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.

Acinetobacter är gramnegativa bakterier som orsakar svåra nosokomiala infektioner hos immunkomprometterade och neutropena patienter. Det har rapporterats att den vanligaste smittkällan är sjukhusutrustning såsom andningsinstrument, där kolonisation av acinetobacter observerats. Av den anledningen att acinetobacter är multiresistenta mot olika antibiotikum medför det att en acinetobact infektioner blir svåra att behandla. Syftet med studien var att undersöka vilka acinetobactarter som orsakat bakteremi i Skåne från 2011-2019 samt undersöka vilka antibiotika de är resistenta för. Dessutom undersöktes hur stor andel av bakteremifynden som är resistenta mot betalaktamantibiotika, och vilka arter som är mest resistenta. För att artidentifiera acinetobacterna användes MALDI-TOF som analysinstrumentet. Resistensbestämningen bestämdes med E-test och diskdiffusionsmetoden, där tolv olika antibiotika testades. Resultatet visade att bland de 107 stammarna var A. Iwoffii störst då den utgjorde 31 st. Dessutom visade resultatet att majoriteten av stammarna var känsliga mot de tolv antibiotikasorterna. Bland de resistenta stammarna var de flesta av arten A. Baumannii som tidigare är kända för att vara multidrug-resistant (MDR). / Acinetobacter are gram-negative bacteria that cause severe nosocomial infections in immunocompromised and neutropenic patients. It has been reported that the most common source of infection is hospital equipment such as respiratory instruments, where colonization of acinetobacter has been observed. Due to the fact that acinetobacter is multi-resistant to various antibiotics, it makes an acinetobact infection difficult to treat. The aim of the study is to investigate which acinetobact species caused bacteremia in Skåne from 2011-2019 and to investigate which antibiotics they are resistant to. In addition, the proportion of bacteremia findings that are resistant to beta-lactam antibiotics was examined, and which species are most resistant. MALDI-TOF was used to identify the species of acientobacter. Twelve different antibiotics were tested with E-test and the disc diffusion method. The results showed that among the 107 strains was A. iwoffii the largest acinetobacter species, which corresponds to 31. In addition, the results obtained showed that the majority of the strains were sensitive to the twelve types of antibiotics. Among the resistant strains were the majority of the species A. Baumannii, which are known to be multidrug resistant (MDR).

Abstract Background: Gastroenteritis is a common infection and the leading cause of morbidity worldwide and is mostly caused by viruses. Outbreaks appear in both developed and developing countries and result in large economic costs. Rapid detection is important for appropriate treatment, control and to prevent the spread of infection.  Objective: Evaluation and performance comparison between the BioFire®FilmArray® Torch System gastrointestinal panel and the Molecular BD MAXTMenteric viral panel to indicate a multiplex method for viral gastroenteritis diagnostic in a routine laboratory setting.  Material and methods: In this study, 58 different samples were used which consisted of selected stool specimens from patients who were tested and treated for gastroenteritis infection at Uppsala Academic Hospital and Norrlands University Hospital in Umeå during 2018-2021, samples from Quality control for molecular diagnostics viral gastroenteritis EQA pilot study during 2018-2019 and cultivated strains of different adenovirus species from 2018. All samples were analyzed with both systems for comparison of detected pathogens.  Results: Sensitivity and specificity values were 95% and 100% respectively for the BioFire®FilmArray®Torch System and 100% and 93.3% for the BD MAXTMSystem.   Conclusions: Bothsystems are rapid and adequate diagnostic tools. The BioFire®FilmArray®Torch System with greater coverage has the ability of detecting more pathogens and is more promising particularly in the occasional infection circumstance. The BD MAXTMSystem demonstrated almost the same results and seems to be a better option in times of an outbreak when the numbers of patients are significantly higher.

Antibiotics are essential drugs in modern medicine. After consumption antibiotics are excreted in unmetabolized form in the urine and reach our sewage treatment plants (STP). STP are not able to degrade all antibiotics leading to release of antibiotics into the aquatic environment. Aquatic animals are thus continuously exposed to antibiotics. This study involved assessment of the toxicity of eight antibiotics previously detected in the river Fyrisån, Uppsala, Sweden, in developing zebrafish (Danio rerio) embryos up to day six of age. The experiments included assessment of embryonal toxicity for the individual antibiotics as well as mixtures of all antibiotics.  The mixtures were based on previously measured concentrations in river Fyrisån and tested in increasing concentrations up to 1000-times higher concentrations. In the toxicity assessment different lethal and sublethal endpoints were observed, such as early movements, heart rate, hatching time and length. These experiments were followed by behaviour study observing the swimming activity during alternating dark-light alternations. Lastly, a bioaccumulation study was performed on mixtures of antibiotics to determine if these antibiotics were bioaccumulative in zebrafish embryos. The results showed that these eight antibiotics, individually and as a mixture did not affect any of the endpoints. As for bioaccumulation, none of the eight antibiotics were bioaccumulating in zebrafish embryos. These antibiotics seem to be non-toxic during fish embryonal development. However, the results cannot determine the long-term effects of antibiotic exposure and thus further studies are needed to assess the potential toxicity of environmentally present antibiotics to fish.

