Neste trabalho, investigou-se o crescimento e as interações de microorganismos, anaeróbios, em batelada com células planctônicas e em reatores diferenciais com biofilme, sob condições termofílicas (55ºC). Foram utilizadas técnicas clássicas para caracterizar os microorganismos anaeróbios estritos e biologia molecular (FISH), com sondas fluorescentes específicas, para caraterizar e quantificar os microrganismos pertencentes ao domínios Archea e Bacteria. Foram realizadas experimentos com meios de cultivo contendo acetato de sódio e propionato de sódio, na presença de sulfato de sódio. Os resultados do FISH mostraram, para os ensaios em batelada com acetato e sulfato, o predomínio de organismos pertencentes ao Domínio Archea (ARC915+MB1174) representados pelo gênero Methanosarcina sp. e bacilos fluorescentes. No biofilme, para a mesma condição, houve predomínio de células do Domínio Bacteria (EUB338) e relacionadas com a oxidação do acetato. Para a condição de cultivo com lactato e sulfato, as células bacterianas semelhantes a Desulfotomaculum sp. predominaram nos reatores em batelada e diferenciais, justificando a elevada porcentagem de células do Domínio Bacteria. Os resultados do NMP mostraram uma maior concentração de anaeróbios totais na condição lactato e sulfato (\'10 POT.11\' - \'10 POT.12\'). Para as arqueas metanogênicas essa concentração foi eveidenciada para a condição de cultivo com acetato e sulfato (\'10 POT.4\'). Os resultados desse trabalho sugeriram que em reatores em batelada, na condição de cultivo com acetato e sulfato, ocorreu equilíbrio entre metanogênese, oxidação de acetato e sulfatogênese, enquanto no biofilme, foram favorecidos os dois primeiros tipos de metabolismo. A condição lactato mais sulfato favoreceu a sulfetogênese em ambos os reatores. No reator diferencial, alimentado com proprionato e sulfato ocorreu fermentação com crescimento sintrófico entre oxidadoras de proprionato, metanogênicas e redutoras de sulfato hidrogenotróficas. / In this work, were investigated the growth and interactions of anaerobic microorganisms, in batch and differential reactors with planktonic cells and biofilm, respectively, in thermophilic conditions (55°C). Classic and molecular biology methods (FISH) were utilized to characterize strict anaerobes, with specific fluorescence probes, to characterize the Archaea and Bacteria Domain cells. Experiments with sodium acetate, s01ium lactate and sodium propionate plus sodium sulfate were realized. To the acetate and sulfate batch assays the results of FISH showed the predominance of Archaea Domain cells (ARC915+MB1174), represented by genera Methanosarcina sp. and fluorescents rods. Under the same conditions the biofilm results showed predominance of Bacteria Domain cells (EUB338) and those related to the acetate oxidation. In lactate plus sulfate condition the bacteria cells, as Desulfotomaculum sp., predominated in batch and differential reactors, justifying the high percentage of Bacteria Domain cells. The MPN results showed a high concentration of total anaerobes under lactate plus sulfate condition (\'10 POT.11\' - \'10 POT.12\'). For methanogenic arquea this concentration was obtained in the acetate plus sulfate condition (\'10 POT.4\'). These results suggest a process equilibrium may have occurred among methanogenesis, acetate oxidation and sulfidogenesis in batch reactors under acetate plus sulfate conditions. However, methanogenesis and acetate oxidation metabolisms predominated in the biofilm. Sulfidogenesis was easier in both reactors, under lactate plus sulfate conditions. In the differential reactors with propionate plus sulfate, the fermentation was in syntrophic co-culture among propionate oxidizing bacteria, methanogenic and hydrogenotrophic sulfate reducing bacteria.

