Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent. In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation. / QC 20100713

Abstract Introduction 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. Method 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. Results 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 Escherichia coli followed by Staphylococcus aureus. In women, Escherichia coli was the most common bacterium, and there was a significant difference between the genders (p= 0.004). In men, Staphylococcus aureus was the dominant species (p= 0.027). Streptococcus pneumoniae was more common in women (p= 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). Escherichia coli 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. Conclusion 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 Escherichia coli and Staphylococcus aureus. 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. / Introduktion 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. Metod 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. Resultat 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 Escherichia coli och Staphylococcus aureus. För kvinnorna var Escherichia coli vanligast och det fanns en signifikant skillnad mellan könen (p= 0,004 ), för männen var Staphylococcus aureus vanligast (p= 0,027). Streptococcus pneumoniae visade högre förekomst bland kvinnorna än männen (p= 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). Escherichia coli 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. Konklusion Incidensen för bakteriemi/fungemi i Gävleborgs län var 235/100 000 invånare. De vanligaste fynden vid säkerställd bakteriemi var Escherichia coli och Staphylococcus aureus. 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.

Cryptosporidium is a protozoan parasite infecting the intestines of its hosts, leading to acute diarrheal disease. Out of 26 recognized species, 14 are known to infect humans. Of most importance, from a human perspective are Cryptosporidium parvum and Cryptosporidium hominis, of which the former is known to have zoonotic potential. Globally, cryptosporidiosis affect people with lowered immune status particularly hard; among children under five it is the most important parasitic cause of gastroenteritis. In the region of KwaZulu-Natal, on the east coast of South Africa, Cryptosporidium is considered endemic. Drinking water is frequently collected from river systems and as Cryptosporidium spp. can be transmitted via contaminated water, this may be one source of infection. Research on the species distribution is important for outbreak investigations and prevention efforts. In water and wastewater such speciation is commonly performed using immunomagnetic separation, an antibody dependent method. There is however a suspicion that these antibodies have less affinity to some species and hence contorts the detected species distribution. An alternative approach is therefore of interest.   In the present study, Cryptosporidium diversity in wastewater collected from four different wastewater treatment plants in KwaZulu-Natal, is evaluated with an optimized antibody-free workflow and a single cell platform. It was shown that the workflow is suitable for complex samples, such as wastewater. Furthermore, diversity was assessed with amplicon sequencing, revealing four different species and genotypes. Further modifications of the methods used could benefit the field of Cryptosporidium research, along with improving global health and preventing disease outbreaks.

Bakterier har alltid haft en stor inverkan på mänskligheten. För att diagnostisera bakteriella sjukdomar och behandla dem krävs identifiering av bakterien eller bakteriens relevanta egenskaper. Transportmedium har utvecklats för att hålla bakterierna vid liv från provtagning till analys. Syftet med studien var att utvärdera bakteriers viabilitet i det vätskebaserade mediet Copan Eswab jämfört med kolmedium (Copan swab). Bakterierna som ingick i studien var Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Niesseria gonorrhoeae och Fusobacterium nucleatum. Förutom jämförande mellan medierna genomfördes en jämförelse mellan Eswab i kyl och i rumstemperatur. Resultaten för H. influenzae (n=9) och N. gonorrhoeae (n=9) visade att Eswab gav lika många eller fler överlevande bakterier. Gällande F. nucleatum (n=9) visade resultaten att fler överlevde i Copan swab (Copanpinnar) de första 28 timmarna, men även att bakterien inte klarar mer än 28 timmar i rumstemperatur. Gällande S. pneumoniae (n=9) och C. jejuni (n=9) gav båda opålitliga svar. Ytterligare mätpunkter och studier krävs för att erhålla mer pålitliga resultat gällande hur länge bakterierna överlever i Eswab. / Bacteria have always had a great influence on mankind. To diagnose any bacterial disease and treat it it’s necessary to identify the bacteria or any relevant attributes. Different types of specimen transport have been developed to keep the bacteria alive from sampling until the analysis is performed. The purpose of the study was to evaluate the viability of bacteria in the fluid-based media Copan EswabTM compared with charcoal medium (Copan swab). Bacteria included in the study were: Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Niesseria gonorrhoeae and Fusobacterium nucleatum. The study also tried to compare how bacteria survived in Eswab which was refrigerated and in Eswab room temperature. Results for H. influenzae (n=9) and N. gonorrhoeae (n=9) showed that an equal amount or more of the bacteria survived in Eswab. More of F. nucleatum (n=9) survived in Copan swab (Copan swab sticks) for the first 28 hours, additionally they showed that the bacteria won’t survive more than 28 hours in room temperature. Regarding S. pneumoniae (n=9) and C. jejuni (n=9) both displayed unreliable results. Overall more measurements and additional studies are needed for more reliable results.

