<p>Extensive use of the sulphonamide drugs against the bacterium <i>Neisseria meningitidis</i> has resulted in drug resistance development. Sulphonamide resistance in <i>N. meningitidis</i> is caused by alterations in the chromosomal <i>folP</i> gene, coding for DHPS (dihydropteroate synthase). One type of resistant DHPS has high sequence divergence compared to DHPS from susceptible strains. This divergent DHPS has a duplication of two amino acids, crucial for resistance, and an altered amino acid in position 68, important for both resistance and substrate binding. When introduced into a susceptible DHPS, these two alterations did not incur resistance and resulted in abnormal substrate binding properties. This indicated that the divergent DHPS was not directly developed by mutations, but rather had been acquired by horizontal transfer of <i>folP</i> from another species.</p><p>Commensal <i>Neisseria</i> species are implied as the origin of the horizontally transferred resistance. Sulphonamide-resistant commensal <i>Neisseria</i> isolates were detected in throat swabs from healthy individuals not exposed to these drugs; however, transformation of resistance from these commensals to <i>N. meningitidis</i> was restricted in the laboratory. A comparison of the genomic region surrounding <i>folP</i> revealed differences in gene organisation and in the DNA uptake sequence between <i>N. meningitidis</i> and distantly related commensals. These differences are likely to restrict transformation between distantly related <i>Neisseria</i> species.</p><p>DHPS participates in the folate biosynthesis pathway. The enzyme preceding DHPS in the pathway, HPPK (hydroxymethyl-dihydropterin pyrophosphokinase), from <i>N. meningitidis</i> was characterised and a method for studying substrate channelling from HPPK to DHPS was developed. The information gained could be exploited in the search for new antibiotics.</p><p>In conclusion, well-adapted sulphonamide-resistant strains of <i>N. meningitidis</i> and commensal <i>Neisseria</i> are established in the bacterial population and resistance can be horizontally spread by natural transformation. This may explain the abundance of sulphonamide-resistant <i>N. meningitidis</i>, although these drugs are no longer used against this bacterium.</p>

Extensive use of the sulphonamide drugs against the bacterium Neisseria meningitidis has resulted in drug resistance development. Sulphonamide resistance in N. meningitidis is caused by alterations in the chromosomal folP gene, coding for DHPS (dihydropteroate synthase). One type of resistant DHPS has high sequence divergence compared to DHPS from susceptible strains. This divergent DHPS has a duplication of two amino acids, crucial for resistance, and an altered amino acid in position 68, important for both resistance and substrate binding. When introduced into a susceptible DHPS, these two alterations did not incur resistance and resulted in abnormal substrate binding properties. This indicated that the divergent DHPS was not directly developed by mutations, but rather had been acquired by horizontal transfer of folP from another species. Commensal Neisseria species are implied as the origin of the horizontally transferred resistance. Sulphonamide-resistant commensal Neisseria isolates were detected in throat swabs from healthy individuals not exposed to these drugs; however, transformation of resistance from these commensals to N. meningitidis was restricted in the laboratory. A comparison of the genomic region surrounding folP revealed differences in gene organisation and in the DNA uptake sequence between N. meningitidis and distantly related commensals. These differences are likely to restrict transformation between distantly related Neisseria species. DHPS participates in the folate biosynthesis pathway. The enzyme preceding DHPS in the pathway, HPPK (hydroxymethyl-dihydropterin pyrophosphokinase), from N. meningitidis was characterised and a method for studying substrate channelling from HPPK to DHPS was developed. The information gained could be exploited in the search for new antibiotics. In conclusion, well-adapted sulphonamide-resistant strains of N. meningitidis and commensal Neisseria are established in the bacterial population and resistance can be horizontally spread by natural transformation. This may explain the abundance of sulphonamide-resistant N. meningitidis, although these drugs are no longer used against this bacterium.