Alteration of gut microbiota has been associated with development of immune mediated diseases, such as allergy. In part, this could be due to the influence of microbes in shaping the immune response. In paper I, we investigated the association of early-life gut colonization with bacteria, and numbers of IL-4, IL-10 and IFN-γ producing cells at two years of age in response to PBMC stimulation with phytohemagglutinin (PHA) in vitro. Early Staphylococcus (S) aureus colonization was directly proportional to increased numbers of IL-4 and IL-10 secreting cells, while early co-colonization with lactobacilli and S. aureus associated with a decrease in IL-4, IL-10 and IFN-γ secreting cells compared to S. aureus alone. This was also confirmed in in vitro stimulations of PBMC with Lactobacillus and/or S. aureus strains, where S. aureus-induced IFN-γ production by Th cells was down regulated by co-stimulation with Lactobacillus. In paper II, we investigated the effects of UV-killed and/or culture supernatant (sn) of Lactobacillus strains and S. aureus strains on IEC and immune cell responses. IEC exposed to S. aureus-sn produced CXCL-1/GRO-α and CXCL-8/IL-8, while UV-killed bacteria had no effect. Further, PBMC from healthy donors exposed to Lactobacillus-sn and S. aureus-sn were able to produce a plethora of cytokines, but only S. aureus induced the T-cell associated cytokines: IL-2, IL-17, IFN-γ and TNF-α; which were down regulated by co-stimulation with any of the different Lactobacillus strains. Intracellular staining verified S. aureus-induced IFN-γ and IL-17 production by Th cells, and increased CTLA-4 expression and IL-10 production by T reg cells. In conclusion, we show that colonization with gut microbiota at early age modulates the cytokine response in infancy. In addition, bacterial species influence cytokine response in a species-specific manner and we demonstrate that lactobacilli modulate S. aureus-induced immune response away from an inflammatory phenotype.

Background: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, which enter the host cells through interactions between the receptor-binding domain (RBD) on the S-protein and the ACE-2 receptor on the host cell. A novel type of vaccine strategy is peptide vaccines, with great potential as a faster and more selective approach to conventional vaccine development. This study focuses on the possibility of generating an antibody response through synthetic peptides harboring B cell epitopes.  Aim: This project aims to investigate the potential of immunogenic peptides to generate an antibody response when used as synthetically produced peptides. As proof-of-concept, the project studies the interactions between previously identified monoclonal antibodies with defined B cell epitopes and the corresponding peptide sequences.  Method: The interactions are evaluated by different ELISA experiments. The candidate peptides are additionally investigated on their binding to polyclonal serum with established S reactive antibodies. Furthermore, the project includes synthesis of one peptide by solid phase peptide synthesis. Results: The ELISA experiments presented no interaction between the synthetic peptides and the monoclonal antibodies or human sera.  Conclusion: The project fulfilled its aim to study the interaction between the B cell epitopes and the monoclonal antibodies. However, no binding was observed. Despite the many advantages in production and stability, development of B cell epitope vaccines come with many challenges. Future will entail if synthetic peptides harboring B cell epitopes can be used as vaccines, or if peptide vaccines will be a focus when a T cell response is to be induced.

Upon encounter of an antigen, epidermis-resident Langerhans cells (LCs) become activated and present the processed antigen to T cells of the draining lymph nodes, resulting in tolerogenic or inflammatory responses. In psoriasis plaques, skin homeostasis is disrupted and replaced by an inflammatory dermatitis. Topical application of the anti-viral compound, imiquimod, induces a psoriasiform inflammatory condition, partly driven by LC production of pro-inflammatory cytokines. Differentiation of the myeloid progenitor cell line, MUTZ-3, produces MUTZ-3 derived Langerhans cells (MUTZ-LCs) which can be used as an in vitro model of LCs. This project aimed to investigate the phenotype and function of imiquimod-treated MUTZ-LCs in monolayer cultures, co-culture with T cells and inserted into a 3D skin model. LC-related surface markers (HLA-DR, CD1a, CD207, CCR7) were upregulated in MUTZ-LCs after 7 days of differentiation with 40 ng/ml GM-CSF, 10 ng/ml TGF-β and 2.5 ng/ml TNF-α. Supernatants of imiquimod-treated monolayer cultures of MUTZ-LCs showed subtle concentrations of IL-6 and TNF-α, but not IL-23. mRNA expression showed no significant upregulation of IL-6, IL-23 or TNF-α after 24 h treatment with imiquimod. The presence of MUTZ-LCs in T cell co-cultures greatly increased the production of IL-2, but did not affect expression of CD25. After 16 h exposure to imiquimod, IL-6, IL-23 and TNF-α could not be detected in culture supernatants of a 3D model consisting of fibroblasts, keratinocytes and MUTZ-LCs. The model was devoid of fibroblasts after 19 days of culture, most likely compromising the immunocompetence, as LC migration in response to activation could not be detected. Further studies could refine and optimize the imiquimod-3D skin model, which has potential as a possible substitute for animal models in psoriasis research.

