Dissertations / Theses on the topic 'Biofilm'

Le développement d’un biofilm est un processus complexe qui se décline en plusieurs étapes. L’adhésion initiale des bactéries au support est suivie de la formation de microcolonies, puis de la synthèse d’une matrice extracellulaire qui englobe les bactéries donnant lieu à un biofilm mature. La dernière étape, sans doute la moins bien décrite jusqu’à présent, consiste en la dispersion du biofilm. Ce phénomène, contrôlé génétiquement, est déclenché en réponse à de nombreux signaux physiques et chimiques, telles que la température, la disponibilité en nutriments ou l’accumulation de molécules signal (peptides autoinducteurs du quorum sensing, monoxyde d’azote...). Les bactéries sessiles synthétisent alors une panoplie d’effecteurs qui permettent à certaines cellules de quitter le biofilm pour coloniser de nouveaux environnements. Dans le cadre d’infections liées aux biofilms, ce processus de dispersion est un mécanisme clé permettant la libération dans l’organisme de bactéries à partir de biofilms souvent constitués sur des matériels médicaux invasifs. Cependant, la physiologie des cellules ainsi dispersées reste relativement méconnue mais doit probablement jouer un rôle majeur dans la colonisation ultérieure et l’infection chez l’hôte. L’objectif de ce travail était de caractériser au niveau transcriptionnel et phénotypique les cellules dispersées de biofilms formés par le pathogène opportuniste Klebsiella pneumoniae, et ceci comparativement aux formes planctoniques et sessiles. Une analyse transcriptionnelle a été réalisée à partir des formes planctoniques (exponentielles et stationnaires), sessiles et dispersées, en combinant un modèle expérimental de culture en flux (flow-cell) et un séquençage global des ARN (RNAseq). Les données obtenues ont indiqué que les bactéries dispersées affichaient un profil transcriptionnel unique et distinct des autres formes bactériennes. De plus, cette analyse transcriptionnelle des formes planctoniques, sessiles et dispersées de biofilm a permis de dresser une carte exhaustive du transcriptome de K. pneumoniae au cours des différentes phases de croissance et de dégager des gènes « signatures » de chacune d’elle. Le profil transcriptionnel unique des bactéries dispersées suggère qu’elles possèdent une physiologie spécifique leur permettant de coloniser efficacement de nouveaux environnements. L’analyse des propriétés des bactéries dispersées a ainsi constitué la deuxième partie du projet, en se focalisant sur les mécanismes clés dans la physiopathologie des infections nosocomiales à K. pneumoniae, à savoir les capacités d’adhésion, de colonisation et de virulence. Le profilage phénotypique a montré que les bactéries dispersées de biofilm colonisaient significativement mieux les surfaces biotiques et abiotiques que des bactéries planctoniques. Cette capacité de colonisation accrue ne s’expliquait ni par une meilleure adhésion initiale des bactéries, ni par une activité métabolique supérieure, mais par une capacité des bactéries dispersées à former très rapidement des microcolonies. À ceci s’ajoutait un pouvoir pathogène augmenté des bactéries dispersées comparativement à la forme planctonique, avec notamment une meilleure résistance à l’activité bactéricide des macrophages. La mise au point d’un modèle murin d’infection pulmonaire adapté a été initiée ; il permettra d’analyser la virulence de ces bactéries dispersées. L’ensemble des résultats indique que les bactéries dispersées de biofilm se placent dans un état unique du cycle de vie bactérien, en étant transcriptionnellement différentes des autres formes bactériennes, et avec des propriétés mixtes, se rapprochant à la fois de la forme planctonique (activité métabolique et taux de croissance élevée) et de la forme sessile (fort pouvoir de colonisation et meilleure résistance à la phagocytose). L’ensemble de ces propriétés confère certainement un rôle déterminant dans le déclenchement d’infections liées aux biofilms
Biofilm development is a complex process involving several steps. The bacterial adhesion to the surface is followed by formation of microcolonies and synthesis of extracellular matrix, which encased bacteria giving rise to mature biofilm. The last step, probably the most poorly described, corresponds to biofilm dispersal. This process is genetically controlled and triggered in response to a wide range of physical and chemical signals, such as temperature, nutrients availability or the accumulation of signal molecules (quorum sensing autoinducers, nitric oxide...). In response to these signals, sessile bacteria synthesize various effectors, which allow a subset of cells to leave the biofilm and colonize new environments. In the context of biofilm-related infections, the biofilm dispersal process is a key mechanism for the release of bacteria from biofilms formed on invasive medicals devices. Little is known about the properties of the resulting biofilm-dispersed bacteria, but they probably play a major role in the subsequent colonization and infection processes within the host. The objective of this work was to characterize the transcriptome and the phenotype of biofilm-dispersed bacteria of the opportunist pathogen Klebsiella pneumoniae comparatively to the planktonic and sessile forms. A transcriptional analysis was carried out using planktonic (both exponential and stationary forms), sessile and biofilm-dispersed bacteria by combining a flow-cell experimental model with global RNA sequencing (RNAseq). Results indicated that biofilm- dispersed bacteria displayed a unique transcriptional pattern in the bacterial lifecycle. Furthermore, analysis of the whole transcriptome of planktonic, sessile and biofilm-dispersed bacteria allowed to emphasize the transcriptional changes occurring in the course of K. pneumoniae lifestyle transitions and to select transcriptional signatures genes for the five bacterial physiological states. The unique transcriptional pattern of biofilm-dispersed bacteria suggests that they display specific physiological characteristics required to colonize new environments. The second part of this work consisted in analyzing the properties of biofilm-dispersed bacteria, by focusing on key mechanisms involved in the physiopathology of K. pneumoniae: adhesion, colonization and virulence. Phenotypic profiling showed that biofilm-dispersed bacteria colonized significantly more biotic and abiotic surfaces than their planktonic counterparts. This increased colonization capacity was not due to an enhanced adhesion or a higher metabolic activity but rather to the intrinsic capacity of the biofilm-dispersed bacteria to form rapidly microcolonies. Besides, biofilm-dispersed bacteria were more pathogenic than their planktonic counterparts showing a better resistance to the bactericidal activity of macrophages. The development of a murine pulmonary infection model is in progress and will enable to assess the virulence of biofilm-dispersed bacteria. Our results indicate that biofilm-dispersed bacteria are placed in a unique state in the bacterial lifecycle, being transcriptionally different from other bacterial forms, and with mixed properties, approaching both the planktonic form (elevated metabolic activity and growth rate) and the sessile form (strong colonization power and high resistance to phagocytosis). These properties certainly play a decisive role in the initiation of biofilm-related infections

Orientador: Livia Maria Andalo Tenuta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: A eficiência anticárie dos dentifrícios fluoretados contendo 1000-1500 µg F/g está bem estabelecida, porém eles têm sido considerados fator de risco para fluorose dental. Para reduzir esse risco, dentifrícios contendo baixa concentração de fluoreto (F) (500-550 µg F/g) têm sido recomendados, mas sua eficiência anticárie ainda não foi demonstrada. Assim, o objetivo deste trabalho foi: 1. comparar a disponibilidade de F na saliva após utilização de dentifrício de baixa concentração de F (BC, 500 µg F/g, NaF), ou dentifrício de concentração convencional (CC, 1100 µg F/g, NaF), seguida ou não de enxágüe; e 2: avaliar in situ o potencial anticariogênico desses dentifrícios, estudando o efeito do F disponível no biofilme dental após a escovação, associado ou não aos produtos formados no esmalte pelo tratamento com os dentifrícios. Em ambos os estudos, foi empregado um delineamento cruzado e duplo cego. No estudo 1, amostras de saliva não estimulada de 5 voluntários foram coletadas antes e imediatamente após a escovação e nos tempos 1, 2, 3, 4, 5, 10, 15, 20, 30, 45 e 60 min após a escovação com BC ou CC, seguida ou não de enxágüe. A área sob a curva da concentração de F na saliva versus tempo foi calculada para determinar a biodisponibilidade de F salivar. Esta foi reduzida em 2,5 x pelo enxágüe pósescovação (p<0,05) e foi semelhante quando BC foi utilizado sem enxágüe e CC foi seguido de enxágüe (p>0,05). No estudo 2, doze voluntários realizaram escovação com dentifrícios contendo concentrações de F distintas (placebo (P) ¿ controle negativo, CC ou BC) e utilizaram um dispositivo palatino contendo blocos de esmalte bovino, previamente tratados ou não com suspensão do respectivo dentifrício. Os blocos foram cobertos com uma placa teste de S. mutans IB 1600 e após 30 min in situ, a placa foi coletada e a concentração de F no fluido foi determinada através de técnica microanalítica com eletrodo íon específico. Um bochecho com sacarose foi realizado como desafio cariogênico e após 45 min os blocos remanescentes e a placa teste foram coletados para avaliação, respectivamente, da perda mineral (simulando o efeito de diferentes espessuras de placa) e da concentração de F no fluido. O pré-tratamento dos blocos de esmalte com os dentifrícios fluoretados isoladamente não impediu a perda mineral em relação ao controle (p>0,05), mas causou aumento na concentração de F no fluido da placa (p<0,05). A escovação com os dentifrícios fluoretados aumentou a concentração de F no fluido da placa, sendo encontrada diferença significativa entre BC e CC (p<0,05), além de uma menor perda mineral em relação ao controle (p<0,05). Adicionalmente, embora a perda mineral tenha sido semelhante para BC e CC na simulação de espessura de placa de até 0,5 mm, ela foi maior para BC na placa mais espessa (1 a 1,5 mm) (p<0,05). Os resultados sugerem que o dentifrício de concentração convencional é mais efetivo do que o de baixa concentração na inibição da perda mineral. Adicionalmente, deve-se estimular o enxágüe da boca após o uso do dentifrício de concentração convencional por crianças de pequena idade
Abstract: The anticaries efficiency of fluoride (F) dentifrices containing 1000-1500 µg F/g is well established, but they are considered a risk factor to dental fluorosis. In order to reduce this risk, low-F concentration dentifrices (500-550 µg F/g) have been recommended, but their anticaries efficiency has not been demonstrated. Thus, this study aimed to: 1. compare salivary F availability after brushing with low- F concentration (LC, 500 µg F/g, NaF) or conventional F concentration (CC, 1100 µg F/g, NaF) dentifrices, followed or not by a water rinse and 2: evaluate in situ the anticaries potential of these dentifrices, studying the anticaries effect of F available on the dental biofilm after brushing, associated or not to F products formed on enamel by F dentifrice application was evaluated. In both studies, a crossover, double blind design was used. In study 1, samples of non-stimulated saliva from 5 volunteers were collected before and immediately after brushing with LC or CC, followed or not by a rinse, and after 1, 2, 3, 4, 5, 10, 15, 20, 30, 45 and 60 min. The area under the curve of salivary F concentration versus time was calculated to determine F bioavailability in saliva. F salivary bioavailability was reduced 2.5 X by the post-brushing rinse (p<0.05) and it was similar when LC was used without rinsing and CC was used followed by a rinse (p>0.05). In study 2, twelve volunteers brushed with dentifrices containing distinct F concentrations (placebo (P) ¿ negative control, LC or CC) and used a palatal appliance containing bovine enamel blocks previously treated or not with a slurry of assigned dentifrice. The blocks were covered with a test plaque from S. mutans IB 1600 and after 30 min in situ, F concentration in the fluid of plaque was assessed. A sucrose rinse was performed as a cariogenic challenge and after 45 min the remaining blocks and plaque test were removed to evaluate, respectively, mineral loss (as a function of plaque thickness) and F concentration in plaque fluid. The isolated effect of the pretreatment of enamel blocks with F dentifrices did not reduced mineral loss when compared to the control (p>0.05), but resulted in higher F concentration in the plaque fluid (p<0.05). Brushing with F dentifrices increased F concentration in the plaque fluid, which was significantly different between LC and CC (p<0.05), and resulted in lower mineral loss when compared to the control (p<0.05). Additionally, although LC and CC did not differ when mineral loss was evaluated on a plaque thickness simulation of up to 0.5 mm, CC was more efficient than LC at thicker plaque (1 to 1.5 mm) (p<0.05). The results suggest that conventional F concentration dentifrice is more efficient than the low-F one in the inhibition of mineral loss. Additionally, post-brushing rinse should be recommended after the use of conventional F concentration dentifrices by young children
Mestrado
Cariologia
Mestre em Odontologia

Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui peut développer deux modes d’infection. Les infections aigües sont associées à la production et à la sécrétion de toxines qui peuvent avoir un effet cytotoxique, alors que dans le contexte d’une infection chronique la bactérie a tendance à s’établir sous la forme d’un biofilm. Un biofilm est une population de microorganismes organisée en une communauté et attachée sur une surface. Cette surface peut être biotique ou abiotique. Les biofilms bactériens ont des caractéristiques intrinsèques qui les rendent plus résistants à des conditions environnementales difficiles (pH, oxygène, UV, flux, etc…). Dans le cadre d’une infection l’établissement du biofilm résulte aussi en une population bactérienne difficile à éliminer par le système immunitaire mais également résistante aux antibiotiques. Des déterminants moléculaires majeurs qui interviennent dans la formation du biofilm sont le flagelle, les pili de type IV et les fimbriae Cup pour l’attachement, ou bien encore les exopolysaccharides qui sont avec l’ADN des composants essentiels de la matrice extracellulaire du biofilm qui englobe la population bactérienne. Chez P. aeuginosa, si l’alginate est le polysaccharide majeur, il a été montré que des souches non-mucoides forment également des biofilms et que dans ce contexte la synthèse et la sécrétion du polysaccharide majeur de la matrice sont dépendantes des gènes pel. Mon travail a consisté dans l’étude de l’organisation structurale du système Pel et le contrôle de son activité
Pseudomonas aeruginosa is a human opportunistic pathogen, and in the course of an infection can develop two lifestyles. One of them is involved in secretion of toxins and results in acute infection, and the other one causes chronic infections and is characterized by formation of a biofilm. A biofilm is a highly organized bacterial population, organized as a complex community that is attached to a surface. Bacterial biofilm shows higher resistance to different enviromental factors including physical factors, antimicrobials or host immune response. Major components taking part in biofilm formation are flagella, type IV pili, cup fimbriae as well as exopolysaccharides. The latter provides a protective matrix for the biofilm, together with proteins and DNA. In non-mucoid strains the major exopolysaccharide of biofilm matrix is synthetised and secreted by pel gene cluster. My work concentrates on the structural organisation of Pel polysaccharide secretion machinery, regulation of its activity as well as detailed characterisation of the Pel polysaccharide itself

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Este estudo avaliou a atividade antimicrobiana de um reembasador resiliente Coe Soft® (RRCS) combinado ao polímero antimicrobiano poli (2 tert-butilaminoetil) metacrilato (PTBAEMA) sobre formação de biofilme de Staphyloccocus aureus, Streptococcus mutans e Candida albicans. Espécimes circulares (15mm x 3mm) do RRCS foram confeccionados (n=27), esterilizados, divididos em três grupos de acordo com as concentrações de PTBAEMA a 0% (controle), 10% e 25% e individualmente inoculados em tubos de falcon contendo 5mL de caldo RPMI para os fungos, TSB para S. aureus e BHI para S. mutans e mantidos em overnight a 37ºC em incubadora com agitação orbital a 75rpm, sendo o S. mutans em microaerofilia. Após a inoculação dos espécimes seguiu-se a formação e maturação do biofilme a 37ºC sob agitação orbital a 75rpm. Em seguida cada espécime foi transferido para tubos contendo PBS e diluições seriadas foram realizadas. Alíquotas dessas diluições foram semeadas em placas de Petri e incubadas a 37ºC por 48h. Os dados obtidos foram transformados em log (UFC+1)/mL, considerando-se α=0,05. Os resultados demonstraram que o grupo contendo 25% de PTBAEMA inibiu completamente a formação de biofilme de S. aureus e S. mutans. Uma redução significativa na contagem de S. aureus e S. mutans (Kruskal- Wallis e Dunn; p=0,001) para o grupo contendo 10% de PTBAEMA foi observada quando comparada aos valores encontrados nos respectivos grupos controle. Para C. albicans não foi encontrada diferença significante entre grupos contendo PTBAEMA e o grupo controle (ANOVA; p>0,05). Conclui-se que os RRCS contendo 10% e 25% de PTBAEMA inibiram a formação de biofilme de S. aureus e S. mutans. Entretanto não teve efeito significante na formação de biofilme de C. albicans.
This study evaluated the antimicrobial activity of the resilient reliner Coe Soft ® (RRCS) combined with antimicrobial polymer poly (2-tert butylaminoethyl) methacrylate (PTBAEMA) on Staphylococcus aureus, Streptococcus mutans and Candida albicans biofilm formation. RRCS circular specimens were prepared (n=27), sterilized, divided into three groups according to PTBAEMA concentrations of 0% (control), 10% and 25% and inoculated into individual falcon tubes containing 5 mL of RPMI broth for fungi, TSB for S. aureus and BHI for S. mutans and kept overnight at 37°C with orbital shaking incubator at 75rpm, and S. mutans in microaerophilic. The specimens’ inoculations were followed by biofilm formation and its maturation at 37°C under orbital shaking at 75rpm. After that, each sample was transferred to tubes containing PBS and serial dilutions were performed. Aliquots of these dilutions were plated in Petri dishes and incubated at 37°C for 48h. The data were transformed into log (CFU +1)/mL, considering α = 0.05. The results showed that the group containing 25% of PTBAEMA inhibited completely biofilm formation of S. aureus and S. mutans. A significant reduction in counts of S. aureus and S. mutans (Kruskal- Wallis and Dunn; p = 0.001) were found in group containing 10% of PTBAEMA when compared to the values in the corresponding control groups. C. albicans had no significant differences between groups containing PTBAEMA and the control group (ANOVA; p> 0.05). It is concluded that the RRCS containing 10% and 25% PTBAEMA inhibited the biofilmformation of S. aureus and S. mutans. However, no significant effect was found on C. albicans biofilm formation.

