Tesis sobre el tema "Spores and Biofilms"

Bacteria are often thought as single cell organisms, however they can develop into morphologically complex multicellular communities composed of different subpopulations of specialized cell types. Biofilm is an example, in which bacteria organize for protection from harmful conditions in the host and to create nutrient-rich areas. In the last years biofilm have been show to comprise an important aspect of microbial persistence in the human gut. Endospore-formers, although thought not to be major constituents of the microbiota in the human intestine, cause several intestinal diseases, usually associated with antibiotic use. Whether these bacteria persist in the intestine in biofilms or as endospores is not totally elucidated since both, biofilms and endospores, are able to resist to antimicrobial agents. Most likely sporulation and biofilm formation are tightly linked processes. For some endosporeformers, spore differentiation is induced by a sub-population of cells within the biofilm. In this work we tackled the link between bacterial biofilms and endosporulation in Bacillus subtilis. We showed that endospores produced in biofilms have higher resistance to UV radiation. We revealed that a gene, remA, conserved among endosporeformers and essential for biofilm formation is expressed during sporulation. remA is expressed in the forespore soon after asymmetric division and in the mother cell after engulfment completion. GerE represses remA expression in the mother cell at late stages of sporulation. Consequently, we found components of the biofilm matrix, TasA and BslA, on the coat of endospores produced in biofilms. We suggest that components of the biofilm matrix may be part of mature endospores. We hypothesize that some of the structural proteins that confer integrity to the matrix biofilm, as TasA, may have a role as a scaffold for the assembly of the endospore surface layers.
A percepção instalada é a de que as bactérias são organismos unicelulares. No entanto, estes organismos são capazes de se organizarem em comunidades multicelulares complexas compostas de subpopulações de células diferenciadas. Os biofilmes são um exemplo deste tipo de organização. Os biofilmes conferem protecção contra as condições desfavoráveis encontradas no hospedeiro, ao mesmo tempo que criam nichos ricos em nutrientes facilitando a implantação da população. Nos últimos anos foi demonstrado que a persistência microbiana no trato gastrointestinal humano se deve em larga medida à formação de biofilmes. Algumas bactérias que podem ser encontradas no trato gastrointestinal humano são ainda capazes de diferenciar um tipo celular altamente resistente a insultos químicos e físicos, o esporo. Nestes casos, não é claro se são os biofilmes ou os endoesporos os principais responsáveis pela persistência destes organismos, já que ambos são resistentes aos antibióticos. Neste trabalho exploramos a ligação genética entre a formação de biofilmes e a esporulação em Bacillus subtilis. Mostramos que os endoesporos produzidos em biofilmes exibem maior resistência aos UV. Mostramos que um gene, remA, conservado em bactérias formadoras de endoesporos e essencial para a formação de biofilmes é expresso durante a esporulação. remA é expresso no pré-esporo após a divisão assimétrica e na célula mãe após o envolvimento do pré-esporo. GerE reprime a expressão de remA na célula mãe em estádios tardios de desenvolvimento. Consequentemente, encontramos componentes da matriz do biofilme no manto de endoesporos maduros. Algumas das proteínas estruturais que conferem integridade à matriz do biofilme, como TasA, poderão servir como base para a montagem das camadas superficiais do esporo.

Le but de cette thèse est de développer des expériences pour comprendre la physique de la motilité dans deux systèmes microbiens, vivant dans le domaine du faible nombre de Reynolds, c’est-à-dire lorsque les forces visqueuses l’emportent sur les forces d’inertie. La première partie de la thèse traite de la croissance de biofilms bactériens sur une surface solide. Les biofilms bactériens sont des communautés de cellules étroitement empilées dans une matrice polymère. Du point de vue physique, ces colonies se comportent comme des gels et la matrice polymérique crée des flux osmotiques qui permettent aux biofilms de se développer et de se déplacer sur une surface en tant que communauté. Ici, je développe une expérience pour explorer la motilité collective des biofilms au contact de gradients externes de pression osmotique. Pour produire des gradients osmotiques stables dans des gels d’agar, je développe une configuration sur mesure au moyen de techniques de millifluidique. Les biofilms répondent au gradient externe en développant une forme asymétrique, conforme aux attentes. La deuxième partie de la thèse aborde le mécanisme de décharge des spores chez les basidiomycètes à phylum fongique. Chez ces espèces, une goutte coalesce avec la spore, ce qui entraîne son écoulement à des accélérations énormes. Cette catapulte à tension superficielle atteint son efficacité maximale lorsque la taille de la goutte est comparable à celle de la spore. J’étudie les morphologies de plusieurs champignons branchiaux, où les spores sont conditionnées à la surface de branchies complexes. Je trouve que pour ces espèces, si les spores sont emballées au maximum comme il est généralement supposé, la taille de la goutte qui coalesce avec la spore doit être bien contrôlée. Cela pose la question de comment est-ce que le champignon peut contrôler un processus purement extracellulaire, face aux fluctuations environnementales
The aim of this thesis is to develop experiments to understand the physics of motility in two microbial systems, living in the realm of low Reynolds number, i.e. when viscous forces dominate over inertial forces. The first part of the thesis discusses the growth of bacterial biofilms over a solid surface. Bacterial biofilms are communities of cells closely packed together inside a polymeric matrix. From the physical viewpoint, these colonies behave as gels and the polymeric matrix creates osmotic fluxes that enable biofilms to grow and move on a surface as a community. Here I develop an experiment to explore biofilm collective motility in contact with external gradients of osmotic pressure. To produce stable osmotic gradients in agar gels, I develop a custom-made setup through millifluidics. Biofilms respond to the external gradient by developing an asymmetric shape, consistent with the expectations. The second part of the thesis discusses the spore discharge mechanism in the fungal phylum Basidiomycetes. In these species, a drop coalesces with the spore, which results in spore discharge at enormous accelerations. This surface tension catapult reaches its maximum efficiency when the size of the drop is comparable to that of the spore. I study morphologies of several gilled mushrooms, where spores are packaged at the surface of complex shaped gills. I find that for those species, drop size must be precisely controlled. This poses the question of how mushrooms may regulate a process that occurs extracellularly, despite fluctuating physical conditions

