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Novel absorber materials are being developed to improve EUV-reticle imaging performance for the next generations of EUV lithography tools. TNO, together with ASML, has developed a compatibility assessment for novel absorber materials, which addresses the risk that exposure of incompatible materials to EUV-radiation and EUV-plasma conditions results in contamination of the optics in the EUV lithography tools. The assessment is divided in two stages to optimize the efficiency of the procedure. Most contamination risks can be addressed cost-efficiently in the first stage with existing vacuum and plasma test facilities. Novel absorber materials can thus be assessed in an early stage of their development without the immediate need for more expensive EUV testing. This stage of the compatibility assessment was executed with an EUV reticle piece with a TaN-based absorber, and results are presented. The TaN-based absorber showed no compatibility issues, as expected. This test procedure now sets the baseline for testing novel absorber materials. 96.000 exposures can be performed in a NXE 3400 EUV lithography tool with a 300W source with absorber materials that successfully passed the first stage of the compatibility assessment. Assuming 96 exposures per wafer, this equals 1000 wafers. Absorber materials that passed the first stage may proceed to the second stage: An accelerated EUV test exposure in the EUV Beamline 2 (EBL2). Each material will be exposed to an EUV-dose equivalent to about half a year of reticle exposure in the NXE 3400 lithography tool with a 300W source. This test is in preparation and expected to be available in the second quarter of 2019.

Bacteria live all around us and in general they live peacefully in balance with us. Sometimes however this balance is disrupted when a bacteria starts to adapt and explore new areas. This is also the case for the gut bacterium Enterococcus faecium. Over the years it has acquired many antibiotic resistances and is causing, at an increasing rate, hospital associated infections. The goal of this research was to unravel which adaptions E. faecium has made that allowed it to become so successful as a hospital associated disease. For this we developed new genetic techniques to study its DNA and more easily manipulate the DNA to get a better understanding of the function of the genes. Our results show that not a singular gene responsible for the improved adaptation but a complex network of genes which are largely present across E. faecium strains. This indicates that this bacterium as a species is already highly suited to survive in hospitals. Further research needs to be conducted to understand in greater detail into how this bacterium survives and aim to discover new methods to prevent it from causing infections in hospitalised patients.

Antibiotics provided humanity resilience to the majority of bacterial infections. An important trade-off is the emergence of antimicrobial resistance, and a diminished and perturbed microbiota, resulting in an increased susceptibility for Clostridioides difficile infections. For both C. difficile and multidrug resistant micro-organisms (MDRO), asymptomatic colonisation of the gut plays an important role in the development of infection. The aim of this thesis is to better understand the role of the microbiota in defence against infections with C. difficile and MDRO. The first part describes the epidemiology and detection of asymptomatically colonized individuals. It concludes that though asymptomatic colonization of MDRO and C. difficile can become a nidus for nosocomial (hospital) infection and transmission, its prevalence is still low in the Netherlands. The second part of this thesis focuses on eradication and/or treatment of these micro-organisms with ‘fecal microbiota transplantation’ of healthy donor feces. It describes the establishment of the Netherlands Donor Feces Bank, and the research, experiences and successes of FMT in the treatment of patients with recurrent C. difficile infections and MDRO. The experience of this thesis may help the establishment, utilization, standardization and maturation of stool banks and research institutes of the next-generation of microbiota modifying therapies.

Antibiotics provided humanity resilience to the majority of bacterial infections. An important trade-off is the emergence of antimicrobial resistance, and a diminished and perturbed microbiota, resulting in an increased susceptibility for Clostridioides difficile infections. For both C. difficile and multidrug resistant micro-organisms (MDRO), asymptomatic colonisation of the gut plays an important role in the development of infection. The aim of this thesis is to better understand the role of the microbiota in defence against infections with C. difficile and MDRO. The first part describes the epidemiology and detection of asymptomatically colonized individuals. It concludes that though asymptomatic colonization of MDRO and C. difficile can become a nidus for nosocomial (hospital) infection and transmission, its prevalence is still low in the Netherlands. The second part of this thesis focuses on eradication and/or treatment of these micro-organisms with ‘fecal microbiota transplantation’ of healthy donor feces. It describes the establishment of the Netherlands Donor Feces Bank, and the research, experiences and successes of FMT in the treatment of patients with recurrent C. difficile infections and MDRO. The experience of this thesis may help the establishment, utilization, standardization and maturation of stool banks and research institutes of the next-generation of microbiota modifying therapies.

