Polymyxins are clinically used antibiotics, discovered in mid-20th century. Once abandoned due to excessive nephrotoxicity, they are now used increasingly to address infections caused by multi-drug... Show morePolymyxins are clinically used antibiotics, discovered in mid-20th century. Once abandoned due to excessive nephrotoxicity, they are now used increasingly to address infections caused by multi-drug resistant Gram-negative bacteria.In this thesis, we describe the development and synthesis of analogues of polymyxin, aimed at reducing its associated nephrotoxicity. Analogues were made by semisynthesis, with modifications introduced mostly in the exocyclic portion of the molecule. Especially the introduction of a disulfide bond within the linked lipid helped in reducing the toxicity of the molecules, as evidenced by testing on proximal tubule epithelial cells. For most potent analogues, the antimicrobial activity was completely retained.In addition, this thesis describes studies on the mechanism of action of polymyxin, mostly based on the full stereoisomer of polymyxin B4. This analogue lacks antimicrobial activity, indicating its original stereochemistry to be of utmost importance for its use as an antibiotic.Hybrids based on polymyxin B derivatives are described, addressing non-conventional targets. A hybrid with vancomycin (typically active on Gram-positive bacteria only) shows activity on Gram-negative bacteria. A polymyxin-based hybrid coupled to a peptide with a beta-hairpin motif addresses Gram-negative bacteria, presumably by binding to outer membrane protein BamA. Show less
The investigations described in this thesis lay out strategies aimed at advancing antibiotic research and development. The examples presented revolve around two main approaches: understanding drug... Show moreThe investigations described in this thesis lay out strategies aimed at advancing antibiotic research and development. The examples presented revolve around two main approaches: understanding drug-target interactions and target identification.Applications of microcalorimetry provide insights into the binding mechanism of known antibiotics and their target within the bacterial membranes. These studies provided the thermodynamic characterization of cell-wall active compounds and their cell-wall precursor or phospholipid targets.Furthermore, by repurposing a small molecule library in a microbial susceptibility screen, the discovery of two new antibiotic leads is described. A suite of target identification methods, including whole genome sequencing and MS-based chemical proteomics, led to the characterization of their mode of action. Structure activity optimization of the leads led to the discovery of a new class of DNA gyrase inhibitors, acting on a so-far unexploited site of this validated bacterial target, as well as the identification of previously unmapped pathways in S. aureus, orchestrated by series of known and unknown enzymes. Show less
Global healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel... Show moreGlobal healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel modes-of-action is therefore of high priority.This thesis contains two drug discovery projects, originating from a antibacterial screen of a compound library. In both projects chemical hits are first structurally optimized, after which their mode-of-action is determined.The first project entails optimizing a hit with potency against MRSA into a submicromolar active antibiotic. By using a chemical proteomics approach, the targets of this compound were elucidated, along with the targets that are most important in its antibacterial activity.The second project concerns Gram-negative bacteria, where a hit molecule is optimized into the conformationally restricted LEI-800. The target of LEI-800 is found to be DNA gyrase, a common antibiotic target. However, it is that LEI-800 inhibits DNA gyrase differently, and more potently, than the status quo. Show less
Aims: Here we used a mature seven -day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or... Show moreAims: Here we used a mature seven -day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or antibiotics or non-contact induction heating, and which combinations could eradicate S. aureus in mature biofilms. Methods: Mature biofilms of S. aureus (ATCC 29213) were grown on titanium alloy (Ti6Al4V) coupons for seven days and were subjected to the following treatments or their combinations: antibiotics, mechanical cleaning, or heat shock by induction heating of 60 & DEG;C for one minute. Experiments were repeated at least five times. Results: In the untreated biofilm, growth up to 1.8x1011 colony-forming units (CFU)/cm2 was observed. Treatment with ciprofloxacin, flucloxacillin, vancomycin, cefuroxime, and amoxicillin all with rifampicin gave 6.0 log, 6.1 log, 1.4 log, 4.8 log, and 3.6 log reduction in CFU/cm2, respectively. Mechanical cleaning alone resulted in 4.9 log reduction and induc-tion heating in 7.3 log reduction. There was an additional effect of ciprofloxacin, fluclox-acillin, and induction heating when used in combinations. There was no additional effect for mechanical cleaning. No bacterial growth could be detected after induction heating followed by seven days of ciprofloxacin with rifampicin. Conclusion: Mechanical cleaning, antibiotics, and non-contact induction heating reduced the bacterial load of mature S. aureus biofilms with approximately 5 log or more as a single treatment. The effect of mechanical cleaning on mature S. aureus biofilms was limited when used in combination with antibiotics and/or induction heating. Show less