Infectious diseases such as malaria and relapsing fever borreliosis (RF), cause severe human mortality and morbidity in developing countries. Malaria, caused by Plasmodium spp. parasites, is estimated by the World Health Organization to cause 1.5-2.7 million deaths annually. RF, caused by Borrelia spirochetes, has the highest prevalence described for any bacterial disease in Africa, with infection outcomes ranging from asymptomatic to fatal. RF borreliosis manifests in humans as a recurring fever and with other symptoms very similar to those of malaria. RF borreliosis has been regarded as a transient infection of the blood. However, B. duttonii exploits the brain as an immunoprivileged site escaping the host immune response while spirochetes in the blood are cleared. To investigate whether residual bacteria are dormant or actively dividing, mice with residual brain infection were administered ceftriaxone, a β-lactam antibiotic interfering with cell wall synthesis. Hence, it only affects actively dividing bacteria. Ceftriaxone eradicated brain RF infection in all treated mice, demonstrating that the bacteria are actively multiplying rather than in a dormant state. The findings support the therapeutic use of ceftriaxone for RF neuroborreliosis since penetration into cerebrospinal fluid is greater for ceftriaxone than for the often recommended doxycycline. The clinical features of malaria and RF are similar and diagnosis is further complicated by the frequently occurring concomitant malaria-RF infections. Therefore, we established a mouse model to study the pathogenesis and immunological response to Plasmodium/Borrelia mixed infection. Interestingly, malaria was suppressed in the co-infected animals whereas spirochete numbers were elevated 21-fold. The immune response in the concomitantly infected mice was polarized towards malaria leaving the spirochetes unharmed. Mice with co-infections also exhibited severe anemia and internal damages, probably attributed to escalating spirochete numbers. A secondary malaria infection reactivated the residual brain RF infection in 60% of the mice. This highlights the importance of co-infections as diagnostic pitfalls as well as the need for novel treatment strategies. Currently there is no commercial malaria vaccine and increasing drug resistance presents an urgent need for new malaria chemotherapeutics. Blood-stage malaria parasites are rapidly growing with high metabolic and biosynthetic activity, making them highly sensitive to limitations in polyamine supply. Disrupting polyamine synthesis in vivo with trans-4-methylcyclohexylamine (4MCHA) eradicated the malaria infection gradually, resulting in protective immunity. This leads the way for further biochemical and pharmacological development of the polyamine inhibitor 4MCHA and similar compounds as antimalarial drugs

Long-term protective immunity depends on proper initiation of professional antigen-presenting cells (APCs). Autoimmune disorders and certain infections can cause disease through modulation of APCs and thereby affecting the outcome of these diseases. This work aimed to investigate the behaviour of different APC subsets during conditions known to cause improper immune responses. In Paper I, the effects of an anti-inflammatory compound called Rabeximod, intended for treatment of rheumatoid arthritis were investigated on different subsets of APCs. The results showed that Rabeximod affected the differentiation and behaviour of inflammatory subsets of dendritic cells (DCs) and macrophages while no effects were observed on anti-inflammatory subsets. Our findings suggest that Rabeximod acts by inhibiting the functionality of inflammatory subsets of APCs. In Paper II, the effects of different malaria derived stimuli such as hemozoin (Hz) and infected red-blood cells (iRBCs) on monocyte-derived dendritic cells (MoDCs) were investigated. Both stimuli triggered activation and migration of MoDCs. MoDCs exposed to iRBCs induced allogeneic T-cell proliferation while those exposed to Hz did not. These results indicate that different malaria derived stimuli may differently affect DCs and that this could lead to improper and inefficient T-cell activation. In Paper III, innate aspects of malarial immunity were compared in children from two sympatric ethnic groups. We observed decreased activation of APCs and severely supressed TLR responses in Dogon children as compared to Fulani. This may indicate an important role for TLR and APC activation in the Fulani, known to be better protected against malaria than the Dogon. In summary, detailed knowledge of APC activation will be helpful in the understanding of specific effector immune responses. This could in turn, improve treatment of inflammatory disorders as well as the generation of efficient vaccines against infectious diseases.