Clostridioides difficile is a Gram-positive gut pathogen capable of causing disease resulting in (mild) diarrhea associated with mild- or severe inflammation of the gut. Patients that have been recently treated with antibiotics, have comorbidities, or the elderly, are most susceptible to developing a C. difficile infection (CDI). Despite this, antibiotic use is the first line therapy for CDI treatment. The antibiotics used in CDI treatment are metronidazol, vancomycin or fidaxomicin. CDI can be difficult to treat and can recur, in which case a fecal microbiota transplantation (FMT) can resolve infection.C. difficile is intrinsically resistant to many different antibiotics. For this reason it is important to find and develop novel antibiotics and treatments for CDI. However, bacteria are capable of adapting to unfavorable environments (such as the presence of antibiotics) and it is crucial to understand how these adaptive processes are regulated. This thesis describes how low concentrations of a novel, experimental antibiotic, ibezapolstat, produces secondary effects that can facilitate C. difficile’s adaptive response. Part of this adaptation is mediated by the general stress response, which is characterized in this thesis. Additionally, this thesis describes two novel ways in which C. difficile can become resistant to metronidazole.

Colistin is increasingly used as a last resort drug to treat infections with multidrug resistant Gram-negative bacteria. Colistin acts specifically on Gram negative bacteria due to the selective presence of lipopolysaccharides in the membranes of Gram-negatives. Through electrostatic interaction with lipopolysaccharides, colistin is able to destabilize the membranes of Gram-negatives. However, due to its previous fall into disuse, there is a gap in the knowledge about the evolution and mechanisms of colistin resistance. In this thesis, we have examined the evolution and mechanisms of colistin resistance in Enterobacteriaceae. We examined the mechanisms of colistin resistance in E. coli strains isolated from bloodstream infections, and diverse Enterobacteriaceae isolated from patients admitted to an intensive care unit, receiving the colistin-containing selective decontamination of the digestive tract treatment. We observed a low prevalence of resistance to this last-resort drug in the Netherlands. In addition, we did not detect clonal relationships between the colistin resistant strains isolated from patients, indicating independent acquisition of colistin resistance in all isolates. Through the construction of mutant E. coli and K. pneumoniae strains, we have determined the contribution of mutations in the PhoPQ, and PmrAB two component regulatory systems to cause colistin resistance. By performing an in vitro evolution experiment by growing colistin-susceptible K. pneumoniae strains under increasing concentrations of colistin, we have determined that the evolution of colistin resistance is these strains occurred through the swift and selective fixation of beneficial mutations in systems associated with LPS modification. The evolution of colistin resistance in K. pneumoniae had a variable effect on virulence characteristics in these strains, including the decreased susceptibility to the human antimicrobial peptide LL-37, increased susceptibility to the humoral complement system, and increased virulence in an C. elegans survival model. Through mass spectrometry analyses of E. coli strains, we observed the modification of lipid A with phosphoethanolamine. In K. pneumoniae, lipid A may be modified through hydroxyl-groups, palmitate-groups, and of 4-amino-4-deoxy-L-arabinose. This thesis shows the diverse evolutionary pathways Enterobacteriaceae may use to become resistant to colistin. We observe that mutations leading to colistin resistance may be selected swiftly, and without a necessary impact on fitness.

In the last decades, the emergence and spread of resistant opportunistic pathogens is compromising the effectiveness of antimicrobial therapies. Understanding the emergence and global spread of drug-resistant microorganisms is thus crucial to combat antimicrobial resistance. The human gut harbors a densely populated and highly diverse microbial ecosystem – the gut microbiota – that can serve as a reservoir for antibiotic resistance genes (‘the resistome’) and for drug-resistant opportunistic pathogens. Critically ill patients that are hospitalized in Intensive Care Units (ICUs) are particularly susceptible to nosocomial infections caused by bacteria that are members of the normal human microbiota. In order to prevent or treat nosocomial infections in these patients, antibiotic therapy is frequently used in patients that are hospitalized in ICUs. Other antimicrobial agents than antibiotics, such as disinfectants, are also important in limiting the prevalence of pathogenic microorganisms and nosocomial infections in clinical settings. However, their effectiveness is similarly compromised by the emergence of pathogens with reduced susceptibility to disinfectants. Previous studies have shown that the gut microbiota is significantly impacted by antimicrobial therapy. Yet, the dynamics of the gut resistome during such therapies and its contribution to the emergence and dissemination of antimicrobial resistance remain poorly understood. In this thesis the dynamics of the gut microbiota and resistome of adult hospitalized patients in an ICU receiving prophylactic and curative antibiotic therapies were investigated using culture-independent techniques. Because gut-associated bacteria and their resistance genes may be shed into the environment through sewage, we also assessed the levels of antibiotic resistance genes in hospital sewage and in subsequent stages of wastewater treatment. The gut microbiota as a putative reservoir for disinfectant resistance genes is also studied. This thesis provides novel insights into the dynamics and diversity of the gut microbiota and the resistome of hospitalized patients, and highlights the significant effects of ICU hospitalization on the gut resistome and microbiota. The expansion of the gut resistome during ICU hospitalization and antimicrobial therapies shown here is a cause for concern. Follow up investigations combining gut microbiology and resistome studies may be be performed to assess the potential transfer of these resistance genes to nosocomial pathogens during and after hospitalization.