Streptococcus pneumoniae has a highly adaptable genome due at least in part to its natural transformability and its ability to recombine with other pneumococci and related species. The emergence of antibiotic resistant clones of S. pneumoniae presents an opportunity to investigate how the genome has altered, spread and diversified within a defined time frame. We postulated that the genomes of epidemic, resistant isolates of S. pneumoniae would carry evidence of the genetic mechanisms that have shaped their evolution. We investigated this using eight to fifteen isolates from each of three S. pneumoniae clones; two multiply resistant clones Spain 9V-3 and Taiwan 19F-14 and one clone that has not acquired multiple resistance, England 14-9. Genome diversity in each of the three clones was investigated using pulsed field gel electrophoresis and multilocus sequence typing. Polymorphisms identified as a result of changes in the size of restriction fragments were found to be caused mainly by genomic rearrangements rather than restriction site mutations. Several deletion/insertion events in addition to one large inversion were identified. A number of polymorphisms correlated with previously known variable regions. Database analysis of multilocus sequence data from all three clones showed that recombination leads to sequence divergence more frequently than de novo mutation, but was significantly less common in England 14-9. The lower frequency of recombination events in England14-9 was in line with a transformation deficiency observed in vitro, and may explain the rare occurrence of penicillin resistance in this clone. Analysis of competence and recombination gene sequences available from databases revealed a potential cause of transformation deficiency: a four amino acid deletion in CelA, involved in DNA uptake and transport. Recombination can act as a DNA repair mechanism, but the significantly low occurrence in England14-9 suggests other mechanisms act to repair severe damage. Although S. pneumoniae does not have a typical SOS response it does possess DNA polymerase IV, encoded by dinB, which is predicted to be involved in error-prone DNA replication and repair of double strand breaks. DinB knockout mutants were created to investigate the effect in isogenic backgrounds. DinB mutants presented a lower frequency of spontaneous rifampicin resistance mutations than wild type 3 isolates. DinB mutants were more sensitive to killing by three different DNA damaging agents as well as by hydrogen peroxide. Isolates of the natural dinB mutant clone Spain 9V-3 were also shown to be more sensitive to DNA damaging agents than clones England 14-9 and Taiwan 19F-14. It is concluded that genetic differences between the three clones investigated do influence their patterns of evolution, and may account for differences in their antibiotic resistance profiles. Furthermore DNA polymerase IV does function as an error prone repair polymerase capable of protecting pneumococci from DNA damage despite the lack of a coordinated SOS response in pneumococci, and the absence of the gene in one successful multi-resistant clone.

Introduction: Streptococcus pneumoniae (pneumococci) is most common and has the highest mortality in community-acquired pneumonia (CAP). In vitro many antibiotics are effective against pneumococci, but recent studies suggest a favor for narrow-spectrum antibiotics. Aim: To investigate difference in mortality from pneumococcal CAP if betalactam antibiotics in monotherapy with a narrow or broad spectrum are selected initially. Methods: A retrospective national register study with cases of pneumococcal CAP 2008-2015. Cohorts of CAP were divided by severety, 0-2 and 3-4 CRB-65-points. Antibiotics and background factors were analyzed. A logistic regression model analyzed potential factors for mortality <30 days. Results: There were 34.299 cases, of which 1592 cases were included, n=1509 with 0-2 CRB-65 and n=83 with 3-4 CRB-65. Overall mortality was 2.5%. 17 out of 1025 (1.7%) died after narrow-spectrum betalactam monotherapy (NSBM), and 23 out of 567 (4.1%) died after broad-spetrum betalactam monotherapy (BSBM). In CAP with 0-2 CRB-65-points, NSBM were associated with less mortality than BSBM (p=0.007, OR 0.39; 95 % CI 0.20-0.78). Age<65 and kidney disease were associated with higher mortality, but in the logistic regression model, these factors were not associated with selected antibiotics and thus not confounders. In CAP with 3-4 CRB-65-points, no significant results were found. Conclusion: NSBM, like penicillins, seems to be safe choice and associated with less mortality than BSBM in less severe pneumococcal CAP. The reasons are unknown, but maybe because of less severe side-effects, better property to the bacteria or better empirical dosage.

