Πολλοί μικροοργανισμοί έχουν την ικανότητα να σχηματίζουν βιοφίλμ στη διεπιφάνεια μεταξύ ενός υδατικού διαλύματος και ενός άλλου ρευστού, στερεού ή πορώδους υλικού. Η ανάπτυξη βιοφίλμ σε πορώδη υλικά έχει σημαντικό ρόλο σε πληθώρα διεργασιών, όπως η βιοαποδόμηση ρύπων στο έδαφος και η βιολογικά ενισχυμένη απόληψη πετρελαίου από ταμιευτήρες. Στην παρούσα εργασία μελετώνται τα φαινόμενα μεταφοράς και η δυναμική συμπεριφορά της ανάπτυξης μικροβιακών βιοφίλμ κατά τη βιοαποδόμηση οργανικών ενώσεων σε πορώδη υλικά. Οι κύριοι στόχοι και τα αποτελέσματα της διατριβής εντάσσονται σε τρεις άξονες. Ο πρώτος άξονας περιλαμβάνει τη διεξαγωγή πειραμάτων ανάπτυξης βιοφίλμ σε γυάλινα δοκίμια πορώδους μέσου για την αποσαφήνιση των μηχανισμών φραξίματος στην κλίμακα των πόρων και την συσχέτιση πορώδους-διαπερατότητας στην κλίμακα του πορώδους μέσου. Ο δεύτερος άξονας περιλαμβάνει την ανάπτυξη μιας μεθοδολογίας για τη μαθηματική περιγραφή: α) της διεργασίας μεταφοράς μάζας δια μοριακής διαχύσεως, και β) της διεργασίας μεταφοράς ορμής κατά τη ροή του εξωκυτταρικού υδατικού διαλύματος, σε κυτταρικά βιολογικά υλικά (βιοφίλμ, μικροβιακά συσσωματώματα, ιστοί). Η μεθοδολογία συνδυάζει τη μέθοδο χωρικής στάθμισης δια συνάρτησης βάρους για την ανάπτυξη των διεπουσών εξισώσεων, με μοντέλα μοναδιαίων κελιών για τον υπολογισμό των συντελεστών που υπεισέρχονται στις διέπουσες εξισώσεις. Ο τρίτος άξονας περιλαμβάνει την ανάπτυξη ενός νέου, ιεραρχικού, υβριδικού εξομοιωτή της δυναμικής συμπεριφοράς μικροβιακών βιοφίλμ σε πορώδη υλικά. Ο εξομοιωτής προβλέπει: α) τη δομική και βιολογική ετερογένεια στην κλίμακα του βιοφίλμ, β) τη μορφή και το ρυθμό ανάπτυξης του βιοφίλμ μέσα στους πόρους ρεαλιστικών πορωδών δομών, και γ) τη συζυγή μεταβολή του πεδίου ροής και της χωρικής κατανομής των συγκεντρώσεων διαλελυμένων χημικών ειδών. / Numerous bacteria are able to form biofilms at the interface between an aqueous solution and another fluid, solid or porous material. Biofilm growth in porous media is of key importance in a variety of processes, such as the biodegradation of pollutants in soil and biologically enhanced oil recovery from subsurface reservoirs. In the present work, the transport phenomena and the dynamics of biofilm growth during the biodegradation of organic pollutants in porous media are studied. The main goals and results of the dissertation are developed along the following axes. The first axis involves the conduction of experiments of biofilm growth in glass models of porous media for the elucidation of the clogging mechanism on the pore scale and the permeability-porosity correlation on the scale of the porous medium. The second axis involves the development of a methodology for the mathematical description of: (a) the diffusive mass transfer process, and, (b) the momentum transfer during the flow of the extra-cellular aqueous solution in cellular biological media (biofilms, microbial flocs, tissues). The methodology combines the spatial averaging method via a weight function for the formulation of the governing equations, with unit cell models for the calculation of the coefficients that enter the governing equations. The third axis involves the development of a novel, hierarchical, hybrid simulator of the dynamic behavior of microbial biofilms in porous materials. The simulator predicts: (a) the structural and biological heterogeneity on the biofilm scale, (b) the pattern and rate of growth rate of biofilms in the pores of realistic porous structures, and (c) the conjugate alteration of the flow field and the spatial distribution of the concentration of solutes.