Orientador: Marcelo Cristianini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Um dos problemas que enfrenta a indústria de laticínios é a recontaminação do leite decorrente da formação de biofilmes em tanques de armazenamento e trocadores de calor. A tecnologia de esterilização de materiais por gás plasma tem sido utilizada com sucesso na esterilização de instrumentos cirúrgicos, oftálmicos e dentários. Os objetivos deste trabalho foram avaliar a eficiência de um sistema de esterilização a gás plasma sob células de Pseudomonas fluorescens aderidas em placas de aço inoxidável utilizando o leite como substrato. Foram avaliadas as variáveis tempo de pré plasma, tempo de exposição ao plasma e potência do mesmo na remoção do biofilme. As placas de aço inoxidável com a bactéria aderida foram submetidas a um tratamento com gás plasma, que foi formado a partir de um produto comercial composto por ácido peracético, peróxido de hidrogênio e ácido acético. Dois sistemas modelo foram utilizados para simular a formação de biofilmes de Pseudomonas fluorescens, um dinâmico e outro estático, simulando trocadores de calor e tanques de armazenamento, respectivamente. Através do modelo estático foi possível obter contagens acima de 105 UFC por placa de aço inoxidável, mesmo após três ciclos de lavagem das placas em água estéril sob agitação. Foi observado um efeito positivo na inativação de P. fluorescens em função do tempo de pré-plasma do sanificante. Exposições acima de 7 minutos foram capazes de produzir reduções superiores a dois ciclos logarítmicos na inativação do microrganismo. Através de um planejamento experimental fatorial 23 foi demosntrado que as variáveis tempo de pré-plasma, tempo de exposição ao plasma e potência do plasma apresentaram efeitos positivos na inativação de Pseudomonas fluorescens. O tempo de exposição (min.) apresentou o maior efeito na destruição da bactéria; mas sendo um pouco superior à potência do plasma (w)
Abstract: One of the problems that food industry is facing is the milk recontamination through biofims formation on storage tanks and heat exchanger. The technology of sterilization for materials through gas plasma has been used with succesfull for cirurgycal, ophathalmic and dentistry equipment. The goals of this work was to evaluate the efficiency of a gas plasma sterilization system on Pseudomonas fluorescens cells adhered on stainless steel plates using milk as substrate. Time of pre plasma, plasma exposition and potency (Watts) were evaluated as independent variables on cell destruction. The stainless steel plates were submitted to gas plasma treatment originated from a commercial product composed of peracetic acid, hydrogen peroxide and acetic acid. Two model systems were used to simulate a biofilm formation of Pseudomonas fluorescens, one dynamic and one static, in order to simulate heat exchangers and storage tanks, respectively. Through static model it was possible to get counts over 105 UFC/plate after washing three times using sterile water under stirring conditions. It was observed a positive effect on P. fluorescens inactivation by pre-plasma in a time dependent way. Expositions over seven minutes were capable to produce reductions higher than two logarithmic cycles on microorganism inactivation. A 23 factorial design indicated that the pre-plasma time, time exposition and potency showed positive effects on Pseudomonas fluorescens inactivation. Time exposition (min) was the most effective variable on bacteria destruction, being a little higher than plasma potency (w)
Mestrado
Mestre em Tecnologia de Alimentos

The aim of this project was to investigate the possible role of the biofilm matrix as a barrier to drug diffusion in Candida biofilms and in mixed species fungal-bacterial biofilms. The penetration of antifungal agents through single- and mixed-species biofilms containing Candida was investigated using a novel filter disk bioassay. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. Drug penetration was more extensive with C. albicans than with the other species and the rates of diffusion of either drug through biofilms of three strains of C. albicans were similar. In all cases, after 3 to 6h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms under these conditions. It has been reported that the production of extracellular matrix by Candida biofilms growing under static incubation conditions is relatively minimal, but increases dramatically when developing biofilms are subjected to a liquid flow. In this study, Candida biofilms were grown under flow conditions in a modified Robbins device (MRD). Biofilms of C. albicans grown in the MRD produced more matrix material than those grown statically, and were significantly more resistant (P<0.001) to amphotericin B. Biofilms of C. tropicalis synthesized large amounts of matrix material even when grown statically, and such biofilms were completely resistant to both amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis RP62A, when grown statically or in the MRD, were also completely resistant to amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis M7, on the other hand, were completely drug resistant only when grown under flow conditions. Overall, these findings demonstrate that the matrix can make a significant contribution to drug resistance in Candida biofilms, especially under conditions similar to those found in catheter infections in vivo, and that the composition of the matrix material is an important determinant in resistance.

Les biofilms constituent un mode de vie microbien extrêmement répandu, aussi bien en milieu naturel que dans les environnements anthropisés. Dans ce dernier cas, les structures morphologique et microbiologique du biofilm vont conditionner son impact sur le système, que cet impact lui soit bénéfique ou préjudiciable. L'objectif de cette thèse est d'approfondir notre connaissance des phénomènes de structuration du biofilm afin, à terme, d'optimiser les performances de procédé. Afin de représenter au mieux les conditions industrielles, des régimes d'écoulement turbulents et des consortia microbiens complexes ont été utilisés. Une première partie se focalise sur l'impact des forces de cisaillement sur l'adhésion microbienne. Les résultats démontrent un changement progressif de la flore bactérienne fixée et de sa distribution spatiale. Dans un second temps, le projet s'est intéressé aux étapes de développement du biofilm et ont permis d'identifier un effet mémoire du biofilm mature. Il s'agit d'une conservation des structures morphologique et microbiologique au cours du temps en dépit d'un changement de régime hydrodynamique. Enfin la dernière partie a consisté en la mise au point d'une méthode de quantification des prédateurs mobiles dans les biofilms. Ces prédateurs participent à la structuration du biofilm et leur quantification peut s'avérer utile dans le contexte de l'épuration des eaux
Biofilms are a biological mode of life widely spread in both natural and engineered environments. In the last case, whether the biofilm is beneficial or detrimental for the process under consideration, both morphology and microbial community of the biofilm determine its impact. The objective of this thesis is to deepen our knowledge of biofilm structuring and, as a further goal, optimize a given process. Turbulent flows and multi-species consortia were used in order to better mimic industrial conditions. The first part of the project focused on the impact of shear stress on microbial adhesion. Results have demonstrated a gradual shift in bacterial communities with shear and a change in the spatial distribution of adhered microorganisms. Secondly, the work dealt with biofilm development. A memory effect, defined as the conservation of initial morphological and microbiological features despite a change in the environmental conditions, has been observed. Finally, a method for quantification of moving predators in mature biofilms has been developed. These predators actively shape the biofilm and their quantification is valuable, especially for wastewater treatment

Orientadores: Reginaldo Bruno Gonçalves, Daniel Saito
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Há um consenso que outros micro-organismos além de Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) e Treponema denticola (Td) estariam correlacionados às periodontites, inclusive algumas espécies ainda não identificadas. Nosso objetivo foi estudar as microbiotas subgengivais de indivíduos com periodontite crônica generalizada (PCG) e periodontite agressiva generalizada (PAG) para avaliar diferenças entre suas microbiotas. MATERIAL E MÉTODOS-Foram selecionados 15 indivíduos com PCG e 14 com PAG. Coletou-se amostra do biofilme subgengival de uma bolsa periodontal profunda (BP-PS ? 7mm) e uma moderada (BM - PS entre 5 e 6 mm) de cada indivíduo. Foi preparado e analisado, por meio de DGGE, o perfil bacteriano entre os grupos. A similaridade e a análise de cluster do padrão de UTO's foram verificadas utilizando-se coeficiente de Jaccard e a construção do dendrograma realizada por UPGMA. Realizou-se também análise clonal direta de 10 amostras de BP de cada grupo e as sequências foram agrupadas em táxons com similaridade >97%. RESULTADOS-DGGE - No perfil de DGGE foi observada uma tendência para a formação de grupos em BP, mas não em BM, com a presença de dois grupos maiores e distintos de oito indivíduos tanto para PCG como PAG, com variação de similaridade intra-grupo entre 53,6-68,4% e 50,2-64,7%, respectivamente. Análise clonal - Foram identificados 109 táxons conhecidos a partir de 987 clones. Ao todo 44 gêneros bacterianos, 28 gêneros comuns aos dois grupos, nove que se apresentaram apenas para PCG e sete para PAG. Entre os dois grupos foram observados 34 táxons comuns, sendo 42 específicos para PAG e 37 para PCG. A espécie Tf foi detectada em 90% dos indivíduos com PCG e 80% com PAG, Pg foi detectada em 70% com PCG e 50% com PAG e Td foi detectada em 40% com PCG e 30% PAG. A espécie Aa foi encontrada em somente 20% de PCG e 30% de PAG. A espécie Filifactor alocis foi observada em altas taxas e prevalência em PCG (58 clones, 90%) e PAG (91 clones, 90%). As espécies encontradas exclusivamente por grupo com prevalência acima de dois pacientes foram: PCG: Treponema lecithinolyticum, Selenomonas dianae, Prevotella pleuritidis, Dialister pneumosintes; e para PAG: Fusobacterium nucleatum ss vincentii, Veillonella parvula, Peptococcus sp. Cepa GEA8, Streptococcus gordonii, Lautropia mirabilis, Gemella sanguinis, Afipia broomeae. Para os filotipos, PCG: Peptostreptococcaceae sp. Clone-MCE10_174, Fusobacterium sp. C-I035, Veillonellaceae sp. C-JS031; PAG: Peptostreptococcaceae sp. C-PUS9170, Treponema sp. CG093. Apesar de não haver exclusividade entre grupos, é de nota os filotipos Synergistes sp. clone W028 (80% e 60%) e o clone D084 (70% e 10%) em PCG e PAG, respectivamente. O filotipo Bacteroidetes sp. AU126 foi encontrado tanto em PCG (60%) como PAG (30%). CONCLUSÃO - O presente trabalho demonstrou por meio de DGGE uma tendência a um perfil microbiano comum entre a maioria das amostras estudadas, entretanto, sem seu completo delineamento como dois grupos distintos microbiologicamente. A análise clonal, apesar de algumas espécies específicas entre grupos, demonstrou pequenas diferenças, sem, entretanto, delinear grupos microbiologicamente específicos.
Abstract: There is an agreement that not only the already known periodontopathogens, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and Treponema denticola (Td) would be involved in periodontitis, but also some others micro-organisms not-yet-identified. The scope of this study is to compare the subgingival microbiota in generalized chronic periodontitis (GCP) or generalized aggressive periodontitis (GAP) subjects. MATERIAL AND METHODS - 15 subjects with GCP and 14 with GAP were enrolled. One subgingival biofilm sample from a periodontal deep pocket (DP) with PD ? 7mm and one from a moderate pocket (MP) with PD from 5 to 6 mm were harvested from each subject. The microbial profiles (OTU's) were compared between groups by DGGE and the similarity OTU profile was analyzed by Jaccard coefficient and the dendrogram and cluster analyses were made by UPGMA. The direct clonal analysis of the 16SrDNA from 10 samples of each group from DP was made. The sequences were grouped in clusters of taxa with > 97% similarity. RESULTS - DGGE - It was observed in the profile a tendency for eight subjects from each group to assemble as clusters in the DP, but not for the MP samples, with similarities between 53.6-68.4% (GCP) and 50.2-64.7% (GAP). Clonal analyses - One-hundred-and-nine already recognized taxa were obtained from 987 clones. From a total of 44 bacterial genera, 28 were common for both groups; nine were exclusive to PCG and seven to PAG subjects. It was found 34 common taxa between GCP and GAP, 37 were specific for GCP and 42 for GAP. The Tf species was found in 90% from GCP subjects and 80% from GAP subjects, Pg was found in 70% from GCP and in 50% from GAP and Td was detected in 40% from GCP and 30% from GAP. The Aa species were found in only 20% GCP subjects and in 30% from GAP. Filifactor alocis species were detected in high prevalence in both GCP (58 clones, 90%) and PAG (91 clones, 90%). The species which were detected exclusively in each group, with 20% prevalence or more were, for GCP: Treponema lecithinolyticum, Selenomonas dianae, Prevotella pleuritidis, Dialister pneumosintes; and GAP: Fusobacterium nucleatum ss vincentii, Veillonella parvula, Peptococcus sp. Cepa GEA8, Streptococcus gordonii, Lautropia mirabilis, Gemella sanguinis, Afipia broomeae. In relation to phylotypes, PCG: Peptostreptococcaceae sp. Clone-MCE10_174, Fusobacterium sp. C-I035, Veillonellaceae sp. C-JS031; PAG: Peptostreptococcaceae sp. C-PUS9170, Treponema sp. C-G093. Despite not been found exclusively for neither GCP nor GAP, the phylotypes Synergistes sp. clone W028 (80% e 60%) and clone D084 (70% e 10%) had a notable presence in GCP and GAP, respectively. The phylotype Bacteroidetes sp. AU126 was found in GCP (60%) and GAP (30%) groups. CONCLUSION - The present study demonstrated by DGGE a slight tendency to the clustering of the microbial profile of some GCP and GAP subjects, although these were not well delineated. The clonal analyses showed some differences, but also could not show GCP and GAP as microbiologic distinct profiles.
Doutorado
Microbiologia e Imunologia
Doutor em Biologia Buco-Dental

Orientadores: Pedro Luiz Rosalen, Marta Cristina Teixeira Duarte
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O objetivo desse trabalho foi avaliar a atividade antimicrobiana, in vitro, de óleos essenciais e frações dos óleos de melhor atividade, contra microrganismos do grupo mutans em estado planctônico. Além disso, os biofilmes de Streptococcus mutans foram submetidos às frações ativas e os óleos de melhor atividade e frações ativas foram avaliados quanto à sua citotoxicidade e caracterizados quimicamente. Para isso, vinte óleos essenciais (OE) foram obtidos por hidrodestilação a partir de plantas pertencentes ao banco de germoplasmas da Coleção de Plantas Medicinais e Aromáticas (CPMA/CPQBA/UNICAMP). Estes OE foram avaliados quanto à sua atividade antimicrobiana por meio dos ensaios: concentrações inibitória (CIM) e bactericida mínima (CBM) contra Streptococcus mutans UA159. Controles positivo (clorexidina 0,12 %) e negativo (propilenoglicol 6,12 % e 25 %) também foram testados...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: The aim of this study was to evaluate the in vitro antimicrobial activity of essential oils (EO) and fractions from highest activity EO against planktonic cells of mutans streptococci. Besides, the biofilms formed by this microorganism were submitted to active fractions and the higher activity EO and active fractions were evaluated regarding their citotoxicity and chemically characterized. For this, twenty essentinal oils were obtained from plants of the "Collectio of Medicinal and Aromatic Plants" (CPMA, CPQBA/UNICAMP), germplasm bank by hydrodistillation. These EO were evaluated by antimicrobial assays: minimum inhibitory (MIC) and bactericidal (MBC) concentrations against Streptococcus mutans UA159. Positive (chlorhexidine 0.12%) and negative (propylene glycol 6.12 % and 25%) controls were also tested...Note: The complete abstract is available with the full electronic document
Mestrado
Farmacologia, Anestesiologia e Terapeutica
Mestre em Odontologia

