’éthylène). Après chaque étape de modification de surface, les matériaux ont été systématiquement caractérisés par goniométrie et spectroscopie à photoélectrons X. Les surfaces d’acier fonctionnalisées par le glycopolymère présentent des propriétés anti-adhésives vis-à-vis de Bacillus subtilis, un effet plus marqué est observé après greffage du glycopolymère déprotégé. Les surfaces de poly(téréphtalate d’éthylène) quant à elles, possèdent des propriétés anti-adhésives leur du greffage du poly(méthacrylate de thymyle) vis-à-vis de Listeria monocytogenes, Staphilococcus aureus et Pseudomonas aeruginosa. Un effet anti-biofilm a également été mis en évidence vis-à-vis de Staphilococcus aureus.En parallèle des homopolymères d’ammonium quaternaire et des copolymères obtenus en incorporant les monomères bioactifs ont été synthétisés. Les propriétés antibactériennes sont testées en milieu planctonique vis-à-vis de Bacillus subtilis. Un degré de polymérisation égal à 78 et les groupements halogénoalcane : iodométhane ou bromoéthane permettent l’obtention de la concentration minimale inhibitrice la plus faible. La présence des monomères bioactifs permettent la diminution de la concentration minimale inhibitrice. Le résultat le plus intéressant est obtenu lors de l’incorporation d’un pourcent de N-(4-hydroxy-3-méthoxy-benzyl)-acrylamide. / Bio-contamination of surfaces and related risks are very important economically and for public health. To prevent this phenomenon, one solution is to modify the properties of the surfaces, in order to give them the wanted functionalities. The goal of this study is the modification of metallic surfaces (steel) or polymer surfaces: poly(ethylene terephtalate) with glycomonomers or bioactives monomers. To reach this objective, a multi-step strategy is applied.The first step enabled the incorporation of reactive species on the surfaces by an acid treatment followed by a reaction with dopamine, or by ammonia plasma treatment. Hydroxyl or amine functional groups are added. Then, an initiator of atom transfer radical polymerization is grafted on surfaces. Monomers are synthesized and conventional polymerization and atom transfer radical polymerization are studied. Optimal conditions are determined and polymerization on surfaces achieved. The last step is the study of the microbiological properties of the synthesized surfaces.Protected and unprotected galactose glycopolymers as well as gaiacyl methacrylate and thymyl methacrylate have been synthesized. Monomers have been polymerized by atom transfer radical polymerization directly to the steel or poly(ethylene therephtalate) surfaces. After each step, materials are analyzed by contact angle measurements and X-ray photoelectron spectrometry.Steel surfaces which are functionalized by a glycopolymer and tested in presence of Bacillus subtilis are found to have antiadhesive properties. A most important effect is observed with the unprotected glycopolymer. Poly(ethylene terephtalate) surfaces have antiadhesive properties in presence of Listeria monocytogenes, Staphilococcus aureus and Pseudomonas aeruginosa when poly(thymyl methacrylate) is grafted. An antibiofilm effect is observed with Staphilococcus aureus.Simultaneously, quaternary ammonium homopolymers and copolymers by integration of bioactive monomers have been obtained. Their antibacterial properties are tested in planctonik conditions in presence of Bacillus subtilis. A degree of polymerization equal to 78 and alkyl halide groups: iodomethane and bromoethane enabled to obtain the lowest minimal inhibitory concentration. Bioactive monomers contributed to emphasize this decrease. The most decreasing effect is obtained when one per cent of N-(4-hydroxy-3-méthoxy-benzyl)-acrylamide is integrated.