Lorsqu'un biofilm se développe dans des conditions statiques, deux populations, sessiles et planctoniques, coexistent et peuvent échanger des cellules. Cependant, l'immigration de cellules planctoniques dans un biofilm n'a, jusqu'à présent, fait l'objet que de peu d'études dans le cas d'un biofilm monoespèce. Chez B. cereus, un pathogène alimentaire, notre équipe a récemment montré que des bactéries planctoniques mobiles peuvent pénétrer en profondeur à l'intérieur d'un biofilm formé en immersion. L'objectif du présent travail était, en partant de ces données, de déterminer le rôle du recrutement dans le développement du biofilm formé en interface air-liquide, et de caractériser ce processus d’un point de vue physiologique et moléculaire. Nous avons montré que la population planctonique est massivement recrutée par le biofilm, mais que, dans nos conditions expérimentales, ce recrutement ne contribue que de façon marginale à la croissance du biofilm. Nous avons mis au point deux dispositifs expérimentaux (en interface air-liquide et en immersion) permettant de quantifier le recrutement, ce qui nous a permis de cribler une banque de mutants, obtenue par mutagenèse aléatoire, pour sélectionner des clones inaptes à être recrutés. Le criblage de 1700 clones a abouti à la sélection d'un gène: Bthur002_62720. La délétion de ce gène par échange allélique affecte fortement la capacité du mutant à être recruté, et la complémentation rétablit le phénotype sauvage. Ce gène code pour une protéine probablement localisée dans l'enveloppe bactérienne. Il est porté par un plasmide, pCT8513, et pourrait être un élément mobile dont l'acquisition augmenterait fortement la capacité de la bactérie réceptrice à être recrutée par un biofilm. Enfin, nous avons mis en évidence le rôle du locus eps dans le recrutement des cellules planctoniques par un biofilm formé en interface air-liquide. Ce locus est homologue du locus epsA-O de Bacillus subtilis, requis chez cette espèce pour la production des exopolysaccharides de la matrice du biofilm. Chez B. cereus, nous avons montré que le locus eps est impliqué dans la formation d’une gaine d’exopolysaccharides faiblement liée à la paroi bactérienne. Cette couche d'exopolysaccharides contribue, avec d'autres exopolysaccharides d'origine inconnue, à la formation de la matrice du biofilm, et joue un rôle important dans l'adhésion de la bactérie sur des surfaces inertes et vivantes. En favorisant l'adhésion de la bactérie sur des surfaces vivantes, le locus eps pourrait faciliter son intégration dans le biofilm. Il pourrait également être impliqué dans le pouvoir pathogène de la bactérie. / When biofilm is developing in static conditions, cell exchanges between sessile and planktonic coexisting population can emerge. Up to now, very few are known about the implication of planktonic cells integration in monospecies biofilm development. in B. cereus, a foodborne pathogen, our team have shown that motility is a key factor for biofilm development and for the deep penetration of motile planktonic bacteria inside a biofilm formed in immersed condition. Based on these data, the purpose of the present work was to determine the role of recruitment in the development of biofilm in air-liquid interface and to characterize this phenomenon physiologically and in a molecular aspect. We showed that a massive planktonic population is integrated in the developing biofilm, however, in our experimental conditions this recruitment contributes only marginally to the biofilm growth. We have developed two recruitment systems (in air-liquid interface and in immersion condition), to quantify recruitment, which has allowed us to screen a library of mutants obtained by random mutagenesis in order to select clones unable to be recruited by a preformed biofilm. Screening of 1700 clones resulted in the selection of a gene: Bthur002_62720. The deletion of this gene by allelic exchange strongly affects the ability of the mutant to be recruited, and complementation restored the wild type phenotype. This gene encodes a protein probably localized in the bacterial envelope. It is carried by a plasmid, pCT8513, and could be a mobile element whose acquisition would greatly increase the ability of the recipient bacterium to be recruited by a biofilm. Finally, we have highlighted the role of the eps locus in the recruitment of planktonic cells in a biofilm formed at air-liquid interface. This locus is homologous to Bacillus subtilis epsA-O locus, required in this species for the production of exopolysaccharides of the biofilm matrix. In B. cereus, we have shown that the eps locus is involved in the formation of an exopolysaccharides sheath weakly bound to the bacterial cell wall. This exopolysaccharides layer contributes with other exopolysaccharides of unknown origin, to the formation of the biofilm matrix, and plays an important role in the adhesion of bacteria on inanimate and living surfaces.By promoting bacterial adhesion on living surfaces; the eps locus could help bacteria integration into the biofilm. It could also be involved in the pathogenicity of bacteria.