Poznavanje i razumevanje adhezivne sposobnosti i formiranja biofilma patogenih bakterija, kao i njihovog odnosa prema faktorima koji mogu stimulisati ili inhibirati razvoj biofilma, je od fundamentalnog značaja za iznalaženje mera za njihovu efikasnu prevenciju i eliminaciju.Imajući u vidu navedenu činjenicu kao i da je Salmonella enterica serotip Enteritidis epidemiološki najfrekventniji serotip cilj ovog istraživanja je bio da se ispita: sposobnost različitih sojeva Salmonella Enteritidis izolovanih iz kliničkog materijala, hrane za životinje i odabranog referentnog soja da formiraju biofilm, adherentnost na površine od stakla i nerđajućeg čelika, sposobnost preživljavanja odabranih biofilm produkujućih sojeva kao i mogućnost primene konfokalne laserske i skening elektronske mikroskopije u vizuelizaciji trodimenzionalne strukture biofilma.Određivanjem morfotipa kolonija na Kongo red agaru na temperaturi inkubiranja od 25°C među testiranim izolatima detektovana su tri morfotipa RDAR (red, dry and rough), BDAR (brown dry and rough) i SAW (smooth and white). Polovina testiranih izolata je eksprimirala RDAR morfotip. Izolati koji su eksprimirali karakterističan morfotip na ovoj temperaturi su formirali na vazduh tečnost međufazi isti tip pelikule.Uporednom analizom rezultata primenjenih skrining testova za utvrđivanje sposobnosti formiranja biofilma pri temperaturi inkubiranja od 25°C ustanovljena je korelacija između pojave određenog morfotipa na Kongo crvenom agaru i sposobnosti formiranja biofilma u kristal violet i pelikula testu. Međutim, sa povećanjem temperature inkubiranja na 37oC, ova korelacija nije ustanovljena, sa izuzetkom izolata SE8.Svi testirani izolati su pokazali sposobnost adherencije na površinu stakla i nerđajućeg čelika, ali u različitoj meri. Na sposobnost adherencije povoljniji uticaj je imala temperatura inkubiranja od 25°C (p<0,05), sa izuzetkom izolata SE3 (p>0,05). Između stepena adherencije izolata na površine stakla i nerđajućeg čelika nisu ustanovljene statistički značajne razlike (p>0,05).Praćenjem stope preživljavanja tokom 28 dana u uslovima isušivanja evidentirana je znatno veća stopa preživljavanja ćelija izolata RDAR morfotipa u odnosu na stopu preživljavanja ćelija BDAR morfotipa (p<0,05). Praćenjem stope preživljavanja tokom 90 dana u uslovima povremene dostupnosti hranljivih materija, zabeležena je veća stopa preživljavanja u odnosu na stopu preživljavanja u uslovima isušivanja. U uslovima povremene dostupnosti hranljivih materija nakon devedeset dana ispitivanja kod obe grupe izolata procenat vijabilnih ćelija je iznosio više od 50%.Primenjenim mikroskopskim tehnikama (CLSM i SEM) omogućena je detaljna vizualizacija formiranih biofilmova. Na modelu izolata SERDAR morfotipa, ustanovljeno je da se formiranje biofilma pod primenjenim eksperimentalnim uslovima, odvija u tri faze: 1) inicijalna adhezija za površinu i formiranje manjih ćelijskih agregata (24h); 2) formiranje većih ćelijskih agregata uz produkciju EPS (48h); 3) sazrevanje biofilma uz značajnu produkciju EPS što omogućava formiranje stabilne trodimenzionalne strukture biofilma (96h).Nasuprot karakteristikama koje bakterije pokazuju tokom rasta u medijumima koji obiluju hranljivim materijama, bakterije u biofilmovima pokazuju drugačije osobine u pogledu ekspresije gena i karakteristika rasta. Zahvaljujući ovim razlikama, bakterije u biofilmovima pokazuju povećanu rezistenciju na antibiotike i dezinficijense, zbog čega se konstantno razvijaju nove kontrolne strategije u cilju iznalaženja potencijalnih bioloških rešenja koja pored različitih enzima, faga, antimikrobnih jedinjenja proizvedenih od strane mikroorganizama uključuju i antimkrobna jedinjenja biljnog porekla kao što su biljni ekstrakti, etarska ulja i različiti začini. Stoga, je u okviru drugog segmenta ovog istraživanja ispitivan hemijski sastav etarskih ulja, antimikrobni efekat etarskih ulja (O. heracleoticum , O. vulgare , Th. vulgaris i Th. serpyllum) i pojedinačnih komponenti etarskog ulja (karvakrola i timola) na bujonske kulture testiranih sojeva Salmonella Enteritidis kao i uticaj odabranih koncentracija etarskih ulja na inicijalnu adheziju i već formirani biofilm odabranih sojeva Salmonella Enteritidis.Etarska ulja je karakterisao visok zbirni udeo glavnih fenolnih komponenti karvakrola i timola: O. heracleoticum (71,6%), O. vulgare (63,6%), Th. vulgaris (59,77%), Th. serpyllum (40,04%). Etarska ulja su ispoljila antimikrobni efekat sledećim redosledom: O. heracleoticum >O. vugare =Th. vulgaris >Th. serpyllum. Antimikrobni efekat etarskih ulja je bio direktno srazmeran zbiru fenolnih komponenti (karvakrola i timola) u etarskom ulju. U odgovoru na tretman etarskim uljima između izolata S. Enteritidis nisu ustanovljene razlike.Etarska ulja, karvakrol i timol su pokazali inhibitorni efekat na inicijalnu adheziju i posledično na formiranje biofilma testiranih izolata S. Enteritidis na dozno zavisan način. Upoređivanjem uticaja etarskih ulja na inhibiciju inicijalne adhezije i metabolitičke aktivnosti ćelija između izolata RDAR i BDAR morfotipa ustanovljene razlike nisu bile statistički značajne (p>0,05).Ispitivanjem uticaja etarskih ulja, karvakrola i timola na ukupnu biomasu biofilma i metabolitičku aktivnost ćelija dokazano je da etarska ulja u primenjenim koncentracijama ispoljavaju uticaj na redukciju ukupne biomase formiranog biofilma i metaboličke aktivnosti bakterijskih ćelija na dozno zavisan način u funkciji vremena. Znatno veća efikasnost primenjenih tretmana je pokazana u slučaju njihove primene na biofilmove formirane od strane izolata BDAR morfotipa (p<0,05).
Knowledge and understanding ability of the pathogenic bacteria that adhere to surface and form biofilm, as well as their relationship between these abilities and factors that stimulate or inhibit biofilm development, are essential to develop strategies for their prevention and elimination.Considering also the fact that Salmonella enterica serotype Enteritidis has been epidemiologically the most frequently found serotype, the aims of this study were to evaluate: biofilm forming ability of several Salmonella Enteritidis strains isolated from clinical material, feed and selected control strain, their ability to adhere to glass and stainless steel surfaces, survival of selected biofilm-producing strains, as well as the possibility of applying confocal laser scanning (CLSM) and scanning electron microscopy (SEM) for visualization of biofilm three-dimensional structure.Determination of colony morphotype on Congo red agar at incubation temperature of 25°C revealed that among all tested isolates three morphotypes were detected: RDAR (red, dry and rough), BDAR (brown dry and rough) and SAW (smooth and white). Half of all tested isolates expressed RDAR morphotype. All isolates that expressed specific morphotype at this incubation temperature also formed the corresponding type of pellicle at air-liquid interface.Comparing the results of the applied assays was ascertained the correlation between specific morphotype on Congo red agar and biofilm forming ability in Cristal violet and pellicle tests, at incubation temperature of 25ºC. In the case of assays conducted at 37°C, this correlation was not established, except for the isolate SE8.All tested isolates showed varying degree of the ability to adhere to glass and stainless steel surfaces. Incubation temperature of 25ºC had more favorable effect on the adherence, with the exception of isolate SE3 (p>0.05). There were no statistically significant differences between adherence ability of all isolates to glass and stainless steel surfaces (p>0.05).Accompaniment of the survival rate during 28 days in the conditions of desiccation, the significantly higher survival rate was obtained for RDAR than BDAR morphotype isolates (p<0.05). Accompaniment of the survival rate during 90 days in the conditions of occasional availability of nutrients, it was detected the higher survival rate than in condition of desiccation. Under these conditions, after 90 days, there were more than 50% of viable cells among both groups of isolates.Applied microscopic techniques (CLSM and SEM) provided detailed visualization of formed biofilms. On model of SERDAR morphotype isolate, it was established that biofilm formation under this experimental conditions has three phases: 1) initial adhesion to the surface and formation of small cell aggregates (24h); 2) formation of large cell aggregates followed with production of extracellular polymer substance (EPS) (48h); 3) maturation of biofilm followed with significant EPS production, which allows formation of stabile three dimensional structure of the biofilm (96h).Contrary to characteristics that bacteria expressed during their growth in the nutrient media, bacteria in biofilms show different properties in terms of genes expression and growth characteristics. Due to these differences, bacteria in biofilms showed higher resistance to antibiotics and disinfectants. For these reasons are being constantly developed new potential biological control strategies that aim at finding the potential biological solutions that besides different enzymes, phages, antimicrobial compounds produced by microorganisms, also include antimicrobial compounds of plant origin, such as extracts, essential oils and different spices.Therefore, the other segment of this research was investigation of the chemical composition and antimicrobial properties of different essential oils (O. heracleoticum, O. vulgare, Th. vulgaris and Th. serpyllum) and their components (carvacrol and thymol), against broth cultures of Salmonella Enteritidis. Also, selected concentrations of essential oils were tested against initial adhesion and preformed biofilm of selected Salmonella Enteritidis isolates.Essential oils were characterized by high amount of phenol compounds carvacrol and thymol: O. heracleoticum (71.6%), O. vulgare (63.6%), Th. vulgaris (59.77%) and Th. serpyllum (40.04%). Essential oils showed antimicrobial potential as follows: O. heracleoticum > O. vugare = Th. vulgaris > Th. serpyllum. Antimicrobial effect was directly proportional to the total content of phenolic components (carvacrol and thymol) in essential oil. Between responses of different S. Enteritidis isolates to essential oil treatment, there was no significant difference.Essential oils, carvacrol and thymol demonstrated inhibitory effect on initial adhesion and consequently, on biofilm formation of S. Enteritidis isolates, in a dose-dependent manner. Comparing influence of essential oil on the inhibition of initial cell adhesion and metabolic activity of cells RDAR and BDAR morphotype, no statistically significant differences were established (p>0.05).Examination of the influence of essential oils, carvacrol and thymol on total biomass of preformed biofilms and metabolic activity of cells, it was revealed that essential oils in applied concentrations cause reduction of total biomass of preformed biofilm and metabolic activity of bacterial cells in a time and dose dependent manner. Applied treatments demonstrated significantly higher efficiency on BDAR morphotype biofilms (p<0.05).

Dans la nature, les micro-organismes sont organisés en communautés agrégées dénommées biofilms, particulièrement adaptées à la survie en milieu hostile. Les difficultés pour prévenir la formation ou éliminer des biofilms matures par des stratégies conventionnelles ont encouragé le développement de nouvelles approches inspirées des mécanismes de compétition entre différents micro-organismes au sein de biofilms naturels. Au cours de ce travail, nous nous sommes intéressés à l'effet anti-biofilm de bactéries bénéfiques appartenant aux genres Lactobacillus et Bifidobacterium. Dans un premier temps, nous avons testé l'effet anti-biofilm de surnageants neutralisés vis-à-vis de deux pathogènes Klebsiella pneumoniae et Staphylococcus epidermidis dans un modèle expérimental statique. Si les extraits des quelques souches de Bifidobacterium testées stimulaient la formation de biofilm par K. pneumoniae sur surface abiotique, la majorité de ceux des 140 souches de Lactobacillus exerçait un effet inhibiteur et nous avons retenu une des souches dont le surnageant de culture entraînait une inhibition majeure (70%), Lactobacillus plantarum CIRM653. Cet extrait s'est également avéré capable de disperser des biofilms préformés à K. pneumoniae sur surface abiotique mais aussi d’inhiber la formation de biofilms sur surface biotique, et ce indépendamment d’un effet bactéricide. La formation de biofilms mixtes formés par L. plantarum et K. pneumoniae dans des modèles expérimentaux cinétiques a permis, comparativement à l'observation de biofilms mono-espèce à K. pneumoniae, de mettre en évidence des défauts de structuration du biofilm associés à une diminution de la biomasse de K. pneumoniae et une augmentation de celle de L. plantarum. Grâce à une approche transcriptionnelle ciblée, nous avons montré que L. plantarum induisait, par le biais de son surnageant, des modifications de l’expression de gènes impliqués dans la formation de biofilm chez K. pneumoniae. Quatre gènes impliqués dans le quorum-sensing (opérons lsr) étaient sous-exprimés et trois gènes de structure du pilus de type 3 étaient sur-exprimés. L'augmentation de la production de pili de type 3 fonctionnels a été validée par Western-blot et des tests d’hémagglutination. Cette surexpression est probablement responsable du niveau élevé des capacités d’adhésion sur surface abiotique d'agrégats de K. pneumoniae issus de la dispersion induite par L. plantarum.Le comportement des deux souches a également été testé in vivo, dans un modèle murin de colonisation intestinale par K. pneumoniae avec administration orale quotidienne de L. plantarum. Le dénombrement du pathogène dans les selles des animaux a montré qu'en présence de L. plantarum, K. pneumoniae maintient des niveaux de colonisation élevés, contrairement au contrôle (sans Lactobacillus) où une diminution graduelle est observée.Enfin, nous avons initié le développement d'un modèle expérimental tripartite permettant d'associer les deux partenaires bactériens avec des cellules épithéliales dans un système en flux continu. La réponse spécifique des cellules eucaryotes a également été abordée : nous avons pu mettre en évidence que L. plantarum exerçait un effet inhibiteur vis-à-vis de la réponse inflammatoire épithéliale pulmonaire induite par K. pneumoniae. En conclusion, la description d'une activité anti-biofilm in vitro ne serait pas synonyme d'une réduction in vivo de la colonisation de surfaces biotiques, mais à une plus grande capacité de dissémination. Ces observations démontrent l’importance d’une expertise précise de l’action des bactéries bénéfiques et de la maitrise du ratio bénéfice-risque pour leur utilisation
In the natural environment microorganisms are organized in aggregated communities called biofilms, which are particularly adapted to the survival in harsh conditions. The difficulties to prevent the formation or elimination of mature biofilms by conventional strategies have encouraged the development of new approaches inspired by competition mechanisms occurring between microorganisms within natural biofilms.In this work, we looked for anti-biofilm effects of beneficial bacteria belonging to Lactobacillus and Bifidobacterium genus. We first tested the anti-biofilm effect of neutralized supernatants against both pathogens Klebsiella pneumoniae and Staphylococcus epidermidis in a static experimental model. The few Bifidobacterium extracts tested led to an increase in biofilm formation by K. pneumoniae on abiotic surface, whereas the majority of the 140 strains of Lactobacillus exerted an inhibitory effect. Lactobacillus plantarum CIRM653 was selected for further experiments because its culture supernatant displayed major inhibition (70%). This extract was also capable of dispersing preformed biofilms of K. pneumoniae on abiotic surface, but also able to inhibit biofilm formation on biotic surface, independently of a bactericidal effect. The formation of mixed biofilm containing L. plantarum and K. pneumoniae in kinetic experimental models highlighted the biofilm structure defects associated with a decrease of K. pneumoniae biomass and an increase of that of L. plantarum, compared to a monospecies K. pneumoniae biofilm. Targeted transcriptional approach was used to assess changes in the expression of genes involved in biofilm formation by K. pneumoniae after contact with L. plantarum supernatant. Four genes involved in quorum-sensing (operons lsr) were under-expressed and three type 3 pili structural genes were over-expressed. The increase of functional surface located type 3 pili was validated by Western blotting and hemagglutination tests. This overexpression was probably responsible for the observed high level of adhesion capacity to abiotic surfaces of K. pneumoniae aggregates recovered after dispersion induced by L. plantarum.The behavior of the two strains was also tested in vivo in a K. pneumoniae murine intestinal colonization model with daily oral administration of L. plantarum. Viable cells counting of the pathogen in the animals’ feces showed that K. pneumoniae maintained high levels of colonization in the presence of L. plantarum, unlike the control (without Lactobacillus) where a gradual decrease was observed.Finally, we initiated the development of a tripartite experimental model allowing the combination of the two bacterial partners with epithelial cells in a continuous flow system. In parallel, the specific response of eukaryotic cells to these bacteria was addressed: L. plantarum exerted an inhibitory effect on the pulmonary epithelial inflammatory response induced by K. pneumoniae.In conclusion, these results highlight the discrepancy between in vitro anti-biofilm activity of L. plantarum and its in vivo behavior leading to increased dissemination of the pathogen. Substantial expertise of beneficial bacteria is therefore necessary to fully assess their benefit-risk ratio

Le Quorum sensing (QS) est un système de communication bactérienne capable de réguler divers processus cellulaires qui dépendent de la densité de la population microbienne. Chez les bactéries à Gram négatif, cela se produit par production de molécules de signalisation auto-inductrices (AI), les acyl homosérine lactones (AHL). La libération d´AHL à l'extérieur de la cellule est détectée par la population bactérienne provoquant en réponse la régulation de l'expression de certains gènes (régulon QS).Notre laboratoire a étudié et identifié un système QS fonctionnel dans la souche Acidithiobacillus ferrooxidans ATCC 23270T. En outre, nous avons montré que des analogues synthétiques d´AHL modulent l´adhésion d´At. ferrooxidansT sur un substrat minéral, tels des coupons de soufre.Dans ce projet de recherche, nous nous proposons d'identifier les gènes qui sont régulés par le QS chez At. ferrooxidansT, en particulier ceux impliqués dans la biogénèse du biofilm. Notre hypothèse est que des analogues synthétiques d’AHL induisent le système QS. Ainsi, nous nous proposons de moduler l´adhésion sur substrat minéral grâce à l'utilisation de ces molécules. L'utilisation de ces AHL permettra de caractériser le régulon QS dans cette souche bactérienne.L'identification d'analogues synthétiques d´AHL qui favorisent l'adhésion à des coupons de soufre nous a permis d'étudier le transcriptome d´At. ferrooxidansT dans des conditions où le régulon QS est stimulé. Puces à ADN d´At. ferrooxidansT avec/sans ces analogues synthétique d´AHLs nous a permis de caractériser le régulon QS et les gènes impliqués dans la biogénèse du biofilm dans les conditions utilisées
Quorum sensing (QS) is a bacterial communication system capable of controlling several cellular processes dependent on the density of the microbial population. In Gram-negative bacteria, it occurs mainly through the production by bacteria of small diffusible signaling molecules, termed autoinducers (AI), of the acyl homoserine lactones type (AHLs). The release of AHLs outside the cell is detected by the bacterial population generating the regulation of the expression of several genes (regulon QS).Our laboratory has studied and identified a functional QS system in the Acidithiobacillus ferrooxidans ATCC 23270T type strain. Besides, by using synthetic analogs of AHLs, we have shown that AHL-type QS molecule analogs modulate adhesion of At. ferrooxidansT to minerals, such as sulfur coupons. In this research, we propose to identify the genes that are regulated by QS in At. ferrooxidansT, particularly those that are associated with biofilm formation. For this, we propose to modulate the adhesion of At. ferrooxidansT to mineral substrate through the use of a synthetic AHL analog. Our working hypothesis postulates that AHLs molecules induce the QS system, and that their use will allow the characterization of the QS regulon of this bacterial strain by transcriptomic analysis.The identification of synthetic AHLs improving adherence of At. ferrooxidansT on sulfur coupons allowed us to study the transcriptome of this organism in conditions in which QS regulon is stimulated. DNA microarrays of At. ferrooxidansT with/without one of these AHLs synthetic analogues allowed us to identify the QS regulon and to determine genes involved in biofilm formation

