University, Diss., 2006--Hannover.

Ulm, Univ., Diss., 2008.

À ce jour, la thèse n’a pas été déposée. L’Université Clermont Auvergne est donc dans l’impossibilité d’en assurer le traitement, la conservation et la diffusion. / To date, this thesis has not been deposited. The Université Clermont Auvergne is therefore unable to ensure its processing, conservation and dissemination.

Pseudomonas aeruginosa is one of the most studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. The transition from a planktonic to a biofilm mode of growth results in diverse changes in gene expression, which causes the attaching cells to become phenotypically and metabolically distinct from their planktonic counterparts. In this study, a proteomic approach was used to study differences in protein profiles obtained from 18-h old P. aeruginosa PAO1 (DSM 1707) planktonic, surface influenced planktonic (SIP) and biofilm populations grown in batch in the absence or presence of a glass wool substratum. Glass wool as an attachment substratum not only supported growth of biofilms, but it also allowed for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterisation. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells and several uniquely expressed proteins were seen on the 2-DE protein maps of the respective populations. Whereas a general down-regulation of protein expression was seen in the biofilm cells, in SIP cells, expression of the proteins was generally up-regulated. The results confirmed that the biofilm population differs from the planktonic population and indicated that the SIP population is not merely a mixture of planktonic and biofilm cells but rather a unique phenotype. Several differentially expressed protein spots were selected and identified using a combination of N-terminal protein sequencing and peptide mass fingerprinting. The proteins comprised mostly of outer membrane or membrane-associated proteins. Based on these analyses, a mutant P. aeruginosa strain, deficient in outer membrane protein OprG, was generated and its ability to form biofilms on a glass wool substratum was compared with that of the wild-type P. aeruginosa strain. The mutant strain was attachment-proficient but biofilm-deficient, suggesting that OprG plays a role in P. aeruginosa biofilm development under the culturing conditions used in this study. / Thesis (PhD (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

No description available.

Enterococci are intestinal facultative anaerobic strains which recognized as opportunistic pathogens. The ability to form biofilms is an important virulence trait that has been reported for Enterococci. Biofilm formation showed differences between E. faecalis strains. However, several factors were involved in this process e.g. the presence of virulence factors, hydrophobicity and heterogeneity. Interestingly, we demonstrated for the first time a biochemical test for a cell surface protein in biofilm formation: addition of the purified N domain of Esp (EspN) to E. faecium E1162Δesp resulted in the restoration of biofilm formation. Streptococcus bovis also, is an intestinal facultative anaerobic bacterium. This organism also has been reported as an opportunistic pathogen causing multiple diseases such as septicemia and endocarditis associated with colorectal cancer (CRC). Although the association of S. gallolyticus infection with CRC is a major issue, the mechanisms behind this link are still unclear. This link between CRC and the virulence of S. bovis strains was studied in more detail in a collaborative project with Dr Harold Tjalsma. The Tjalsma group mainly focussed on host-pathogen interactions, whereas we analysed biofilm formation of S. bovis strains as well as their pathogenicity using the in vivo C. elegans infection model. Our biofilm showed that S. bovis strains form biofilms particularly well on collagen-rich surfaces at least indicate why there is this association. C. elegans experiments also showed that pathogenicity of S. bovis strains is more similar to E. faecalis than to E. faecium in which both S. bovis and E. faecalis have a slow mode of killing that is absent in E. faecium. Full genome sequences of S. gallolyticus UCN34 strain have revealed the presence of a number of potential collagen-binding proteins (e.g., gallo_2179) that are related to the MSCRAMMs family. However, we successfully cloned the gallo_2179 gene in an enterococcal expression vector, and demonstrated transcribed in E. faecalis. Unfortunately, this strain did not form better biofilms on a collagen surface, suggesting either that not sufficient amount of the protein was made, or that the protein is not functional in E. faecalis. In addition, a bioinformatics analysis was performed to identify putatively secreted proteins in S. gallolyticus. Proteins that were expected to be found include for instance three collagen-binding proteins, amylase, tannase and beta lactamase.

No description available.