Bacteria in nature mostly exist in biofilms, which are structured adherent communities encased in polymeric matrices. In the human body, most biofilms are composed of commensal microorganisms with the gastrointestinal tract being the most heavily colonized site. Bacterial attachment to the overlying mucus gel layer of the intestinal epithelium is fundamental to the establishment of a stable commensal microflora. However the interaction of bacteria with the complex mucus gel is poorly described. Moreover, the complexity and diversity of the gut microbiota is itself an obstacle to studying its biology. Microbiota functions are the product of communities of bacteria and interactions between multiple species. New approaches are needed to study this aspect of even the most well-studied member of the human gut microbiota, Escherichia coli. This thesis was devoted to the exploration of the transcriptional response of E. coli facing different elements of human gut following 3 main objectives. First, the initial part of my work was related to the conception and optimization of appropriate genetic tools to both track E. coli within the multispecies context that constitute human gut commensals, and survey the expression of genes of interest. Use of the Green Fluorescent Protein (GFP) genes allowing enhanced fluorescence and shortened half-life has permitted significant progress both in whole cell tagging as well as transcriptional reporting, while the red fluorescent counterparts were disappointing. Second, using the subset of tools that has been validated to be reliable, influence of mucin on the biofilm formation ability of E. coli has subsequently been studied. I have shown that mucin promotes E. coli biofilm formation through transcriptional modulation of surface adhesion structures such as curli and type 1 pili. Third, concurrently, E. coli's population relationship to commensal bacteria (K. pneumoniae and E. faecalis) was investigated and demonstrated, with the possible influence of surface adhesion structures such as curli as the biological focus. The results suggest that curli production in biofilm increases the fitness of E. coli when co-cultured with K. pneumoniae while promoting synergistic interaction between E. coli and E. faecalis. The implication based on the data is discussed. This work improves the understanding of E. coli response to the gut environment, and provides foundations to build more powerful tools for further investigations.

El problema que trae aparejado la falta de un diagnóstico certero y rápido de los patógenos respiratorios en pacientes fibroquísticos tiene un fuerte impacto en el tratamiento antimicrobiano, en el control de infecciones y principalmente en la calidad de vida y sobrevida de estos pacientes. En el caso particular de los organismos del complejo Burkholderia cepacia, que colonizan a estos pacientes, son ampliamente reconocidas las dificultades y limitaciones de los métodos bioquímicos tradicionales para su identificación. En los últimos años se han puesto muchos esfuerzos en desarrollar nuevos métodos de identificación rápidos, sencillos y de bajo costo que permitan la diferenciación e identificación inequívoca de bacilos no fermentadores, pertenecientes y no pertenecientes al complejo Burkholderia cepacia. Este trabajo de tesis fue concebido con la idea de avanzar en el conocimiento general sobre las técnicas de identificación, las características genotípicas y fenotípicas de organismos del complejo B. cepacia y a su vez resolver problemas de diagnóstico del sector hospitalario y epidemiológicos de nuestro país.

A library of 18 coliform bacteria strains was obtained from different sampling points in the drinking water distribution system in Lexington, KY, over a three month period in 2006. To investigate the cause of the coliform occurrence we conducted a microbial source tracking study using phenotypic (API 20E, Biolog, and Vitek) and genotypic (pulsed field gel electrophoresis (PFGE) and ribotyping) analyses to determine the degree of genetic variation among isolates. Characterization of isolates by PFGE and ribotyping showed that coliform events in the distribution system were related and a regrowth problem may exist due to biofilm formation. The ability of a persistent Enterobacter cloacae strain to adhere and form biofilm was found to depend on environmental conditions such as temperature, pipe material, soiled surface, chlorine and nutrient levels with higher temperatures and nutrient levels promoting adherence. Considerable variation in adherence and biofilm formation was observed among representative Enterobacter isolates.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

This thesis is an investigation of methods to remediate methicillin-resistant Staphylococcus pseudintermedius (MRSP) biofilms through conventional and microfluidic-based in vitro assays. MRSP biofilm related infections are a major concern for veterinary clinicians as they may complicate remediation by the immune system or antimicrobials. Novel antimicrobials that have been found to reduce biofilm growth in other staphylococci were assessed in both mono- and combination therapy against MRSP biofilm. Quantitative assay results (p < 0.05) suggest fosfomycin alone and in combination with clarithromycin can significantly reduce biofilm formation. Morphological examination using scanning electron microscopy and atomic force microscopy further demonstrated the effectiveness of fosfomycin alone on biofilm formation on orthopaedic screws and mica sheets. Fabricated microfluidic assays were utilized to assess multiple concentrations of antimicrobial therapy against pre-formed biofilm under physiologically relevant conditions in a quick and repeatable manner. Results demonstrated the usefulness of microfluidic platforms in determining minimum biofilm eradication concentrations.

