The emergence of biofilms:Computational and experimental studies

The response of biofilms to any external stimuli is a cumulative response aggregated from individual bacteria residing within the biofilm. The organizational complexity of biofilm can be studied effectively by understanding bacterial interactions at cell level. The overall aim of the thesis is to explore the complex evolutionary behaviour of bacterial biofilms. This thesis is divided into three major studies based on the type of perturbation analysed in the study. The first study analyses the physics behind the development of mushroom-shaped structures from the influence of nutrient cues in biofilms. Glazier-Graner-Hogeweg model is used to simulate the cell characteristics. From the study, it is observed that chemotaxis of bacterial cells towards nutrient source is one of the major precursors for formation of mushroom-shaped structures. The objective of the second study is to analyse the impact of environmental conditions on the inter-biofilm quorum sensing (QS) signalling. Using a hybrid convection-diffusion-reaction model, the simulations predict the diffusivity of QS molecules, the spatiotemporal variations of QS signal concentrations and the competition outcome between QS and quorum quenching mutant bacterial communities. The mechanical effects associated with the fluid-biofilm interaction is addressed in the third study. A novel fluid-structure interaction model based on fluid dynamics and structural energy minimization is developed in the study. Model simulations are used to analyse the detachment and surface effects of the fluid stresses on the biofilm. In addition to the mechanistic models described, a separate study is carried out to estimate the computational efficiency of the biofilm simulation models

The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation

The widespread use of antibiotics in the past 80 years has saved millions of human lives, facilitated technological progress and killed incalculable numbers of microbes, both pathogenic and commensal. Human-associated microbes perform an array of important functions, and we are now just beginning to understand the ways in which antibiotics have reshaped their ecology and the functional consequences of these changes. Mounting evidence shows that antibiotics influence the function of the immune system, our ability to resist infection, and our capacity for processing food. Therefore, it is now more important than ever to revisit how we use antibiotics. This review summarizes current research on the short-term and long-term consequences of antibiotic use on the human microbiome, from early life to adulthood, and its effect on diseases such as malnutrition, obesity, diabetes, and Clostridium difficile infection. Motivated by the consequences of inappropriate antibiotic use, we explore recent progress in the development of antivirulence approaches for resisting infection while minimizing resistance to therapy. We close the article by discussing probiotics and fecal microbiota transplants, which promise to restore the microbiota after damage of the microbiome. Together, the results of studies in this field emphasize the importance of developing a mechanistic understanding of gut ecology to enable the development of new therapeutic strategies and to rationally limit the use of antibiotic compounds

Dissecting regulatory mechanisms of quorum sensing pathways in Bacillus subtilis

Living organisms generally share a small number of characteristics, among which include maintaining homeostasis, growth, and responding to changing environments. Wherever we find life, we typically observe this life performing these tasks. Likely no environment is truly barren, so organisms must be able to continue living in crowded conditions. Humans use their senses to determine the quality of their local environment. Individuals use languages, written, spoken and digital to communicate these findings to their neighbors. Bacteria have evolved complex systems to sense these conditions, and to trigger appropriate developmental programs to help them survive, grow, and respond in changing environments. Bacteria both produce and sense signals about these density-dependent conditions in a process called quorum sensing. Chapter 1 provides an introduction to the mechanisms utilized by bacteria referred to as quorum sensing. An overview is given of the history of the study of these mechanisms, as well as a review of molecules and strategies from both Gram-negative and Gram-positive organisms. Also discussed here are mechanisms of quorum quenching used by organisms in quorum sensing pathways. Next, we discuss in some detail the molecular mechanisms used by Bacillus subtilis to regulate pathways under control of the quorum response. Chapter 2 describes work looking to further explain the mechanism of ComA activation. In this chapter, we use a genetic screen to identify constitutive mutants of ComA. We then characterize these mutants for their regulation by ComP and RapC and for their ability to bind DNA. These results were used in an attempt to generate a computational model of ComA activation. We take preliminary steps in validating this model by logically creating and testing combination mutants of ComA. The role of acetyl-phosphate in ComA activation is also briefly explored. Chapter 3 explores the role of Rap proteins in regulating the biological processes of genetic competence, sporulation, motility and biofilm formation. We were able to characterize several Rap proteins as novel regulators of these pathways. We also were able to show that Rap60 uses a separate surface for interaction with ComA as compared to canonical Rap protein anti-activators of genetic competence

Investigation of the regulation mechanisms for bioplastics production from industrial residues

Dissertação para obtenção do Grau de Mestre em BiotecnologiaThe current high demand for plastics has become unsustainable. Polyhydroxyalkanoates are biopolymers stored by bacteria that can potentially replace modern plastics due to: wide range of applications; biodegradability; use of renewable resources as feedstock. High costs of current Polyhydroxyalkanoates production can be reduced using mixed cultures of organisms. Activated sludge from wastewater treatment plants is selected for Polyhydroxyalkanoates production through the imposition of cycles of intermittent feeding. In this study, the acclimation of activated sludge using synthetic volatile fatty acids (VFAs) as substrate resulted in a culture rich in Paracoccus spp. and unidentified filamentous bacteria. Low cost substrates such as sugarcane molasses (SM) or cheese whey (CW) can be employed as feedstock for further cost reduction. This requires an additional step before the microbial selection to ferment the feedstock into VFAs. In this work, the feedstock was changed from SM to CW. The population fed with SM was rich in Actinomycetaceae, while the population fed with CW was rich in Streptococcaceae, affecting the VFA composition. Consequently, the PHA-storing population and the polymer were affected. In the fermented SM (fSM) phase, the population was rich in Azoarcus (41.5 - 64.6%) and in the fCW phase the population was more diverse. Changing the pH in the fermentation reactor also affected the selection stage with an increase in Thauera and Azoarcus and a decrease in Paracoccus. A significant unidentified population of one layer sheet- forming bacteria was observed. Lastly, the occurrence of cell-to-cell communication (QS) in the selection stage was investigated. Possibly, QS molecules were detected when the carbon source was depleted. All steps of polyhydroxyalkanoate production are interconnected and for optimization, all stages must be studied and improved. Moreover, if QS proves to be involved in polyhydroxyalkanoate storage, the addition of QS molecules to the process may be explored for further optimization