Il existe de nombreuses technologies de décontamination, néanmoins les spores et les biofilms bactériens demeurent une préoccupation majeure dans de nombreux domaines, tels que le secteur hospitalier, alimentaire et de la biodéfense car elles sont résistantes. Une mousse novatrice contenant un biocide (l’hypochlorite de sodium ou le peroxyde d’hydrogène) et un agent stabilisant (le Xanthane) a été étudiée pour répondre à ce besoin. Cette mousse a la capacité d’être mise en oeuvre de différentes façons sur le terrain par : pulvérisation au sol ; talochage et pulvérisation sur les murs ; remplissage de pièces entières (murs et sols). Le travail de thèse est d’évaluer les modes d’action biocide de ces mousses sur les spores et les biofilms. Afin d’étudier le mode d’action de ces mousses des protocoles expérimentaux ont été mis au point sur les spores et les biofilms suivant leurs futures mises en oeuvre (horizontale, verticale et remplissage) et suivant différents facteurs environnementaux pouvant influencer leur efficacité de décontamination (températures, salissures, matériaux, …). L’ensemble de ce travail de thèse a permis de distinguer l’intérêt de la mousse au Xanthane contenant NaOCl 5% par rapport à celle H2O2 12% pour répondre aux besoins spécifiques de la décontamination des agents de la menace biologique. Cette mousse permet une décontamination rapide de 7 logs de spores en 30 minutes pour chacune des trois voies de mise en oeuvre à 20°C. De plus, elle permet la destruction de biofilms contenant 107 de bactéries/cm² en 1 heure maximum sur un support horizontal et par remplissage. Cette mousse NaOCl est suffisamment mature pour pouvoir réaliser un futur transfert industriel
Several decontamination technologies exist, however bacterial spores and biofilms remain a concern in a lot of fields, like hospital, alimentary and military. A new foam containing a biocide (sodium hypochlorite or hydrogen peroxide) and a stabilizing agent (Xanthan) has been studied to answer this problematic. This foam can be used in different ways on the field following contaminations: grounds’ spraying, walls’ covering and spraying, full pieces’ filling (walls and ground). The goal of this thesis is to evaluate the biocide efficiency of these foams on spores and biofilms. We optimized experimental protocols in order to study mechanisms of foams’ action on spores and biofilms based on theirs future applications (horizontal, vertical and filling) and depending on different environmental factors which may impact foam decontamination efficiencies (materials, temperatures, soil, …). This thesis work enabled to highlight the Xanthan foam containing 5% NaOCl from the one including 12% H2O2 in military sector. This foam allows a rapid decontamination, about 7 logs of spores in 30 minutes, for each of the three ways of use at 20°C. Moreover, the destruction of biofilms containing 107 logs of bacteria/cm² was achieved in 1 hour on a horizontal support by filling. This NaOCl foam is ready to be used for industrials

Les contaminants tels que les spores / biofilms sont problématiques dans nombreux secteurs de l'industrie alimentaire. En effet, même après les procédures d'hygiène, ces biofilm/spores pouvaient être retrouvés sur toutes les surfaces en contact direct ou non direct avec les aliments (Bénézech & Faille, 2018). Les risques associés aux biofilms peuvent être contrôlés soit en limitant le nombre des cellules adhérentes, soit en facilitant l'élimination des bactéries adhérentes. Le nettoyage en place (CIP) est une pratique de nettoyage courante et est largement utilisée dans les industries alimentaires ; cependant, il reste à un certain niveau une procédure de forte consommation d'eau. De plus, certaines études ont présenté quelques espèces bactériennes qui ont survécu même après le CIP et peuvent-être une source probable de contamination du produit.D'un autre côté, un fluide biphasique tel que la mousse peut exercer la même contrainte de cisaillement en paroi générée par un fluide monophasique, avec moins d'eau consommée. La mousse avec ses propriétés telles que le cisaillement peut être un paramètre clé pour un nettoyage mécanique des systèmes fermés tels que les tuyaux à faible consommation d'eau.Dans cette étude, nous avons étudié l'effet de l'écoulement de mousse dans les tuyaux et comparé son efficacité avec des conditions du type CIP standard sur le détachement des spores et des biofilms. La première approche consistait à travailler avec différents régimes d'écoulement de mousse (1D, 2D, 3D tout en augmentant la vitesse de 2 à 6 cm.s-1) ayant différentes qualités de mousse (quantité d'air: 50%, 60%, 70%) sur différentes espèces de micro-organismes où l'encrassement a été effectué soit en utilisant des spores de B. amyloliquefaciens 98/7 ou B. cereus 98/4 qui montrent une différence par leur caractère hydrophobe / hydrophile. Quant à P. fluorescens pf1, il a été utilisé comme un bon formateur de biofilm (biofilm de 24 heures) largement utilisé dans l'industrie alimentaire. L'encrassement a été réalisé à la position verticale ou à l'horizontale induisant des biofilms avec différentes structures. Les résultats du nettoyage de la mousse ont été comparés aux résultats des conditions de type CIP (la même action mécanique moyenne et la même concentration de tensioactif).La deuxième approche consistait à soumettre le flux de mousse à différentes singularités (réduction progressive de l'expansion soudaine - coudes) tout en travaillant avec un régime d'écoulement de mousse (1D 50%) et une espèce (B. amyloliquefaiciens 98/7 spores) pour mettre en évidence tout changement dans le flux de mousse sur l’efficacité de nettoyage.La troisième approche portait sur le travail avec une espèce (B. amyloliquefaciens 98/7) considérée comme un bon « outil microbien », en produisant la mousse à partir des différents surfactants (SDS, Capstone® FS 30, Ammonyx® LO). Ces derniers diffèrent par leurs propriétés chimiques (non ionique, anionique et zwitterion) et donc permettant de produire des mousses avec des propriétés physiques différentes en termes de taille, de nombre et de répartition des bulles, et de profil d'écoulement.Par rapport aux précédents travaux sur la caractérisation de mousse en écoulement, il a été possible de mettre en évidence le rôle potentiel de la variation de la contrainte de cisaillement en parois parallèlement à la variation d'épaisseur du film liquide au niveau des parois avec le passage des bulles, sur l'efficacité de nettoyage. De plus, selon des travaux antérieurs, l’existence des forces capillaires exercées sous les débits les plus faibles, la nature hydrophile / hydrophobe des spores et la structure du biofilm pourraient expliquer au moins en partie l'efficacité surprenante de l'élimination des spores par la mousse
Contaminants such as spores/biofilms are problematic in many food industry sectors. Indeed even after hygiene procedures, biofilms/spores could be found on every surface that is in direct contact or not with food (Bénézech & Faille, 2018). Risks associated with microorganisms can be controlled either by limiting the number of adherent cells or by facilitating the removal of adherent bacteria. Even though Cleaning in Place (CIP) is widely used and it is a common cleaning practice in food industries; however, it remains at some level a high- water consumption procedure. In addition, some studies, presented some bacterial species that still survived even after CIP and maybe a probable source of product contamination. On the other hand, a double phase fluid such as foam can impose the same wall shear stress with less water being consumed. Foam with its properties such as shearing can be key a parameter for a mechanical cleaning of closed systems such as pipes with a lower consumption of water.In this study we investigated the effect of flowing foam in pipes and compared its efficiency with standard CIP like conditions on the detachment of spores and biofilms. The first approach was working with different foam flow regimes (1D, 2D, 3D while increasing the velocity from 2 to 6 cm s-1) having different foam qualities (amount of air: 50%, 60%, 70%) on different species of microorganisms where fouling was performed either by using spores of B. amyloliquefaciens 98/7 or B. cereus 98/4 that shows a difference by their hydrophobic/hydrophilic character. As for P. fluorescens pf1 it was used as a good biofilm former (24 hrs. biofilm) widely encountered in the food industry. Fouling was performed either vertically or horizontally