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. 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 Helicobacter pylori was generally higher than for other studied bacteria such as Enterobacteriaceae; ¼ of the isolates displayed a mutation frequency higher than Enterobacteriaceae defective mismatch repair mutants and could be regarded as mutator strains. In H. pylori, 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. 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.

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. 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. 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. 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.

Idag lever cirka 33 miljoner människor med HIV/Aids. Totalt har omkring 30 miljoner människor avlidit sedan första Aids- fallet diagnostiserades år 1981. I västvärlden har HIV-smittade en tät kontakt med sjukvården eftersom de går på regelbundna kontroller, därmed är sjuksköterskor en yrkeskategori som träffar dessa patienter ofta. I utvecklingsländerna ser det ofta tyvärr annorlunda ut, HIV-smittade har ingen eller gles kontakt med sjukvården. Syftet med studien var att belysa sjuksköterskans upplevelser i omvårdnaden av HIV/Aids patienter. Uppsatsen genomfördes som en litteraturstudie där 14 vetenskapliga artiklar granskades, bearbetades och analyserades. Resultatet visade att sjuksköterskornas upplevelser av att vårda HIV/Aids patienter varierade. Positiva upplevelser kunde bland annat vara att arbetet var utvecklande och sjuksköterskorna kände sig viktiga för patienterna. Negativa upplevelser var bland annat rädsla och brist på resurser. Att vårda patienter med HIV/Aids kräver god kunskap om både sjukdomen och adekvata omvårdnadsåtgärder. Mer utbildning inom området efterlyses i sjuksköterskans grundutbildning. Förslag till vidare forskning är att undersöka vad sjuksköterskorna anser att man kunde göra för att ta bort de felaktiga negativa bilderna av sjukdomen och komma till rätta med de brister som finns idag. / Today about 33 million people have conflicted HIV/Aids. A total of about 30 million people have died of Aids since the first case was diagnosed in 1981. In the Industrial states people infected with HIV have close contact with medical care units, since they attend regular examinations. Due to this, nurses often come in contact with these patients. In the developing countries it is unfortunately not like that, HIV-infected has sparse or none contact with medical units. The purpose of this study was to illustrate nurses' experiences when caring for patients diagnosed with HIV/Aids. The study was carried out as a literature study where 14 scientific articles were reviewed, processed and analyzed. The result shows that nurses' experiences during their care of HIV/Aids patients varies. Among the positive experiences could be noticed that nurses felt that their work was important and was developing them. Among the negative experiences occurred fear and lack of resources. Medical care of patients with HIV/Aids demands good knowledge about the disease and adequate nursing care. Additional education is needed during nurses' basic education. A suggestion concerning further research includes researching what suggestions nurses have on how to correct faulty views of the disease and how to correct areas that need improvement.

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. 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 Escherichia coli 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. 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. 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.

Postoperativ sårinfektion är en komplikation som var tionde patient drabbas av efter ett kirurgiskt ingrepp. Det medför inte bara lidande och förlängd vårdtid för patienten utan kan även vara direkt livshotande. Den förlängda vårdtiden medför dessutom kostnader för samhället, och resurser skulle kunna användas till annan vård. Syftet med litteraturstudien var att beskriva sådana omvårdnadsåtgärder som sjuksköterskan kan vidta för att förebygga postoperativa sårinfektioner. Evidensbaserade omvårdnadsåtgärder som, var för sig minskar risken för att patienten ska drabbas av en postoperativ sårinfektion, och tillsammans utgör grunden för en säker vård. Databassökning av vetenskapligt material inom området ligger till grund för resultatet. Genom aktuell forskning presenteras och lyfts olika omvårdnadsåtgärder som reducerar risken för patienten att drabbas av en postoperativ sårinfektion. Viktiga omvårdnadsåtgärder är: korrekt hårborttagning, bibehållen normotermi, dusch med desinfektion, glukoskontroll, administrering av antibiotikaprofylax och postoperativ sårvård. Ny forskning inom området efterfrågas för att kunna följa utvecklingen, eftersom den befintliga är publicerad för många år sedan. För att patienten ska kunna erbjudas en säker vård behövs kontinuerlig utbildning under sjuksköterskeutbildningen men även i den kliniska verksamheten. Regelbunden uppföljning och utvärdering bör också ske i den kliniska verksamheten för att omvårdnadsåtgärderna ska vara effektiva. / Postoperative surgical wound infection is a complication that every tenth patient suffering after a surgical procedure. The consequences are the suffering and prolonged length of stay for the patient and can also be directly fatal. The prolonged duration of treatment is a high cost in society and resources could be used for other care. The purpose of literature review was to describe nurse’s interventions, which can be taken to prevent postoperative surgical wound infections. Evidence-based care interventions which reduce the risk of the patients suffering a postoperative surgical wound infection and together they represent a safe care. The result is based on search in databases for scientific materials in the subject area. Through current research highlights interventions which reduce the risk of the patient suffering a postoperative wound infection. Essential nursing interventions which are identified as: Hair removal, warming, shower with disinfectant, glucose monitoring, administration of antibiotic prophylaxis and wound care. New research in this area is requested to follow the developments since the current research is getting old. If the care should be safe for patient there must be education in nursing training as well as in the clinical work. Continuous follow-up should also occur in the clinical work in order to get feedback if the nursing interventions are effective.