Macrophages are one of the three main human cell types infected by HIV-1. They are highly plastic cells requiring a dynamic actin cytoskeleton for their role in development, homeostasis, tissue repair and immunity. For HIV-1, disrupting actin in macrophages is detrimental in that it leads to a complete block of viral uptake and reduces reverse transcription but, significantly, not fusion. Rho GTPases (Rac1, RhoA and Cdc42) regulate many aspects of actin dynamics including those required for endocytosis. Using a pharmacological approach, it was shown that Rac1 along with Rho GTPase effectors Pak1 and N-WASP are important for productive HIV-1 entry in macrophages. However, pharmacological inhibitors aren’t available for many host factors and may have off-target effects. To overcome this, expression of dominant negative (DN) Rho GTPases was attempted in human stem cell-derived macrophages (esMDMs). While DN Rac1 expressing esMDMs were successfully generated, this was not possible for the other two. DN Rac1 expressing esMDMs, as expected, had less filamentous actin and reduced dextran uptake compared to control esMDMs. In contrast to the pharmacological studies, HIV-1 infection studies in Rac1 DN esMDMs revealed a significant increase in HIV-1 fusion, reverse transcription and nuclear import, which could be due to reduced filamentous actin leading to a slower rate of endocytosis thereby allowing more time for viral fusion within endocytic vesicles. Surprisingly, reduced HIV-1 gene expression was observed in Rac1 DN esMDMs. This was corroborated by transfection studies implicating Rho GTPases in LTR driven gene expression. To overcome the ineffectiveness of RhoA and Cdc42 DN constitutive gene expression in esMDMs, an inducible lentiviral gene expression system based on the use of a constitutive promoter and a FLEx switch mediating irreversible DNA inversions was generated. The novel FLEx vector was the first system shown to induce transgene expression in esMDMs albeit at a very low efficiency.

<p>Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein.</p><p>Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated <i>Escherichia coli</i> individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ.</p><p>Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA.</p><p>We have here compared how an SD⁺ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD⁺ is confirmed. A downstream SD⁺ gives decreased gene expression. If an SD⁺ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD⁺ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD⁺ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD⁺ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD⁺or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.</p>