inducing biofilms with different structures. Results from foam cleaning were compared with CIP like conditions results (the same mean mechanical action, and the same concentration of surfactant). The second approach was subjecting foam flow to different singularities (sudden expansion gradual reduction – bends) while working with one foam flow regime (1D 50%) and one species (B. amyloliquefaciens 98/7 spores) to highlight any changes in the foam flow cleaning efficiency. The third approach was working also with one species (B. amyloliquefaciens 98/7) considered as a good “microbial tool” producing foam from the use of different surfactants (SDS, Capstone® FS 30, Ammonyx® LO) that differs by their chemical properties ( nonionic, anionic and zwitterion) thus producing different foams having different physical properties in terms of bubbles size , number and repartition, and flow pattern. Comparing to previous related works on foam flow characterization, it was possible to highlight the potential role on the cleaning efficiency of the Wall Shear Stress variations in parallel to the liquid film thickness variation at the wall with the bubbles' passage. In addition, according to previous work, the possible capillary forces exerted under the lowest flow rates and considering the hydrophilic/hydrophobic nature of the spores, in addition to biofilm structure would explain at least partly the surprising efficiency in the spores' removal by foam

Les contaminants tels que les spores / biofilms sont problématiques dans nombreux secteurs de l'industrie alimentaire. En effet, même après les procédures d'hygiène, ces biofilm/spores pouvaient être retrouvés sur toutes les surfaces en contact direct ou non direct avec les aliments (Bénézech & Faille, 2018). Les risques associés aux biofilms peuvent être contrôlés soit en limitant le nombre des cellules adhérentes, soit en facilitant l'élimination des bactéries adhérentes. Le nettoyage en place (CIP) est une pratique de nettoyage courante et est largement utilisée dans les industries alimentaires ; cependant, il reste à un certain niveau une procédure de forte consommation d'eau. De plus, certaines études ont présenté quelques espèces bactériennes qui ont survécu même après le CIP et peuvent-être une source probable de contamination du produit.D'un autre côté, un fluide biphasique tel que la mousse peut exercer la même contrainte de cisaillement en paroi générée par un fluide monophasique, avec moins d'eau consommée. La mousse avec ses propriétés telles que le cisaillement peut être un paramètre clé pour un nettoyage mécanique des systèmes fermés tels que les tuyaux à faible consommation d'eau.Dans cette étude, nous avons étudié l'effet de l'écoulement de mousse dans les tuyaux et comparé son efficacité avec des conditions du type CIP standard sur le détachement des spores et des biofilms. La première approche consistait à travailler avec différents régimes d'écoulement de mousse (1D, 2D, 3D tout en augmentant la vitesse de 2 à 6 cm.s-1) ayant différentes qualités de mousse (quantité d'air: 50%, 60%, 70%) sur différentes espèces de micro-organismes où l'encrassement a été effectué soit en utilisant des spores de B. amyloliquefaciens 98/7 ou B. cereus 98/4 qui montrent une différence par leur caractère hydrophobe / hydrophile. Quant à P. fluorescens pf1, il a été utilisé comme un bon formateur de biofilm (biofilm de 24 heures) largement utilisé dans l'industrie alimentaire. L'encrassement a été réalisé à la position verticale ou à l'horizontale induisant des biofilms avec différentes structures. Les résultats du nettoyage de la mousse ont été comparés aux résultats des conditions de type CIP (la même action mécanique moyenne et la même concentration de tensioactif).La deuxième approche consistait à soumettre le flux de mousse à différentes singularités (réduction progressive de l'expansion soudaine - coudes) tout en travaillant avec un régime d'écoulement de mousse (1D 50%) et une espèce (B. amyloliquefaiciens 98/7 spores) pour mettre en évidence tout changement dans le flux de mousse sur l’efficacité de nettoyage.La troisième approche portait sur le travail avec une espèce (B. amyloliquefaciens 98/7) considérée comme un bon « outil microbien », en produisant la mousse à partir des différents surfactants (SDS, Capstone® FS 30, Ammonyx® LO). Ces derniers diffèrent par leurs propriétés chimiques (non ionique, anionique et zwitterion) et donc permettant de produire des mousses avec des propriétés physiques différentes en termes de taille, de nombre et de répartition des bulles, et de profil d'écoulement.Par rapport aux précédents travaux sur la caractérisation de mousse en écoulement, il a été possible de mettre en évidence le rôle potentiel de la variation de la contrainte de cisaillement en parois parallèlement à la variation d'épaisseur du film liquide au niveau des parois avec le passage des bulles, sur l'efficacité de nettoyage. De plus, selon des travaux antérieurs, l’existence des forces capillaires exercées sous les débits les plus faibles, la nature hydrophile / hydrophobe des spores et la structure du biofilm pourraient expliquer au moins en partie l'efficacité surprenante de l'élimination des spores par la mousse
Contaminants such as spores/biofilms are problematic in many food industry sectors. Indeed even after hygiene procedures, biofilms/spores could be found on every surface that is in direct contact or not with food (Bénézech & Faille, 2018). Risks associated with microorganisms can be controlled either by limiting the number of adherent cells or by facilitating the removal of adherent bacteria. Even though Cleaning in Place (CIP) is widely used and it is a common cleaning practice in food industries; however, it remains at some level a high- water consumption procedure. In addition, some studies, presented some bacterial species that still survived even after CIP and maybe a probable source of product contamination. On the other hand, a double phase fluid such as foam can impose the same wall shear stress with less water being consumed. Foam with its properties such as shearing can be key a parameter for a mechanical cleaning of closed systems such as pipes with a lower consumption of water.In this study we investigated the effect of flowing foam in pipes and compared its efficiency with standard CIP like conditions on the detachment of spores and biofilms. The first approach was working with different foam flow regimes (1D, 2D, 3D while increasing the velocity from 2 to 6 cm s-1) having different foam qualities (amount of air: 50%, 60%, 70%) on different species of microorganisms where fouling was performed either by using spores of B. amyloliquefaciens 98/7 or B. cereus 98/4 that shows a difference by their hydrophobic/hydrophilic character. As for P. fluorescens pf1 it was used as a good biofilm former (24 hrs. biofilm) widely encountered in the food industry. Fouling was performed either vertically or horizontally inducing biofilms with different structures. Results from foam cleaning were compared with CIP like conditions results (the same mean mechanical action, and the same concentration of surfactant). The second approach was subjecting foam flow to different singularities (sudden expansion gradual reduction – bends) while working with one foam flow regime (1D 50%) and one species (B. amyloliquefaciens 98/7 spores) to highlight any changes in the foam flow cleaning efficiency. The third approach was working also with one species (B. amyloliquefaciens 98/7) considered as a good “microbial tool” producing foam from the use of different surfactants (SDS, Capstone® FS 30, Ammonyx® LO) that differs by their chemical properties ( nonionic, anionic and zwitterion) thus producing different foams having different physical properties in terms of bubbles size , number and repartition, and flow pattern. Comparing to previous related works on foam flow characterization, it was possible to highlight the potential role on the cleaning efficiency of the Wall Shear Stress variations in parallel to the liquid film thickness variation at the wall with the bubbles' passage. In addition, according to previous work, the possible capillary forces exerted under the lowest flow rates and considering the hydrophilic/hydrophobic nature of the spores, in addition to biofilm structure would explain at least partly the surprising efficiency in the spores' removal by foam