La bactérie aquatique Shewanella oneidensis est capable, en condition statique et en présence d'oxygène, de former un biofilm à l'interface air-liquide, appelé pellicule. Mon travail a porté sur la biogenèse de la pellicule.Il a été montré dans le groupe que le régulateur de réponse du système chimiotactique, la protéine CheY3, était impliqué dans la biogenèse de la pellicule. Cette protéine est essentielle dans les étapes précoces et tardives de sa formation alors que son partenaire habituel, CheA3, semble ne jouer un rôle que dans les étapes tardives. Mon travail s'est focalisé sur la recherche de partenaires de CheY3.J'ai introduit une banque d'ADN génomique de S. oneidensis dans la souche ΔcheY3 et j'ai cherché des gènes dont la surexpression permettait de restaurer la formation de la pellicule. Cette approche a révélé deux gènes pdgA et pdgB. J'ai montré que les protéines PdgA et PdgB étaient capables de synthétiser du di-GMPc, suggérant que ce messager secondaire est impliqué dans la biogenèse de la pellicule. L'hydrolyse du di-GMPc par des enzymes dédiées empêche en effet sa formation.J'ai montré que l'opéron mxd, contrôlant la synthèse d'exopolysaccharides dans les biofilms de surface, était impliqué dans la formation de la pellicule. La première protéine codée par cet opéron, MxdA, est capable de lier le di-GMPc. Des expériences de pontage chimique et de double hybride ont révélé que MxdA, CheY3, PdgA et PdgB, formaient un réseau de régulation gouvernant la biogenèse de la pellicule.J'ai montré que les systèmes à deux composants BarA/UvrY et ArcS/ArcA contrôlant la transcription de l'opéron mxd sont aussi impliqués dans la formation du biofilm flottant
The aquatic bacterium Shewanella oneidensis is able to form, under static conditions and in the presence of oxygen, a biofilm at the air-liquid interface, called pellicle. My work was focused on the biogenesis of this pellicle.It was previously shown in the team that, surprisingly, the CheY3 protein, the response regulator of the chemotactic regulatory system, is involved in the biogenesis of the pellicle. This protein was shown to be essential both in early and late steps of pellicle formation whereas its usual partner, the kinase CheA3, seems to play a role in the late steps only. I was therefore looked for the partners of the CheY3 protein for pellicle formation.For this purpose, I have introduced a multi-copy genomic library in the ΔcheY3 strain and searched for genes whose overexpression allowed pellicle restoration. Strikingly, this approach revealed two genes pdgA and pdgB. Interestingly, we showed that PdgA and PdgB proteins are able to synthesize c-di-GMP, suggesting a role for this second messenger in pellicle biogenesis. Indeed, c-di-GMP hydrolysis by dedicated enzymes blocks pellicle formation.We also showed that the mxd operon, controlling the exopolysaccharides synthesis in biofilm associated with a solid surface, is also involved in pellicle formation. Moreover, the first protein encoded by this operon, MxdA, is able to bind c-di-GMP. Cross-linking and bacterial two-hybrid experiments revealed that MxdA, CheY3, PdgA and PdgB, form a complex regulatory pathway governing the biogenesis of the pellicle.Finally, we have shown that the two-component systems BarA/UvrY and ArcS/ArcA, controlling the mxd transcription, are also involved in pellicle formation

L’écosystème buccal est un environnement complexe dans lequel cohabitent plus de 700 espèces de bactéries différentes. Un déséquilibre au sein de ce biofilm est à l’origine des principales maladies de la cavité buccale : les maladies carieuses et parodontales.Pendant plusieurs années, l’étude de l’écosystème buccal s’est faite par une approche réductionniste : les microbiologistes étudiaient les espèces bactériennes individuellement. Cette stratégie a permis d’examiner et de comprendre tous les différents composants de cet écosystème, sans pour autant pouvoir appliquer les conclusions de ces études au biofilm buccal dans son ensemble. En effet, les bactéries ne se comportent pas de la même façon lorsqu’elles sont à l’état planctonique, ou lorsqu’elles sont organisées en biofilm.La flore microbienne buccale est reconnue comme étant l’une des plus complexes dans le corps humain. La multitude d’espèces en présence complique l’étude de ce biofilm. En effet, sa reproduction in vitro est rendue difficile par la complexité des relations entre chacune des espèces. De plus, son recueil, et ses décomptes qualitatif et quantitatif restent très délicats.Dans la littérature sont décrits plusieurs modèles de biofilm.Les modèles pluri-espèces dynamiques in vitro ont l’avantage de se rapprocher des conditions retrouvées in vivo, permettant un certain flux de milieu, le contrôle de paramètres tels que le pH et la température, ainsi que l’élimination des déchets produits.Cependant, ces modèles restent très onéreux et difficiles de mise en place, ce qui complique l’étude du biofilm buccal. Egalement, l’identification des bactéries mises en présence reste un sujet d’étude délicat : en effet, les méthodes traditionnelles de culture montrent des limites, et ne permettent pas une analyse quantitative des résultats, essentielle à la compréhension des phénomènes mis en jeu dans cet écosystème.Le but de notre travail ici est la mise en place de modèle de biofilm pluri-espèces dynamique, fiable et reproductible, facile à mettre en place et moins onéreux que ceux décrits dans la littérature. Ce modèle de biofilm doit permettre l’étude de l’influence de variations de conditions environnementales sur ce dernier, ainsi que celle de candidats probiotiques ayant déjà prouvé leur efficacité sur des supports in vitro statiques. Enfin, toujours dans une optique de simplification, les différentes méthodes d’identification des biofilms formés sont comparées (méthodes de culture traditionnelle, PCR conventionnelle, spectrométrie de masse MALDI-TOF, et qPCR), afin d’établir un protocole d’identification reproductible permettant une analyse qualitative et quantitative des résultats
The oral ecosystem presents a great complexity since it can harbor more than 700 different bacterial species. Most of them are organized in a biofilm on both the dental and the mucosal surfaces. Studying this complex environment is of utmost importance because a rupture in its stability can lead to the preeminence of pathogenic microorganisms, causing dental decay, gingivitis and periodontitis.For many years, the study of the oral ecosystem was conducted throught a reductionist approach: microbiologists studies bacterial strains individually. This strategy allowed the understanding of all different components of this ecosystem, but lacked the transposition of its conclusions to the study of a whole complex oral biofilm. As a matter of fact, bacteria don’t behave the same way in a planktonic state or when they are organized in a biofilm.The oral microflora is known to be one of the most complex floras hosted by the human body. The multitude of strains hardens its study. Indeed, its in vitro reproduction is made as complex as the different interactions occurring between each strain. Moreover, harvesting and quantitative and qualitative analysis of such biofilms remain very delicate procedures.Several biofilm models have been described in the literature. In vitro dynamic multispecies models share the same asset: to closely mimic in vivo conditions. They allow a medium flow, and parametrical controls such as pH, temperature; and waste removal. However, those models are very expensive and difficult to master. Also, bacterial identification is still a tough matter : traditional culture methods have shown their limits, and don’t allow a quantitative analysis, which is essential to understand the phenomenons occurring in this ecosystem.The aim of our work was to set up a dynamic multispecies oral biofilm, both reliable and reproducible, easy to set up and less expensive than those previously described in the literature. This model shall allow the study of environmental conditions variations and the efficiency of probiotic candidates that already showed their efficacy on static supports.Lastly, we compared different biofilm identification methods, traditional culture, conventional PCR, MALDI-TOF mass spectrometry, and quantitative PCR, in order to establish a reproducible identification protocol allowing both quantitative and qualitative analysis

Les infections ostéo-articulaires requièrent souvent un acte chirurgical couplé à une antibiothérapie prolongée, en lien avec la capacité des bactéries en cause à former des biofilms sur les prothèses. La présence d’antibiotiques à des concentrations sub-inhibitrices (sub-CMI) peut stimuler la capacité des bactéries à former des biofilms, ce qui complexifie la problématique. Notre étude avait pour but de caractériser in vitro le comportement biofilm d’une souche clinique de Staphylococcus aureus issue d’une infection ostéo-articulaire en présence de 12 antibiotiques préconisés dans le traitement des infections à staphylocoques, à différentes concentrations (dont des concentrations sub-CMI).Un dénombrement des bactéries viables a été effectué à partir de biofilms formés en présence de chacun des 12 antibiotiques à différentes concentrations (incluant des concentrations sub-CMI), ainsi que dans les suspensions présentes autour de ces mêmes biofilms. La présence de 7 d'entre eux (ceftaroline, daptomycine, gentamicine, fosfomycine, ofloxacine, rifampicine et vancomycine) entraînait une inhibition de la formation de biofilm à des concentrations inférieures ou égales aux concentrations critiques. L'action de la fosfomycine s'étendait même à des concentrations sub-CMI. Seules la daptomycine et la gentamicine étaient capables d’agir à la fois sur les bactéries sessiles et sur les bactéries non-adhérentes en suspension.Quant aux biofilms mâtures de cette même souche préalablement formés en absence d'antibiotiques, seules des concentrations 800 à 51200 fois supérieures à la CMI -concentrations largement incompatibles avec une utilisation thérapeutique - entraînaient leur éradication.Dans la deuxième partie de ce travail, nous avons concentré nos efforts sur l'action de la fosfomycine, choisie en fonction de son effet sur la formation de biofilm et de son intérêt thérapeutique. Une analyse transcriptomique des cellules présentes dans les biofilms formés en présence de fosfomycine et de cellules issues de biofilms matures traités ultérieurement à la fosfomycine a montré que la présence de cet antibiotique induisait majoritairement une sous expression de gènes codant des protéines impliquées dans le métabolisme et le transport des nucléotides, des acides aminés et des carbohydrates. Des gènes codant des adhésines et des protéines impliquées dans la synthèse de la capsule (ScdA) étaient également sous-exprimés. De manière moins importante, l'expression de gènes codant des molécules impliquées dans la synthèse du peptidoglycane (MGT et MurA) et des autolysines était également diminuée. Le ralentissement métabolique et les modifications induites au niveau des membranes par la présence de l'antibiotique seraient responsables du changement des capacités d'adhésion de la bactérie.L'impact de la fosfomycine à une concentration sub-CMI sur les caractéristiques des bactéries viables isolées dans la suspension autour des biofilms a également été déterminé. De façon surprenante, ces bactéries montrent une capacité accrue à former du biofilm par rapport à celles issues de l'environnement d'un biofilm non soumis à l'action de l'antibiotique, en lien probablement avec un accroissement d'épaisseur de leur couche de peptidoglycane.En conclusion, ces données obtenues in vitro devront être confirmées dans des modèles d'infections expérimentales in vivo. Malgré tout, elles soulignent la pertinence de l’utilisation de la fosfomycine dans la prévention des infections ostéo-articulaires liées à S. aureus, à condition d'éradiquer en parallèle les formes non adhérentes
Osteoarticular infections (OAI) often require a surgical procedure with prosthesis removal followed by long-term complex antibiotherapy. The ability of Staphylococcus aureus to adhere and produce biofilm on the surface of implanted material contributes to treatment failures and microbiological relapses. In addition, biofilm formation can be induced by some antibiotics at sub-minimal inhibitory concentrations (sub-MICs). The present study characterizes in vitro the effects of 12 antibiotics on biofilm formed by a strain of methicillin-susceptible Staphylococcus aureus isolated from an osteo-articular infection.The influence of these antibiotics was assessed on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the biofilm biomass and in the suspensions (unattached cells) surrounding the biofilm. Biofilm formation was prevented in presence of ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin at the highest concentrations tested. Only fosfomycin showed inhibition properties also at sub-MICs. Unattached and sessile viable bacteria were undetectable to daptomycin and gentamicin at the highest concentrations tested.Determination of the minimum biofilm eradication concentrations (MBECs) indicated that in vitro eradication of 24h-old biofilms required concentrations at least 800 times higher than the planktonic MIC, concentrations obviously not compatibles with classical therapeutic doses.In the second part of this work, we focused our study on the action of fosfomycin, because of its effect on biofilm formation and its therapeutic interest. A transcriptome analysis was performed with sessile cells from both biofilm formed in the presence of sub-MIC of fosfomycin and cells from pre-formed 24h-old biofilm treated by fosfomycin at sub-MBEC. Fosfomycin induced mostly down regulation of genes assigned to nucleotide, amino acid and carbohydrate transport and metabolism. Adhesins and capsular biosynthesis proteins (ScdA) encoding genes were also down regulated. To a lesser extent, peptidoglycan biosynthesis proteins (MGT and MurA) and autolysins encoding genes were found down regulated. Metabolic slowdown and cell membrane modifications induced by fosfomycin are likely to be responsible for the impairment of bacterial adhesion capacity.The action of fosfomycin at sub-MIC on unattached cells surrounding biofilm was also analyzed. Surprisingly, they displayed higher capacity to form new biofilm than their counterparts obtained without fosfomycin, probably associated with their large peptidoglycan layer.In conclusion, these data underline the relevance of the use of fosfomycine in preventing osteo-articular infections due to S. aureus and should be assessed in in vivo experiments. However, simultaneous eradication of unattached cells should also be considered due to the high capacities of these cells to disseminate and establish new biofilm, a real risk of treatment failure

Orientadores: Karina Cogo Müller, Francisco Carlos Groppo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-25T11:18:29Z (GMT). No. of bitstreams: 1 Graziano_TalitaSignoreti_M.pdf: 1603562 bytes, checksum: 9522f75e1f8c1147c84c0fde100549ed (MD5) Previous issue date: 2014
Resumo: As estatinas são um grupo de fármacos que atuam como inibidores competitivos da enzima 3-Hidroxi-3-MetilGlutaril Coenzima-A Redutase (HMG-CoA redutase). Além de atuarem como importantes agentes hipolipemiantes, também apresentam outros efeitos, chamados de pleiotrópicos. Diversos estudos têm explorado um possível efeito protetor das estatinas atuando na redução na morbidade e mortalidade de várias doenças infecciosas. A atividade antimicrobiana das estatinas tem sido reportada por estudos in vivo e in vitro. O objetivo desse estudo foi avaliar os efeitos das estatinas sobre o crescimento e viabilidade de bactérias aeróbias patogênicas, e o efeito da sinvastatina sobre o biofilme de Staphylococcus aureus. Culturas das espécies de Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli e Enterococcus faecalis foram avaliadas na forma planctônica quanto à sensibilidade à atorvastatina, pravastatina e sinvastatina, através do teste de Concentração Inibitória Mínima (CIM). Além disso, diante da atividade apresentada pela sinvastatina contra S. aureus, foi determinada a ação dessa droga sobre a viabilidade celular através dos testes de Time-kill e Efeito pós-antibiótico (EPA). Também foi verificado um possível efeito sinérgico entre a sinvastatina e vancomicina. Por fim, a ação da sinvastatina foi avaliada contra biofilmes de S. aureus. Os valores de CIM da sinvastatina para o microrganismo S. aureus foram: 15,65 µg/ml (ATCC 29213) e 31,25 µg/ml (ATCC 33591, 43300, 14458 e 6538). A sinvastatina apresentou um perfil bacteriostático, e na concentração de 4xCIM seu EPA foi similar ao da vancomicina. Não foi encontrado nenhum tipo de interação entre a associação de sinvastatina e vancomicina. Entretanto, a sinvastatina foi capaz de reduzir a formação do biofilme nas concentrações entre 1/8CIM à 4xCIM. Além disso, na concentração 4xMIC foi capaz de diminuir a viabilidade, biomassa e a produção de polissacarídeos extracelulares e aumentar a produção de polissacarídeos intracelulares de biofilmes maduros de S. aureus. A produção de proteínas pelo biofilme não foi alterada. Em conclusão, os resultados encontrados mostram que a sinvastatina possui um grande potencial a ser explorado, principalmente em relação ao descobrimento de novos antimicrobianos
Abstract: Statins are drugs that competitively inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA). Besides their important lipid-lowering action, they also are pleiotropic agents. Several studies have explored a possible protective effect of statins to reduce the morbidity and mortality of various infectious diseases. The antimicrobial activity of statins has been reported by in vivo and in vitro studies. The aim of this study was to evaluate the effects of statins on the growth, viability and biofilm formation of pathogenic aerobic bacteria. The Minimum Inhibitory Concentrations (MIC) of atorvastatin, pravastatin and simvastatin against planktonic cells of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis strains were obtained. Since simvastatin showed activity against S. aureus, its effects on cell viability were evaluated in a time-kill and post-antibiotic effect (PAE) assays. A possible synergistic effect between simvastatin and vancomycin was also assessed. In addition, the effect of simvastatin against biofilms of S. aureus was tested. The MIC values of simvastatin for S. aureus were: 15.65 µg/ml (ATCC 29213) and 31.25 µg/ml (ATCC 33591, 43300, 14458 and 6538). Simvastatin showed a bacteriostatic profile, and in a 4x>MIC concentration the PAE was similar to vancomycin. No synergistic effect was found between simvastatin and vancomycin. Simvastatin was able to reduce the formation of biofilms in concentrations ranging from 1/8MIC to 4xMIC. In addition, the 4xMIC was able to decrease the viability, biomass and production of extracellular polysaccharides and increase the production of intracellular polysaccharides on mature biofilm of S. aureus. The protein production on biofilm was not altered in the presence of simvastatin . In conclusion, our results showed that simvastatin has a great potential to be explored, especially in relation to the development new antimicrobial agents
Mestrado
Farmacologia, Anestesiologia e Terapeutica
Mestra em Odontologia

The Problem: Biofilms are a community of bacteria that cause infections which are resistant to the immune system and antimicrobial treatments, posing a significant threat for patients with implantable and indwelling medical devices. Purpose: The purpose of this research is to effectively treat biofilms utilizing electric currents assisted by antibiotics. Method: Evaluated the impact of direct electric current with or without vancomycin against Staphylococcus epidermidis biofilms. Results: (1) Electric current reduced the S. epidermidis biofilm and (2) increased the effectiveness of vancomycin. (3) Older biofilms had an increased resistance to vancomycin treatments. (4) Higher electric current intensities and (5) longer duration treatments were more effective against biofilms. Conclusion: Electric current increased the effectiveness of vancomycin against S. epidermidis biofilms.

