Plasmids are small circular DNA molecules within bacterial cells that are separated from the bacterial chromosome and replicate independently. Also, they play a crucial role in the dissemination of antibiotic resistance genes among bacteria through horizontal gene transfer. They can be present in many copies within host cell, which is known as plasmid copy number. Plasmids can regulate their own copy number by different mechanisms. Additionally, the selective pressure can also play a pivotal role in determining plasmid copy number. The presence of antibiotics in the surrounding environment can drive variations of plasmid copy number. In this study, we examined plasmid copy number variations of multidrug resistance plasmids in presence of antibiotics by using EvaGreen® - based multiplexed digital droplet PCR. We could observe that cultures of Klebsiella pneumoniae and Escherichia coli harboring multidrug resistance plasmids grown in presence of sub-MIC concentrations of the antibiotics did not show high variations in plasmid copy numbers. On the other hand, mutants of K. pneumoniae selected for increased antibiotic resistance showed high increases in copy number of a multidrug-resistance plasmid.

Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.

Flaviviruses are important emerging and re-emerging arthropod-borne pathogens that cause significant morbidity and mortality in humans. It consists of globally distributed human pathogens such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZIKV). Depending on type, flaviviruses can cause a variety of symptoms ranging from haemorrhage to neurological disorders. Virus infection is detected by host pattern recognition receptors (PRRs), and through downstream signalling it leads to the production of interferons (IFNs). These IFNs then act in an autocrine or paracrine manner on the cells to induce various IFN-stimulated genes (ISGs), which have antiviral roles. However, the amount of IFN produced depends on the nature of the PRRs used by host cells to detect a particular virus. Although there are many PRRs present in the host cells, their relative contribution in different cell types and against a specific virus may vary. In the first study, we determined the importance of IPS-1 signalling in immunity and pathogenicity of tick-borne flaviviruses. This is an adaptor protein for cytoplasmic RIG-I-like receptors. Using IPS-1-deficient mice, we showed its importance against TBEV and Langat virus (LGTV) infection (the LGTV model virus belongs to the TBEV serogroup). Absence of IPS-1 leads to uncontrolled virus replication in the central nervous system (CNS), but it has only a minor role in shaping the humoral immune response at the periphery. LGTV-infected IPS-1-deficient mice showed apoptosis, activation of microglia and astrocytes, an elevated proinflammatory response, and recruitment of immune cells to the CNS. Interestingly, we also found that IFN-b upregulation after viral infection was dependent on IPS-1 in the olfactory bulb of the brain.  Thus, our results suggest that local immune microenvironment of distinct brain regions is critical for determination of virus permissiveness. Interferons can upregulate several ISGs. Viperin is one such ISG that has a broad-spectrum antiviral action against many viruses. However, the importance of cell type and the significance of viperin in controlling many flavivirus infections in vivo is not known. Using viperin-deficient mice, we found that viperin was necessary for restriction of LGTV replication in the olfactory bulb and cerebrum, but not in the cerebellum. This finding was also confirmed with primary neurons derived from these brain regions. Furthermore, we could also show the particular importance of viperin in cortical neurons against TBEV, WNV, and ZIKV infection. The results suggested that a single ISG can shape the susceptibility and immune response to a flavivirus in different regions of the brain. Although viperin is such an important ISG against flaviviruses, the exact molecular mechanism of action is not known. To understand the mechanism, we performed co-immunoprecipitation screening to identify TBEV proteins that could interact with viperin. While viperin interacted with the prM, E, NS2A, NS2B, and NS3 proteins of TBEV, its interaction with NS3 led to its degradation through the proteosomal pathway. Furthermore, viperin could reduce the stability of other viperin-binding TBEV proteins in an NS3-dependent manner. We screened for viperin activity regarding interaction with NS3 proteins of other flaviviruses. Viperin interacted with NS3 of JEV, ZIKV, and YFV, but selectively degraded NS3 proteins of TBEV and ZIKV, and this activity correlated with its antiviral activity against these viruses. The last study was based on in vivo characterization of the newly isolated MucAr HB 171/11 strain of TBEV which caused unusual gastrointestinal and constitutional symptoms. This strain was compared with another strain, Torö-2003, of the same European subtype of TBEV but isolated from the different focus. Here we found unique differences in their neuroinvasiveness and neurovirulence, and in the immune response to these two strains. In summary, my work shed some light on the interplay between tick-borne flavivirus and the innate immune system. I have shown two examples of CNS region-specific differences in innate immune response regarding both in