Background: Although antibiotic treatment is recommended for acute exacerbations of chronic obstructive pulmonary disease (AECOPD), its value in real-world settings is still controversial.... Show moreBackground: Although antibiotic treatment is recommended for acute exacerbations of chronic obstructive pulmonary disease (AECOPD), its value in real-world settings is still controversial. Objectives: This study aimed to evaluate the short- and long-term effects of antibiotic treatment on AECOPD outpatients. Methods: A cohort study was conducted under the PharmLines Initiative. We included participants with a first recorded diagnosis of COPD who received systemic glucocorticoid treatment for an AECOPD episode. The exposed and reference groups were defined based on any antibiotic prescription during the AECOPD treatment. The short-term outcome was AECOPD treatment failure within 14-30 days after the index date. The long-term outcome was time to the next exacerbation. Adjustment for confounding was made using propensity scores. Results: Of the 1,105 AECOPD patients, antibiotics were prescribed to 518 patients (46.9%) while 587 patients (53.1%) received no antibiotics. The overall antibiotic use was associated with a relative risk reduction of AECOPD treatment failure by 37% compared with the reference group (adjusted odds ratio [aOR] 0.63 [95% CI: 0.40-0.99]). Protective effects were similar for doxycycline, macrolides, and co-amoxiclav, although only the effect of doxycycline was statistically significant (aOR 0.53 [95% CI: 0.28-0.99]). No protective effect was seen for amoxicillin (aOR 1.49 [95% CI: 0.78-2.84]). The risk of and time to the next exacerbation was similar for both groups. Conclusion: Overall, antibiotic treatment, notably with doxycycline, supplementing systemic glucocorticoids reduces short-term AECOPD treatment failure in real-world outpatient settings. No long-term beneficial effects of antibiotic treatment on AECOPD were found for the prevention of subsequent exacerbations. Show less
The ongoing increase in antimicrobial resistance combined with the low discovery of novel antibiotics is a serious threat to our health care. Genome mining has given new potential to the field of... Show moreThe ongoing increase in antimicrobial resistance combined with the low discovery of novel antibiotics is a serious threat to our health care. Genome mining has given new potential to the field of natural product discovery, as thousands of biosynthetic gene clusters (BGCs) are discovered for which the natural product is not known.Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a highly diverse class of natural products. The large number of different modifications that can be applied to a RiPP results in a large variety of chemical structures, but also stems from a large genetic variety in BGCs. As a result, no single method can effectively mine for all RiPP BGCs, making it an interesting source for new molecules.In this thesis, new methods are explored to mine genomes for the BGCs of novel RiPP variants, with a focus on discovering RiPPs that have new modifications. RRE-Finder is a new tool for the detection of RiPP Recognition Elements, domains that are often found in RiPP BGCs. DecRiPPter is another tool that employs machine learning models to discover new RiPP precursor genes encoded in the genomes. Both tools can be used to prioritize novel RiPP BGCs. Two candidate BGCs are characterized, one of which could be shown to specify a new RiPP, validating the approach. 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
Scope: The Dutch Working Party on Antibiotic Policy constituted a multidisciplinary expert committee to provide evidence-based recommendation for the use of antibacterial therapy in hospitalized... Show moreScope: The Dutch Working Party on Antibiotic Policy constituted a multidisciplinary expert committee to provide evidence-based recommendation for the use of antibacterial therapy in hospitalized adults with a respiratory infection and suspected or proven 2019 Coronavirus disease (COVID-19).Methods: We performed a literature search to answer four key questions. The committee graded the evidence and developed recommendations by using Grading of Recommendations Assessment, Development, and Evaluation methodology.Questions addressed by the guideline and