Motility and chemotaxis have been implicated in the process of biofilm formation in a wide range of species. Using a combination of microscopy and image analysis, genetics, microbiology and biochemistry, the initial approach of Rhodobacter sphaeroides cells to a solid surface has been characterised. Interestingly, these data suggest that for R. sphaeroides alterations in motility and swimming behaviour may result in differences in biofilm formation simply by changing the number of cells which reach the surface. This is in contrast to a few other well-studied species where the motility apparatus, the flagellum, has been shown to play an active role in surface sensing and the transition to biofilm growth. Tracking swimming cells and measuring surface attachment revealed that changes in motility affect the ability of cells to attach to a surface, with non-motile cells attaching least and mutants with frequent stops attaching less than smooth swimming cells with few stops. Tracking attaching cells and classifying their method of attachment revealed that flagellar tethering is not essential for R. sphaeroides attachment. Competition assays with fluorescently labelled strains showed that the initial imbalance between motile and non-motile cells remains as microcolonies develop over 48 hours,and the proportion of non-motile cells remains fairly constant. Development on a surface over 48 hours was similar for motile and non-motile strains, including aflagellate strains, once attached. Using parameters calculated by tracking swimming cells to calculate the effective diffusion coefficient in a simple model of cell movement suggested that motion alone could explain the differences in attachment without assuming different cell properties. In particular, aflagellate strains might be hindered from surface attachment by their reduced motility alone. This is interesting since some other bacterial species use the flagellum as a surface sensor.