<p><strong>Abstract</strong></p><p><strong>Introduction</strong></p><p>Occurrence of bacteraemia and fungemia is a serious condition with high mortality and the incidence is increasing worldwide. The aim of this study was to survey the occurrence of bacteria and fungi in blood cultures from adult patients domiciled in the county of Gävleborg during one year and also to calculate the incidence and mortality in the same geographical area.</p><p><strong>Method</strong></p><p>This is a descriptive epidemiologic study, based on all episodes of blood cultures analyzed at the Microbiology laboratory, Gävle hospital during 2005. Patients from 20 years of age, domiciled in the county of Gävleborg at the date of drawing the blood culture, where included in the study. Criteria of exclusion were negative blood cultures and cultures which were classified as contaminants.</p><p><strong>Results</strong></p><p>Altogether there were 4 564 blood cultures analyzed, resulting in 524 (11 %) positive cultures for further study. There were 442 patients (48 % women) involved in 499 episodes with confirmed bacteraemia or fungemia. Gram positive bacteria represented 52 %, gram negative 45 % and fungi 3 %. The most frequently isolated bacterium was <em>Escherichia coli </em>followed by <em>Staphylococcus aureus. </em>In women, <em>Escherichia coli </em>was the most common bacterium, and there was a significant difference between the genders (<em>p= </em>0.004). In men, <em>Staphylococcus </em><em>aureus </em>was the dominant species (<em>p= </em>0.027<em>)</em>. <em>Streptococcus pneumoniae </em>was more common in women (<em>p= </em>0.005). The incidence of bacteraemia and fungemia in the county of Gävleborg was 235/100 000 inhabitants above the age of 20 (women, 223/100 000 men, 247/100 000). The incidence increased with age and the mean age was 70.2 years. The mortality within 30 days after the last positive blood culture was 22 % (97 patients). <em>Escherichia coli </em>was the most common bacteria diagnosed among those who died. The mortality in fungemia was 66 %. There was no significant difference in incidence or mortality between the two provinces Gästrikland and Hälsingland. Patients with bacteraemia and fungemia were initially cared for at all medical care units at the three hospitals in the county.</p><p><strong>Conclusion</strong></p><p>The incidence of bacteraemia/fungemia in the county of Gävleborg was 235/100 000 inhabitants. The most common bacteria in patients with confirmed bacteraemia were <em>Escherichia coli </em>and <em>Staphylococcus aureus. </em>Increasing age was a contributing risk factor. Patients with fungemia had considerably higher mortality compared to patients with bacteraemia. There where no significant differences in mortality between the two provinces.</p> / <p><strong>Introduktion</strong></p><p>Fynd av bakterier, bakteriemi, och svampar, fungemi, i blodbanan är ett allvarligt tillstånd med hög mortalitet och incidensen ökar i världen. Syftet med denna studie var att kartlägga vilka bakterier och svampar som förekom i alla blododlingar tagna under ett år från vuxna patienter i Gävleborgs län, samt att analysera incidens och mortalitet för bakteriemi och fungemi i länet.</p><p><strong>Metod</strong></p><p>Det är en deskriptiv epidemiologisk studie som utgår från analyserade blododlingar under år 2005 vid Enheten för Klinisk Mikrobiologi Laboratoriemedicin vid Gävle sjukhus. Till studien inkluderades personer från 20 års ålder som var mantalsskrivna i Gävleborgs län det datum som blododlingen utfördes. Exklusionskriterierna var negativa odlingssvar och svar som bedömdes som kontamination.</p><p><strong>Resultat</strong></p><p>Totalt analyserades 4 564 blododlingar, av vilka 524 (11 %) var positiva och bearbetades i denna studie. Det blev 442 patienter (48 % kvinnor) med 499 episoder av säkerställd bakteriemi eller fungemi. De grampositiva bakterierna stod för 52 %, gramnegativa bakterier 45 % och svampar 3 %. De enskilt vanligaste bakterierna var <em>Escherichia coli </em>och <em>Staphylococcus aureus. </em>För kvinnorna var <em>Escherichia coli </em>vanligast och det fanns en signifikant skillnad mellan könen (<em>p= </em>0,004 ), för männen var <em>Staphylococcus aureus </em>vanligast (<em>p= </em>0,027<em>)</em>. <em>Streptococcus pneumoniae </em>visade högre förekomst bland kvinnorna än männen (<em>p= </em>0,005). Incidensen för bakteriemi och fungemi i Gävleborgs län var 235/100 000 invånare äldre än 20 år (kvinnor, 223/100 000 och män, 247/100 000). Incidensen ökade med åldern och medelåldern var 70,2 år. Mortaliteten inom 30 dagar efter utförd blododling var 22 % (97 patienter). <em>Escherichia coli </em>var vanligast hos de avlidna. För patienter med fungemi var mortaliteten 66 %. Det påvisades ingen signifikant skillnad beträffande incidens eller mortalitet mellan länets båda landskap Gästrikland och Hälsingland. Patienter med bakteriemi och fungemi vårdades initialt på samtliga vårdenheter på länets tre sjukhus.</p><p><strong>Konklusion</strong></p><p>Incidensen för bakteriemi/fungemi i Gävleborgs län var 235/100 000 invånare. De vanligaste fynden vid säkerställd bakteriemi var <em>Escherichia coli </em>och <em>Staphylococcus aureus</em>. Ökande ålder var en riskfaktor. Patienter med fungemi hade avsevärt högre mortalitet än de med bakteriemi. Ingen skillnad påvisades mellan de två landskapen beträffande mortalitet.</p>