Recommendations: We assessed evidence on the risk of bacterial infections in hospitalized COVID-19 patients, the associated bacterial pathogens, how to diagnose bacterial infections and how to treat bacterial infections. Bacterial co-infection upon admission was reported in 3.5% of COVID-19 patients, while bacterial secondary infections during hospitalization occurred up to 15%. No or very low quality evidence was found to answer the other key clinical questions. Although the evidence base on bacterial infections in COVID-19 is currently limited, available evidence supports restrictive antibiotic use from an antibiotic stewardship perspective, especially upon admission. To support restrictive antibiotic use, maximum efforts should be undertaken to obtain sputum and blood culture samples as well as pneumococcal urinary antigen testing. We suggest to stop antibiotics in patients who started antibiotic treatment upon admission when representative cultures as well as urinary antigen tests show no signs of involvement of bacterial pathogens after 48 hours. For patients with secondary bacterial respiratory infection we recommend to follow other guideline recommendations on antibacterial treatment for patients with hospital-acquired and ventilator-associated pneumonia. An antibiotic treatment duration of five days in patients with COVID-19 and suspected bacterial respiratory infection is recommended upon improvement of signs, symptoms and inflammatory markers. Larger, prospective studies about the epidemiology of bacterial infections in COVID-19 are urgently needed to confirm our conclusions and ultimately prevent unnecessary antibiotic use during the COVID-19 pandemic. (C) 2020 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Show less
Bacterial vaginosis (BV) is perceived as a condition of disrupted vaginal microbiota, but remains of unknown aetiology. In this study, vaginal microbiota composition was determined in twenty-one... Show moreBacterial vaginosis (BV) is perceived as a condition of disrupted vaginal microbiota, but remains of unknown aetiology. In this study, vaginal microbiota composition was determined in twenty-one women with BV, before and after treatment with metronidazole or clindamycin. Microbiota composition varied greatly between women and defining a (un)healthy vaginal microbiota state remains elusive, challenging BV diagnosis and treatment. While relative abundance ofLactobacillusincreased after antibiotic treatment in two-third of women, its abundance was not associated with treatment outcome. Instead, remaining complaints of abnormal vaginal discharge were more common after metronidazole treatment and associated with increased relative abundance ofUreaplasma. Show less
The aim of this thesis was to stimulate rational and effective use of antimicrobials, by addressing the first two cornerstones: (1) refining stewardship of existing antimicrobials and (2) re... Show moreThe aim of this thesis was to stimulate rational and effective use of antimicrobials, by addressing the first two cornerstones: (1) refining stewardship of existing antimicrobials and (2) re-introducing old antibiotics within the framework of antimicrobial stewardship. The overall aim is to contribute to antimicrobial stewardship and to explore the value of the re-introduction of old antibiotics that are currently scarcely used. The basic step is the in vitro relationship expressed as minimal inhibitory concentration (MIC) for a given bacteria for a given antibiotic. The next step is the in vivo situation. This thesis concentrates on the in vivo situation. Show less
Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render... Show moreAntibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections. Show less
Renal clearance is responsible for the elimination of a large number of water-soluble drugs and metabolites and is therefore of large importance when characterizing the pharmacokinetics of drugs.... Show moreRenal clearance is responsible for the elimination of a large number of water-soluble drugs and metabolites and is therefore of large importance when characterizing the pharmacokinetics of drugs. Renal clearance includes glomerular filtration, tubular secretion and reabsorption and each of these processes is subject to different developmental changes. To estimate the renal clearance of drugs in children, a thorough understanding of these developmental changes in the different subprocesses contributing to renal function is needed. Therefore the aim of the research described in this thesis was to characterize the developmental changes in renal function over the entire pediatric age range. To this end, a system-based pharmacology approach was applied implicating that within the models for the different subprocesses contributing to renal function a distinction was made between system-specific and drug-specific properties. The transition to a more system-based pharmacology approach and the combination of different strategies (extrapolation to other drugs, adult data or non-clinical data) will result in an approach focusing on the underlying system instead of focusing on the drugs and may facilitate development of pharmacokinetic models and evidence-based dosing regimens in the pediatric population. Show less