Staphylokokken sind in erster Linie Saprophyten, welche die Haut und Schleimhäute des Menschen besiedeln und dort eine wichtige Rolle für das Gleichgewicht der gesunden Mikroflora spielen. Gleichzeitig haben sie sich aber auch zu den häufigsten Verursachern nosokomialer Infektionen entwickelt. Vor allem S. aureus und S. epidermidis verursachen Erkrankungen, die von leichten Haut- und Wundinfektionen bis hin zu lebensbedrohlichen Infektionen wie Pneumonien, Sepsis oder Endokarditis reichen. Infektionen durch S. epidermidis treten dabei meist in Verbindung mit Fremdkörpern wie Kathetersystemen, künstlichen Gelenken und Herzklappen auf. In diesem Zusammenhang scheint vor allem die Fähigkeit, einen Biofilm auf diesen Fremdkörpern bilden zu können eine große Rolle für die Pathogenese zu spielen. Die Therapie von Staphylokokken-Infektionen wird zunehmend durch die ausgeprägte Antibiotikaresistenz dieser Erreger erschwert, die sich mittlerweile auch auf Reserveantibiotika wie Daptomycin, Synercid® (Quinupristin/Dalfopristin) oder Linezolid erstreckt. In der Vergangenheit wurde gezeigt, dass subinhibitorische Konzentrationen des Streptogramin-Antibiotikums Synercid® die Biofilmbildung in S. epidermidis induzieren. Darüber hinaus mehren sich die Hinweise auch bei anderen Bakterien, dass geringe Antibiotika-Konzentrationen, wie sie in natürlichen Habitaten wie zum Beispiel im Boden vorkommen, wichtige Signale bei der Kommunikation von Bakterien untereinander darstellen und möglicherweise eine Funktion bei der Modulation des Metabolismus im Kampf um Nährstoffe ausüben. In dieser Promotionsarbeit sollte daher exemplarisch, mit Hilfe der erst seit kurzem zur Verfügung stehenden DNA-Mikroarray-Technik, der Effekt von subinhibitorischen Synercid®-Konzentrationen auf die globale Genexpression von S. epidermidis und S. aureus analysiert werden. Dabei stand vor allem die Wirkung auf die Biofilmbildung und die Identifikation neuer, an der Biofilmbildung beteiligter Komponenten im Mittelpunkt. Die Ergebnisse zeigen, dass subinhibitorische Synercid®-Konzentrationen bei S. aureus, im Gegensatz zu S. epidermidis, zu einer reduzierten Biofilmbildung führen. Bei beiden Arten ist jedoch die unterschiedliche Biofilmbildung durch eine ausgeprägte Veränderung der allgemeinen Genexpression begleitet. Vor allem war eine starke Induktion von Genen zu beobachten, die für Proteine des Translationsapparates kodieren. Außerdem waren Gene des Nukleotid-Stoffwechsels, der Aminosäure-Synthese und des Kohlenstoff-Metabolismus unterschiedlich reguliert. Des Weiteren konnte mit Hilfe von HPLC-Analysen sowohl bei S. epidermidis als auch bei S. aureus eine reduzierte Konzentration des Signalmoleküls Guanosin-3-5-bisphosphat (ppGpp) festgestellt werden. ppGpp spielt vor allem bei der Kontrolle und Anpassung des Stoffwechsels an Wachstumsbedingungen, wie zum Beispiel das Nährstoffangebot, eine wichtige Rolle. Die Ergebnisse legen nahe, dass die Inaktivierung der ribosomalen Proteine durch Synercid® die reduzierte ppGpp-Konzentration bedingt, was zum einen zur verstärkten Bildung ribosomaler Proteine führt und zum anderen den Zellstoffwechsel in einer Weise beeinflusst, der eine vermehrte extrazelluläre Polysaccharid-Synthese und damit Biofilmbildung ermöglicht. Zusammenfassend zeigen die hier vorgestellten Daten, dass die unterschiedliche Beeinflussung der Biofilmbildung bei S. epidermidis und S. aureus sehr wahrscheinlich durch verschiedene Regulationswege des ica-Operons und/oder unterschiedliche katabolische Fähigkeiten bedingt werden. Die Ergebnisse belegen weiterhin, dass die Wirkung von Antibiotika weit über deren bekannte inhibitorische Effekte hinausgeht und vor allem den Stoffwechsel der Bakterien dramatisch beeinflußt. Die Konsequenzen dieser Befunde sowohl für das Verständinis von mikrobiologischen Konsortien in Ökosystemen als auch für die Anwendung von Antibiotika in der Medizin sind noch nicht abzusehen, bieten jedoch eine interessante Grundlage für weitere experimentelle Arbeiten. / Staphylococci are common inhabitants of the human skin and mucosal membranes, as well as the most prevalent causes of nosocomial infections. The infections mainly caused by S. aureus and S. epidermidis range from mild skin infections to life-threatening infections, like pneumonia, bacteremia or endocarditis. Especially S. epidermidis is the primary pathogen involved in medical device associated infections. In this context, the ability to form a biofilm seems to be crucial. The prevalent antibiotic resistances of Staphylococci poses an increasing challenge to medicine in recent years. The remaining effective antibiotics to treat staphylococci infections are last resort antibiotics, such as daptomycine, Synercid® and linezolid. However, staphylococci isolates with intermediate and high resistances towards these antibiotics have already been reported. There are several lines of evidence that, beside their killing activity, antibiotics have a modulating effect on the metabolism of bacteria when applied in low concentrations. Hence, the impact of this natural compounds on bacteria have to be analysed in more detail in order to provide insights into their mode of action and how bacteria respond to these compounds. Former studies showed, that biofilm formation of staphylococci is influenced by environmental factors, such as temperature, osmolytic stress and the availability of glucose or oxygen, as well as by subinhibitory concentrations of antibiotics. In this context it was shown, that low concentrations of tetracycline, erythromycine and Synercid® induce biofilm formation of S. epidermidis. The aim of this study was to investigate the effect of subinhibitory concentrations of Synercid® on biofilm formation and global gene expression of S. epidermidis and S. aureus. For this purpose DNA microarrays were used to identify and characterise the mechanisms by which Synercid® alters biofilm formation. Furthermore, this studies should lead to the identification of new factors involved in biofilm formation of staphylococci. The data presented here, demonstrate that subinhibitory concentrations of Synercid® reduce biofilm formation in S. aureus, while inducing it in S. epidermidis. The altered biofilm formation is accompanied by a strong impact on the overall gene expression of both species. Especially the expression of genes encoding proteins of the translational apparatus were increased. In addition, genes encoding proteins of the nucleotide metabolism, amino acid biosynthesis and carbohydrate utilisation were affected. Furthermore, HPLC analyses revealed a decreased concentration of the small nucleotide signal molecule guanosine-3-5-bisphosphate (ppGpp) in S. aureus and S. epidermidis under the influence of Synercid®. ppGpp is involved in the control and adaptation of cellular metabolism to changing growth conditions. We hypothesise that the reduced concentrations of ppGpp is due to the inactivation of ribosomal proteins, as a result of Synercid® binding, and mediates on the one hand the increased production of proteins of the translation apparatus and on the other hand effects the cellular metabolism in a way that enable the cells to produce more extracellular polysaccharides resulting finally in an enhanced biofilm formation. In summary, the data presented here show that the differential effect of Synercid® on biofilm formation of S. epidermidis and S. aureus is most likely due to a differential regulation of the ica operon and/or different catabolic abilities. Moreover the data suggest that the effect of antibiotics is more than the sole interaction with a target structure, but influence gene expression and metabolism of bacteria more globally. The consequences of these findings for the comprehension of the interplay in microbial communities as well as for the clinical use of antibiotics is far from being understood. However, the results of this work might provide an interesting basis for future experimental work elucidating these interferences.