One of the oldest and most exciting questions in science is: are we alone in the universe? During the last four billion years of Earth’s history, countless organisms have inhabited almost every environmental niche on the planet, from the deepest sea to driest deserts. However, so far no extraterrestrial life has been found. Studying the propensity for life on our neighboring planet, Mars,helps us understanding its potential for past and present life, and guides future missions. Liquid water is a prerequisite for life as we know it and recently, evidence of transient night time liquidbrines on the surface of present day Mars have been theorized. These brines may be hyper-salinewith high ionic strengths and varying pH-values. Halobacterium salinarum is an extremophilic (saltloving) halophilic archaeon whose natural habitat includes hyper-saline brines, desiccating conditions and exposure to high fluences of solar UV radiation. Herein, we report the response of Hbt.salinarum following exposure to simulated Martian conditions, with regard to survival and DNAintegrity. The simulated conditions include the synthetic Martian Brine Analogues (MBAs), diurnalnocturnaltemperature cycling, prolonged desiccation and Mars-like solar UV (200-400 nm) radiation.We also addressed the prolific space hardware contaminant, Bacillus subtilis whose endospores show substantial resistance against space conditions. The ambition was to investigate potentia linterplanetary forward contamination by Hbt. salinarum, should it have bacterial spores available as nutrients in the Martian brines. Halophiles are some of our best candidates for studying unicellular life on Mars and other bodies where liquid water is also stabilized by high salt concentrations.Moreover, Hbt. salinarum was able to survive over one month in the Martian brines, albeit with growth limited to one particularly hospitable brine. It displayed survival in the brines at relevant temperatures and with diurnal-nocturnal cycling but only when first desiccated to remove preventwater crystal formation. The radiation resistance was highly dependent on the choice of brine inwhich Hbt. salinarum was confined and desiccated. Even in the hospitable brines, the halophile lost over 90% of its viable population following irradiation equal to one Martian day, in our experimentalsetup. The inter-brine difference in DNA fragmentation following irradiation confirmed the differencein survival. Hbt. salinarum was subsequently unable to digest B. subtilis endospores for nutrient exploit and responded no differently than when nutrient-deprived. Surprisingly, the addition of otherwise available nutrients in the brines caused a hurried decrease in survival, with the exception of the hospitable brine. Despite its extremophilic and polyextremotolerant character, Hbt. salinarumis unlikely to survive, not to mention thrive, in a combination of all tested stressors.

Human C-reactive protein (CRP), injected intravenously into mice or produced inside mice by a human transgene, protects mice from death following administration of lethal numbers of Streptococcus pneumoniae. The protective effect of CRP is due to reduction in the concentration of bacteria in the blood. The exact mechanism of CRP-dependent killing of pneumococci and the partners of CRP in this process are yet to be defined. The current efforts to determine the mechanism of action of CRP in mice are directed by four known in vitro functions of CRP: 1. the ability of pneumococcal C-polysaccharide-complexed CRP to activate complement pathways, 2. the ability of CRP to bind to Fcγ receptors on phagocytic cells, 3. the ability of CRP to bind to immobilized complement regulator protein factor H which can also be present on pneumococci, and, 4. the ability of CRP to interact with dendritic cells. CRP-treated dendritic cells may well be as host-defensive as CRP alone. An interesting condition for the protective function of CRP is that CRP must be given to mice within a few hours of the administration of pneumococci. CRP does not protect mice if given later, suggesting that CRP works prophylactically but not as a treatment for infection. However, full knowledge of CRP may lead to the development of CRP-based treatment strategies to control pneumococcal infection. Also, because CRP deficiency in humans has not yet been reported, it becomes important to investigate the deficiency of the mechanism of action of CRP in CRP-positive individuals.

The overall aim of this thesis is to improve knowledge about the prevention of infectious and bleeding complications in patients with hematological malignancies, primarily in those with chronic lymphocytic leukemia (CLL) and myelodysplatic syndrome (MDS). Hypogammaglobulinemia, impaired production of immunoglobulins (Ig), is an established risk factor for infection, but the impact of IgG pure subclass deficiency (IgG subclass deficiency with adequate production of IgG, IgA, and IgM) has been debated. In a retrospective single institution study, we concluded that pure IgG subclass deficiency in CLL patients is rare and is not associated with an increased risk of infection. Hence, routine analysis of IgG subclasses in patients with CLL is not warranted. There is no consensus on recommending vaccination against Streptococcus pneumoniae to CLL patients mainly because comparative studies are lacking. In our randomized trial, the efficacy of a conjugated pneumococcal vaccine on immune response was superior or equal to a polysaccharide vaccine for all pneumococcal serotypes common for the two vaccines. A conjugate pneumococcal vaccine should therefore be included in vaccination programs for patients with CLL. Bronchoalveolar lavage (BAL) is a well-established invasive method to identify the cause of pulmonary infiltrates in immunocompromised patients. In a retrospective trial, we have studied the diagnostic yield of BAL in patients with hematological malignancies. We concluded that BAL is highly useful in either verifying or excluding some of the important respiratory tract infections affecting these patients, particularly invasive pulmonary aspergillosis (IPA) and Pneumocystis jirovecii pneumonia (PJP). However, standardized procedures for BAL sampling should be continually revised to avoid unnecessary microbiological tests. Thrombocytopenia, an adverse prognostic factor in patients with MDS, can be aggravated by azacitidine, first-line treatment for high-risk MDS. Eltrombopag, a thrombopoietin-receptor agonist (TPO-R), alleviates thrombocytopenia in patients with immune thrombocytopenic purpura (ITP). In a phase I clinical trial, we concluded that the combination of eltrombopag and azacitidine in high-risk MDS patients with thrombocytopenia is feasible and well tolerated in doses up to 200 mg eltrombopag daily.