Introduction : La chronicité de la rhinosinusite, sa résistance aux antibiotiques, et ses exacerbations aiguës laissent croire que les biofilms sont impliqués dans la rhinosinusite chronique. Objectifs : Nous avons évalué la capacité des bactéries Pseudomonas aeruginosa, staphylocoques à coagulase négative et Staphylococcus aureus à former des biofilms par un essai in vitro, et si cette capacité de formation a un lien avec l’évolution de la maladie. Nous avons évalué in vitro l’effet de la moxifloxacine, un antibiotique utilisé dans le traitement de la rhinosinusite chronique sur des biofilms matures de Staphylococcus aureus. Méthodes : Trent et une souches bactériennes ont été isolées de 19 patients atteints de rhinosinusite chronique et qui ont subit au moins une chirurgie endoscopique des sinus. L’évolution de la maladie a été notée comme "bonne" ou "mauvaise" selon l’évaluation du clinicien. La production de biofilm a été évaluée grâce à la coloration au crystal violet. Nous avons évalué la viabilité du biofilm après traitement avec la moxifloxacine. Ces résultats ont été confirmés en microscopie confocale à balayage laser et par la coloration au LIVE/DEAD BacLight. Résultat et Conclusion : Vingt deux des 31 souches ont produit un biofilm. La production d’un biofilm plus importante chez Pseudomonas aeruginosa et Staphylococcus aureus était associée à une mauvaise évolution. Ceci suggère un rôle du biofilm dans la pathogenèse de la rhinosinusite chronique. Le traitement avec la moxifloxacine, à une concentration de 1000X la concentration minimale inhibitrice réduit le nombre des bactéries viables de 2 à 2.5 log. Ces concentrations (100 µg/ml - 200 µg/ml) sont faciles à atteindre dans des solutions topiques. Les résultats de notre étude suggèrent que l’utilisation de concentrations supérieure à la concentration minimale inhibitrice sous forme topique peut ouvrir des voies de recherche sur de nouveaux traitements qui peuvent être bénéfiques pour les patients atteints de forme sévère de rhinosinusite chronique surtout après une chirurgie endoscopique des sinus. / Introduction: The role of biofilms in chronic diseases is increasingly recognized. Chronic rhinosinusitis, with its chronic indolent course, resistance to antibiotics, and acute exacerbations, has an evolution that parallels that of other biofilm-related diseases. Objectives: 1-To develop an in vitro method to assess the biofilm formation capacity. 2- To determine whether biofilm-forming capacity of bacteria demonstrated in chronic rhinosinusitis has an impact on persistence of the disease following endoscopic sinus surgery. 3- To determine the in vitro activity of moxifloxacin against Staphyylococcus aureus in biofilm form. Method: Thirty-one bacterial strains recovered from 19 patients with chronic rhinosinusitis at least one year post-endoscopic sinus surgery. Evolution of disease was assessed by questionnaire and endoscopy as favorable or unfavorable. The bacteria were cultured on a 96-well culture plaque and a semi-quantitative method using crystal violet to quantify biofilm production was used. Confirmation of the effect of the antimicrobial agents on viability was performed with confocal laser microscopy, using a LIVE/DEAD BacLight staining. Results: Twenty-two of 31 samples produced a biofilm thicker or equal to the positive control. Biofilm formation was associated with a poor evolution for Pseudomonas aeruginosa and Staphylococcus aureus, but not for coagulase-negative staphylococci. Biofilm treated with moxifloxacin at 1000X (0.1mg/ml – 0.2 mg/ml) gave a 2 to 2.5 log reduction in number of viable bacteria. Conclusion: We have shown that Crystal violet method is able to detect biofilm formation. There is a correlation between in vitro biofilm production by Pseudomonas aeruginosa and Staphylococcus aureus and unfavorable evolution after endoscopic sinus surgery, suggesting a role for biofilm in chronic rhinosinusitis. Increased concentrations of moxifloxacin, easily attainable in topical solutions have a potential role in the management of biofilm infections.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Silymarin is a flavonolignan complex isolated from milk thistle seeds of Silybum marianum being used in the treatment of injury related to oxidative stress, including liver and neurological diseases, as Parkinson disease. Although silymarin has been reported to possess a variety of pharmacological properties including anti-inflammatory, anticarcinogenic and neuroprotective effects, information regard its antimicrobial and drug modulator potential against microbial resistance is scanty in the literature. In addition, the possible involvement of antioxidant activity in its neuroprotective effect, and on the activity of important enzymes of the central nervous system (i.e., Na+/K+-ATPase and monoamine oxidase (MAO)) have not yet been completely elucidated. Therefore, the objective of this study was to investigate the effect of silymarin on the activity of the enzymes Na+/K+-ATPase and MAO as well as its ability in to modulate the action of antimicrobials in vitro. The results demonstrated that silymarin scavenged the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and, also reduced significantly the Fe2+ (10 μM) and SNP (sodium nitroprusside, 5 