Microbes live in diverse, challenging and competitive environments. To survive and propagate microbes must be able to sense and respond to environmental fluctuations, such as changes in pH, nutrient status or temperature. As such, bacteria have a number of signal transduction mechanisms at their disposal that allow them to detect a range of different stimuli, integrate different signals and react to them appropriately. The work presented in this thesis aimed to understand more about the signalling cascades that the Gram positive soil-dwelling bacterium <em>Bacillus subtilis</em> uses to mediate its transition from a motile lifestyle that requires rotating helical flagella, to a sessile lifestyle called the biofilm, where cells adhere to a surface and are encased in a self-produced extracellular polymeric matrix. Bacterial tyrosine phosphorylation is required for <em>B. subtilis</em> biofilm formation and has been suggested to also play a role in regulating the putative motility protein, YvyG. This led to the hypothesis that tyrosine phosphorylation might play a role in both motility and biofilm formation. The first part of this thesis investigates this hypothesis and successfully ascribes a function to YvyG as an orthologue of a flagellar type 3 secretion system chaperone that is essential for flagellar assembly. Crucially this work provides further evidence that the <em>B. subtilis</em> flagellum is regulated by both conserved and species-specific means. These experiments led to YvyG being re-named as FlgN. Despite previous work suggesting that phosphorylation of YvyG was important for protein function and localisation, the data presented here found no evidence of this, and therefore indicate that the impact of bacterial tyrosine phosphorylation must be assessed in vivo before any significance can be drawn from the identification of such modifications by in vitro approaches. The second part of this study examines the role of the DegS-DegU two component signal transduction system in mediating the transition from motility to biofilm formation. DegS-DegU is required for both motility and biofilm formation, and previous work indicated that DegS-DegU may sense flagellar assembly. The data presented show that upon an inhibition of flagellar rotation DegU~P levels are increased, as inferred from accepted proxies. This could conceivably be the first step in biofilm formation to allow cells to sense and respond to a surface and change their gene expression profile. The <em>B. subtilis </em>flagellum therefore acts as a mechanosensor to control the DegS-DegU two component system. Collectively, the work presented here contributes to our understanding of how <em>B. subtilis</em> regulates flagellar assembly, and further enhances our knowledge of how bacteria are able to use their flagella not only as devices for propulsion, but also to sample changes in the extracellular environment.

