The bacterial cell wall is a nearly universal structure that offers protection and gives the cell its shape. However, environmental stressors, such as cell wall-targeting antibiotics and... Show moreThe bacterial cell wall is a nearly universal structure that offers protection and gives the cell its shape. However, environmental stressors, such as cell wall-targeting antibiotics and hyperosmotic conditions, can induce bacteria to shift to a wall-deficient state. It is unknown whether the lack of this cell wall ‘barrier’ can enable DNA exchange via horizontal gene transfer (HGT), a process that facilitates the spread of antibiotic resistance amongst bacteria. The research in this thesis confirms that wall-deficiency enables HGT in the actinobacterium Kitasatospora viridifaciens. Specifically, we show that wall-deficient L-forms take up extracellular material including DNA, sugar polymers and lipid nanoparticles via an endocytosis-like mechanism, which in case of DNA uptake leads to genetic transformation. Moreover, wall-less cells exchange genomic DNA in a DNAse-resistant manner, most likely via cell-cell fusion and/or production of extracellular vesicles. We also isolated vesicle-producing actinobacteria from a wastewater treatment plant, an environment that is considered a hotspot for the spread of antibiotic resistance genes. Lastly, growth of K. viridifaciens under hyperosmotic stress conditions leads to genetic instability, which together with HGT can contribute to enhanced genome plasticity. In summary, this thesis provides important insights into the consequences of bacterial cell wall-deficiency for HGT and evolution. Show less
With the rise of multi-drug resistant bacterial pathogens, the possibility of a post-antibiotic world is quickly becoming reality. It is therefore vital that research is focussed on overcoming the... Show moreWith the rise of multi-drug resistant bacterial pathogens, the possibility of a post-antibiotic world is quickly becoming reality. It is therefore vital that research is focussed on overcoming the most challenging bacterial resistance mechanisms. To this end, the aim of the work described in this thesis was to develop novel strategies to combat resistant bacteria, with particular focus on the threat posed by gram-negative bacteria. Show less
Sepsis is a life-threatening condition caused by a dysregulated host response to infection, it is associated with significant morbidity, mortality, and with a high financial burden on global... Show moreSepsis is a life-threatening condition caused by a dysregulated host response to infection, it is associated with significant morbidity, mortality, and with a high financial burden on global healthcare systems. Bacterial infections are the primary cause of sepsis, but the growing prevalence of antimicrobial resistance complicates the effectiveness of antimicrobial treatments. Moreover, limited understanding of the host immune response during sepsis hinders the discovery of valuable biomarkers and drug targets. As such, there is an urgent need to improve the treatment of sepsis. To tackle this challenge, we have concentrated our efforts on optimizing current treatment strategies and on facilitating the discovery of novel host inflammatory response directed therapeutics. In this thesis, we have utilized quantitative pharmacological modeling approaches to assess the adequacy of current dose regimens and to evaluate antibiotic pharmacokinetic variability, thereby optimizing antimicrobial therapies for sepsis. Additionally, our researches had aimed to deepen our understanding of the underlying dynamics of sepsis pathology, enabling the identification of promising biomarkers and therapeutic targets for sepsis. Our work demonstrated how quantitative modeling strategies can support the design of optimized treatment strategies, and how systematic model-based integration of disease mechanisms can help to overcome the translational challenges in sepsis drug development. Show less
Explaining treatment response variability between and within patients can support treatment and dosing optimization, to improve treatment of individual patients. This thesis discussed multiple... Show moreExplaining treatment response variability between and within patients can support treatment and dosing optimization, to improve treatment of individual patients. This thesis discussed multiple aspects of treatment variability and the associated statistical learning techniques which can be used to explain and/or predict part of that variability. Even though in recent times the availability of several high-throughput measurement technologies has created many new opportunities to develop improved treatment strategies, deriving actionable insights from such data remains a challenge. To this end, the use of longitudinal and high-dimensional data analysis techniques is needed to explore omics data for explaining treatment response and clinical course, and to answer clinical questions from routine healthcare data from hospitals and research institutes. Show less
Introduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis... Show moreIntroduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis drive the conformational changes in MutS thatare needed for the mismatch repair cascade.Chapter 2 focuses on the final stages of the DNA mismatch repair pathway, which are the resection and subsequent resynthesis of the mismatch containing strand.Chapter 3 presents an example of how DNA polymerases can be targeted for the development of novel antibiotics against Mycobacterium tuberculosis (Mtb).Chapter 4 describes a new instrument named the Puffalot, developed for the preparation of cryo-EM grids, which aims to improve the reliability of cryo-EM sample preparation.Discussion provides a summary of the scientific findings described in this thesis in light of the published literature as well as an overview of the future directions and perspectives. Show less
Antibiotic resistance, caused by widespread use of antibiotics, leads to bacterial infections that are difficult, if not impossible, to treat and is a major worldwide health concern. Currently... Show moreAntibiotic resistance, caused by widespread use of antibiotics, leads to bacterial infections that are difficult, if not impossible, to treat and is a major worldwide health concern. Currently Methicillin-resistant Staphylococcus aureus (MRSA) is the most commonly identified antibiotic-resistant pathogen in clinical medicine worldwide. The spread of MRSA highlights the urgent need for alternative therapies, such as vaccination.Wall teichoic acids (WTAs), prime constituents of the Gram-positive cell wall, can function as effective antigenic epitopes and are therefore promising candidates for the development of a conjugate vaccine against S. aureus infections. WTAs are anionic poly-ribitol phosphate (RboP) chains attached to the peptidoglycan and they have a fundamentol role in the physiology in the bacteria.Since isolation from the bacteria of WTAs leads to heterogenous mixtures of fragments and bacterial contaminations, organic synthesis is the method of choice to generate WTA-fragments with pre-defined substitution patterns in higher purity and in larger amounts, allowing detailed immunological studies that can aid in future vaccine development.This Thesis presents methods to synthesize various WTA-fragments from Staphylococcus aureus and Enterococcus faecalis and their applications. Show less
The soil-dwelling, filamentous bacteria of the genus Streptomyces are renowned for their production of useful secondary metabolites including antibiotics. The work described in this thesis provides... Show moreThe soil-dwelling, filamentous bacteria of the genus Streptomyces are renowned for their production of useful secondary metabolites including antibiotics. The work described in this thesis provides new insights on the role and regulation of antibiotic production and resistance in these bacteria. It shows that antibiotic resistance is already beneficial at sub-inhibitory antibiotic concentrations. Resistance can even readily evolve at such low concentrations, thereby possibly explaining the level of resistance seen in pristine environments. Antibiotic producers can benefit from spatial structure, as present in the soil, through the preferential allocation of resources and this enables invasion from low frequencies. Streptomyces do not produce all antibiotics continuously, but antibiotic production is instead tightly regulated in response to environmental cues, including those produced by competitors. Streptomyces are most likely to induce antibiotic production in response to a competitor that shares similar secondary metabolite clusters, indicating a possible role for shared signalling. Besides changes in antibiotic production, other responses to competition are revealed on a transcriptomic level, including an increased expression of developmental genes, suggesting earlier sporulation. Show less
Fernandez, J.; Prado, V.; Trebicka, J.; Amoros, A.; Gustot, T.; Wiest, R.; ... ; European Fdn Study Chronic Liver 2019
Streptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with... Show moreStreptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with the life cycle. This thesis shows the identification of S. roseifaciens, a novel species with an uncommon, verticillate spore morphology and a unique household of SsgA-like proteins. Analyses of the peptidoglycan composition show that S. coelicolor show a pattern of 3-3 cross-linking befitting a tip-growing organism and change in composition between vegetative mycelium and spores. Kitasatosporae carry meso-DAP in the peptidoglycan of vegetative mycelium and LL-DAP in the peptidoglycan of spores. In line with this difference, the peptidoglycan architecture of these two growth stages undergoes such radical changes that they would seem to be from different species. S. coelicolor is naturally vancomycin resistant, but the addition of D-alanine and disruption in a single gene increases vancomycin sensitivity by a thousandfold. A knockout mutant of the alanine racemase, alr, requires exogenous addition of D-alanine. The Alr crystal structure of S. coelicolor and the D-cycloserine producer S. lavendulae were compared as to look for possible mechanisms for D-cycloserine resistance. Show less
The thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new... Show moreThe thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new and improved diagnostic routes for the typing of bacteria and their antibiotic resistance. The research is application driven and the focus is on utilizing high-end mass spectrometric instrumentation in diagnostic clinical microbiology, in a complimentary nature to already established techniques. Show less
This thesis describes the structural and biochemical characterization of the β-lactamase BlaC from Mycobacterium tuberculosis (Mtb), and the Alr and YlmE proteins from Streptomyces coelicolor A3(2)... Show moreThis thesis describes the structural and biochemical characterization of the β-lactamase BlaC from Mycobacterium tuberculosis (Mtb), and the Alr and YlmE proteins from Streptomyces coelicolor A3(2).Mtb is the main cause of tuberculosis. The inherent production of BlaC by Mtb makes the antibiotic treatment of tuberculosis particularly difficult because BlaC renders Mtb naturally resistant to β-lactam antibiotics. One possible way to circumvent this BlaC-mediated resistance is the co-administration of β-lactamase inhibitors, thus preventing antibiotics’ hydrolysis. The crystal structure of BlaC was determined in complex with the β-lactamase inhibitors clavulanic acid, sulbactam, tazobactam, and avibactam, and new BlaC-inhibitors covalent adducts were visualized. The affinity of BlaC for the inhibitors was further studied using catalytically inactive mutants of the enzyme.In parallel, the Alr and YlmE proteins from S. coelicolor A3(2) were studied. Alr and YlmE are putatively involved in the racemization of L-Ala into D-Ala. The latter is an essential peptidoglycan building block, and ensures cell wall compaction and bacterial survival. The structural and biochemical characterization of the heterologous, purified Alr and YlmE proteins showed that while Alr is indeed involved in Ala racemization, YlmE is not. Our findings revealed a possible new, surprising role for YlmE in nucleic acid binding. Show less
Keijzer, J. de; Mulder, A.; Ru, A.H. de; Soolingen, D. van; Veelen, P.A. van 2017
Aim of this thesis was to provide evidence for the clinical implication of biomarkers in blood and urine, as well as genetic markers, for the prediction of the severity and course of febrile UTI.... Show moreAim of this thesis was to provide evidence for the clinical implication of biomarkers in blood and urine, as well as genetic markers, for the prediction of the severity and course of febrile UTI. Furthermore, this thesis focused on optimization of antimicrobial treatment of febrile UTI. The main results are: 1. Recent hospitalization, indwelling urinary catheter and especially individual fluoroquinolone (FQ) use are independent risk factors for a FQ-resistant Escherichia coli febrile UTI. 2. Women with febrile UTI, including postmenopausal women and those with comorbidities, can be safely and successfully treated with a 7-day course of oral ciprofloxacin. In men, however, treatment duration should be at least 14 days. 3. Diabetes mellitus does not affect the clinical presentation and course of febrile UTI; concurrent illnesses and higher age of the diabetic population attribute to a more complicated course. 4. MR-proADM more accurately predicts a complicated course of disease than currently available inflammatory biomarkers. Importantly, biomarkers derived directly from host defense mechanisms are not suitable to distinguish between febrile UTI patients with and without bacteremia. 5. MP-TF activity is related to disease severity and bacteraemia in febrile E. coli UTI and may contribute to the prothrombotic state in gram-negative sepsis. Show less