The development of biofilms on mild and stainless steel surfaces in pure and mixed batch cultures of the bacterial species Pseudomonas fluorescens and Desulfovibrio desulfuricans and the role of these biofilms in corrosion of steel has been investigated. Early events leading to the formation of biofilms have been elucidated by studying the attachment of bacterial cells to steel using epifluorescence microscopy. To identify the nature of the bacterial surface components involved in the initial adhesion to mild steel, lectins, their sugar inhibitors and saccharolytic and proteolytic enzymes have been employed. Polyclonal antibodies have been raised against bacteriallipopolysaccharides (LPS) and their influence on bacterial adhesion assessed. LPS have been analysed chemically by gas-chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to determine their ~arbohydrate composition and fatty acid content. On the basis of the results obtained the ~nvolv~~~nt of glucose and N-acetylglucosamine, present in O-antigenic fractions of LPS, 10 the lOlnal attachment of the two bacterial species to mild steel is suggested. Both types of carbohydrates are likely to be involved in early attachment of Pseudomonas to mild steel, whereas only a polymeric fonn of N-acetylglucosamine seems to participate in adhesion of Desulfovibrio. The subsequent biofilm development on steel surfaces and their accompanying corrosion h~s been monitored by scanning electron microscopy (SEM). SEM studies reveal very different patterns of bacterial biofilms on mild and stainless steel and show varied degrees of corrosion occurring on these surfaces. Thin and patchy Pseudomonas biofilms are accompanied by little corrosion whilst thick. more continuous, Desul/ovibrio biofilms are associated with higher levels of corrosion. Energy dispersive X-ray analysis (BOAX) of corrosion products present on steel surfaces indicates ferrous sulphides as the major components in Desul/ovibrio biofilms. The corrosion of steel in bacterial cultures has also been investigated by kinetic polarisation measurements. The results obtained from cathodic and anodic polarisation curves, combined with SEM and EDAX analyses confmn the SEM observation. Stainless steel is not subjected to any great degree of fouling or corrosion under the chosen experimental conditions. The EPS associated with biofilms and released into the liquid phase of the culture media (free EPS) has been characterised. Proteins and carbohydrates in these polymers are detected colorimenically and by SDS-gel electrophoresis. Uronic acids, found in biofilmbound BPS. are not detected in free EPS. The GC-MS and GC-FIO analyses have aided in establishing types and quantities of neutral carbohydrates present in bacterial exopolymers and show that the neutral sugar composition of free and surface-associated BPS is not identical for a given bacterial culture. The biofilm-bound BPS are believed not to play a major role in corrosion of mild steel but to provide additional mechanisms in its facilitation. No correlation between levels of free BPS and corrosion of steel is found.

Oral bacteria are the main cause of common oral diseases such as caries, periodontal infection and root canal infections. Bacteria in nature survive predominantly as biofilms, which are complex microbial communities composed of populations of microorganisms adhered to living or non-living surfaces and embedded in a self-produced matrix of extracellular polymeric substance (EPS). Various biofilm models have been developed to simulate the real situation in nature. In this thesis, I studied a new in vitro biofilm model, and examined the antimicrobial efficacy of current as well as newly developed endodontic irrigants/protocols against planktonic and biofilm bacteria. Single and multispecies biofilms were grown on sterile hydroxyapatite and dentin discs coated with bovine dermal collagen Type I . Transmission electron microscopy (TEM) was used to examine the biofilm microorganisms. The antimicrobial effect of sodium hypochlorite (NaOCl), iodine potassium iodide (IPI), chlorhexidine (CHX) and a new disinfecting agent (QMiX- a mixture of CHX, EDTA, and a detergent) was evaluated. The antimicrobial strategies included in the studies were photoactivated disinfection (PAD) and its experimental modifications. Biofilms at different stages of maturation were exposed to various antibacterial agents, and the killing of biofilm bacteria was observed using viability staining and confocal laser scanning microscopy (CLSM). The new in vitro biofilm model had similarities to in vivo biofilms, as described in the literature. This biofilm model reached maturation between two and three weeks. Mature biofilms were less sensitive to disinfecting agents than young biofilms. The time required for the biofilms to become resistant to disinfecting agents (maturation) was not dependent on the source of biofilm bacteria or the type of disinfectant used. Modified photoactivated disinfection was up to almost twenty two times more effective in killing biofilm bacteria than conventional PAD and up to almost eight times more effective than the commonly used endodontic irrigants. A new endodontic irrigant, QMiX was more effective in killing planktonic and biofilm bacteria than 2% CHX, BioPure MTAD (a mixture of tetracycline isomer, an acid, and a detergent), and 1% and 2% NaOCl.The new biofilm model seems promising for testing and developing efficient methods to eradicate oral biofilm bacteria.

Bacterial biofilms are the cause of many diseases linked to persistent bacterial infection. When remaining in the root canal space following endodontic treatment, bacteria can lead to a secondary infection, resulting in treatment failure and the subsequent need for re-intervention. Currently, there are no methods in use to detect bacterial presence within the root canal space in a fast and reliable manner. Such methods would be especially beneficial in determining the end point of root canal treatments. Here, we make use of molecular fluorescent dyes and fluorescent microscopy/spectroscopy to optically detect remnant live bacteria within the root canal space. Calcein AM was evaluated as a fast acting vital cell stain, suitable for rapid staining of stressed and mature biofilms. Calcein AM staining produced fluorescence only when vital bacteria were present and showed significantly higher ratios for vital/non-vital bacteria as well as vital bacteria/ substrates when compared to other stains tested (p < 0.001). Detection was achieved with an ex-situ approach, where root canals were sampled with endodontic paper points for subsequent staining and analysis using a spectrometer coupled to a fluorescence microscope. Detection of vital cells in a stressed in-vitro endodontic biofilm was shown to be more sensitive than culturing: fluorescence detection was achieved for up to 40 seconds of 1% NaOCl exposure, compared to 15 seconds for culturing. The methodology was then applied to clinical trial involving 53 teeth and 114 roots. Detection immediately preobturation was achieved in 18.4% of roots and in 35.8% of teeth. The feasibility of remote in-situ spectral analysis was shown with preliminary studies using fluorescent beads as well as in-vitro studies using a 200 μm diameter probe on biofilms. Ratiometric analysis of live/dead stained nutritionally starved endodontic biofilms provided a simple alternative to conventional live/dead staining. Using software for spectral unmixing provided a method for fast and objective detection. The creation of a prototype, which couples a fluorescence spectrometer to a wide-field microscope, allowed for a successful patient trial, demonstrating the potential of this system to be miniaturised and commercialised for a chair-side use.

Initial work involved the isolation and characterization of bacteria found on the surfaces in a range of food processing environments. The attachment characteristics of three species of bacteria (S.liquefaciens, S.cohnii and P.fragi) isolated from the same production surface were examined. Mixed culture biofilm development was also modelled using these three organisms. The species were found to have differing attachment abilities and therefore cell surface characteristics such as outer membrane protein and lipopolysaccharide profiles, exopolysaccharide production, and cell surface hydrophobicity and charge were examined to explain these differences. All of these cell surface characteristics were subject to considerable variation in response to the environmental conditions. The outer membrane protein and lipopolysaccharide profiles did not show any relationship to attachment levels, and similarly exopolysaccharide production did not relate to the levels of attachment observed, although exopolysaccharide production was found to be particularly associated with attached cells. The attachment of S. cohnii and P jragi correlated to cell surface hydrophobicity, whilst the attachment of S.liquefaciens was dependent upon cell surface charge.

Campylobacter jejuni is the largest contributor to bacterial gastroenteritis in the developed world. In addition to intestinal infections, this organism is implicated in the autoimmune neurodegenerative condition known as Guillain–Barré syndrome. C. jejuni is a common commensal organism of warm-blooded animals and avians that is passed to humans through contaminated food products or water. Biofilms are a specialised method of growth seen in many microbes and provide a means for bacteria to remain viable in harsh environmental conditions. The biofilms of C. jejuni have been hypothesised to play an important role in the transmission of infection. Herein, we aimed to investigate factors involved in C. jejuni biofilm formation as well as detailing the development of novel applications to established techniques. The first chapter of this thesis aimed to evaluate the current understanding of biofilm formation in C. jejuni. We explored factors which play important roles in biofilm formation such as chemotaxis, glycosylation, microbial metabolism, quorum sensing and stress response regulators. These elements revealed that processes which are involved in the biofilm formation of C. jejuni are frequently at odds with those required for virulence and pathogenicity. This is contrary to biofilms of other organisms such a Pseudomonas aeruginosa which require biofilms for infection. We propose that biofilms of C. jejuni play a more important role in the transmission of infection rather than pathogenesis. We then present a novel improvement to an existing method for quantification of biofilm formation. Currently, methods for quantification of biofilm formation rely on the dissolution of stained biofilm formed in microtiter plates. Solubilisation of the stain provides spectroscopic data which allows for comparisons between the relative levels of biofilm formed. The solvents used in current assays are ineffective at solubilising formed biofilm and give inconsistent results. This second chapter details the development of an alternate solvent which was able to provide complete dissolution of stained biofilm and, thus, improved the accuracy and robustness of plate-based biofilm assays. This new solvent was effective against a range of bacterial species and provides a marked improvement to microtiter plate-based methods for assessing biofilm formation. The third chapter details a combinatorial approach to expression studies in C. jejuni biofilms focusing on the role of chemotaxis signal transduction. RNA sequencing was used to examine relative differences in transcript levels between planktonic C. jejuni and those contained in a biofilm. This was combined with proteomic data obtained using iTRAQ to provide a complete profile of differential gene expression. Differential expression was observed in approximately 600 genes, with 300 displaying upregulation during biofilm formation (e.g. iron metabolism, glycan production and cell division) and 300 showing downregulation (such as metabolism and amino acid metabolism). Large tracts of the chemotaxis pathway displayed downregulation, which we investigated further using mutant C. jejuni strains deficient in the key chemotactic proteins CheV and CheW. Both of these mutant strains exhibited a significant increase in the ability to form biofilm through increased establishment of microcolonies. The absence of these signal transduction proteins also had a marked effect on motility and autoagglutination. We hypothesized that the diminished motility of the mutant strains allows for an increase in autoagglutination and, thus, more efficient microcolony formation which presents a potential pathway for modulating biofilm formation in C. jejuni. The next chapter presents a role for N-linked protein glycosylation in C. jejuni biofilm formation. Phenotypic analysis of mutant strains deficient in key enzymes, PglB and PglF, shows that in the absence of protein glycosylation, there is a substantial increase in the ability for C. jejuni to form biofilms. This appeared to be a result of increased autoagglutination leading to an increase in microcolony formation, a mechanism similar to that observed in CheV and CheW. The architecture of the formed biofilms also appears to have been altered; SEM analysis of biofilms shows a filamentous appearance in mutant strains. This may present a further means for regulating biofilm formation in C. jejuni. The work contained in this thesis identified a number of important factors which play a role in the biofilm formation of C. jejuni. It also demonstrates that coupling transcriptomic and proteomic analysis can provide a comprehensive profile of differential gene expression during biofilm formation. Many of the approaches and techniques detailed are also novel applications which have seldom been applied to the field of biofilms. These insights aim to improve not only the understanding of bacterial biofilm formation as a whole, but also the methods in which we can study them.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
Full Text

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
Introdução: Um biofilme é uma comunidade estruturada de células bacterianas envolvidas numa matriz constituída por substâncias poliméricas e aderido a uma superfície sólida. Esta comunidade permite um modo protegido de crescimento que permite a sobrevivência dos constituintes celulares em ambientes hostis e fornece uma tolerância aumentada a agentes antimicrobianos. Objectivo: Tentar compreender a forma como os constituintes celulares se organizam e ter um melhor conhecimento das formas de resistência antimicrobiana características da organização em biofilmes, como também de rever os métodos actualmente usados para a desinfecção no tratamento endodôntico e abordando métodos alternativos ainda em estudo. Materiais e métodos: Para tal realizou-se uma pesquisa bibliográfica nos principais motores de busca: Pubmed, B-On, SciELO, Science Direct, como também no repositório da Universidade Fernando Pessoa e da Faculdade de Medicina Dentária do Porto utilizando as palavras-chave “Biofilms”, “Apical Periodontitis”, “Enterococcus faecalis” e “Biofilm Treatment “ que foram associadas de várias formas. Desta pesquisa efectuada, entre Junho de 2014 e Julho de 2014, foram escolhidos 117 artigos em Português e Inglês dos quais foram usados 89. Resultados: A forma como actualmente procedemos à desinfecção do sistema de canais radiculares, passando pela instrumentação mecânica e irrigação química não é totalmente satisfatória no que toca a uma total erradicação dos microorganismos devido a várias limitações como a complexidade anatómica dos canais e a ecologia presente no interior dos mesmos. Conclusões: De futuro, terão que ser desenvolvidas outras estratégias antimicrobianas para suplementar as existentes. Embora estas pareçam promissoras in vitro elas carecem de estudos in vivo, os quais serão necessários no futuro para ultrapassar as várias limitações presentes no sistema de canais radiculares. Introduction: A biofilm is a structured bacterial cell community enveloped in a matrix composed of polymeric substances and attached to a solid surface. This community allows for a protected way of growth that permits the survival of the cellular components in hostile environments and provides a higher tolerance to antimicrobial agents. Objective: Trying to understand the way cellular components are organized and have a better knowledge of the antimicrobial resistances that are characteristic of the way biofilms are structured, as well as to review the currently used methods for disinfection in an endodontic treatment and to address alternative methods still in study. Materials and Methods: To this end a bibliographic research was performed on the main search engines: Pubmed, B-On, SciELO, Science Direct, and also on Universidade Fernando Pessoa and Faculdade de Medicina Dentária do Porto’s repository using “Biofilms”, “Apical Periodontitis”, “Enterococcus faecalis” and “Biofilm Treatment“ as key-words that were associated in many forms. From this research performed between June 2014 and July 2014, were selected 117 articles in Portuguese and English and from those, 89 were used. Results: The way that we currently proceed regarding the disinfection of the root canal system using mechanical instrumentation and chemical irrigation is not fully satisfactory, when it comes to the total eradication of the microorganisms present due to several limitations like the complexity of the root canal anatomy and the ecology present inside the root canal. Conclusions: In the future, other antimicrobial strategies will have to be developed to supplement the currently used ones. Although these look promising in vitro they lack in vivo studies, that will be necessary in the future to overcome the several limitations present in the root canal system.

Les interactions allélopathiques sont définies comme la libération par un organisme de composés chimiques inhibant les compétiteurs. Dans les milieux aquatiques, elles sont reconnues comme un facteur de la dynamique des communautés de phytoplancton. Malgré les conditions favorables créées par l'organisation des microorganismes en biofilms, l'allélopathie a été beaucoup moins étudiée dans les communautés de microorganismes phototrophes benthiques. L'objectif de cette thèse était d'étudier des interactions allélopathiques, au travers d'un cas particulier, entre les microorganismes dans les biofilms phototrophes d'eau douce. Ainsi le travail s'est focalisé sur les effets allélopathiques d'une algue verte filamenteuse, Uronema confervicolum Lagerheim, sur des diatomées benthiques. L'hypothèse principale est que, dans certaines conditions, U. confervicolum libère suffisamment de composés allélopathiques pour inhiber significativement le développement des diatomées épiphytes. Cette hypothèse soulève trois questions (i) quels sont les composés allélopathiques, (ii) leur production dépend-elle des conditions environnementales et (iii) quel est l'effet des composés allélopathiques produits sur les diatomées ? Cette interaction a été étudiée au travers de multiples approches. L'activité d'extraits de biomasse algale et de filtrat de culture a été testée sur la croissance, la photosynthèse, l'adhésion et la motilité des diatomées. Les techniques de fractionnement guidé par bioessais et de profilage métabolomique ont été utilisées pour identifier les composés allélopathiques produits. Les effets des facteurs environnementaux (limitations en nutriment, apports de lumière et de gaz) et de la phase de croissance sur la production et la libération de différents composés allélopathiques ont également été étudiés. Une étude approfondie de l'effet sur les diatomées des composés allélopathiques anti-adhésion a été réalisée en combinant le profilage métabolomique, la transcriptomique et la microscopie électronique à balayage. Les extraits d'U. confervicolum ont montré un effet négatif sur la croissance, la photosynthèse, l'adhésion et la motilité des diatomées. Deux acides gras polyinsaturés (acides linoléique et a-linolénique) ont été identifiés comme des composés allélopathiques inhibant la croissance et la photosynthèse des diatomées. L'inhibition de l'adhésion des diatomées est, par contre, causée par d'autres composés allélopathiques non identifiés. L'augmentation de l'intensité lumineuse a accru la production de composés allélopathiques, à l'instar des composés défensifs des plantes. Cependant, aucune des théories classiques sur les défenses chimiques des plantes n'a permis d'expliquer l'ensemble des résultats obtenus sur les questions de régulation. En plus de l'inhibition de l'adhésion, le filtrat d'U. confervicolum inhibe la formation de la matrice constituée de substances polymériques extracellulaires, impacte le métabolisme énergétique et induit une modification globale du transcriptome et du métabolome des diatomées. Une partie des gènes et métabolites les plus fortement impliqués dans la réponse des diatomées à l'inhibition de l'adhésion ont été identifiés. Ce travail a permis de mieux évaluer l'activité allélopathique de l'algue verte filamenteuse U. confervicolum et son impact potentiel sur la structure et le fonctionnement des biofilms phototrophes. D'autres approches seront nécessaires, à l'avenir, pour mieux comprendre le rôle global de l'allélopathie dans des biofilms phototrophes complexes
Allelopathic interactions are defined as the release, by an organism, of chemicals that inhibit competitors. In aquatic environments, they are recognised as a driver of phytoplankton community dynamics. Despite the favourable conditions created by the organisation of microorganisms in biofilms, allelopathy has been much less studied in benthic phototrophic microorganism communities. The objective of this thesis was to increase our comprehension of allelopathic interactions amongst phototrophic microorganisms in biofilms by analysing several aspects of a specific allelopathic interaction. The model chosen was the allelopathic effect of Uronema confervicolum Lagerheim, a filamentous green alga isolated from river biofilms, on diatoms from the same environment. The main hypothesis was that, at least in specific environmental conditions, U. confervicolum produce and release sufficiently large amounts of allelopathic compounds to inhibit significantly the development of epiphytic diatoms. Three questions arised from this hypothesis: (i) what are the allelopathic compounds implied, (ii) do their production and release depend on environmental factors and (iii) what is the effect of these compounds on epiphytic diatoms? This interaction has been studied using multiple approaches. Activity of algal biomass extracts and culture filtrates have been tested on diatom growth, photosynthesis, adhesion and motility. Bioassay-guided fractionation and metabolomic profiling have been used to identify the allelopathic compounds produced. The effect of environmental factors (nutrient limitation, light and gas supply) and growth phase on the production and release of the different allelopathic compounds have also been studied. In-depth study of the effect of anti-adhesion allelochemicals on diatom by combining metabolomic profiling, transcriptomics and scanning electron microscopy have been carried out. The extracts of U. confervicolum exhibited negative effects on diatoms growth, photosynthesis, adhesion and motility in bioassays. Two polyunsaturated fatty acids (linoleic and a-linolenic acids) have been identified as allelopathic compounds affecting growth and photosynthesis but not adhesion ability of diatoms. The inhibition of diatom adhesion was caused by other allelopathic compounds, unidentified so far. Increasing light intensity enhanced the production of U. confervicolum allelopathic compounds as predicted by plant defence theories, but the regulation of allelopathic activity was not completely explainable by any classic plant defence theory. Finally, beside an inhibition of diatom adhesion U. confervicolum filtrate was also found to inhibit the formation of diatom extracellular polymeric substances matrix, affect energy metabolism and induce global modification of diatom transcriptome and metabolome. Some of the genes and metabolites implied in the response of diatoms to adhesion inhibition have been identified. These results indicate the strong potential of the filamentous green alga U. confervicolum to impact biofilm community by its allelopathic activity. Results on the allelopathic activity of this alga and the effect on diatoms in laboratory experiments provide the basis for further experiments to access the role of allelopathy in complex biofilms. These findings will encourage researchers working on biofilms to consider allelopathy when phototrophic biofilm ecology is studied