Objective: To assess biofilm proliferation within the sealer-dentin interfaces of methacrylate resin-based sealers, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP). Methods: Standardized human root specimens were filled with the test materials and were aged for 1 week, 1, 3 or 6 months in saline (n=3/group). Monoclonal biofilms of Enterococcus faecalis were grown on the specimens for 7 days in continuous media reactor. The extent of biofilm proliferation of E. faecalis within the sealer-dentin interface for each material at each incubation period was assessed using fluorescence microscopy of dihydroethidium-stained specimens. Results: TE had less biofilm proliferation than EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Conclusion: Self-etch and epoxy resin-based sealers were more susceptible to interfacial biofilm proliferation than total-etch system at shorter incubation periods.

No description available.

<p>Aim: To determine whether there was any significant difference in the antimicrobial activity of 4 herbal toothpastes against cultures of 3 primary plaque colonizers (Streptococcus mutans, Streptococcus sanguinis and a non-specific &alpha / -heamolytic streptococcus).</p>

Actinobacillus pleuropneumoniae (App) est l’agent étiologique de la pleuropneumonie porcine, une infection pulmonaire contagieuse chez les porcs. Parmi les nombreux mécanismes de virulence retrouvés chez les bactéries, la formation de biofilms joue souvent un rôle important dans la pathogenèse. Il a été récemment démontré qu’App avait la capacité de former des biofilms in vitro. Dans notre laboratoire, la formation de biofilms par App a été évaluée en microplaques dans différents milieux de culture. Nous avons démontré que la souche de référence de sérotype 1 est capable de former des biofilms. Le but de ce travail est d’identifier des gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. L’objectif de cette étude était de générer une banque de mutants d’App 4074NalR à l’aide du transposon mini-Tn10. Cette banque de 1200 mutants a été criblée à l’aide du modèle in vitro de formation de biofilms en microplaques et en tubes : 24 mutants démontrant une formation de biofilms modifiée par rapport à la souche mère App 4074NalR ont été sélectionnés et identifiés, nous permettant ainsi de localiser le site d’insertion du transposon. Une analyse a permis d’identifier de nouveaux gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. Notre criblage a permis d’identifier 16 gènes connus impliqués dans la formation de biofilms chez App (hns) ou chez d’autres pathogènes (potD2, ptsI, tig and rpmF) mais également de nouveaux gènes impliqués dans la formation de biofilm (APL_0049, APL_0637 and APL_1572). Une caractérisation plus poussée de ces gènes nous permettra d’améliorer la compréhension des mécanismes impliqués dans la formation de biofilm chez App. / A. pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a contagious pulmonary infection in swine. Among the numerous virulence mechanisms found in bacteria, the formation of biofilms often plays an important role in pathogenesis. It has been recently demonstrated that App has the ability to form biofilms in vitro. In our laboratory, the formation of biofilms by App has been evaluated in microplates under different growth conditions. We showed that the reference strain of serotype 1 is capable of forming biofilms when cultured in a specific growth medium. The objective of this work is to identifiy genes implicated in the biosynthesis and regulation of biofilm formation in App. The objective of this study was to generate a mutant library of App using the mini-Tn10 transposon. A total of 1200 mutants has been screened with the help of in vitro models for biofilm formation which use microtiter plates or test tubes; 24 mutants exhibited modified biofilm formation when compared to the parental strain 4074NalR. The selection and identification of these mutants allowed the identification of the insertion site of the transposon. Analysis revealed novel genes implicated in biosynthesis and regulation of the biofilm formation in App. Our screen allowed the identification of genes already associated in biofilm formation of App (hns) or other pathogens (potD2, ptsI, tig and rpmF). Genes (APL_0049, APL_0637 and APL_1573) that have not yet been associated with biofilm formation were also identified. Further characterization of the genes mentioned above would permit a greater understanding of the mechanisms implicated in biofilm formation of App.