Abstract The Balance Balanus albicostatus is the dominant species of fouling organism in Kaohsiung Port. In different harbor district environments In Kaohsiung Port, one can discriminate the population of B. albicostatus into three different groups¡Gthe estuary area of Love River, the outer harbor area, and the harbor area. The variation of estuary area¡¦s (Jhongjheng Bridge Station on Love River) population is the highest with the averaged survival rate of 53.8¡Ó21.7%. The population in the harbor area (The First Harbor Mouth station) is more stable with the averaged survival rate of 62.5¡Ó8.1%. The composition of population in the eliminated area in all stations after one year is similar to the previous one. It is suggested that barnacle population in Kaohsiung port can recover from environmental disturbance throughout larval recruitment within a year. Based on the results from the growth experiment of the settled barnacle¡¦s larvae, it was indicated that the growth and survival rate of the settled larvae was least affected by the recruitment of any kinds of settled larvae including barnacle itself. To understand the influence of environmental factors on the hatching of egg and development of larvae, we cultivated the larvae of B. albicostatus under the control of temperatures and salinities in the laboratory. The hatching rate of egg and times required for larvae to metamorphosis at each stage is the shortest under the condition of high temperature and high salinity. The larvae can metamorphose into cyprid in five days after hatching at the high temperature (35¢J), while it is prolonged to 19 days after hatching under the low temperature (15¢J) and low salinity (22 psu). The settlement rate of cyprids is better under high salinity (33 psu). To understand the effects of biofilm on settlement of cyprids, the biofilms are cultured under the conditions of the non-filtered, 80£gm, 20£gm, and 1£gm filtered seawater. When the biofilms have grown for 5 days, the settlement rate of cyprids on the biofilm cultured by the filtered seawater is higher than those on the non-filtered biofilms and alcohol-sterilized ones. At the day 12, the 1£gm filtered treatments had a greatest quantity of settled larvae while the number of settled cyprids in the other treatments decreased. The number of settled cyprids on panels with biofilms cultured for 15 days in non-filtered seawater and the one with biofilms fallen off for more than 75% of the total area did not significantly differ from the one with biofilms cultured in the seawater filtered with the 1£gm mesh. This showed that biofilms cultured under different filtered conditions affect the settlement of cyprids over time. It is concluded that the population of barnacle Balanus albicostatus, based on survival rate and growth, can be grouped into three different groups in the Kaohsiung Port. From the laboratory experiments, it is showed that the type of biofilm and the aging processes over time can affect the settlements of cyprids. It therefore suggested that the larval recruitments of the barnacle B. albicostatus can be affected by the types of biofilm on the different substrates, and consequently the larval recruitments in the different environmental conditions in Kaohsiung Port.

Most bacteria live in surface-attached colonies known as biofilms, which contain distinct cell types embedded in a self-produced extracellular network of polymers. Differentiation into functionally-distinct sub-populations of cells, or phenotypes, is primarily a result of nutrient availability and extracellular signals. These inputs change over time, leading to spatial and temporal patterns in the relative populations of phenotypes. Although transitions between phenotypes have been investigated in single cells, the triggers for this process within an intact biofilm are not well understood. / Engineering and Applied Sciences

No description available.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

The anaerobic, cellulolytic bacterium Clostridium thermocellum formed biofilms on cellulose consisting of a single layer of cells which did not secrete an extracellular polymeric matrix. Sporulation occurred under normal growth and was believed to assist with biofilm translocation to new substrates. Although the cell-substrate distance was less than 210 nm, the biofilm layer lost up to 29% of hydrolyzed oligomeric products when reactors were loaded with extreme concentrations of cellulose (up to 200 g/litre). This effect was much less severe at lower cellulose concentrations. Of the total cellulose carbon, 4% (gC/gC) was utilized for cell mass production and up to 75% was converted into primary metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). Increasing the starting cellulose concentration shifted the ethanol-to-acetic acid ratio from 0.91 g/g to 0.41 g/g. Such high substrate loadings and metabolite shifts have not been previously reported and may be of interest for consolidated bioprocessing strategies. Cellulose conversion was initially limited by microbial growth, with a biofilm development rate estimated at 0.46h-1 to 0.33h-1 and where up to 20% of the substrate was consumed. Subsequently, substrate-limited conditions determined the rate kinetics. Surface accessibility for microbial colonization was the dominant rate limiting factor, while mass imposed constraints very late towards the end-point fermentation. CO2 was found to be an excellent reporter molecule for cellulose consumption and biofilm growth. Online CO2 tracking may also be used to assess the digestibility of substrates with unknown surface properties. A mathematical model that described biofilm growth, substrate consumption and product formation was found to have an excellent fit with experimental data of CO2 production which reinforced the previous findings on the cellulolytic biofilm form and function. Together, these results demonstrate that biofilms are undeniably the key to understanding the effective microbial conversion of cellulosic substrates.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.

No description available.