Malaria, caused by the Plasmodium parasite and transmitted by mosquitoes, kills almost half a million people each year. Drug resistance in both the parasite and its vector make preventative measures increasingly important, and a fully protective vaccine is absolutely necessary to eradicate the disease. However, genetic diversity of the parasite makes vaccine development difficult. One of the best vaccine candidates is MSP2, a surface protein present during the blood stage of P. falciparum infection. Antibodies, which are important for natural immunity, have been shown to bind MSP2 and prevent parasite infection of blood cells. The purpose of this study was to analyze MSP2 sequence diversity in a cohort of patients infected while traveling or living in sub-Saharan Africa, and to investigate patient antibody responses to MSP2 variants infecting other individuals. Parasite isolates from our cohort were made up of 47% 3D7 alleles and 53% FC27 alleles. Protein sequences showed similar levels of conservation within allelic families, and blocks of conserved amino acids between different variants suggest there may be epitopes that can induce antibody production targeting multiple variants. Antibody reactivity tests suggest the variable region of MSP2 is important for antibody binding to variants of the same allelic type, while the conserved region is important for reactivity to different allelic types. This thesis gives evidence to the importance of including epitopes from conserved and variable regions of both MSP2 allelic families in order to induce strain-transcending immunity against P. falciparum malaria. / A genomic surveillance platform for indel-rich genes from Plasmodium spp. using long-read amplicon sequencing