μM) induced lipid peroxidation in rat brain homogenate. Silymarin protected against the oxidation of thiol groups (protein and non-protein) induced by the pro-oxidants, and avoided the reduction in the activity of catalase caused by pro-oxidants at a concentration of 30 μg/mL. The incubation of different concentrations of silymarin increased the activity of Na+/K+-ATPase enzyme and reduced the activity of the enzymes MAO-A and MAO-B. However, the inhibition of MAO-B was more pronounced. The evaluation of the kinetic parameters demonstrated that Silymarin did not alter significantly the Km values for MAO-A and MAO-B, but caused a decrease in Vmax values for both isoforms of the enzyme. With regard to the antimicrobial activity, silymarin and its major active constituent silybin, did not demonstrate antibacterial and antifungal activities not relevant from a clinical point of view (with values of MIC - minimal inhibitory concentration- > 500 μg/mL). However, silybin showed clinically significant antibacterial activity against Escherichia coli with MIC/8 = 64 μg/mL. The combination of sylimarin and silybin demonstrated synergistic activity modulating the efficacy of antibiotics drugs (amikacin, gentamicin, ciprofloxaxin ou imipenem) or antifungal (mebendazole ou nystatin), particularly from the class of aminoglycosides, against multiresistant strains Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. However, silybin antagonized the antibacterial effect of gentamicin and imipenem against P. aeruginosa. Similarly, silymarin and silybin had antagonistic effect with nystatin against Candida albicans, Candida tropicalis and Candida kruzei. In conclusion, the results showed that silymarin alters the activities of Na+/K+-ATPase and monoamine oxidase, indicating that its neuroprotective effect is not only associated to its antioxidant capacity. The potential of silymarin and silybin to modulate the effects of the drugs suggests an alternative to control bacterial infections caused by antibiotics resistance. / A Silimarina é um complexo de flavonolignanas isolado das sementes de Silybum marianum, sendo utilizada no tratamento de distúrbios relacionados ao estresse oxidativo, incluindo hepatopatias e doenças neurológicas, como a Doença de Parkinson. Embora a silimarina seja referida por possuir uma variedade de propriedades farmacológicas, incluindo efeitos anti-inflamatório, anticancerígeno e neuroprotetor, informações sobre o seu potencial antimicrobiano e modulador de fármacos contra a resistência microbiana são escassas na literatura. Além disso, o possível envolvimento da ação antioxidante no seu efeito neuroprotetor, e sobre a atividade de enzimas importantes do Sistema Nervoso Central (como a Na+/K+-ATPase e a monoamina oxidase - MAO), ainda não foi completamente elucidado. Portanto, o objetivo deste estudo foi investigar o efeito da silimarina na atividade das enzimas Na+/K+-ATPase e MAO bem como a sua capacidade de modular a ação de antimicrobianos in vitro. Os resultados demonstraram que a silimarina sequestrou o radical DPPH (2,2-difenil-1-picrilidrazina) e também reduziu significativamente a peroxidação lipídica em homogeneizado de cérebro de ratos induzida por nitroprussiato de sódio (5 M) e Fe+2 (10 M). A silimarina protegeu contra a oxidação dos grupos tióis proteicos (e não proteicos) induzida pelos pró-oxidantes e evitou a diminuição na atividade da catalase causada pelos pró-oxidantes, na concentração de 30 g/mL. A incubação de diferentes concentrações de silimarina aumentou a atividade da enzima Na+/K+-ATPase e reduziu a atividade das enzimas MAO-A e MAO-B. No entanto, a inibição da MAO-B foi mais pronunciada. A avaliação dos parâmetros cinéticos demonstrou que a silimarina não alterou de forma significativa os valores de Km para a MAO-A e MAO-B, mas causou diminuição nos valores de Vmax para as duas isoformas da enzima. No que diz respeito à atividade antimicrobiana, a silimarina e o seu principal componente ativo, a silibinina, não demonstraram atividade antibacteriana e antifúngica clinicamente relevante (com valores de CIM - concentração inibitória mínima superiores a 500 μg/mL). No entanto, a silibinina apresentou atividade antibacteriana clinicamente significativa contra Escherichia coli com CIM/8 de 64 μg/mL. A combinação de silibinina ou silimarina demonstrou efeito sinérgico modulando a eficácia de fármacos antibióticos (amicacina, gentamicina, ciprofloxaxino ou imipenem) ou antifúngicos (mebendazol ou nistatina) particularmente da classe dos aminoglicosídeos, contra as cepas multirresistentes de E. coli, Pseudomonas aeruginosa e Staphylococcus aureus. No entanto, a silibinina antagonizou o efeito antibacteriano da gentamicina e do imipenem contra P. aeruginosa. Da mesma forma, a silimarina e a silibinina tiveram efeito antagônico com a nistatina contra Candida albicans, Candida tropicalis e Candida kruzei. Em conclusão, os resultados mostraram que a silimarina altera as atividades da Na+/K+-ATPase e MAO, indicando que o seu efeito neuroprotetor não está apenas associado à sua capacidade antioxidante. O potencial da silimarina e da silibinina para modular o efeito de fármacos antimicrobianos sugere uma alternativa para o controle das infecções bacterianas provocadas pela resistência aos antibióticos.