La caractérisation expérimentale et numérique du comportement rhéologique d'une mousse aqueuse s'écoulant à l'intérieur d'un tuyau horizontal avec et sans singularités (demi-expansion soudaine, et chicane) a été étudiée. Différentes conditions d'écoulement ont été étudiées en faisant varier les qualités de mousse (55-85%), et trois nombres de Reynolds (32, 65, et 97). Les mesures de la pression, de la répartition de la vitesse locale et de la contrariante et de l'épaisseur des films liquides au niveau de la paroi à l'aide respectivement de capteurs de pression, de la PIV, de la polarographie et de la conductimétrie ont montré une réorganisation de la mousse en aval du changement de géométrie, avec un film liquide plus épais au fond du conduit, des bulles de plus grande taille au sommet, ainsi qu'une plus grande fraction de vide de la mousse augmentant de la partie inférieure à la partie supérieure de la section du conduit. En outre, la mousse présenterait un caractère visco-élastique comparable à celui d'un liquide monophasique non newtonien. Des simulations de dynamique des fluides ont été entreprises pour prédire ce comportement rhéologique de la mousse, les deux modèles Herschel-Bulkley et Bingham ont été testés en tenant compte de la présence d'un film liquide sous-jacent au fond du canal. La comparaison entre les résultats expérimentaux et numériques a montré que, quelle que soit la qualité de la mousse, le modèle de Herschel-Bulkley pouvait décrire avec précision le comportement rhéologique de la mousse dans les différentes conditions d'écoulement analysées.Le deuxième objectif était d'étudier la capacité d'un écoulement de mousse humide (qualité de 50%) à nettoyer des surfaces en acier inoxydable contaminées par des micro-organismes. Pour cela, deux types de contamination ont été étudiés, des gouttelettes contenant des spores de Bacillus subtilis (soit hydrophiles Bs PY79 ou hydrophobes Bs PY79 spsA), et des biofilms produits par trois souches de bactéries (Escherichia coli SS2, Bacillus cereus 98/4, et Pseudomonas fluorescens Pf1). Différentes conditions d'écoulement ont été réalisées en faisant varier les contraintes de cisaillement pariétale (2.2 - 13.2 Pa), et la taille des bulles (0.18-0.34 mm) dans un conduit sans changement géométrique, afin d'identifier les mécanismes de libération de la contamination et ainsi mieux contrôler et optimiser le processus de nettoyage. Les résultats montrent que, par rapport au NEP conventionnel, le flux de mousse a éliminé efficacement les spores ainsi que les biofilms. De plus, la combinaison d'une contrainte de cisaillement pariétale élevée et de bulles de petite taille (<0,2 mm) s'est avérée prometteuse pour améliorer l'efficacité du nettoyage des spores. D'autre part, une nette amélioration de l'élimination des biofilms a été observée en augmentant la contrainte de cisaillement pariétale. La caractérisation de la mousse et des phénomènes d'interface a indiqué que des mécanismes tels que la fluctuation des contraintes de cisaillement locales, ou de l'épaisseur du film liquide entre les bulles et la paroi induite par le passage des bulles, l'imbibition de la mousse et le balayage de la contamination dans le film liquide, pourraient participer largement aux mécanismes d'élimination. Enfin, l'étude d'analyse du cycle de vie a démontré que le nettoyage à la mousse peut être une technique appropriée pour réduire la consommation d'eau et d'énergie (7 et 8 fois moins) présentant moins d'impacts environnementaux que les procédés CIP, avec environ 70%. Enfin, le nettoyage à la mousse peut être une méthode alternative, qui peut améliorer l'efficacité et réduire l'impact environnemental.D'autres activités menées au cours de la période de doctorat liées à la conception hygiénique sont présentées en soulignant le rôle des contaminants (spores et biofilms), du matériau (autre que l'acier inoxydable) et de la géométrie (conduits ou conception plus complexe) dans le contrôle de l'hygiène
In this research, experimental and numerical characterization of the rheological behavior of an aqueous foam flowing inside a horizontal pipe with and without singularities (presence of half-sudden expansion, and a fence) were investigated. Different conditions of foam flow were studied by varying the foam qualities (from 55% to 85%), and three Reynolds numbers (32, 65, and 97). Measurements of the pressure measurements, and at the wall the local velocity repartition and the thickness of the liquid films using respectively pressure sensors, Particle Image Velocimetry, and a conductimetry technique shown a reorganization of the foam downstream the geometry change, with a thicker liquid film at the duct bottom, larger bubble sizes at the top, as well as a larger foam void fraction increased from the bottom to the top part of the duct section. In addition, foam would present a visco-elastic character comparable to a non-Newtonian monophasic liquid. Computational Fluid Dynamics simulations were undertaken to predict this rheological behavior of the foam, the two models Herschel-Bulkley and Bingham were tested taken into account the presence of an underlying liquid film at the bottom of the channel . Comparison between experimental and numerical results showed that regardless of the foam quality, Herschel-Bulkley model could accurately describe the rheological behaviour of the aqueous foam under the different flow conditions analysed.The second target was to investigate the ability of a wet foam flow (quality of 50%) to clean stainless-steel surfaces contaminated by microorganisms. For this purpose, two different contamination patterns were studied, droplets containing Bacillus subtilis spores (either hydrophilic Bs PY79 or hydrophobic Bs PY79 spsA), and biofilms produced by three bacteria strains encountered in food industry production plants (Escherichia coli SS2, Bacillus cereus 98/4, and Pseudomonas fluorescens Pf1). Different flow conditions were performed by varying the wall shear stresses (2.2 - 13.2 Pa), and bubble sizes (0.18-0.34 mm) in a straight duct with no geometrical changes, in order to identify the mechanisms of contamination release and thus better control and optimize the foam cleaning process. Results show that compared to conventional cleaning-in-place, foam flow effectively removed Bs spores as well as Bc-98/4, Ec-SS2, and Pf1 biofilms. Moreover, the combination of high shear stress at the wall and small bubble sizes (<0.2 mm) showed promise for improving the cleaning efficiency of spores. On the other hand, a clear improvement of the biofilm removal was observed when increasing the mean wall shear stress. The characterization of the foam and the interface phenomenons (using polarography, conductimetry, and bubble size analysis methods) indicated that mechanisms such as fluctuation in local wall shear stresses, or in the liquid film thickness between the bubbles and the steel wall induced by bubble passage, foam imbibition, and sweeping of the contamination within the liquid film could participate largely to the removal mechanisms. Finally, the life cycle assessment study demonstrated that foam flow cleaning could be a suitable technique to reduce water and energy consumption (7 and 8 times less, respectively) presenting less environmental impacts than CIP processes, with about 70%. Lastly, foam flow cleaning can be an alternative method, which can improve efficiency and reduce environmental impact. Additional activities conducted during the PhD period related to hygienic design are presented highlighting the role of the contaminants (spores and biofilms), the material (other than stainless steel) and the geometry (ducts or more complex design) in hygiene monitoring