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. 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.

The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations. In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings. To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations. How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2. Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results. In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.

The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

Bacterioplankton are keystone organisms in marine ecosystems. They are important for element cycles, by transforming dissolved organic carbon and other nutrients. Bacterioplankton community composition and productivity rates change in surface waters over spatial and temporal scales. Yet, many underlying biological processes determining when, why and how bacterioplankton react to changes in environmental conditions are poorly understood. Here, I used experiments with model bacteria and natural assemblages as well as field studies to determine molecular, physiological and ecological responses allowing marine bacteria to adapt to their environment. Experiments with the flavobacterium Dokdonia sp. MED134 aimed to determine how the metabolism of bacteria is influenced by light and different organic matter. Under light exposure, Dokdonia sp. MED134 expressed proteorhodopsin and adjusted its metabolism to use resources more efficiently when growing with lower-quality organic matter. Similar expression patterns were found in oceanic datasets, implying a global importance of photoheterotrophic metabolisms for the ecology of bacterioplankton. Further, I investigated how the composition and physiology of bacterial assemblages are affected by elevated CO2 concentrations and inorganic nutrients. In a large-scale experiment, bacterioplankton could keep productivity and community structure unaltered by adapting the gene expression under CO2 stress. To maintain pH homeostasis, bacteria induced higher expression of genes related to respiration, membrane transport and light acquisition under low-nutrient conditions. Under high-nutrient conditions with phytoplankton blooms, such regulatory mechanisms were not necessary. These findings indicate that open ocean systems are more vulnerable to ocean acidification than coastal waters. Lastly, I used field studies to resolve how bacterioplankton is influenced by environmental changes, and how this leads to seasonal succession of marine bacteria. Using high frequency sampling over three years, we uncovered notable variability both between and within years in several biological features that rapidly changed over short time scales. These included potential phytoplankton-bacteria linkages, substrate uptake rates, and shifts in bacterial community structure. Thus, high resolution time series can provide important insights into the mechanisms controlling microbial communities. Overall, this thesis highlights the advantages of combining molecular and traditional oceanographic methodological approaches to study ecosystems at high resolution for improving our understanding of the physiology and ecology of microbial communities and, ultimately, how they influence biogeochemical processes.

The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.

In most bacterial pathogens, the genome is comprised within a single circular chromosome which is typically organized by the origin-to-terminus axis that divides the chromosome into equally-sized arms of replication (replichores). This similarity in length is presumed to be required for the synchronization of the two replication forks to meet at the terminus for efficient chromosome segregation. Transfer of genes between organisms, different from the route of parent to offspring, is called horizontal gene transfer (HGT). Acquiring foreign DNA through HGT is an important factor for the evolution of virulence in bacteria since it provides access to new features such as new toxins and antibiotic resistance genes. Chromosomes of many pathogenic bacteria such as Salmonella spp. carry such horizontally-transferred DNA fragments called pathogenicity islands. However, after such HGT events, the existing organization of chromosome can be disrupted and an imbalance between the two halves of the circular chromosome might occur. The predicted outcome of a replichore imbalance is the retardation of growth which in turn might result in the out-competition by other faster-growing bacteria in the environment. For that reason, we have investigated the association of the fitness cost and the replichore imbalance with isogenic strains with varying degrees of inter-replichore inversions. Our results showed that there is a correlation between the magnitude of replichore imbalance and fitness cost, for example 2.49-fold imbalance (one replichore 2.49-fold longer than the other) resulted in 11% reduction of fitness in comparison with balanced replichores. Therefore, our data suggest that the replichore imbalance could be utilized to predict the fitness cost of HGT events.