O objetivo desse trabalho foi construir o fago recombinante D29::gfp e testar a sua utilização como um agente revelador da viabilidade bacilar na determinação da concentração inibitória mínima (GIM) aos principais fármacos administrados no tratamento da tuberculose. O fago recombinante contém o promotor hsp70 e o gene da proteína verde fluorescente (gfp) e foi construído através da restrição pela Spe I em uma região intergênica próxima a extremidade coesiva direita no genoma do fago D29. O promotor hsp70 e gfp clonados no pYL GFP foram amplificados pela PCR utilizando iniciadores com sítios para Spe I. O DNA do fago D29 digerido pela Spe I foi ligado com o fragmento hsp 70- gfp empregando a T 4 DNA ligase e os produtos da reação de ligação foram transformados de acordo com o protocolo de encapsulamento. A infecção do M.smegmatis com esse fago recombinante induziu a expressão da proteína verde fluorescente (GFP). Para avaliar o uso do fago recombinante em teste de sensibilidade aos fármacos anti-tuberculose, 100 isolados clínicos foram testados quanto ao perfil de sensibilidade a isoniazida (H), rifampicina (R), estreptomicina (S) e etambutol (E), utilizando o método das proporções em Lowenstein-Jensen (L-J), técnica em microplaca com a resazurina (REMA) e técnica em microplaca com D29::gfp. Os resultados do REMA demonstraram que 30 isolados clínicos foram sensíveis à H e 58 (66 %) isolados clínicos foram resistentes, dentre os quais a CIM foi 1 µg/mL ou maior para 41 (71 %). A CIM da R para 49 (56%) dos isolados clínicos resistentes foi de 0,5 µg/mL para 17 (35%). A CIM da S para 33 (37%) dos isolados clínicos resistentes foi de 2 µg/mL para 13 (40%) e CIM do E para 34 (39%) dos isolados clínicos resistentes foi de 16 µg/mL ou maior para 19 (56%). A caracterização molecular pela PCR IS6110 identificou 88 isolados clínicos como M.tuberculosis e pelo PRA hsp65, sete isolados clínicos foram M.kansasii, quatro foram M.abscessus e um M.szulgai. Após empregar o fago recombinante como um agente indicador da viabilidade bacilar para testar a atividade dos fármacos anti-tuberculose conclui-se que a expressão da proteína verde fluorescente foi inespecífica e não reprodutiva, não justificando o seu uso para determinar a CIM para os principais fármacos administrados no tratamento da tuberculose. / The objective of this work was to construct the recombinant phage D29::gfp and to use this phage as an indicator agent of cell viability in a minimal inhibitory concentration (MIC) assay for the mains drugs used for tuberculosis treatment. The recombinant phage contains the mycobacteria-specific hsp70 promoter controlling the green fluorescent protein gene (gfp) and was constructed by Spe I restriction in the intergenic region next to the right cohesive termini of the D29 phage genome. An hsp 70 promoter and gfp previously cloned in p YL GFP was amplified by PCR using primers with Spe I sites. The Spe I-restricted D29 phage DNA was ligated with the hsp 70-gfp fragment using T4 DNA ligase and ligated product was transformed using the packing protocol. Infection of M.smegmatis with this recombinant phage indicated the expression of green f1uorescent protein (GFP). To use the recombinant phage for assaying the activity of anti-TB drugs, 100 clinical isolates was tested for susceptibility to isoniazid (H), rifampicin (R), streptomycin (S), and ethambutol (E) using both the proportion method on Lowenstein-Jensen (L-J) medium, resazurin microtiter assay plate (REMA), as well as a microplate assay using D29::gfp. The REMA plate method showed that 30 clinical isolates were susceptible to H and 58 (66%) clinical isolates were resistant, where the MICs were 1 µg/mL or higher for 41 (71%). The R MICs for 49 (56%) resistant clinical isolates were 0,5 µg/mL for 17 (35%). The S MICs for 33 (37%) resistant clinical isolates were 2 µg/mL for 13 (40%) and E MICs for 34 (39%) resistant clinical isolates were 16 µg/mL or higher for 19 (56%). Molecular characterization by PCR IS6110 showed that 88 clinical isolates were M.tuberculosis and by PRA hsp65 were seven clinical isolates were M.kansasii and four was M.abscessus, and one M.zulgai. After using the recombinant phage as an indicator agent of cell viability for assaying the activitity of anti-TB drugs we can conclude that the expression of green fluorescent protein was non-specific and not reproducible, rendering it not useful for the determination of the MIC of the principal drugs used for the treatment of tb.