<p>Becoming antibiotic resistant is often associated with fitness costs for the resistant bacteria. This is seen as a loss of competitiveness against the antibiotic-sensitive wild-type in an antibiotic-free environment. In this study, the physiological alterations associated with fitness cost of antibiotic resistance <i>in vitro</i> (in the laboratory medium), and <i>in vivo</i> (in a mouse infection model), are identified in the model system of fusidic acid resistant (Fus^R) <i>Salmonella</i> <i>enterica</i> serovar Typhimurium.</p><p>Fus^R mutants have mutations in <i>fusA</i>, the gene that encodes translation elongation factor G (EF-G). Fus^R EF-G has a slow rate of regeneration of active EF-G·GTP off the ribosome, resulting in a slow rate of protein synthesis. The low fitness of Fus^R mutants <i>in vitro</i>, and <i>in vivo</i>, can be explained in part by a slow rate of protein synthesis and resulting slow growth. However, some Fus^R mutants with normal rates of protein synthesis still suffer from reduced fitness <i>in vivo</i>. We observed that Fus^R mutants have perturbed levels of the global regulatory molecule ppGpp. One consequence of this is an inefficient induction of RpoS, a regulator of general stress reponse and an important virulence factor for <i>Salmonella</i>. In addition, we found that Fus^R mutants have reduced amounts of heme, a co-factor of catalases and cytochromes. As a consequence of the heme defect, Fus^R mutants have a reduced ability to withstand oxidative stress and a low rate of aerobic respiration.</p><p>The pleiotropic phenotypes of Fus^R mutants suggest that antibiotic resistance can be associated with broad changes in bacterial physiology. Knowledge of physiological alterations that reduce the fitness of antibiotic-resistant mutants can be useful in identifying novel targets for antimicrobial agents. Drugs that alter the levels of global transcriptional regulators such as ppGpp or RpoS deserve attention as potential antimicrobial agents. Finally, the observation that Fus^R mutants have increased sensitivity to several unrelated classes of antibiotics suggests that the identification of physiological cost of resistance can help in optimizing treatment of resistant bacterial populations.</p>

<p>Antibiotic resistance is of current concern. Bacteria have become increasingly resistant to commonly used antibiotics and we are facing a growing resistance problem. The present thesis was aimed at studying the impact of antibiotic treatment on pathogenic bacteria as well as on the normal human microbiota, with focus on resistance development.</p><p>Among the factors that affect the appearance of acquired antibiotic resistance, the mutation frequency and biological cost of resistance are of special importance. Our work shows that the mutation frequency in clinical isolates of <i>Helicobacter pylori</i> was generally higher than for other studied bacteria such as <i>Enterobacteriaceae; </i>¼ of the isolates displayed a mutation frequency higher than<i> Enterobacteriaceae </i>defective<i> </i>mismatch repair mutants and could be regarded as mutator strains.</p><p>In <i>H. pylori</i>, clarithromycin resistance confers a biological cost, as measured by decreased competitive ability of the resistant mutants in mice. In clinical isolates, this cost could be reduced, consistent with compensatory evolution stabilizing the presence of the resistant phenotype in the population. Thus, compensation is a clinically relevant phenomenon that can occur in vivo.</p><p>Furthermore, our results show that clinical use of antibiotics selects for stable resistance in the human microbiota. This is important for several reasons. First, many commensals occasionally can cause severe disease, even though they are part of the normal microbiota. Therefore, stably resistant populations increase the risk of unsuccessful treatment of such infections. Second, resistance in the normal microbiota might contribute to increased resistance development among pathogens by interspecies transfer of resistant determinants.</p>

<p>In recent years molecular biology has become an integral part of the clinical laboratory. With an ever increasing number of methodologies and applications being presented each year it has increased our knowledge of how bacteria cause disease as well as our ability to predict disease outcome. </p><p>The main focus of this thesis has been to develop methods for identifying biomarkers and prediction methods for bacterial infectious diseases by taking advantage of the ever increasing possibilities of molecular biology. We applied cutting edge techniques in order to establish novel platforms for identifying and characterizing biomarkers of disease. </p><p>In paper one we describe a novel approach to measure levels of antibiotic resistance and viability of C. trachomatis, a method that is a clear improvement over existing techniques. In the second paper we describe the development of two assays designed to type pertussis toxin subunit 1 in circulating strains, in order to facilitate multi center studies for vaccine escape surveillance. In paper three we develop a novel microarray application designed to identify a large number of bacterial traits of H. pylori simultaneously with human genetic polymorphisms in order to identify a collection of risk factors that could be used as a prediction tool for gastric cancer risk. In the last paper we define the “antigenome” of H. pylori and identified 14 promising, previously unreported antigens as well as a number of potential biomarkers.</p><p>The platform technologies described in this collection of papers will hopefully help us identifying novel ways of fighting and predicting bacterial disease in future studies. </p>