Neste trabalho, investigou-se o crescimento e as interações de microorganismos, anaeróbios, em batelada com células planctônicas e em reatores diferenciais com biofilme, sob condições termofílicas (55ºC). Foram utilizadas técnicas clássicas para caracterizar os microorganismos anaeróbios estritos e biologia molecular (FISH), com sondas fluorescentes específicas, para caraterizar e quantificar os microrganismos pertencentes ao domínios Archea e Bacteria. Foram realizadas experimentos com meios de cultivo contendo acetato de sódio e propionato de sódio, na presença de sulfato de sódio. Os resultados do FISH mostraram, para os ensaios em batelada com acetato e sulfato, o predomínio de organismos pertencentes ao Domínio Archea (ARC915+MB1174) representados pelo gênero Methanosarcina sp. e bacilos fluorescentes. No biofilme, para a mesma condição, houve predomínio de células do Domínio Bacteria (EUB338) e relacionadas com a oxidação do acetato. Para a condição de cultivo com lactato e sulfato, as células bacterianas semelhantes a Desulfotomaculum sp. predominaram nos reatores em batelada e diferenciais, justificando a elevada porcentagem de células do Domínio Bacteria. Os resultados do NMP mostraram uma maior concentração de anaeróbios totais na condição lactato e sulfato (\'10 POT.11\' - \'10 POT.12\'). Para as arqueas metanogênicas essa concentração foi eveidenciada para a condição de cultivo com acetato e sulfato (\'10 POT.4\'). Os resultados desse trabalho sugeriram que em reatores em batelada, na condição de cultivo com acetato e sulfato, ocorreu equilíbrio entre metanogênese, oxidação de acetato e sulfatogênese, enquanto no biofilme, foram favorecidos os dois primeiros tipos de metabolismo. A condição lactato mais sulfato favoreceu a sulfetogênese em ambos os reatores. No reator diferencial, alimentado com proprionato e sulfato ocorreu fermentação com crescimento sintrófico entre oxidadoras de proprionato, metanogênicas e redutoras de sulfato hidrogenotróficas. / In this work, were investigated the growth and interactions of anaerobic microorganisms, in batch and differential reactors with planktonic cells and biofilm, respectively, in thermophilic conditions (55°C). Classic and molecular biology methods (FISH) were utilized to characterize strict anaerobes, with specific fluorescence probes, to characterize the Archaea and Bacteria Domain cells. Experiments with sodium acetate, s01ium lactate and sodium propionate plus sodium sulfate were realized. To the acetate and sulfate batch assays the results of FISH showed the predominance of Archaea Domain cells (ARC915+MB1174), represented by genera Methanosarcina sp. and fluorescents rods. Under the same conditions the biofilm results showed predominance of Bacteria Domain cells (EUB338) and those related to the acetate oxidation. In lactate plus sulfate condition the bacteria cells, as Desulfotomaculum sp., predominated in batch and differential reactors, justifying the high percentage of Bacteria Domain cells. The MPN results showed a high concentration of total anaerobes under lactate plus sulfate condition (\'10 POT.11\' - \'10 POT.12\'). For methanogenic arquea this concentration was obtained in the acetate plus sulfate condition (\'10 POT.4\'). These results suggest a process equilibrium may have occurred among methanogenesis, acetate oxidation and sulfidogenesis in batch reactors under acetate plus sulfate conditions. However, methanogenesis and acetate oxidation metabolisms predominated in the biofilm. Sulfidogenesis was easier in both reactors, under lactate plus sulfate conditions. In the differential reactors with propionate plus sulfate, the fermentation was in syntrophic co-culture among propionate oxidizing bacteria, methanogenic and hydrogenotrophic sulfate reducing bacteria.