<p>Cancer or malignant tumors has a high death frequency in many countries. Nowadays many research facilities are dedicated to find new substances and techniques which would lead to better cancer therapies. Seven years ago a research team from Finland made a remarkable connection between vitamin D deficiencies and an increased chance of getting prostate cancer. The research investigating this statement has lead to findings of a new non-classical effect of the calcium controlling vitamin, 1,25(OH)2D3. This effect involves anti-proliferatory effects and more importantly apoptotic effects resulting in the hope of finding a new drug that can cure prostate cancer with the smallest amount of harm to the body.</p><p>In an attempt to find out if the signalling pathway of this apoptotic effect is fast or slow, an experiment designed to detect when the apoptotic protein caspase-3 is induced has been performed. Cells from the cell line LNCaP has been cultured and incubated with 1,25(OH)2D3 and after 0min - 48h an assay was performed to detect the relative amounts of caspase-3 present in every sample. The optimal time period (48h) was then subjected to three different concentrations of 1,25(OH)2D3 and read in the same way as the previous samples. The results showed an increase in caspase-3 expression as early as 2 min, but disappear to be seen again at 24h and are more profound in 48h samples. The caspase-3 expression was also seen to form a possible exponential curve in dose-response.</p>

An exogenous retrovirus (XRV) that integrates into a germ cell may be inherited as a Mendelian gene; it becomes an endogenous retrovirus (ERV). The human genome consists of up to 8% HERVs. The gammaretroviral (ERV class I) HERV-H, with 926 members, is the largest ERV group. Despite millions of years since integration, it has polymorphic envelope open reading frames in at least three loci. Selections for functional envelopes are indicated on chromosomes 1 and 2. However, envelopes were present only in a fraction of the total HERV-H. Mutated polymerases, indicating old ERVs, contradicted relatively intact long terminal repeats. To explain this, we formulated a “Midwife” element theory where proteins are complemented in trans. A phylogenetic analysis did not support separate HERV-H and -F groups. The new taxonomy included HERV-H like (RGH2-like and RTVLH2-like subgroups) and Adjacent HERV-H like. A bioinformatic reconstruction of a putative ancestral HERV-H exposed novel traits. Two nucleocapsid zinc fingers and a pronounced nucleotide bias for C in the HERV-H like were unique among the gammaretroviruses. Two recently integrated gammaretroviral groups (PtNeo-I[PTERV1] and -II) were found in chimpanzees but not in humans. The PtNeo groups were most similar to baboon ERVs and a macaque sequence, but neither to other chimpanzee nor to any human gammaretroviruses. The pattern was consistent with cross-species transfer via predation. To advance the retroviral taxonomy, we projected structural markers over sequence phylogenetic trees. A number of markers were useful to distinguish between genera and to delineate groups. Basic retroviral knowledge is vital to understand emerging infections. Phylogenetic analyses of taxonomically improved sequences, facilitates the search for common retroviral denominators to target. This thesis provided new insights in retroviral evolution and taxonomy using the ERVs, with special focus on the large gammaretroviral HERV-H group, as an additional source of information next to that of XRVs.

Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.

New strategies are needed to counter the growing problem of bacterial resistance to antibiotics. One such strategy is to design compounds that target bacterial virulence, which could work separately or in concert with conventional bacteriostatic or bactericidal antibiotics. Pilicides are a class of compounds based on a ring-fused 2-pyridone scaffold that target bacterial virulence by blocking the chaperone/usher pathway in E. coli and thereby inhibit the assembly of pili. This thesis describes the design, synthesis, and biological evaluation of compounds based on the pilicide scaffold with the goal of improving the pilicides and expanding their utility. Synthetic pathways have been developed to enable the introduction of substituents at the C-2 position of the pilicide scaffold. Biological evaluation of these compounds demonstrated that some C-2 substituents give rise to significant increases in potency. X-ray crystallography was used to elucidate the structural basis of this improved biological activity. Furthermore, improved methods for the preparation of oxygen-analogues and C-7 substituted derivatives of the pilicide scaffold have been developed. These new methods were used in combination with existing strategies to decorate the pilicide scaffold as part of a multivariate design approach to improve the pilicides and generate structure activity relationships (SARs). Fluorescent pilicides were prepared using a strategy where selected substituents were replaced with fluorophores having similar physicochemical properties as the original substituents. Many of the synthesized fluorescent compounds displayed potent pilicide activities and can thus be used to study the complex interactions between pilicide and bacteria. For example, when E. coli was treated with fluorescent pilicides, it was found that the compounds were not uniformly distributed throughout the bacterial population, suggesting that the compounds are primarily associated to bacteria with specific properties. Finally, by studying compounds designed to inhibit the aggregation of Aβ, it was found that some compounds based on the pilicide scaffold inhibit the formation of the functional bacterial amyloid fibers known as curli; these compounds are referred to as 'curlicides'. Some of the curlicides also prevent the formation of pili and thus exhibit dual pilicide-curlicide activity. The potential utility of such 'dual-action' compounds was highlighted by a study of one of the more potent dual pilicide-curlicides in a murine UTI model were the compound was found to significantly attenuate virulence in vivo.

<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>