Introduktion: Alzheimers sjukdom är en neurodegenerativ sjukdom som orsakas av ansamling av amyloid-β (Aβ) i hjärnan. Prevalensen av Alzheimers sjukdom ökar, och symtom inkluderar minnesförlust, ångest, depression, förvirring, nedsatt omdöme och desorientering. Ålder och genetiska varianter är två riskfaktorer för att utveckla Alzheimers sjukdom. Det finns två modeller som förklarar hur sjukdomen kan uppstå, 1) den amyloida hypotesen som beskriver hur deposition av Aβ leder till ökad aggregation av proteinet tau vilket orsakar celldöd och neurodegeneration, och 2) den kolinerga hypotesen vilken beskriver att Aβ-plack minskar produktionen av acetylkolin vilket leder minskad aktivitet i kolinerga nerver. Det är främst entorhinala cortex och hippocampus som drabbas. Diagnostisering görs genom medicinska och neurologiska undersökningar och genom standardiserade test/instrument. I dagläget kan symtom av Alzheimers sjukdom behandlas med acetylkolinesterasinhibitorer och memantin. Ett annat behandlingssätt är att använda monoklonala antikroppar som riktats mot Aβ för att minska belastningen av Aβ i hjärnan. För att bedöma effekt av sådana läkemedel används bedömningsmetoder baserade på kognitiva och funktionella tester.  Syfte: Syftet med detta arbete var att undersöka effektivitet och säkerhet av anti-Aβ antikroppar för behandling av Alzheimers sjukdom, vilket gjordes genom att analysera kognitiv förmåga, biomarkörer och biverkningar.  Metod: Detta arbete är en litteraturstudie som baserades på fem läkemedelsstudier vilka erhölls från databasen PubMed. Sökord som användes vid litteratursökning var ”aducanumab”, ”lecanemab”, ”donanemab”, ”crenezumab” och ”bapineuzumab”.  Resultat: Hög dos aducanumab i studien EMERGE och lecanemab visade statistiskt signifikant förändring på alla utfallsvariabler, och analys av biomarkörer visade minskad amyloid-belastning i hjärnan. Donanemab visade statistiskt signifikant skillnad på den primära utfallsvariabeln och på analys av biomarkörer, men resultat på sekundära utfallsvariabler var inte statistiskt signifikanta. Crenezumab visade endast statistiskt signifikant förändring på den primära utfallsvariabeln i CREAD2. Bapineuzumab visade ingen statistiskt signifikant skillnad på någon utfallsvariabel eller på förändringar i biomarkörer. De resultat som var statistiskt signifikanta indikerade minskad kognitiv försämring hos patienterna. ARIA var en vanlig biverkning hos alla läkemedel utom crenezumab, men förekomsten av ARIA var i de flesta fall mild till måttlig. Andra vanliga biverkningar inkluderade infusionsrelaterade reaktioner, huvudvärk och fall.  Slutsats: Baserat på resultaten från detta arbete dras slutsatsen att aducanumab, lecanemab och donanemab var de läkemedel med högst effektivitet. Framtiden ser mest lovande ut för aducanumab och lecanemab med anledning av positiva resultat på primära och sekundära utfallsvariabler och biomarkörer samt FDAs godkännande av läkemedlen i USA. Förekomsten av ARIA påverkar säkerheten av läkemedlen och därför behöver fler studier genomföras för att undersöka deras säkerhet ytterligare. / Background: Alzheimer’s disease is a neurodegenerative disease that is caused by accumulation of amyloid-β (Aβ) in the brain. The prevalence of Alzheimer’s disease is increasing, and symptoms of the disease include memory loss, anxiety, depression, confusion, impaired judgment and disorientation. Age and genetic variants are the two main risk factors for developing Alzheimer’s disease. There are two models which describe the development of the disease, 1) the amyloid hypothesis which describes how the deposition of Aβ leads to increased aggregation of the protein tau, which causes neuronal cell death and neurodegeneration, and 2) the cholinergic hypothesis which describes that Aβ plaques decrease the production of acetylcholine, this causes less activity in cholinergic neurons. The two areas in the brain which are mainly affected by neurodegeneration are the entorhinal cortex and the hippocampus. Diagnosing Alzheimer’s disease is done by medicinal and neurological assessments and by using standardized tests/instruments. Currently only symptomatic treatments for Alzheimer’s disease are available; acetylcholine esterase inhibitors and memantine. Another treatment method is using monoclonal antibodies against Aβ to decrease the Aβ load in the brain. To assess the effectiveness of these drugs assessment methods based on cognitive and functional tests can be used.  Aim: This study aimed to analyse the efficacy and safety of anti-Aβ antibodies as a treatment for Alzheimer’s disease, which was done by analysing cognitive ability, biomarkers and adverse events.  Method: This literature study was based on 5 clinical randomized controlled trials which were obtained from the PubMed database. Keywords that were used in the searches were “aducanumab”, “lecanemab”, “donanemab”, “crenezumab” and “bapineuzumab”.  Results: High-dose aducanumab in the study EMERGE and lecanemab showed statistically significant differences on all endpoints, and analysis of biomarkers showed a decrease in amyloid load in the brain. Donanemab showed statistically significant differences on the primary endpoint and analysis of biomarkers but results on secondary endpoints were not statistically significant. Crenezumab only showed statistically significant change on the primary endpoint in CREAD2. Bapineuzumab did not show statistically significant differences on any endpoint or on changes in levels of biomarkers. Statistically significant results on primary and secondary endpoints indicated decreased cognitive impairment among the patients. ARIA was a common adverse event in all drugs, with exception of crenezumab, but the occurrence of ARIA was in most cases mild to moderate. Other common adverse events were infusion-related reactions, headaches and falls.  Conclusion: With consideration of the results of this paper a conclusion can be drawn that aducanumab, lecanemab and donanemab have been shown to be effective on primary endpoints and analysis of biomarkers. The drugs that seem the most promising are aducanumab and lecanemab, mainly because they showed efficacy on both primary and secondary endpoints, and biomarkers and because of the FDA’s recent approval of both drugs in the US. The occurrence of ARIA is something that affects the safety of these drugs and because of this more studies are needed to further assess their safety.

Oral health is a part of general health and the two are connected in many ways as well as impact each other. Oral diseases are some of the most common chronic diseases of mankind. Diseases such as caries and periodontitis are two of the most common ones affecting the oral cavity. Bacteria associated with these two diseases are Streptococcus mutans and Aggregatibacter actinomycetemcomitans respectively. Our hypothesis is that extracts from guava leaf or matcha tea affect adhesion of A. actinomycetecomitans or S. mutans to human epithelium. The aim of this study is to investigate a DNA-based method for studying attachment of bacteria to epithelial cells. Two different concentrations of the two bacterial species, high and low, were treated with matcha or guava leaf extract and adhesion on the human epithelial cell cultures was analyzed. The results were then analyzed using qPCR-based methods to test the amount of bacterial adhesion to the epithelial cells. Furthermore, the results showed that matcha was more effective for inhibition of bacterial adhesion than guava leaf.In conclusion, our results show that bacterial adhesion of A. actinomycetecomitans and S. mutans to human epithelial cells can be quantified by DNA-based methods, and the adhesion altered by plant extracts.