Thesis (PhD(Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa. / AFRIKAANSE OPSOMMING: Patogene Flavobacterium spp. veroorsaak ernstige infeksie uitbrake in ’n verskeidenheid gekweekte vissoorte, wat jaarliks tot groot ekonomiese verliese in die akwakultuur bedryf lei. Die vermoë van Flavobacterium johnsoniae-tipe isolate om as oppervlak-gehegde gemeenskappe (biofilms) in akwakultuur sisteme te groei bedreig visgesondheid oor verlengde periodes. Die vermoë van 28 F. johnsoniae-tipe isolate om biofilms te vorm is vergelyk met hul vermoë om chitien te degradeer, die profiel van hul ekstrasellulêre koolhidraat komplekse (EKK) en bandpatrone verkry met puls-veld jel elektroforese (PVJE). Fisiologiese veranderinge in die proteoom van 5-dagoue planktoniese-, biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 is met twee-dimensionele (2-D) jel elektroforese geanaliseer. Sewentien differensieël uitgedrukte en 14 uniek uitgedrukte proteïene is deur middel van matriks-geassisteerde laser desorpsie ioniserings-tyd van vlug-massa spektrometrie (MGLDI-TVV MS) geïdentifiseer. Twee-en-dertig differensieël uitgedrukte gene in 5-dag-oue biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 was deur middel van suppressie afgetrokke hibridisasie (SAH) geïdentifiseer. Beduidende negatiewe korrelasies is tussen die chitin-degraderings vermoë en EKK en die biofilm-vormings kapasiteit van 24-uur-oue biofilm kulture van F. johnsoniae-tipe isolate waargeneem. Resultate verkry met PVJE het die heterogene samestelling van F. johnsoniae-tipe isolate uitgewys. ‘n Beduidende positiewe korrelasie is tussen PVJE groeperings en vis gasheer spesie waargeneem. Differensieël en uniek uitgedrukte gene geidentifiseer in die planktoniese-, biofilm- en agar oppervlak-geassosieerde fases is deur middel van 2-D/MS asook differensieël uitgedrukte gene geïdentifiseer in die biofilm en agar oppervlakgeassosieerde fases deur middel van SAH was as betrokke by aanpassing/beskerming, metaboliese prosesse, membraan/vervoer/ beweeglikheid en transkripsie/translasie gekategoriseer. Sover bekend is hierdie die eerste beskrywing van differensieël uitgedrukte gene en geenprodukte van F. johnsoniae-tipe isolate afkomstig van geinfekteerde vis in Suid Afrika.

Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 tese_marcia_magalhaes_mata.pdf: 1998479 bytes, checksum: c18127c54931180d99c33eb9ceb106cd (MD5) Previous issue date: 2013-06-24 / L. monocytogenes is the etiologic agent of listeriosis, a severe food-borne disease. This pathogen has also variable ability to adhere to food-processing surfaces. Thus, the aims of this study was at first to evaluate the influence of the temperature (04-10-25-37°C) and time of incubation (24-48-168h) on the formation of attached cells by L. monocytogenes strains of diverse origins, serotypes and lineages using a colorimetric microtitre plate method. After this, comprehensive proteomics experiments using label-free 1D- liquid chromatography/tandem mass spectrometry (1D-LC/tandem MS) were performed to determine if the global proteomic responses of L. monocytogenes strains (Siliken and F2365) is altered markedly as attached cells compared to its planktonic state when growth media and temperature are the same. Our results showed that attached cells produced by different origins of L. monocytogenes did not change significantly when subjected to experimental conditions, unlike what was observed with attached cells produced by different serotypes and lineages of L. monocytogenes, which were clearly affected by environmental conditions.such as temperature and time of incubation. The ability of lineage II and serotype 1/2a and 1/2b to form large amount of attached cells when compared with the others in specific conditions indicates that risks from Listeria adherence must be taken seriously in sensitive food environments in order to find safer alternatives to prevent contamination and further dissemination of listeriosis. Only 8 proteins demonstrated substantial changes in common between both strains and temperatures in attached cells compared to their planktonic counterparts. They are: GroEL, DnaK, PtsH, PdxS, Pgi, RpsB, RpsD, and RpsP. Moreover, it was observed that the cell surface protein BapL abundance, though low, was not enhanced in attached cells suggesting its role in adherence could be a generalized contribution to the cell wall hydrophobicity. Interestingly, our experiment suggest that at 25°C the attached cells in both strains undergo flagella synthesis repression. Also, Sig B Regulon can be associate with an enhanced general stress response occurs in lineage II Strain (Siliken) but not in lineage I Strain (F2365) and could relate to the consequences of attachment. The temporal survey-based approach demonstrates clearly that high coverage represents a powerful means to investigate dynamic responses in L. monocytogenes from a functional genomics perspective / L. monocytogenes é o agente etiológico da listeriose, uma doença severa de origem alimentar. Esse patógeno também é capaz de se aderir a uma grande variedade de superfícies do processamento de alimentos. Sendo assim, os objetivos deste estudo foram primeiramente, avaliar a influência da temperatura (04-10-25 e 37°C) e tempo de incubação (24-48-168h) na formação de células aderidas de cepas de L. monocytogenes de diferentes origens, sorotipos e linhagens utilizando o método colorimétrico em placas de microtitulação. Após, experimentos de proteômica abrangentes que não utilizam marcadores, como a Cromatografia líquida de 1D/ espectrometria de massa em tandem (1D-LC/tandem MS) foram realizadas para determinar se o perfil proteico global de células planctônicas e de células aderidas de cepas de L. monocytogenes (Siliken e F2365) foi alterado significativamente quando os meios de crescimento e de temperatura de incubação foram os mesmos. A partir dos resultados obtidos verificou-se que as células aderidas formadas por L. monocytogenes de diferentes origens não sofreram alterações significativas quando submetidas às condições experimentais, diferentemente do que foi observado com as células aderidas formadas por L. monocytogenes de diferentes sorotipos e linhagens, as quais foram claramente afetadas pelas condições do ambiente. A habilidade da linhagem II e dos sorotipos 1/2a e 1/2b de formar grande quantidade de células aderidas quando comparadas com as demais, em condições específicas, indica alto risco de contaminação e disseminação da listeriose, bem como a sobrevivência e persistência deste micro-organismo no ambiente. Com base na análise proteômica, apenas oito proteínas demonstraram alterações substanciais em comum entre ambas as cepas e temperaturas em células aderidas comparadas com suas respectivas células planctônicas. São elas: GroEL, DnaK, PtsH, PdxS, Pgi, RpsB, RpsD, and RpsP. Verificou-se também que a abundância da proteína de superfície celular BapL, embora baixa, não foi aumentada em células aderidas sugerindo que o seu papel na adesão pode ser uma contribuição generalizada para a hidrofobicidade da parede celular. De forma muito interessante, nosso experimento sugere que células aderidas a 25°C por ambas as cepas levam a uma síntese de repressão flagelar. E ainda, Sig B Regulon pode estar associado com o aumento em geral da resposta ao estresse ocorrido na cepa de linhagem II (Siliken) mas não na cepa de linhagem I (F2365) o que pode estar relacionado com as consequências da adesão. A técnica utilizada no experimento demonstrou claramente que com alta abrangência é possível estudar proteomas bacterianos representando assim uma ferramenta poderosa para investigar respostas dinâmicas em L. monocytogenes através de uma perspectiva de genômica funcional.

This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.