Objective: To quantify and assess the antibacterial effect of different medicaments on young and aged biofilms, and to modify the medicaments in order to increase their antibacterial effect. Hypotheses: Microbes in aged biofilms grown from a mixture of oral bacteria are more resistant to the antimicrobial activity of calcium hydroxide than microbes in young biofilms. Biofilms are less resistant to calcium hydroxide combined with other antimicrobial agents than to pure calcium hydroxide. Methodology: Collagen coated hydroxyapatite disks were immersed in plaque suspension solution and incubated for one and three weeks to grow young and aged biofilms, respectively. The tested medicaments were calcium hydroxide, iodine potassium iodide, cetrimide, and the following combinations: iodine potassium iodide + cetrimide, calcium hydroxide, calcium hydroxide + iodine potassium iodide, calcium hydroxide + cetrimide, calcium hydroxide + iodine potassium iodide + cetrimide. After exposure to the medicaments for one day, one week, and two weeks, biofilms on disks were stained with a LIVE/DEAD viability stain and imaged using confocal laser scanning microscopy. The three-dimensional reconstructions of the images were done and proportions of green and red fluorescence were measured and statistically analyzed. Results: Aged biofilms were thicker than the young biofilms. All tested medicaments showed reduced antibacterial activity on the aged biofilms compared to young biofilms. Combining iodine potassium iodide to cetrimide had an additive effect and mixed with calcium hydroxide showed stronger antibacterial effect than calcium hydroxide alone. Conclusions: Aged biofilms are more resistant to antibacterial agents than young biofilms. Combining iodine potassium iodide and cetrimide to calcium hydroxide resulted in an antibacterial effect that was stronger than using calcium hydroxide alone.
Dentistry, Faculty of
Graduate

Biofilm formation is a global, $multi-billion phenomenon spanning a plethora of stakeholders. This thesis investigates critical fundamental aspects of biofilm formation on steel and evaluates the efficacy of a novel, environmentally sustainable and multifunctional inhibitor compound developed through a broader Australian Research Council Discovery Project collaboration. Focused on sustainable and effective biofilm disruption, results from this thesis are used to expand fundamental knowledge and generate a targeted approach to biofilm mitigation that improves biocide function.

Bacteria can actively communicate and interact with each other to establish multicellular communities. Many of these processes involve functional differentiation of cells into specialised subpopulations by expression of varying genetic programmes. This leads to division of labour between the arising subpopulations of cells in the community. One type of such community is the biofilm, which is composed of microbial cells enclosed in a biopolymeric matrix. Such biofilms can be formed in a large range of environments from sea beds to animal tissues. Bacillus subtilis is a soil dwelling Gram-positive rod that was shown to closely interact with plants and establish a protective symbiosis by formation of biofilms on the roots. The biofilm matrix synthesised by B. subtilis is composed of the exopolysaccharide, for which the chemical structure is not yet established, and a protein TasA that forms amyloid-like fibres spanning between the cells and anchored to the cells by an accessory protein TapA. A third protein of unknown function, YuaB, has also been shown to be necessary for establishment of a biofilm. However, the mechanism of function for YuaB has not been elucidated. The data presented in this report focus on the role played by YuaB during formation of the biofilm. By analysis of cell differentiation patterns YuaB was shown to be required for maturation of the biofilm. The localisation of YuaB is identified in two “subtypes” of biofilm, a biofilm pellicle floating on the air-liquid interface and complex colonies formed on solid surfaces. This is achieved using a combination of biofilm fractionation combined with Western blotting and a newly developed method for immuno-fluorescent labelling of biofilm proteins. YuaB acts in synergy with the exopolysaccharide and TasA, as both components of the biofilm matrix are synthesised in the absence of YuaB but the biofilm is not made. The initial structural characterisation of YuaB is presented based on in silico predictions and physiological and biophysical analysis of the mutations introduced into the sequence of YuaB. The experimental data is concluded with a hypothesis that YuaB forms a hydrophobic protective layer necessary for support of the structure of the matured biofilm.

Le phénomène de biofouling affecte toute surface immergée dans un milieu aqueux, le recouvrant par un biofilm que ce soit dans le milieu médical ou marin. Afin de lutter contre ce phénomène, lors de ce travail, des matériaux biosourcés à base d’alginate ont été élaborés. Pour des applications dans le milieu médical, des films d’hydrogel de surface lisse ou poreuse et des films déposés sur acier par électrophorèse ont été élaborés. La présence de cuivre et zinc sous leur forme ionique Cu2+ et Zn2+ a été démontrée. Tous les films à base de cuivre et de zinc ont montré des propriétés antibactériennes contre des souches de bactéries pathogènes. Des volumes d’hydrogel d’alginate à base de calcium, cuivre et zinc ont été aussi élaborés pour des applications dans le monde marin. La stabilité des différents matériaux a été étudiée dans différents milieux et à différentes températures. La propriété antimicrobienne de ces hydrogels a été démontrée sur deux souches de microalgues ainsi que sur quatre souches de bactéries marines sans toxicité. Finalement, le retardement de la formation de biofilms sur la surface d’acier inoxydable a été étudié par la méthode d’OCP en présence de matériaux à base de zinc
Biofouling is a phenomenon that affects every surface that is immersed in an aquatic medium covering it by a biofilm either in the medical or marine field. In order to prevent this phenomenon, in this work, biosourced alginate materials were elaborated. For applications in the medical field, hydrogel films of smooth or porous surface and films coating stainless steel by electrophoresis were elaborated. The presence of copper and zinc in their ionic form Cu2+ and Zn2+ was demonstrated.All the copper and zinc based films showed antibacterial properties on pathogenic bacterial strains.Alginate hydrogels in bulk based on calcium, copper and zinc were also elaborated for applications in the marine field. Stability of the different materials was studied in different mediums and at different temperatures. The antimicrobial property of these hydrogels based on copper and zinc was demonstrated on two microalgae stains as well as on four marine bacteria strains with no sign of toxicity. Finally, the delay in the formation of biofilms on stainless steel surfaces was studied by the OCP method in the presence of zinc based materials

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Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde
S. epidermidis é o principal agente de infecções associadas a dispositivos médicos implantados, sendo sua habilidade para formar biofilme em superfícies inertes o fator determinante para a persistência desse micro-organismo. Neste estudo avaliamos 52 isolados clínicos desta espécie quanto à susceptibilidade a antimicrobianos, produção de biofilme/natureza química, presença de genes relacionados à virulência (atlE, capB,aap, embp, bhp, IS256 e IS257), e o efeito de concentrações subinibitórias (sub-CIMs) de etanol e clorexidina na produção de biofilme. Além disso, algumas das amostras biofilme-positivas foram estudadas quanto ao efeito de sub-CIMs destes antissépticos na expressão de icaA, icaR, sigB e sarA. Mais de 60% das amostras apresentaram resistência para ≥ 10 drogas e as amostras produtoras de biofilme mostraram, no geral, maior percentual de resistência a antimicrobianos. No teste em placa de microtitulação (MTP), 23 amostras foram produtoras de biofilme, sendo 14 de natureza polissacarídica, 8 proteica e 3 indeterminada. No teste em Ágar Vermelho Congo, somente amostras produtoras de biofilme polissacarídico apresentaram reação positiva. Genes do operon ica foram detectados em 23 isolados, sendo 17 destes classificados como produtores e 6 como não produtores de biofilme no MTP. A frequência dos outros genes relacionados à produção de biofilme foi: embp (69%), aap (29%) e bhp (12%), não sendo detectada correlação entre estes e a produção de biofilme do tipo PIA-independente. Os genes aap (29%) e IS256 (23%) mostraram correlações significativas com: produção de biofilme, presença de ica, perfil biofilme+/ica+, e produção de nível forte de biofilme. O gene IS256 foi ainda correlacionado significativamente com resistência a alguns antimicrobianos. Sub-CIMs de etanol (2 e/ou 4%) determinaram aumento na produção de biofilme em 15 das 17 amostras PIA-dependentes e nas 8 PIA-independentes, mas não induziram produção de biofilme em amostras originalmente não produtoras. Ao contrário do etanol, sub-CIMs de clorexidina não somente não induziram produção, como determinaram redução da produção de biofilme nas amostras biofilme-positivas. Nas amostras PIA-dependentes, o etanol (1%) acarretou aumento da expressão relativa de icaA e redução da expressão de icaR, além de aumento da expressão dos reguladores globais (sarA e sigB), enquanto a amostra PIA-independente mostrou redução na expressão destes reguladores globais. Ao contrário do etanol, a clorexidina (0,5 μg/mL) determinou aumento da expressão de icaR e redução de icaAnas amostras PIA-dependentes, além de redução na expressão de sarA e sigB na amostra PIA-independente. Os resultados indicaram que a produção de biofilme mostrou-se associada com alguns dos potenciais marcadores de virulência, sendo também evidenciada associação de alguns desses marcadores com resistência a certos antimicrobianos. As amostras PIA-dependentes foram prevalentes, destacando-se, porém, o encontro de número expressivo de amostras PIA-independentes. Os genes aap,embp e bhp não se mostraram correlacionados com a produção de biofilme proteico, indicando existência de outros mecanismos envolvidos na formação desse tipo de biofilme. Nas amostras PIA-dependentes, etanol e clorexidina mostraram efeitos opostos na expressão de icaA e icaR, corroborando dados fenotípicos previamente obtidos, e enfatizando a necessidade de ampliação do estudo da clorexidina, tendo em vista o potencial de aplicação prática deste achado.
S. epidermidis is the main agent of infections associated with implanted medical devices, being its ability to form biofilms on inert surfaces the determinant factor for the persistence of this microorganism. Fifth two clinical isolates of this species were evaluated for susceptibility to antimicrobials, biofilm production/chemical nature, presence of genes related to virulence (atlE, capB, aap, embp, bhp, IS256 andIS257), and the effect of subinibitory concentrations (sub-MICs) of ethanol and chlorhexidine in biofilm production. Moreover, some of biofilm-positive samples were studied for the effect of sub-MICs of these antiseptics in the expression of icaA, icaR, sigB and sarA. Over 60% of the samples showed resistance to ≥ 10 drugs and biofilm producers showed, in general, a higher percentage of antimicrobial resistance. In microtiter plate test (MTP), 23 strains were biofilm producers, being 4 of polysaccharide nature, 8 proteinaceous and 3 undetermined. In Congo Red Agar test, only biofilm polysaccharide producer strains showed a positive reaction. ica operon genes were detected in 23 isolates, being 17 of these classified as producers and 6 as non-biofilm producers in MTP. The frequency of other production-related biofilm genes was: embp (69%), aap (29%) and bhp (12%), no being detect a correlation between them and the production of PIA-independent biofilm. The aap (29%) and IS256 (23%) genes showed significant correlations with: biofilm production, presence of ica biofilm, biofilm+/ica+ profile, and strong level of production of biofilm. The IS256 gene was also significantly correlated with resistance to some antibiotics. Sub-MIC of ethanol (2 and / or 4%) led to an increase in biofilm production in 15 of 17 samples PIA-dependent and in the 8 PIA-independent, but did not induce biofilm production in not originally producing samples. Unlike ethanol, sub-MICs of chlorhexidine not only did not induce production as determined reduction of biofilm production in biofilm-positive samples. In PIA-dependent strains, ethanol (1%) caused an increase in the relative expression of icaAand reduced expression of icaR, in addition to increased expression of global regulators (sarA and sigB), while the PIA-independent strain showed reduction in the expression of these global regulators. Unlike ethanol, chlorhexidine (0.5 mg/mL) determined increased expression of icaR and reduction of icaA in PIA-dependent strains, besides a reduction in the expression of sarA and sigB in the PIA-independent strain. The results indicated that biofilm production was associated with some of potential virulence markers, and also evidenced some combination of these markers and resistance to certain antibiotics. The PIA-dependent strains were prevalent, highlighting, however, the encounter of significant number of PIA-independent strains. The aap, embp and bhp genes were not correlated with the production of proteinaceous biofilm, indicating the existence of other mechanisms involved in the formation of such biofilms. In PIA-dependent strains, ethanol and chlorhexidine showed opposite effects on the expression of icaA and icaR, corroborating phenotypic data previously obtained, and emphasizing the need to expand the study of chlorhexidine, in view of the potential of practical application of this finding.

Bacillus thuringiensis est capable de former un biofilm à l’interface air-liquide dans des tubes en verre en condition statique. Pendant la formation du biofilm, deux populations coexistent : une population sessile qui flotte sur le milieu de culture et une population planctonique, située dans le milieu de culture sous la pellicule. En utilisant des méthodes spectrophotométriques, nous avons suivi l’évolution de la croissance des bactéries planctoniques et du biofilm de la souche B. thuringiensis 407. Les résultats obtenus montrent qu’au moment où la biomasse sessile augmente rapidement, la population planctonique chute brutalement et descend jusqu’à une valeur proche de zéro. Cette chute de la population planctonique n’est pas observée avec le mutant spoOA de la souche 407 ou avec des souches incapables de former un biofilm, et ne peut pas être attribuée à la sédimentation ou à la lyse cellulaire. Elle est donc consécutive à un recrutement massif des cellules planctoniques par le biofilm en formation. Nous avons visualisé, par microscopie à epi-fluorescence, l’intégration des bactéries planctoniques de la souche 407 dans son biofilm préformé. Les bactéries recrutées sont localisées dans des zones restreintes du biofilm, où la densité des cellules sessiles est faible, ce qui révèle une distribution spatiale hétérogène des cellules immigrantes au sein du biofilm. Pour identifier les mécanismes impliqués dans le recrutement des cellules planctoniques dans le biofilm, nous avons criblé une banque de mutants de la souche 407 obtenus par mutagenèse aléatoire, pour leur capacité à intégrer un biofilm pré-existant. L’un des mutants de la banque, fortement affecté dans sa capacité à intégrer un biofilm, est touché dans le gène Bthur62720. Celui-ci est porté par le plasmide BTB-9P et code pour une protéine de 21 kDa. Cette protéine est sans homologue, et présente un peptide signal, un domaine N-terminal de fonction inconnue et un domaine C-terminal membranaire. En utilisant deux méthodes, l’immunocytochimie et la fusion traductionnelle avec la GFP, nous avons montré que cette protéine est pariétale, polaire et que son domaine N-terminal est cytoplasmique. A l’aide d’un marqueur des phospholipides chargés, le 10-N-nonyl acridine orange, nous avons montré que la délétion de Bthur62720 désorganise la distribution des radeaux lipidiques, qui apparaissent essentiellement polaires chez la souche 407 sauvage. De plus, cette délétion affecte fortement la nage linéaire, mais pas la culbute, ni la présence de flagelles. Ces résultats nous permettent de formuler l’hypothèse que Bthur62720 stabilise les radeaux lipidiques aux pôles. La localisation polaire de ces radeaux, en permettant la formation des complexes de chémorécepteurs, serait nécessaire pour assurer la nage linéaire
Bacillus thuringiensis is able to produce a pellicle at the air-liquid interface in glass tubes under static conditions. During biofilm formation, two populations coexist: a sessile floating population and a planktonic population, located in the culture medium beneath the pellicle. Using spectrophotometric measurements, we followed the growth of both populations during the B. thuringiensis 407 pellicle formation. Our results show that while the biofilm biomass increases rapidly, the planktonic population growth drops sharply. This decrease is not observed with the 407 spoOA mutant or for strains unable to form a biofilm, and cannot be attributed to cell lysis or cell sedimentation. Therefore, it is the result of a massive integration of planktonic cells in the preformed pellicle. We also visualized, using epi-fluorescence microscopy, the integration of planktonic bacteria of the 407 strain in its preformed biofilm. The recruited cells are located in restricted areas of the biofilm, where the density of sessile cells is low, revealing a heterogeneous spatial distribution of the immigrant cells within the biofilm. To identify the mechanisms involved in the recruitment of planktonic cells in the biofilm, we screened a bank of mutants of the 407 strain, obtained by random mutagenesis, for their ability to integrate a pre-existing biofilm. One of the mutants in the library is strongly affected in its ability to integrate a biofilm. This deficiency is caused by the disruption of the Bthur62720 gene, which is carried by the BTB-9p plasmid and encodes a 21 kDa protein. This protein has no homolog and in silico analysis predict a signal peptide, a N-terminal domain of unknown function and a C-terminal membrane domain. Using immunocytochemistry and translational fusion assays with GFP, we showed that this protein is parietal, polar and that its N-terminal domain is cytoplasmic. With a specific dye of charged membrane phospholipids, 10-N-nonyl acridine orange, we showed that the deletion of Bthur002_62720 disorganizes the lipid rafts distribution, which appear essentially polar in wild type strain 407. Moreover, this deletion strongly affects linear swimming, but not bacterial tumbling or the presence of flagella. These results allow us to hypothesis that Bthur62720 stabilizes the lipid rafts located at the cell poles. The polar localization of these rafts, required for the clustering of chemoreceptors, would be necessary to ensure a normal chemotaxis function and thus, bacterial swimming