Orientador: Mirna Lucia Gigante
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O objetivo geral deste trabalho foi avaliar o efeito de diferentes matrizes na adesão e formação de biofilme em aço inoxidável por Bacillus cereus, bem como avaliar a eficiência dos procedimentos de higienização no controle de biofilmes de esporos desse micro-organismo. Nas duas primeiras etapas, avaliou-se a capacidade de adesão e formação de biofilme por B. cereus em aço inoxidável, com e sem prévio condicionamento da superfície, utilizando-se água, leite UHT desnatado e integral como matrizes e quatro diferentes tipos de inóculos, pool de células vegetativas de B. cereus isolados da indústria láctea, pool de esporos de B. cereus isolados da indústria láctea, células vegetativas da cepa de B. cereus ATCC 14579 e esporos da cepa de B. cereus ATCC 14579. Na terceira etapa do trabalho avaliou-se a influência da matriz condicionante (água e leite UHT integral), do meio de inoculação do pool de esporos de B. cereus (água e leite UHT integral) e do tempo de exposição (5 min (0,08h), 10, 24, 48 e 72 horas) sobre a adesão e formação de biofilme por B. cereus em aço inoxidável. Na quarta etapa, avaliou-se a eficiência de nove procedimentos de higienização na remoção dos biofilmes formados pelo pool de esporos de B. cereus em aço inoxidável. Todos os experimentos foram repetidos três vezes e os dados estatisticamente avaliados. A hidrofobicidade e o potencial zeta das superfícies dos esporos também foram avaliados. Os resultados das duas primeiras etapas indicaram que o pool de esporos de B. cereus isolados de indústria láctea apresentou a maior capacidade de adesão e formação de biofilme em aço inoxidável quando comparado aos outros tipos de inóculos, em todas as condições avaliadas. O maior grau de adesão de esporos de B. cereus (4,93 log UFC/cm2) foi observado ao se utilizar leite integral como matriz condicionante do aço inoxidável. Entretanto, comparando-se todas as matrizes, a menor adesão (3,01 log UFC/cm2) foi observada quando o pool de esporos de B cereus foi veiculado no leite integral sem prévio condicionamento da superfície. Na terceira etapa do trabalho observou-se que a adesão e formação de biofilme pelo pool de esporos de B. cereus foi maior quando inoculados em água, independente das matrizes de condicionamento. A adesão de B. cereus aumentou 1,02 e 0,3 log UFC/cm2 ao longo do tempo de exposição, quando o pool de esporos de B. cereus foi inoculado em água e leite integral, respectivamente. O biofilme de esporos veiculados na água apresentou maior resistência aos procedimentos de higienização. A sanitização com hipoclorito de sódio foi mais eficiente na remoção dos biofilmes quando comparada ao ácido peracético. O pool de esporos de B. cereus isolados da indústria láctea foi altamente hidrofóbico e apresentou carga negativa em uma ampla faixa de pH, com ponto isoelétrico de aproximadamente 3,0. Os esporos de B. cereus isolados da indústria láctea apresentaram maior capacidade de adesão ao aço inoxidável quando comparados aos outros inóculos avaliados, o que pode estar relacionado à alta hidrofobicidade e a baixa carga de superfície dos esporos
Abstract: The aim of this study was to evaluate the effect of different matrices on the adhesion and biofilm formation by Bacillus cereus on stainless steel, and to evaluate the effectiveness of sanitation procedures for controlling biofilm from spores of this microorganism. The first two parts were carried out in order to evaluate the adhesion and biofilm formation by B. cereus on stainless steel, with and without previous conditioning of the surface, using water, skim and whole UHT milk as matrices and four different types of inocula: a pool of B. cereus vegetative cells isolated from dairy industry, a pool of B. cereus spores isolated from dairy industry, vegetative cells of B. cereus ATCC 14579, and spores of B. cereus ATCC 14579. The third part of the study evaluated the effect of the conditioning matrix (water and whole UHT milk), the inoculation medium of pool of B. cereus spores (water and whole UHT milk) and exposure time (5 min (0.08h), 10, 24, 48 and 72 hours) on the adhesion and biofilm formation by B. cereus on stainless steel. In the fourth part, the effect of nine sanitation procedures on the removal of B. cereus spores biofilm was evaluated. All experiments were repeated three times and data were statistically evaluated. Hydrophobicity and zeta potential from spore¿s surface were also evaluated. Regarding the results to the first and second parts, the pool of B. cereus spores isolated from dairy industry had the highest ability of adhesion on stainless steel when compared to the other inocula, for all tested conditions. After stainless steel surface conditioning with whole milk, B. cereus spores showed the highest adhesion (4.93 log CFU/cm2). However, lower adhesion (3.01 log CFU/cm2) was observed when B. cereus spores were delivered in whole milk as compared to the other matrices, without previous conditioning of the surface. The results of the third part indicated that the adhesion and biofilm formation by the pool of B. cereus spores was higher when they were inoculated in water, regardless of the conditioning matrix. B. cereus spores adhesion increased by 1.02 and 0.3 log CFU/cm2 over exposure time, when the pool of B. cereus spores was inoculated into water and whole milk, respectively. Biofilm of B. cereus spores inoculated in water showed the highest resistance against all tested sanitation procedures. Sodium hypochlorite was the most effective sanitizer for removing all biofilms when compared to the peracetic acid. The pool of B. cereus spores isolated from dairy industry was highly hydrophobic and showed a negative charge at a wide pH range, with an isoelectric point of about 3.0. B. cereus spores isolated from dairy industry showed the highest ability to adhere on stainless steel when compared to the other inocula, which is possibly related to its higher hydrophobicity and lower spore surface charge
Doutorado
Tecnologia de Alimentos
Doutora em Tecnologia de Alimentos

Durante la mia attività di tesi mi sono focalizzata sulla caratterizzazione funzionale di una sorgente plasma surface Dielectric Barrier Discharge (sDBD) impiegata per l’inattivazione di spore batteriche di Geobacillus stearothermophilus, e per l’eradicazione di biofilm batterici di Staphylococcus aureus e Pseudomonas aeruginosa. Durante la prima attività è stata compiuta la caratterizzazione elettrica della sorgente sDBD e sono state condotte analisi di temperatura all’interno del volume di trattamento della sorgente. In seguito sono stati eseguiti test biologici per dimostrare l’efficacia del plasma nell’inattivazione di spore. I risultati ottenuti dimostrano come aumentando la potenza impiegata per generare la scarica di plasma aumenta anche il potere sterilizzante della sorgente e come il valore massimo di temperatura raggiunto sia rimasto al di sotto del valore di soglia. Nella seconda fase i biofilm di S. aureus e P. aeruginosa sono stati trattati tramite sorgente sDBD e l’efficacia del trattamento è stata valutata calcolando la Log Reduction. In seguito la morfologia dei biofilm è stata analizzata tramite il microscopio a scansione elettronica (SEM). I risultati mostrano come il trattamento di biofilm con sorgente plasma sDBD sia efficace sia per ceppi batterici gram positivi sia gram negativi. Le immagini al SEM hanno confermato l’effetto battericida dal plasma.