The significant rise in the number of micro-breweries in North America has increased the need for efficient on-site industrial wastewater facilities. Brewery wastewater is considered to be a high strength food industry wastewater with high variability in terms of both organic and hydraulic loading. Small breweries require cost-effective, reliable, and simple to operate treatment technologies to properly manage their brewery wastewaters. Moving bed biofilm reactor (MBBR) technology has shown promise at the lab-scale and full-scale with respect to brewery effluent treatment. MBBR systems have the capability for short hydraulic retention times (HRT), high organic loading rates, as well as increased treatment capacity and stability due to biofilm retention, all within a compact reactor size when compared to other aerobic and attached growth treatment options. Two MBBR systems utilizing two different carrier types (Kaldnes K5 and Kontakt), and a suspended growth (SG) control reactor, were used in this study to investigate the impacts of surface area loading rate (SALR) and HRT on attached growth (AG) and SG kinetics and carrier type for brewery wastewater at 2000 mg-sCOD/L. An increase in SALR from 10-55 g-sCOD/m2/d while at an HRT of 12 hr resulted in no significant impact in total volumetric removal rates between the MBBR systems and the SG control reactor; however, MLSS concentrations were lower for the MBBR systems at SALRs below 55 g-sCOD/m2/d, which indicated AG contribution. Over 92% soluble chemical oxygen demand (sCOD) removal was achieved at each SALR in each of the three reactors. These results indicated that the reactors were substrate limited and SG controlled. Due to the SG dependency, the difference between the two types of carriers was indeterminate. A decrease in HRT from 12-3 hr while maintaining an SALR of 40 g-sCOD/m2/d resulted in a shift from SG to AG dependency in the MBBR systems. The total volumetric removal rates for the MBBR systems were significantly higher at HRTs of 3 and 4 hr as compared to the SG control reactor. The AG volumetric removal rates from both MBBR systems were highest at an HRT of 3 and 4 hr. At an HRT of 12 hr all three reactors maintained over 92% sCOD removal; however, at an HRT of 4 hr the SG control reactor dropped to 88% and at 3 hr to 61%, whereas the MBBR systems maintained 95% removal at an HRT of 4 hr and only decreased to 73% at 3 hr. These results indicated that the MBBR systems were more effective at lower HRT than the SG control reactor, with no significant difference observed between the two carrier types tested. Biofilm morphology and viability from each of the two carriers utilized in the study of moving bed biofilm reactor (MBBR) treatment of brewery wastewater were investigated using stereoscopy and confocal laser scanning microscopy (CLSM) in combination with live/dead cell staining. Both carriers demonstrated thicker and more viable biofilms at high SALR and denser and less viable biofilms at low SALR. At lower HRT, the carriers reacted differently resulting in thicker, but less dense biofilms on the Kontakt carriers and thinner, but more dense biofilms on the K5 carriers. However, no trend in cell viability was observed with change in HRT. Although the systems were suspended growth (SG) dominated, based on the MBBR kinetics and carrier biofilm morphology and cell viability, either carrier would be a viable choice for an MBBR treating brewery wastewater at HRTs between 4 to 12 hr and SALRs between 10-55 g-sCOD/m2/d.