Dans cette thèse, nous nous intéressons aux mécanismes précurseurs de la formation d'un biofilm dans le cas de la cyanobactérie Synechocystis sp. PCC 6803, en tant que micro-organisme modèle. Nous avons en premier lieu observé la motilité de cellules qui sédimentent puis diffusent librement sur une surface en verre. De plus, nous avons relevé les détails de la motilité des bactéries, qui est intermittente : des périodes de mouvements directionnels (les ``runs'') alternent avec des périodes de mouvements localisés (les ``tumbles''). Le coefficient de diffusion résultant décroît avec le temps, avant de se stabiliser; en revanche des souches mutantes relarguant moins de substance extracellulaire gardent un coefficient de diffusion constant au cours de l'expérience. Nous proposons un modèle de ralentissement basé sur une marche aléatoire à "temps continu" influencée par le recouvrement progressif de la surface par les exopolysaccharides secrétés par les bactéries. Les bactéries pourraient ainsi "reconnaître" le type de surface sur lequel elles évoluent et adapter leur motilité en conséquence, ce qui constitue une étape préliminaire dans la formation d'un biofilm. Nous avons vérifié ce point avec une étude de la diffusion sur des surfaces de différentes rigidités. L'expérience est cette fois prolongée pour étudier l'effet de la dureté du substrat sur la morphologie du biofilm. La proportion de bactéries non motiles est plus importante sur les surfaces molles. Cet effet à l'échelle de la particule conditionne en grande partie la morphologie des micro-colonies émergentes après plusieurs jours de culture, avec davantage de micro-colonies et une densité cellulaire plus hétérogène sur les surfaces molles. Nous construisons un modèle qui prend en compte la division cellulaire et le ralentissement de la dynamique individuelle, et permettant de conclure sur le lien entre rigidité de surface, dynamique cellulaire, et formation de micro-colonies. Le troisième chapitre expérimental aborde la réponse du système à des changements de conditions lumineuses, d'abord de manière isotrope puis en introduisant une lumière directionnelle. En conditions isotropes, les échelons d'intensité lumineuse perturbent les temps caractéristiques de ``run'' et de ``tumble''. Nous analysons ces variations dans le cadre de la théorie de la réponse linéaire. Nos données suggèrent qu'il est possible de décrire la réponse à une perturbation d'intensité lumineuse de manière similaire à des "stimuli" chimiques, avec la même fonction réponse. Sous un flux lumineux directionnel, nous observons une phototaxie complexe pour laquelle une fraction des cellules a un mouvement aléatoire pendant qu'une autre est sensible à l'anisotropie de l'éclairage. L'orientation des déplacements s'effectue dès l'introduction du flux lumineux, mais les temps de ``run'' et de ``tumble'' continuent d'évoluer pendant la période du flux lumineux suivant une tendance proche de celle observée en conditions isotropes. Ces résultats mettent en évidence le couplage entre l'intensité lumineuse et l'anisotropie de l'éclairage dans les mécanismes responsables de la phototaxie
In this work, we focus on the early stages of biofilm formation, using the cyanobacterium Synechocystis sp. PCC 6803 as a model micro-organism. First, we observe the motility of cells that have just reached the surface after sedimentation, and are then let free to diffuse on a glass surface. The resulting diffusion coefficient decreases with time, until it reaches a plateau value; whereas mutant strains secreting less extracellular substance do not exhibit such a slowdown. To explore the mechanism at work, we investigate the details of bacterial motility, which is intermittent: periods of directional movements (``runs'') alternate with periods of localized movements (``tumbles''). We propose a slowdown model based on a continuous-time random walk, influenced by the progressive surface coverage with the exopolysaccharides secreted by the bacteria. They could then ``recognize'' the surface onto which they are diffusing and adapt consequently their motility, which establishes a preliminary step for the biofilm formation. We have adressed this issue by studying diffusion onto surfaces with different stiffnesses. The experimentation is extended in order to analyze the effect of surface toughness on the biofilm morphology. The proportion of non-motile bacteria is higher on softer surfaces. This effect on the individual particle affects the shape of emergent microcolonies after several days of growth, with more microcolonies and a more heterogeneous surface density on soft surfaces. We build a model that takes into account cellular division and individual dynamics slowdown that enables us to point out the relation between surface rigidity, cellular dynamics, and microcolonies formation. The third experimental chapter tackles the response of the system to changes in light conditions, first in an isotropic way, then by introducing a directional light. For isotropic conditions, steps of light intensity disrupt the characteristic ``run'' and ``tumble'' times. We analyze these variations in the framework of the linear response theory. Our data suggest that it is possible to describe the response to light disruptions in the same way as what has been done for chemical ``stimuli'', with a similar response function. With a directional luminous flux, we observe complex phototaxis, in which some fraction of the cellular population displays random movements, whereas another one is sensitive to the lighting anisotropy. The displacements are oriented as soon as the luminous flux is switched on, and the ``run'' and ``tumble'' times also respond this change. Our results show that light intensity triggers bacterial motility, whether it is oriented or not

Orientador: Márcia Regina de Moura[UNESP] Aouada
Resumo: O presente trabalho é uma investigação das propriedades de embalagens ativas beneficiadas com a substância Curcuma longa derivada do açafrão. Essa substância apresenta propriedade antioxidativa, que tem despertado interesse da indústria de fármacos, e que vem sendo bastante utilizada na indústria de alimentos como corante natural e tempero. O propósito do trabalho é a síntese e caracterização da solução base para a produção dos filmes ativos contendo composição inédita de Curcuma longa, carboximetilcelulose (CMC) e nanopartículas de quitosana (NSQ). A produção de filmes e revestimentos poliméricos para a indústria alimentícia é alvo de constantes pesquisas, devido a necessidade de diminuir o volume de embalagens plásticas descartadas e otimizar as propriedades e validade dos alimentos. Dentre a imensa variedade de polímeros a carboximetilcelulose é muito favorável a produção de filmes pelo seu caráter atóxico, biodegradabilidade e baixo custo. A quitosana assim como o CMC é uma substância com ampla aplicação nos campos da farmacologia, tecnologia de biomateriais, biomedicina, agricultura e indústrias cosmética e alimentícia. Os filmes foram produzidos com carboximetilcelulose, nanopartículas de quitosana e Curcuma longa de acordo com o método “casting”. As nanopartículas foram obtidas pelo método de gelificação ionotrópica. Foram realizadas análises: térmicas, mecânicas, permeabilidade ao vapor de água, espectroscopia do infra- vermelho, ângulo de contato, microscopia eletrôn... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present work is an investigation of the properties of active packaging benefited with the substance Curcuma longa derived from saffron. This substance has antioxidative properties, which has aroused interest in the drug industry and is widely used in the food industry as a natural dye and seasoning. The purpose of the work is the synthesis and characterization of the base solution for the production of active films containing novel composition of Curcuma longa, carboxymethylcellulose (CMC) and chitosan nanoparticles (NSQ). The production of films and polymer coatings for the food industry is the subject of constant research, due to the need to reduce the volume of discarded plastic packaging and to optimize the properties and validity of the food. Among the immense variety of polymers the carboxymethylcellulose is very favorable the production of films by its nontoxic character, biodegradability and low cost. Chitosan as well as CMC is a substance with broad application in the fields of pharmacology, biomaterial technology, biomedicine, agriculture and cosmetic and food industries. The films were produced with carboxymethylcellulose, chitosan nanoparticles and Curcuma longa according to the casting method. The nanoparticles were obtained by ionotropic gelation. Thermal, mechanical, water vapor permeability, infrared spectroscopy, contact angle and scanning electron microscopy were performed. The nanoparticles were characterized by zeta potential and presented spherical sh... (Complete abstract click electronic access below)
Mestre

Orientador: Cinthia Pereira Machado Tabchoury
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O biofilme dental exposto a sacarose in situ por 13 dias ou mais apresenta altas concentrações de polissacarídeos extra-celulares (PEC), altas contagens da lactobacilos e baixa concentração inorgânica. Entretanto, essas mudanças e suas conseqüências em estágios inferiores de formação de biofilme são desconhecidas. Assim, o objetivo deste estudo foi avaliar a composição microbiológica e bioquímica do biofilme dental formado na presença de sacarose ou glicose + frutose em diferentes tempos, com a finalidade de observar a dinâmica de maturação do biofilme e sua relação com a desmineralização do esmalte. Doze voluntários adultos utilizaram em 3 fases cruzadas de 14 dias, um dispositivo intra-oral palatino contendo 6 blocos de esmalte dental humano, os quais foram expostos 8 vezes ao dia aos seguintes tratamentos: água destilada e deionizada (T1), solução de glicose 10% + frutose a 10% (T2) ou solução de sacarose a 20% (T3). O biofilme foi coletado após 3, 7 e 14 dias de formação e avaliado quanto à composição microbiológica e bioquímica. A análise microbiológica consistiu nas contagens de microrganismos totais (MT), estreptococos totais (ET), estreptococos do grupo mutans (EM), lactobacilos (LB), %EM/MT, %EM/ET e LB/MT. As variáveis bioquímicas avaliadas foram Ca, F, Pi, polissacarídeos intra (PIC) e extracelulares (PEC) no biofilme. Nos espécimes dentais, a perda mineral do esmalte seccionado longitudinalmente foi determinada. Maior desmineralização foi encontrada nos blocos submetidos ao T3 do que nos tratados com T1 e T2 (p < 0,05), sendo a perda mineral considerada significante a partir de 7 dias (p < 0,05). As concentrações de F, Ca e Pi no biofilme dental foram menores no T2 e T3 do que no T1 (p < 0,05), ), sendo que para F e Ca não houve diferença entre os tempos (p > 0,05) e para Pi, 7 e 14 dias mostraram maiores concentrações que no biofilme de 3 dias (p < 0,05). As concentrações de PIC foram significantemente maiores no T2 e T3 do que no T1 (p < 0,05), havendo um aumento com 7 e 14 dias, enquanto as de PEC foram maiores no T3 do que no T1 e T2 (p < 0,05), não mostrando diferença entre os tempos. Em relação à composição microbiana, os resultados mais evidentes foram em relação à contagem de LB e %LB/MT que apresentaram-se maiores no biofilme tratado com T2 e T3 do que no T1, entretanto essa diferença só foi observada a partir do 7o. dia (p < 0,05). Os resultados sugerem que mudanças na composição do biofilme formado na presença de sacarose já são evidentes a partir do 3o. dia de formação, entretanto a perda mineral só é significativa com 7 dias
Abstract: Dental biofilm exposed in situ to sucrose for 13 days or longer presents high concentration of extracellular polysaccharide (EPS), high lactobacilli counts and low inorganic concentration. However, these changes and their consequences at earlier stages of biofilm formation are unknown. Thus, the aim of this study was to evaluate the microbiological and biochemical composition of dental biofilm formed in the presence of sucrose or glucose + fructose at different periods, in order to observe its dynamic of maturation and its relationship with enamel demineralization. Twelve adult volunteers wore, for 3 crossover phases of 14 days, an intra-oral palatal appliance containing 6 human enamel blocks, which were exposed 8 times/day to the following treatments: distilled and deionized water (T1), 10% glucose + 10% fructose solution (T2) or 20% sucrose solution (T3). The biofilm was collected after 3, 7 and 14 days of formation and evaluated with regard to microbiological and biochemical composition. Microbiological analyses consisted in counts of total microorganisms (TM), total streptococci (TS), mutans streptococci (MS), lactobacilli (LB), %MS/TM, %MS/TS and %LB/TM. The biochemical variables evaluated were F, Ca, Pi, intra (IPS) and extracellular (EPS) polysaccharides. In dental specimens, enamel cross-sectional mineral loss was determined. Higher mineral loss was found in enamel blocks treated with T3 than those exposed to T1 and T2 (p < 0.05), however only with 7 days the mineral loss was considered significant (p < 0.05). The concentrations of F, Ca and Pi in dental biofilm were lower in T2 and T3 than in T1 (p < 0.05). Also, for F and Ca no difference was observed among the periods (p > 0.05) and for Pi, 7 and 14-day biofilm showed higher concentrations than 3-day biofilm (p < 0.05). IPS concentrations were significantly higher in T2 and T3 than in T1 (p < 0.05), showing increased concentrations with 7 and 14 days (p < 0.05), whereas EPS concentration did not show statistical difference among the periods (p > 0.05), but showed higher values in T3 than in T1 and T2 (p < 0.05). With respect to microbiological composition, the most evident results were related to LB counts and % LB/TM that showed higher values in T2 and T3 than in T1 (p < 0.05), but this difference was observed only with 7-day biofilm (p < 0.05). The results suggest that the changes on composition of biofilm formed under sucrose exposure are evident at 3 days of formation, however the mineral loss is only significant with 7 days of biofilm formation
Mestrado
Cariologia
Mestre em Odontologia

Orientador: Tomomasa Yano
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Escherichia coli Patogênica Extra-intestinal (ExPEC) forma um grupo bacteriano heterogêneo quanto a fatores de virulência envolvidos em sua patogenicidade e locais de infecção no hospedeiro. Nosso trabalho teve como objetivo estudar as características fenotípicas e genotípicas da formação do biofilme e filogenia de amostras de Escherichia coli associada à sepse humana (SEPEC), assim como seus padrões de adesão e invasão às células renais (Vero) e endoteliais (Hec-1B). SEPEC mostrou alta prevalência na capacidade de formar biofilme (82,2%) "in vitro", em superfícies de plásticos, sugerindo envolvimento das fimbria tipo 1 (fimH), antígeno 43 (ag43) e antígenos de curli (csgA). No estudo filogenético, 26 (53,2%) amostras de SEPEC foram classificadas como pertencentes ao grupo D, 19 (38,8%) ao grupo B2, 2 ao grupo A (4,0%) e 2, classificadas como pertencentes ao grupo B1 (4,0%). Outras adesinas, invasinas e toxinas como mat (adesina regulada pela temperatura e associada à meningite), iha (Adesina regulado por ferro), neuC (polissacarídeo capsular K1), gimB (ilha de patogenicidade associada à meningite neonatal), ibeA (relacionada à invasão do endotélio cerebral) e tia (locus de invasão toxigênico em E. coli Enterotoxigênica), foram detectadas em freqüências de 38,8%, 32,7%, 24,5%, 12,2%, 10,2%, 18,4%, 69,4% e 57,1%, respectivamente. No teste de adesão, 100% das amostras de SEPEC aderiam e invadiram tanto células Vero como Hec-1B. Através de microscopia eletrônica de transmissão, foram verificados pontos da adesão bacteriana em células Hec-1B, com formações de pedestais entre a membrana plasmática e as células bacterianas. A microscopia eletrônica de varredura, em células Vero, demonstrou pontos da adesão de SEPEC sobre a superfície celular, com a formação de microcolônias envoltas por exopolissacarídeos (EPS)
Abstract: Extraintestinal Pathogenic Escherichia coli (ExPEC) form a heterogeneous group as the bacterial virulence factors involved in its pathogenicity and local infection in the host. Our work aimed study the phenotypic and genotypic characteristics of biofilm formation and phylogeny of sepsis-associated Escherichia coli Human (SEPEC), as well as their patterns of invasion and adhesion to kidney (Vero) and endothelial (Hec-1B) cells. SEPEC showed high prevalence in the ability to form in vitro biofilm (82.2%) on plastic surfaces, suggesting involvement of type 1 fimbriae (fimH), antigen 43 (ag43) and curly fimbriae (csgA). In phylogenetic analysis, 26 (53.2%) SEPEC strains were classified as belonging to group D, 19 (38.8%) to group B2, 2 in group A (4.0%) and 2, classified as belonging to group B1 (4.0%). Others adhesins, toxins and invasin mat (adhesin temperature- egulated associated with meningitis), iha (iron-regulated adhesin), neuC (capsular polysaccharide K1), gimB (pathogenicity island associated with neonatal meningitis), ibeA (brain microvascular endothelial cells invasion) and tia (toxigenic invasion locus from Enterotoxigenic E. coli), were detected at frequencies of 38.8%, 32.7%, 24.5%, 12.2%, 10.2%, 18, 4%, 69.4% and 57.1%, respectively. In the adhesion test, 100% of SEPEC strains adhered and invaded both Vero cells and Hec-1B. Through transmission electron microscopy, were found parts of bacterial adhesion in Hec-1B cells, with the formation of pedestals between the cytoplasmic membrane and bacterial wall-cell. The scanning electron microscopy in Vero cells, showed the accession of points SEPEC on the cell surface, with the formation of microcolonies surrounded by exopolysaccharides (EPS)
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular

O objetivo deste estudo foi avaliar o efeito do hipoclorito de sódio e a clorexidina sobre biofilme dental formado in situcom relação a: concentração do hipoclorito de sódio (1%, 2.5% e 5%) e clorexidina 2%, tempo de exposição à solução irrigadora (5, 15 e 30 minutos.), volumes das soluções (500 µl e 1 mL), espessura do biofilme e área de limpeza segundo analise morfométrica. Foram utilizados 120 blocos de dentina bovina esterilizada, colocados em um aparelho intraoral e utilizados por um voluntário durante 3 dias. Transcorrido o período experimental as amostras foram retiradas e coradas com 50 µl de laranja de acridina para determinar a espessura do biofilme pre irrigação por meio do Microscopio confocal de varredura laser (CLSM). Foram conformados 12 grupos experimentais com 10 blocos cada um e irrigados com NaOCl e clorexina. Dez amostras foram irrigadas com 500 µl (N=5) e 1mL (N=5) de NaOCl 1% por: 5 min (G1), 15 min (G2) e 30 min (G3). Dez amostras foram irrigadas com 500 µl (N=5) e 1mL (N=5) de NaOCl 2.5% por: 5 min (G4), 15 min (G5) e 30 min (G6). Dez amostras foram irrigadas com 500 µl (N=5) e 1mL (N=5) de NaOCl 5% por: 5 min (G7), 15 min (G8) e 30 min (G9). Dez amostras foram irrigadas com 500 l (N=5) e 1mL (N=5) de Clorexidina 2% por: 5 min (G10), 15 min (G11) e 30 min (G12). Para cada sub grupo experimental (N=5) se deixou um sexto bloco o qual foi irrigado com água destilada estéril para procedimentos de controle. Nos grupos de 15 e 30 minutos a solução de NaOCl e clorexidina foi renovada a cada 5 minutos. Os segmentos de dentina foram lavados com 200 µl de água destilada estéril para eliminar resíduos não aderidos e corados com 50 µl de Laranja de acridina para determinar a espessura do biofilme após irrigação por meio do CLSM. Foram encontrados altos valores de dissolução do biofilme e dentina limpa após contato com NaOCl a 5% durante 5 e 15 min. e com todos os grupos de NaOCl durante 30 min. O uso de Clorexidina a 2% não dissolveu o biofilme e nem aumentou a limpeza dentinária quando comparado com o NaOCl (P < 0.05).
Introduction: The aim of this study is to evaluate the biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. Methods: 120 bovine dentin specimens were infected intraorally using a removable orthodontic device. 30 samples were used for each irrigant solution; 2% Chlorhexidine, 1%, 2.5% and 5.25% of sodium hypochlorite (NaOCl). The solutions were used for 5, 15 and 30 minutes and 2 experimental volume 500µl and 1mL. The samples were stained using the acridine orange dye before and after the experiments and evaluated using a confocal microscope. The percentage of biofilm, isolated cells and no colonized dentin was measured using a grid system. Differences in the reduction or increase of the studied parameters was assessed using non-parametric methods ( P < 0.05). Results: The higher values of biofilm dissolution and clean dentin were found in the 30 minutes NaOCl groups and in the 5 and 15 minutes of 5.25% NaOCL. The use of 2% chlorhexidine solution does not improve the biofilm dissolution neither increases the cleaning of the dentin in comparison to the NaOCl solutions (P < 0.05). Conclusions: 2% chlorhexidine does not dissolve the biofilms. 30 minutes of sodium hypochlorite are necessary to have the higher values of biofilm dissolution and to increase the cleaning of the dentin independently of the concentration in comparison to the 5 min and 15 min contact time.

Neste trabalho foram preparados materiais híbridos do tipo silicatos organicamente modificados (ormosils) contendo fosfotungstato, [PW12O40]-3 e dopados com nanopartículas de TiO2. O objetivo é obter uma ação sinérgica destes dois fotocatalisadores na prevenção de formação de biofilmes e/ou sua fotodegradação. O fotocatalisador principal no sistema é o fosfotungstato, sendo o co-adjuvante o TiO2. Sendo assim, procurou-se manter a concentração deste no menor nível possível. Os materiais foram caracterizados por espectroscopias vibracionais, espectroscopia de fotoelétrons de raios X (XPS), Fluorescência de Raios X, Microscopia de força atômica e Microscopia eletrônica de varredura (MEV). Os filmes não mostraram eficácia na fotodegradação de biomoléculas como fosfolipídios encontrados na membrana celular. Os ensaios de inibição de crescimento de biofilmes de Escherichia coli sobre os ormosils mostraram que a maior inibição de bactérias é do filme contendo maior teor de nanopartículas de titânia portanto, são bons candidatos para filmes e revestimentos bactericidas/bacteriostáticos a serem usados em máscaras respiratórias, revestimentos de superfícies em salas de cirurgia e em filtros de ar em sistemas fechados (sistemas de ar condicionado e ventilação em geral).
This thesis deals with hybrid materials named organically modified silicates (Ormosils) with phosphotungstate, [PW12O40]-3 , and doped with TiO2 nanoparticles. The aim was to achieve a synergic action between both photocatalysts resulting on a more efficient coating for inhibition of the biofilm growing and/or its photodegradation. The photocatalyst in main system is the phosphotungstate, being the co-adjuvant the TiO2. Therefore, we tried to maintain the concentration of this at the lowest level possible The materials were characterized by vibrational spectroscopies, X-Ray Photoelectron Spectroscopy, X- ray Fluorescence Spectroscopy, Atomic Force Microscopy and Scanning Electron Microscopy. The films were unable to photodegradate biomolecules films such as phospholipids as well as they display interesting inhibition capacity against formation of biofilm of E.Coli bacteria. The tests of inhibition of growth of Escherichia coli biofilms on the ormosils showed that a greater inhibition of bacterias exists in the film containing higher content of nanoparticles of titania. Therefore, they are good candidates for bactericidal films and coatings to be used in respirators, surface coatings in surgery rooms and air filters in closed systems (systems of air conditioning and ventilation in general).

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Devido ao aumento das infecções causadas por Candida albicans e espécies não-albicans, surge a necessidade de novas estratégias terapêuticas, como a identificação de cepas bacterianas com características probióticas. Assim, o objetivo foi avaliar a ação antimicrobiana de cepas clínicas de Lactobacillus paracasei, Lactobacillus fermentum e Lactobacillus rhamnosus sobre diferentes espécies de Candida (C. albicans, C. glabrata, C. krusei e C. tropicalis). Foram utilizadas cepas de Lactobacillus isoladas da cavidade bucal de indivíduos livres de cárie e cepas de Candida isoladas de lesões de candidose orofaríngea obtidas em estudos anteriores. Para estudo in vitro, foram formados biofilmes monoespécies de Candida (Grupo controle) e biofilmes mistos de Candida e Lactobacillus, nos quais os lactobacilos foram acrescentados antes da cepa de Candida (Grupo Profilático), após a cepa de Candida (Grupo Terapêutico) ou ao mesmo tempo da cepa de Candida (Grupo Simultâneo). Após 48 h de incubação, foi realizada a contagem do número de células viáveis de Candida (UFC/mL). Para estudo in vivo, os micro-organismos foram inoculados em larvas de G. mellonella e o desenvolvimento da candidose foi monitorado pela curva de sobrevivência, estudando-se os grupos experimentais descritos acima. Nos resultados in vitro, foi verificado que os efeitos inibitórios mais significativos de Lactobacillus sobre Candida ocorreram nos grupos profiláticos. As cepas de C. tropicalis e C. krusei foram mais sensíveis a ação de Lactobacillus do que C. albicans e C. glabrata. Além disso, foi verificado que as três espécies de Lactobacillus estudadas (L. paracasei, L. rhamnosus e L. fermentum) tiveram ação inibitória sobre Candida. Em relação ao estudo in vivo, também foi verificado que os grupos profiláticos tiveram maiores efeitos inibitórios sobre a candidose experimental em relação aos grupos terapêuticos, levando a um aumento significativo nas taxas de sobrevivência das larvas. Em relação as cepas estudadas, os efeitos profiláticos de Lactobacillus sobre a candidose, foram encontrados, de forma semelhante, para todas as cepas de Candida e Lactobacillus estudadas. Assim, concluiu-se que as cepas clínicas de L. paracasei 28.4, L. rhamnosus 5.2 e L. fermentum 20.4 foram capazes de inibir a formação de biofilme e desenvolvimento de candidose causados por C. albicans, C. krusei, C. tropicalis e C. glabrata, principalmente quando Lactobacillus foi administrado de forma profilática.
Due to the increase of infections caused by Candida albicans and non-albicans species, there is a need for new therapeutic strategies, such as the identification of bacterial strains with probiotic characteristics. The objective of this study was to evaluate the antimicrobial action of Lactobacillus paracasei, Lactobacillus fermentum and Lactobacillus rhamnosus strains on different Candida species (C. albicans, C. glabrata, C. krusei and C. tropicalis). We used Lactobacillus strains isolated from the oral cavity of caries-free and Candida strains isolated from lesions of oropharyngeal candidiasis. For in vitro study, monospecies of Candida (Control Group) and mixed biofilms of Candida and Lactobacillus were prepared, in which the lactobacillus were attached before the Candida strain (Profile Group), after a Candida (Therapeutic Group) strain or same time as the Candida strain (Simultaneous Group). After 48 h of incubation, we counting the number of viable Candida cells (CFU/mL). For in vivo study, we inoculate the microorganisms in larvae of G. mellonella and the development of the application for monitoring by survival curve, studying the experimental groups described above. In the in vitro results, we find that the most significant inhibitory effects of Lactobacillus on Candida occurred in the prophylactic groups. The strains of C. tropicalis and C. krusei were more sensitive to the action of Lactobacillus than C. albicans and C. glabrata. In addition, it we verified that the three species of Lactobacillus studied (L. paracasei, L. rhamnosus and L. fermentum) had an inhibitory action on Candida. In relation to the in vivo study, we verified that the prophylactic groups had more inhibitory effects on an experimental candidose in relation to the therapeutic groups, leading to a significant increase in the survival rates of the larvae. In relation to the studied strains, the prophylactic effects of Lactobacillus on candidiasis were similarly for all Candida and Lactobacillus strains studied. Thus, it concluded that the clinical strains of L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 were able to inhibit a biofilm formation and development of Candida caused by C. albicans, C. krusei, C. tropicalis and C. glabrata, especially when Lactobacillus was given prophylactically. Keywords:

Microbiology and Immunology
M.S.
Long-term Stationary Phase Behavior of Streptococcus pyogenes Biofilms Department of Microbiology and Immunology Streptococcus pyogenes is the etiological agent of many human diseases ranging from mild superficial skin infections and pharyngitis to life-threatening necrotizing fasciitis. There can be several complications as a result of S. pyogenes infection including post-streptococcal glomerulonephritis and rheumatic fever, which leads to rheumatic heart disease. Despite the significant virulence associated with the pathogen, the bacteria can also persist asymptomatically in human host carriers. S. pyogenes is characterized by significant strain-to-strain variation with many single nucleotide polymorphisms and differences in genetic content of up to 33% of the genome. Active infection is associated with the rapid growth of the pathogen, whereas survival or carriage is associated with slow growth. Our laboratory has demonstrated that during survival in long-term stationary phase cultures and in eukaryotic cells, S. pyogenes diversifies into a mixed population. Isolates from this population show diversification in their proteome, in metabolism, and in virulence factor transcription patterns. These are stable, heritable changes with unique mutations in global gene regulators in some isolates, suggesting that an accumulation of genetic mutations leads to diversification. There are two proposed modes of survival in the human host; by taking residence intracellularly in host cells and as biofilms. Previous studies showed that isolates surviving within eukaryotic cells acquire heritable changes in metabolism and virulence factor expression. Biofilms are highly organized structures formed by many bacteria, which provide resiliency to harsh environmental conditions. It has been demonstrated that S. pyogenes form biofilms in vivo and in vitro, and up to 90% of clinical isolates can form biofilms. Considering the resiliency of biofilms, and the organized roles played by individual cells in biofilms, we hypothesized that biofilms may provide S. pyogenes with a niche for persistence and diversification. Despite the capacity for survival of planktonic cells, we have found that viable cells could not be isolated from static biofilms after 10 days. No metabolic variants were found among biofilm isolates prior to loss of biofilm viability. Biofilm structure was examined using confocal microscopy to image cells after LiveDead® staining. These experiments revealed that the biofilms lost viability rapidly, and also appeared to disperse. Dispersion of 2-day old biofilms could be induced with culture supernatants collected from 7-day old planktonic cells. Overall, the results of these studies suggest that secreted factors from late stationary phase cultures induce biofilm dispersion and biofilms do not serve as a niche for long-term survival and diversification of S. pyogenes. Therefore, S. pyogenes biofilms may be more critical for initial colonization of the oropharynx. These studies may provide a valuable insight to the role of biofilms in S. pyogenes infections.
Temple University--Theses

Streams experiencing a recurrent non-flow phase (i.e., flow intermittency) are characteristic of world regions with arid and semi-arid climates, where Mediterranean regions are part of. During non-flow streambed sediments, and consequently, microorganisms inhabiting these sediments are exposed to desiccation. These microorganisms, assembled in biofilms, lead a substantial part of the ecosystem processes. They recycle the carbon materials, intervene in the nutrient cycles and are on the base of the food web, fueling energy to the higher trophic levels. The aim of this tesis is to understand how biofilms respond to flow intermittency in order to unravel the consequences of the increasing spatial and temporal extent of flow intermittency, as a consequence of the global change, on the biogeochemical cycles and on the ecosystem functions in temporary streams. Structural and functional biofilm responses were analyzed at the cellular level (algae and bacteria), as well as at the whole biofilm responses (autotrophic vs heterotrophic processes) in two different field studies
Els rius que experimenten una fase sense cabal (intermitència fluvial) són característics de les regions del món amb climes àrids i semi-àrids, com ara les regions de la Mediterrànies. Durant la fase seca es produeix la dessecació de la llera del riu i conseqüentment els microorganismes que creixen sobre aquests sediments estan exposats a la dessecació. El conjunt d’aquests microorganismes es coneix com a biofilm, el qual juga un paper clau en el processament de la matèria orgànica i en els cicles del carboni i nutrients, A més són a la base de la xarxa tròfica aportant energia als nivells tròfics superiors. L'objectiu principal d'aquesta tesi és entendre el funcionament del biofilm quan es dona la fase seca, pas clau per entendre i predir les implicacions que tenen els períodes creixents sense cabal en els cicles biogeoquímics i en el funcionament de l’ecosistema. Les respostes estructurals i funcionals del biofilm des d’un punt de vista cel·lular (algues i bacteris), així com també en el conjunt del biofilm (processos autotròfics i heterotròfics) es van investigar mitjançant dos estudis de camp

Bacterial surface associated communities, so called biofilms, located on the inside of drinking water distribution pipes, are believed to be responsible for lowering the drinking water quality by releasing bacteria into the water stream. As there were some indications that a strong electric field along the pipes inner walls could deter and possibly kill bacteria trying to adhere to the surface, it was the aim of this thesis to investigate that effect and its possible application in tap water. A closed system was set up with three cylindrical containers, made from short pieces of the pipe intended to be used in water distribution systems, with isolated electrodes on the inside. The voltage (0 – 10 kV) as well as pulse length (0 – 110 ms) and pulse repetition rate (1 Hz or 5 Hz) was varied in order to find the optimal settings. Through the system, liquid acetate enriched minimal media inoculated with Comamonas dentitrificans 110 as model organism was being pumped. Each run lasted for a week, after which the biofilm in the containers were stained with 1 % crystal violet, and the biofilm formation analyzed. The above system was not ideal for studying any effects of the electric field; the results were inconclusive though a trend showing biofilm deterrence could be seen, with long pulses at a high pulse repetition rate being the optimal setting. Bacterial concentrations and ion strengths in the three container system could not be kept comparable to that in distribution pipes, so a lone container, was set up in an open system, with only tap water (adjusted to 30°C) running through it for 4 months, in order to assess the biofilm thickness and the speed at which it forms. A thin but evenly distributed biofilm was seen on the inside of the tap water container indicating that further testing could be done in tap water with a very low risk of biofilm not forming inside the control container. To clarify how the electric field affected biofilm, small volumes of cultures containing detached pieces of biofilm were put into a 1 ml cuvette over which an electric field similar to that in the big system was applied. Electrolysis was made using similar solutions so that comparisons could be made. Little disturbance could be observed in the structures when applying an electric field but the effects seen in these test was very subtle compared to electrolysis were almost all biofilm fragment had lost their initial structure. Whether strong electric fields will prevent biofilm formation is unclear, it seems unlikely, but not impossible. The field will have a wider range in tap water and maybe boost the effect of chlorine, but will also go up against much tougher bacteria.

S. aureus is a natural commensal of the human host and an opportunistic pathogen that causes a wide range of diseases. Biofilm formation is an important S. aureus virulence determinant. S. aureus colonisation of medical devices and host tissues as a biofilm can impede treatment as genetically and metabolically diverse cells in the different layers of the film can prevent the penetration or activity of the therapeutic agent used. Biofilm formation is a multi-factorial process which can be influenced by many environmental factors. A major environmental stress encountered by bacteria in vivo is severe iron-restriction. However, pathogenic bacteria can use low iron concentrations as a signal to up regulate factors responsible for virulence. This work demonstrates for the first time that S. aureus biofilm formation is iron regulated. It demonstrates that biofilms formed in low iron are dependant on the proteins Eap and Emp which are also iron regulated and are positively regulated by the ferric uptake regulator, Fur. This work has also identified that PNAG, which currently has a controversial role in biofilm formation, was capable of compensating for the loss of Eap and Emp in low iron when it was over expressed, but that wild type levels of PNAG appeared to have a limited role. Nevertheless, the ica operon responsible for the production of PNAG was essential for the expression of Eap and Emp. In addition, Sae, Fur, Agr, SarA and Hfq were shown to all have interlinking roles in the expression of these and other proteins associated with virulence and low iron biofilm formation. The identification of the importance of Eap and Emp in low iron biofilm formation and the regulatory network controlling their expression may have implications on the development of new therapeutic agents essential for the prevention and treatment of S. aureus infection.