Introduction : La chronicité de la rhinosinusite, sa résistance aux antibiotiques, et ses exacerbations aiguës laissent croire que les biofilms sont impliqués dans la rhinosinusite chronique. Objectifs : Nous avons évalué la capacité des bactéries Pseudomonas aeruginosa, staphylocoques à coagulase négative et Staphylococcus aureus à former des biofilms par un essai in vitro, et si cette capacité de formation a un lien avec l’évolution de la maladie. Nous avons évalué in vitro l’effet de la moxifloxacine, un antibiotique utilisé dans le traitement de la rhinosinusite chronique sur des biofilms matures de Staphylococcus aureus. Méthodes : Trent et une souches bactériennes ont été isolées de 19 patients atteints de rhinosinusite chronique et qui ont subit au moins une chirurgie endoscopique des sinus. L’évolution de la maladie a été notée comme "bonne" ou "mauvaise" selon l’évaluation du clinicien. La production de biofilm a été évaluée grâce à la coloration au crystal violet. Nous avons évalué la viabilité du biofilm après traitement avec la moxifloxacine. Ces résultats ont été confirmés en microscopie confocale à balayage laser et par la coloration au LIVE/DEAD BacLight. Résultat et Conclusion : Vingt deux des 31 souches ont produit un biofilm. La production d’un biofilm plus importante chez Pseudomonas aeruginosa et Staphylococcus aureus était associée à une mauvaise évolution. Ceci suggère un rôle du biofilm dans la pathogenèse de la rhinosinusite chronique. Le traitement avec la moxifloxacine, à une concentration de 1000X la concentration minimale inhibitrice réduit le nombre des bactéries viables de 2 à 2.5 log. Ces concentrations (100 µg/ml - 200 µg/ml) sont faciles à atteindre dans des solutions topiques. Les résultats de notre étude suggèrent que l’utilisation de concentrations supérieure à la concentration minimale inhibitrice sous forme topique peut ouvrir des voies de recherche sur de nouveaux traitements qui peuvent être bénéfiques pour les patients atteints de forme sévère de rhinosinusite chronique surtout après une chirurgie endoscopique des sinus. / Introduction: The role of biofilms in chronic diseases is increasingly recognized. Chronic rhinosinusitis, with its chronic indolent course, resistance to antibiotics, and acute exacerbations, has an evolution that parallels that of other biofilm-related diseases. Objectives: 1-To develop an in vitro method to assess the biofilm formation capacity. 2- To determine whether biofilm-forming capacity of bacteria demonstrated in chronic rhinosinusitis has an impact on persistence of the disease following endoscopic sinus surgery. 3- To determine the in vitro activity of moxifloxacin against Staphyylococcus aureus in biofilm form. Method: Thirty-one bacterial strains recovered from 19 patients with chronic rhinosinusitis at least one year post-endoscopic sinus surgery. Evolution of disease was assessed by questionnaire and endoscopy as favorable or unfavorable. The bacteria were cultured on a 96-well culture plaque and a semi-quantitative method using crystal violet to quantify biofilm production was used. Confirmation of the effect of the antimicrobial agents on viability was performed with confocal laser microscopy, using a LIVE/DEAD BacLight staining. Results: Twenty-two of 31 samples produced a biofilm thicker or equal to the positive control. Biofilm formation was associated with a poor evolution for Pseudomonas aeruginosa and Staphylococcus aureus, but not for coagulase-negative staphylococci. Biofilm treated with moxifloxacin at 1000X (0.1mg/ml – 0.2 mg/ml) gave a 2 to 2.5 log reduction in number of viable bacteria. Conclusion: We have shown that Crystal violet method is able to detect biofilm formation. There is a correlation between in vitro biofilm production by Pseudomonas aeruginosa and Staphylococcus aureus and unfavorable evolution after endoscopic sinus surgery, suggesting a role for biofilm in chronic rhinosinusitis. Increased concentrations of moxifloxacin, easily attainable in topical solutions have a potential role in the management of biofilm infections.