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2021
In the model organism Bacillus subtilis, the surface of the spore consists of a protein coat that affords protection against noxious chemicals and lytic enzymes and modulates spore germination and binding to cells and abiotic surfaces. The coat, formed by over 80 proteins, is divided into an inner layer, an outer layer and a crust. The coat proteins are synthesized in the mother cell and are recruited to the surface of the developing spores through specific protein-protein interactions and also according to successive waves of gene expression. SafA is a morphogenetic protein responsible for the assembly of the inner coat layer. SafA exists in three forms, the full-length protein, SafAFL, SafAC30 which corresponds to the C-terminal moiety of the protein, and SafAN21, corresponding to the N-terminal moiety of the protein. The C30 region is responsible for recruiting the inner coat proteins, whereas the N-terminal region carries the signals for localization at the spore surface. Among the proteins that SafA recruits to the inner coat is a transglutaminase, called Tgl, in a process termed substrate-driven localization. Assembly of SafA is auto-regulatory in that once assembled, Tgl introduces ε-(γ glutamyl)lysil bonds into SafA. SafAC30 forms an oblong hexamer, (SafAC30)6 and this species is cross linked in vitro by Tgl. Strikingly, the activity of Tgl decreased with increasing SafAC30 concentration, suggesting both that Tgl cross-linked itself to SafAC30 and a mechanism for controlling the activity of the enzyme in vivo. Here we overproduced and purified SafAFL and SafAN21 and tested for cross-linking by purified Tgl. Although Tgl proved to be much less efficient in cross-linking SafAN21 than SafAFL or SafAC30, its activity was inversely proportional to the concentration of all three substrates. Thus, in vitro, all three forms of SafA have the potential of controlling the activity of Tgl in a concentration-dependent manner. That SafAN21 is a poor substrate for Tgl, in spite of the fact that it contains several glutamine and lysine residues, suggests that this region of SafA is not involved in cross-linking to itself or to Tgl in vivo and raises the possibility that it may be involved in cross-linking to another protein or directly to the cortex peptidoglycan. Moreover, we found that formation of (SafAC30)6 involves disulfide bonds and that Tgl exerts a “spotwelding” activity on this species, i.e., not all the monomers in (SafAC30)6 are cross-linked by Tgl. Two lysine residues in SafAC30, K177 and K318 are important for this activity but K318 makes a more important contribution. Since K318 is close to two of the four cysteine residues in SafAC30, C323 and C325, and also in the close vicinity of several glutamine residues, a model is proposed in which disulfide bond formation at this position nucleates further cross-linking of (SafAC30)6 by Tgl. Veg, an 86 amino acid protein, was known to influence biofilm formation and spore germination. The veg gene is downstream of yabG, a gene that is part of a genomic signature for sporulation. yabG codes for a cysteine protease required for processing of the Tgl substrates prior to their cross-linking by Tgl. YabG also functions in the assembly of the spore surface layers in the human pathogen Clostridioides difficile. Here, we have shown that the overexpression of veg during the late stages of sporulation in B. subtilis impairs assembly of the abundant CotB and CotG, the latter a critical determinant of the structural organization of the outer coat. The assembly of other proteins, such as the peptidoglycan hydrolase YdhD and of a putative L-cysteine binding protein was also affected. Veg bears a Sm-like motif characteristic of proteins that regulate mRNA stability and translation in all domains of life. Molecular modelling studies suggest that, like the Sm proteins, Veg forms an heptamer with a central channel and a positively charged surface that may mediate interactions with single-stranded nuclei acids. Veg may thus define a novel level of control in spore coat assembly, influencing mRNA stability and/or translation and we propose that it functions in the same way in biofilm development. We overproduced and purified the C. difficile Veg protein, an 11 kDa protein with significant similarity to B. subtilis Veg. The structural and biochemical characterization of Veg will provide important insights into its function and its suspected functional relationship to YabG.

Thermophilic spore-forming bacteria such as Geobacillus spp. are common contaminants of milk powder processing plants. Their spores can remain viable throughout the entire dairy powder industrial process, including pasteurisation (72 °C for 15s) and the even hotter evaporator sections. Geobacillus spp. can form biofilms in dairy processing equipment that, over extended run times (e.g., >16 h), can deposit unacceptably high spore loads in end-product. While not dangerous to human health, this potentially leads to quality defects and price reductions, requiring that the process be stopped and the plant fully cleaned. The objective of this study was to: a. investigate the kinetics of Geobacillus growth, biofilm development and spore formation as a function of temperature and water activity, b. to develop an understanding of how these factors influence thermophile attachment and biofilm formation on stainless steel and affect the time before spore release into milk being processed into milk powder, and, based on this knowledge, c. explore options to extend run times of dairy powder plants Growth studies were undertaken, and a stainless-steel, laboratory bench-scale flow-through reactor was built and used, to investigate: 1. The effect of temperature (45 to 75 °C), media composition and water activity (0.959 to 0.992) on growth rates of 16 Geobacillus spp. originally isolated from milk powder processing plants; 2. the kinetics of attachment, biofilm formation and eventual release of new spores from spores inoculated into the flow-through reactor system via milk and with regard to spore inoculum levels, milk flow rates and temperature; 3. the effect on Geobacillus spp. growth of temperature step changes in the flow-through-system, and 4. the potential to disperse biofilms on stainless steel surfaces in the flow-through reactor using exogenously provided nitric oxide (NO). Growth rates of the 16 strains of Geobacillus spp. were modelled as function of temperature using a four-parameter square-root (‘Ratkowsky’) model. The model was developed to predict the growth of Geobacillus spp. under time-varying temperature conditions and to identify temperatures optimal for growth and biofilm formation. Over 300 growth curves were generated at temperatures in the range 45 to 75 °C, using different incubation methods, enumeration methods and growth media, although not all data sets were used because many were deemed to be unreliable due to insufficient or erratic growth of the spore-forming thermophiles under apparently well-controlled growth conditions, a phenomenon reported anecdotally by others. The studies showed that growth occurred in the temperature range 45 to ~70 °C with fastest growth occurring at ~60 °C. Consistent with published reports the generation time at 60 °C was estimated to be ~ 22 – 25 min. Studies at different water activities (a\(_w\)) suggested that the minimum water activity for growth was ~0.975. The results also showed that growth rate variability of all 16 strains is large compared to growth rate variability reported for non-spore forming cells. Nonetheless, no strain had growth rates that were systematically different to that of the pooled data. Growth rates observed for G. stearothermophilus W14 were representative of the average response of all strains and this strain was selected for use in subsequent studies. Growth rate data of G. stearothermophilus strain W14 under either anaerobic (100% N\(_2\)) or aerobic (20% O\(_2\)) conditions were also generated using a bioreactor (fermenter apparatus) at 55 °C, 60 °C, 65 °C, and 70 °C. The bioreactor was employed mainly to enable a comparison of aerobic and anaerobic growth rates by reliably producing anaerobic conditions. G. stearothermophilus strain W14 showed fastest growth at ~60 °C in anaerobic conditions with a doubling time of 26 min for vegetative cells, which was similar to growth rates under analogous aerobic conditions. The kinetics of cell and spore attachment to stainless steel were studied by inoculating the flow-through system with spores of G. stearothermophilus W14 in the milk flowing through the system and monitoring the change over time in vegetative cells and spores in the milk leaving the system (i.e., the ‘effluent’). Studies were conducted at temperatures from 45 to 70 °C. At near optimal temperatures (i.e., 60 – 65 °C) viable cell and spores counts initially decreased in the milk effluent but began to increase consistently after 3-6 h indicating attachment, germination and proliferation of cells and production of spores. Different milk flow rates applied to the system (5, 10, 20, 40 mL/min) showed no significant differences in the time for the generation of vegetative cells or spores. Spore inocula, fed into the system as pulses (1 h) or continuously added (usually for ~24 h but up to 40 h in some experiments), showed significant differences in the time to attachment and detectable proliferation, and as a function of temperature. In the pulsed system, the spore counts increased above the inoculum level after ~8 h of milk flow at near optimal temperatures, whereas in the continuous system levels in the effluent milk increased after ~4 h. In the pulsed system, a lower spore inoculum fed to the system (<10\(^2\) CFU/mL), resulted in a longer time before spore counts increased in the effluent (>8 h). Swabbing internal surfaces of the flow-through equipment at the end of “runs” at 45 °C showed early stage biofilm growth (>10\(^3\) CFU/cm\(^2\)) whereas runs at 65 °C showed high counts (>10\(^8\) log CFU/cm\(^2\)). The flow-through system was a useful way to study, under ‘commercially-relevant’ conditions, the attachment, growth and sporulation of thermophilic spore formers on warm stainless steel surfaces in milk powder plants and provided a system to study potential interventions against biofilm formation by thermophilic spore-forming bacteria. Based on Knight et al. (2004), who reported a method to minimise biofilm build up using temperature cycling to interrupt the growth cycle of non-spore forming bacteria in whole milk processing, temperature cycling studies were undertaken using the flow-through system to evaluate whether powder plant run times could be extended using the same approach. A number of experiments were conducted with temperature of the system altered systematically during the “run”. In some runs, there was reduced attachment and outgrowth of thermophilic spore-forming bacteria, suggesting that temperature cycling could extend run times if applied at the sites of most rapid attachment, growth and biofilm formation. The result showed that the temperature cycling should include temperatures near the limits of the temperature growth range of G. stearothermophilus to be able to significantly retard growth. Together with the temperature model, these data can be used as a foundation to estimate the expected benefits of manipulation of temperatures of milk powder processes. Nitric oxide (NO) has been reported to disrupt bacterial biofilms. Its potential for use to disrupt G. stearothermophilus biofilms in milk processing equipment was also studied using the flow-through system. At realistic and commercially relevant levels, NO did not significantly delay the time for unacceptable spore levels to occur in the milk effluent, although there was evidence of a reduction in final spore loads. However, recent studies have suggested that NO, while effective against Gram-negative biofilms, will not be ineffective against spore-forming Gram-positive bacterial biofilms. In short, the application of NO to extend run times is not supported, however, by the results of this study. Considering the ecology and physiology of G. stearothermophilus, and related species, the results of this study have reinforced that thermophile contamination during dairy powder processing will likely continue to be a difficult problem to address. This is because of the ability of spores to survive processing and cleaning, the rapid growth rate of the microorganism, its ability to form biofilms, and the inevitable production of spores in biofilms. Cell differentiation, including spore-formation, occurs through quorum sensing (QS) mechanisms and appears to be a ‘bet-hedging’ mechanism. It is concluded that thorough sanitation procedures will still be required at the end of each processing session to minimise residual fouling on stainless steel because the time to unacceptable spore loads also depends on initial contamination levels. While temperature cycling produced some effects, under some circumstances, further research is required to determine whether this approach can be manipulated and optimised to achieve commercially significant extension of powder plant run times.

Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs
Meeresschwamm-assoziierte Actinomyceten stellen ein Reservoir für verschiedene natürliche Produkte mit neuartigen biologischen Aktivitäten dar. Ihr antibiotisches Potenzial gegenüber einer Reihe von Gram-negativen und -positiven Bakterien ist bereits intensiv erforscht worden. Wenig ist allerdings über ihre antiinfektive und antivirulente Wirksamkeit gegenüber menschlichen Pathogenen bekannt. Ziel dieser Doktorarbeit war es, die antiinfektiven Fähigkeiten (anti-Shiga-Toxin und anti-Biofilm) der aus Schwämmen isolierten Actinobakterien zu untersuchen. Hierfür wurden bioaktive Metabolite der Actinobakterien identifiziert und isoliert und abschließend wurde ihr Wirkmechanismus mit Hilfe einer Transkriptomanalyse charakterisiert. Diese Arbeit ist in drei Studien gegliedert, welche alle zum Ziel hatten die antiinfektive Wirksamkeit von aus Actinomyceten gewonnenen Extrakten und Komponente(n), welche möglicherweise als zukünftige Therapeutika dienen könnten, zu untersuchen. Die erste Studie befasst sich mit den anti-Shiga-Toxin Effekten der Meeresschwamm- assoziierten Actinomyceten. Durchfallinfektionen stellen in vielen Entwicklungsländern aber auch in Industrieländern eine große Gefahr dar. Durchfallerkrankungen die durch enterohämorrhagische Escherichia coli (EHEC) hervorgerufen werden, können sich zu lebensbedrohlichen Komplikationen wie Gastroenteritis