Since a decade, Clostridium difficile infection (CDI) has increased progressively in incidence and severity of disease. Currently, CDI is considered the leading cause of nosocomial diarrhoea,... Show moreSince a decade, Clostridium difficile infection (CDI) has increased progressively in incidence and severity of disease. Currently, CDI is considered the leading cause of nosocomial diarrhoea, associated with an increased duration of hospitalization, healthcare expenses, morbidity and mortality. This thesis describes our findings with outbreak control, diagnosis, identification of specific risk factors and treatment of CDI after the discovery of the emergence of C. difficile PCR-ribotype 027 in the Netherlands. The studies illustrate the role of antibiotics in relation to persistence, severeness and spreading of CDI. Antibiotics are shown to be a primary risk factor for the development of (ribotype-specific) CDI and an essential part of the outbreak control measures (__bundle-approach__), namely antibiotic stewardship. The use of antibacterials is a risk for selection of novel endemic C. difficile strains in e.g. animals, which introduce an increasing risk of alternative zoonotic transmission routes. Except for very mild CDI, which is clearly induced by usage of specific antibiotics, antibacterial treatment is advised. This thesis reviews the comparative effectiveness of the currently available treatment modalities, thereby providing evidence-based recommendations for CDI remedies. Treatment options include: oral and non-oral antibiotics, toxin-binding resins and polymers, immunotherapy, probiotics, faecal or bacterial intestinal transplantation. Show less
Group B Streptococcus (GBS, Streptococcus agalactiae) has been recognized as an important cause of perinatal morbidity and mortality. The frequency of GBS colonization ranges from 10% to 35% in... Show moreGroup B Streptococcus (GBS, Streptococcus agalactiae) has been recognized as an important cause of perinatal morbidity and mortality. The frequency of GBS colonization ranges from 10% to 35% in women of reproductive age. GBS colonization can be transient, intermittent or persistent. Vertical transmission of GBS from mother to child occurs during labor. Studies on vertical GBS transmission in colonized mothers during labor report incidences of colonization of the infant between 16 and 69%. Early-onset group B streptococcal disease (GBS-EOD) occurs in approximately 1% of newborns who are colonized with GBS. Established risk factors for acquiring GBS-EOD are prolonged rupture of membranes, preterm labor, intrapartum fever, GBS bacteriuria during pregnancy or a previous child with GBS-EOD. Intrapartum antibiotic prophylaxis (IAP) given to women at risk of transmitting GBS to their baby may prevent GBS-EOD. Identification of mothers at risk may be performed by screening (taking a culture during pregnancy to detect maternal colonization) and/ or by identifying pregnancies with one or more of the established risk factors for GBS-EOD. Since the overall effect of the Dutch guideline on the incidence of GBS-EOD is disappointing, adaptation of the Dutch guidelines should be reconsidered. The aim of this thesis is to contribute to the information needed for the establishment of an optimal prevention strategy for GBS-EOD. In this thesis, studies on prevalence of GBS carriage, risk factors for GBS-sepsis in relation to GBS carriage, timing of GBS cultures, association of GBS carriage and preterm labor and resistance of GBS for antibiotics are combined. Show less
This Thesis deals with the design, synthesis and biological evaluation of peptide-based drugs. In chapters 1-4, the development of peptides derived from natural gluten which can serve as drugs to... Show moreThis Thesis deals with the design, synthesis and biological evaluation of peptide-based drugs. In chapters 1-4, the development of peptides derived from natural gluten which can serve as drugs to combat the symptoms of celiac disease is described. These symptoms are caused by a misdirected immune response towards dietary gluten in genetically predisposed individuals. Peptides which can inhibit this immune response possibly can be used as an addition to the gluten-free diet, which is the only therapy available today. In Chapters 5 and 6, the synthesis and biological evaluation analogs of the antibiotic peptide Gramicidin S is described. Gramicidin S is a naturally occurring antibiotic peptide. It is very effective against bacteria, but also exhibits toxicity towards human red blood cells which limits its use to topical applications. Analogs of this natural peptide may lead to efficient antibiotics which are broadly applicable. Show less