Within the five experimental chapters of this doctoral thesis (i) the eubacterial diversity of the microbiota of chronic wounds and healthy skin was investigated, (ii) biofilm formation and associated coaggregation interactions of wound and skin-associated bacterial isolates was examined, (iii) formulation of media which reproduced some aspects of the nutritional conditions of wounds and healthy skin were developed, (iv) novel wound biofilm models were developed and validated and (v) microbial population interactions associated with healthy skin and chronic wounds were investigated using a novel model system. (i) The microbial diversity of chronic wounds and contralateral skin swabs was investigated using culture, denaturing gradient gel electrophoresis (DGGE) and microscopy. Intrapersonal analysis identified that non-infected wounds had a proportionally higher incidence of bacteria which were identified on contralateral healthy skin according to DGGE analysis when compared to infected wounds indicating that taxonomically distinct consortia are associated with infection. Microcolonies and putative biofilms structures were identified in both culture-defined infected and non-infected wounds indicating that the presence of biofilms may not be linked to infection. (ii) By assessing pair-wise combinations of skin and wound-associated bacteria, the role of coaggregation in the formation of wound polymicrobial communities was assessed using a quantitative spectrophotometric assay. Aggregation interactions were weak or not detectable, apart from those associated with Corynebacterium xerosis. This bacterium produced a high autoaggregation score (c. 50%). The limited coaggregation interactions suggest that coaggregation may be comparatively unimportant in the development of wound biofilms. (iii) In order to facilitate the development of biofilm models specific to chronic wounds, the formulation of representative growth media is important in order to reproduce the in situ nutrient environment. Therefore complex, artificial sweat and serum media broadly reflective of the nutrient availability in wounds and healthy skin were developed and validated based upon their ability to support realistic phenotypes (assessed by proteomics) and the growth of a range bacterial isolates. Developed media maintained the sessile growth the test bacteria and produced broadly similar proteomic profiles to foetal calf serum. (iv) Two novel model systems were developed to study cross-sectional population interactions and to investigate longitudinal population development of wound consortia and biofilm formation. A fine celled foam (FCF) multi-well wound model and a multiple membrane FCF model maintained dynamic steady state of axenic and mixed populations of bacteria associated with chronic wounds and supported the development of biofilms. (v) The FCF multi-well wound model was used to investigate population interactions in environments broadly reflective of healthy skin and wounds. When grown in artificial sweat prior colonisation with Staphylococcus saprophyticus resulted in a significant reductions in methicillin resistant Staphylococcus aureus (99%) and P. aeruginosa (75%) whilst prior colonisation by C. xerosis resulted in a significant reduction in P. aeruginosa (91%) only. However no significant reductions in pathogenic bacteria were noted in artificial serum indicating colonisation resistance could be simulated in the model and the outcome of immigration was markedly influenced by the species of established bacterium and nutrient availability.

Nitrogen removal from wastewater is important for the revention of significant health and environmental impacts such as eutrophication. Nitrogen removal is achieved by the combined action of nitrification and denitrification. Nitrification is performed by autotrophic, slow growing microorganisms that require oxygen and are inhibited in the presence of denitrifiers when oxygen and COD are available due to competition for oxygen. Denitrification however, performed by relatively fast growing heterotrophic bacteria, is inhibited by oxygen and requires COD. This implies that nitrification and denitrification are mutually exclusive. The supply of oxygen to a fresh wastewater, high in ammonia and COD, causes waste of both oxygen and COD. Conservation of COD is therefore critical to efficient wastewater treatment. The approach investigated in this study to achieve complete nitrogen removal was to physically separate the nitrification and denitrification biomasses into separate bioreactors, supplying each with appropriate conditions for growth and activity. A storage driven denitrification sequencing batch biofilm reactor (SDDR) was established which exhibited a high level of COD storage (up to 80% of influent COD) as poly-B-hydroxybutyrate capable of removing >99% of nitrogen from wastewaters with a C/N ratio of 4.7 kg COD/kg N–NO3 –. The SDDR was combined in sequential operation with a nitrification reactor to achieve complete nitrogen removal. The multiple stage, multiple biomass reactor was operated in sequence, with Phase 1 - COD storage in the storage driven denitrification biofilm; Phase 2 - ammonia oxidation in the nitrification reactor; and Phase 3 - nitrate reduction using the stored COD in the storage driven denitrification reactor. The overall rate of nitrogen removal observed was up to 1.1 mmole NH3 L–1 h–1 and >99% of nitrogen could be removed from wastewaters with a low C/N ratio of 3.9 kg COD/kg N–NH3. The multiple stage, multiple biomass system was limited in overall nitrogen removal the reduction in pH caused by nitrification. A parallel nitrification-denitrificatio (PND) reactor was developed in response to the pH control issue. The PND reactor was operated with Phase 1 – COD storage in the storage driven denitrification biofilm and Phase 2 – simultaneous circulation of reactor liquor between the denitrification and nitrification biofilms to achieve complete nitrogen removal and transfer of protons. The PND reactor performed competitively with the multistage reactor (removal of >99% nitrogen from wastewaters with feed ratios of 3.4 kg COD/kg N–NH3) without the need for addition of buffering material to oderate the pH.