oder dem hämolytisch urenischen Syndrom (HUS) weiterentwickeln. Das von den EHEC Stämmen produzierte Shiga-Toxin (Stx) stellt hierbei den Haupt Virulenz Faktor dar, welcher die eukaryotische Proteinsynthese menschlicher Zellen negativ beeinflusst, was wiederum den Zelltod durch Apoptose zur Folge hat. Die Behandlung der EHEC-Patienten mit Antibiotika wird nicht empfohlen, da dies zu einem Anstieg von freigesetztem Stx der zersetzen Bakterien führen könnte, wodurch das Risiko für die Entwicklung des HUS ansteigt. Aus diesem Grund werden effektive Medikamente dringen benötigt, welche die Stx Produktion blockieren ohne das Wachstum der Bakterien zu beeinflussen. In dieser Studie wurden 194 Extrakte und einige isolierte Komponenten von aus Schwämmen gewonnenen Actinomyceten auf ihren negativen Einfluss auf die Stx Produktion des EHEC Stammes EDL933 mit der Hilfe des Ridascreen® Verotoxin ELISA Kits untersucht. Es konnte gezeigt werden, dass die Zugabe der Extrakte keinen signifikanten Einfluss auf die Stx Produktion hatte. Strepthonium A auf der anderen Seite, welches aus Streptomyces sp. SBT345 isoliert wurde (vom mediterranen Schwamm Agelas oroides) konnte die Stx Produktion von EDL933 bei einer Konzentration von 80 µM reduzieren ohne das Wachstum des EHEC Stammes zu beeinflussen. Die Struktur von Strepthonium A wurde mittels spektroskopischer Analyse (1D- und 2D-NMR), sowie mittels ESI-HRMS und ESI-HRMS2 Experimenten entschlüsselt. Basierend auf diesen Ergebnissen könnte Strepthonium A eine mögliche Alternative oder Zusatz in der Behandlung einer EHEC Infektion darstellen. In der zweiten Studie wurde der Einfluss der Meeresschwamm-assoziierten Actinomyceten auf die Biofilmbildung von Staphylokokken bewertet. Medizinische Produkte wie Kontakt Linsen, metallische Implantate, Katheter, Herzschrittmacher, usw. stellen optimale ökologische Nischen für die Ausbildung von bakteriellen Biofilmen dar, wodurch Infektionen im Menschen hervorgerufen werden können. Bakterien in einem Biofilm sind deutlich toleranter gegenüber der Immunantwort ihres Wirtes sowie gegenüber konventionellen Antibiotika und sind daher schwer zu bekämpfen. In dieser Studie wurde das anti-Biofilm Potential eines organischen Extrakts der flüssigen Fermentation von Streptomyces sp. SBT343 (vom mediterranen Schwamm Petrosia ficiformis) ermittelt. In vitro Ergebnisse zeigten, dass das organische Extrakt anti-Biofilm (gegenüber Staphylococci) Fähigkeiten besitzt und nicht toxisch für Maus Makrophagen (J774.1), Fibroblasten (NIH/3T3) und humane korneale Epithelzellen ist. Zudem konnte gezeigt werden, dass das SBT343 Extrakt die Ausbildung eines Biofilms von Staphylokokken auf den Oberflächen von Polystyrol, Glass und Kontaktlinsen unterbinden konnte ohne das bakterielle Wachstum zu beeinflussen. Die hochauflösende Fouriertransformation-Massenspektrometrie (HR-MS) Analyse konnte die Komplexität sowie die chemische Vielfalt an Komponenten im Extrakt aufzeigen. Eine vorläufige, physio-chemische Charakterisierung deutet darauf hin, dass die aktive Komponente im Extrakt hitzestabil und nicht proteinartiger Natur ist. Abschließend konnte durch Fraktionierungsexperimente gezeigt werden, dass die biologische Aktivität auf synergistischen Effekten mehrerer Komponenten im Extrakt beruht. In einer dritten Studie wurden 50 organische Extrakte, welche aus fester und flüssiger Fermentierung von 25 verschiedenen aus Meeresschwämmen isolierten Actinomyceten gewonnen wurden, auf anti-Biofilm-Aktivität untersucht. Hierbei wurde die anti-Biofilm Aktivität des organischen Extrakts der Festkultur von Streptomyces sp. SBT348 (vom mediterranen Schwamm Petrosia ficiformis) identifiziert. Eine Bioassay gestützte Fraktionierung führte zu der Identifikation der aktiven Fraktion Fr 7 im SBT348 Extrakt. Durch weitere Aufreinigung des Extrakts mit einer semipräparativen HPLC, konnten die bioaktiven Sub-Fraktionen SKC1, SKC2, SKC3, SKC4 und SKC5 isoliert werden. Die Sub- Fraktion SKC3 hatte den stärksten anti-Biofilm Effekt und bestand aus einer reinen Verbindung mit BIC90 und MIC Werten von 3,95 µg/ml und 31,25 µg/ml gegen S. epidermidis RP62A. SKC3 zeigte weder erkennbare Toxizität gegenüber Zelllinien in vitro noch gegenüber den Larven der großen Wachsmotte Galleria melonella in vivo. SKC3 war Hitze- und Enzym-resistent, was auf eine nicht proteinartige Natur hindeutet. Eine HR-MS Analyse ergab, dass die Masse von SKC3 1258,3 Da beträgt. Die Strukturanalyse von SKC3 durch 1D und 2D-NMR ist zurzeit in Bearbeitung. Um weiteres Verständnis über den anti-Biofilm Wirkmechanismus von SKC3 auf S. epidermidis RP62A zu erlangen, wurde eine RNA Sequenzierungsanalyse durchgeführt. Die Transkriptomanalyse zeigte, dass SKC3 von RP62A nach einer 20-minütigen Inkubationszeit erkannt wird und dass SKC3 den zentralen Metabolismus des Staphylokokken Stammes nach 3 h negativ beeinflusst. Zusammengenommen deuten die Ergebnisse darauf hin, dass SKC3 als Leitstruktur für die Entwicklung neuer anti- Staphylokokken Medikamente dienen könnte. Zusammenfassend heben die Ergebnisse dieser Arbeit die antiinfektiven Eigenschaften der Meeresschwamm-assoziierte Actinomyceten hervor und bieten eine Möglichkeit für die Nutzung dieser in Wirkstoffentwicklungsprogrammen

The growth of thermophiles during the manufacture of milk powder leads to a progressive increase in the number of thermophilic bacteria contaminating the final product. The limited residence time of the milk in the plant during milk powder manufacture and the concentration effect of converting milk into milk powder cannot explain the number of thermophiles found in the final product. This suggests that thermophiles are attaching to the large surface area of stainless steel found within a milk powder plant and then growing and developing into biofilms, with individual cells and/or biofilm fragments sloughing off into the product line and thus contaminating the final product. The aim of the present study was to investigate the attachment mechanisms that enable the thermophile Anoxybacillus flavithermus (B 1 2) to attach to stainless steel surfaces. Passing a B 1 2 culture through a column of stainless steel chips, collecting the first cells to pass through, re-culturing and repeating the process six times, resulted in the isolation of a mutant, labelled X7, with lO-fold reduced ability to attach to stainless steel as well as a reduced ability to attach to plastic and glass. A comparison of bacterial cell surface properties indicated that X7 was less hydrophobic than its parental strain B 1 2 . Cell surface charge measurements also suggest that X7 has less net negative surface charge. Disruption of extracellular polysaccharides and DNA appeared to have no effect on the attachment process. Removal of surface proteins caused a reduction in attachment of B 1 2 and X7 as well as a reduction in surface hydrophobicity suggesting surface protein involvement in both. Analysis by two-dimensional gel electrophoresis of lysozyme/mutanolysin extracted surface proteins revealed two proteins expressed at reduced levels in X 7 compared with B 1 2 . One protein was identified by mass spectrometry as the cytoplasmic enzyme Formate acetyltransferase. The role of Formate acetyltransferase and the second unidentified protein on the attachment process of Anoxybacillus flavithermus remains unclear.