IFN induction pathways and antiviral effectors. Furthermore, we have investigated the in vivo pathogenesis of a strain of TBEV that caused unusual gastrointestinal and constitutional symptoms. / Flavivirus finns spridda över hela världen och orsakar miljontals infektioner varje år. Några av de medicinsk mest viktiga flavivirusen är fästingburen encefalit virus (TBEV), West Nile virus (WNV), Japansk encefalit virus (JEV), gula febern (YFV) och Zika virus (ZIKV). Dessa virus kan orsaka olika komplikationer till exempel blödarfeber och hjärninflammation. Vid en infektion så upptäcker värdcellen virusinfektionen med hjälp av speciella receptorer, så kallade PRRs. Dessa finns i alla celler och känner igen viruskomponenter som normalt inte finns i en oinfekterad cell. När PRRs detekterar en virusinfektion svarar cellen med att tillverka ett signal protein interferon (IFN). IFN skickas ut ur cellen och hämmar virusinfektioner genom att sätta igång ett försvarsprogram i andra celler bestående av hundratals försvarsproteiner som kan motverka virusinfektionen. Vilka PRRs som behövs för att detektera ett virus är olika vid olika virusinfektioner. I första studien fann vi att IPS-1 är av yttersta vikt för skydda mot fästingburna flavivirus. IPS-1 är ett så kallat adapter protein som behövs för att två PRRs, RIG-I och MDA-5, ska kunna förmedla signaler som leder till IFN tillverkning. Med hjälp av möss som saknar IPS-1 fann vi att IPS-1 behövs för att tillverka IFN protein och skydda mot fästingburna flavivirus. IPS-1 var särskilt viktigt för interferon produktion inom luktloben i hjärnan. Därför kunde vi dra slutsatsen att immunresponsen regleras olika inom olika delar av hjärnan. Ett försvarsprotein som visat sig vara särskilt viktig vid virusinfektion är viperin. Viperin har visat sig kunna hämma en rad olika virus men den specifika rollen av viperin in vivo vid flavivirus infektion var inte fullt känd. Vi fann att viperin behövs för att hämma LGTV i lukloben och storhjärnan men inte i lillhjärnan. Vi kunde bekräfta detta med hjälp av primära nervceller isolerade från dessa hjärnregioner. Vi fann även att viperin var av yttersta vikt för att kontrollera TBEV, WNV och ZIKV infektion i nervceller från hjärnbarken (del av storhjärnan). Därför kunde vi dra slutsatsen att ett enskilt försvarsprotein kan avgöra mottagligheten mot flavivirus inom olika hjärnregioner. Trots att viperin är så viktig för att skydda mot flavivirus så vet vi inte hur viperin åstadkommer detta. Därför ville vi undersöka hur viperin kan förmedla sin antivirala effekt. Vi fann att viperin kan binda till flera TBEV proteiner, men att viperin specifikt kan bryta ner ett virusprotein som heter NS3. NS3 är väldigt viktigt för att flavivirus ska kunna etablera en infektion och kunna föröka sig. Eftersom vi visste att viperin kan hämma andra flavivirus ville vi veta om viperin även förstör NS3 från JEV, ZIKV och YFV. Vi upptäckte att viperin kunde binda till NS3 hos alla dessa flavivirus men att viperin specifikt förstörde TBEV och ZIKV NS3, intressant nog så kunde viperin endast hämma dessa virus infektioner men inte JEV och YFV. I den sista studien ville vi karaktärisera en ny TBEV stam som bara orsakar magoch tarmbesvär men inga neurologiska symptom. TBEV har aldrig tidigare visat sig kunna orsaka detta och därför ville vi undersöka saken vidare. Vi fann att denna TBEV stam skiljde sig mot en närbesläktad stam genom att orsaka en starkare immunrespons men mildare sjukdomsförlopp. Sammanfattningsvis har jag undersökt samspelet mellan fästingburna flavivirus och det medfödda immunförsvaret. Jag har även visat att immunresponsen regleras olika inom olika hjärnregioner, både beträffande IFN inducering och antivirala proteiner. Vidare har jag hittat mekanismen för hur viperin proteinet hämmar TBEV och ZIKV, vilket var genom att förstöra NS3. Dessutom har jag karaktäriserat sjukdomsförloppet hos möss efter infektion med en ovanlig TBEV stam som orsakar mag och tarm besvär.

The genetic code in our cells is built up by deoxyribonucleic acid (DNA) with a sequence that is individual and unique to each person. A cell’s origin can be decided by comparing an established DNA profile with a known profile. The most publicly known application is in the forensic field and its use for identification and for establishing a connection between perpetrators and victims or crime scenes. DNA profiling is also commonly used for kinship investigations. The information embedded in the DNA is also used for diagnostic purposes in conventional medicine. Generating DNA profiles is a well-established procedure, which is used daily and for many purposes. An amount of approximately 150-1500 cells is required to be able to establish a full DNA profile using current methods. There are several situations where the amount of material is limited. To enable analysis where the testing material is limited it is of great value to develop a method that can perform these analyses on minute amounts of cells. If there were a method for generating DNA profiles from single cells then mixed samples from crime scenes would be separable. In tumour biology it is also of interest to obtain information from single cells. The aim with the thesis was to establish the smallest amount of cells needed for a full DNA profile. The thesis started with analyses on extracted DNA. During several experiments dilution series were made to investigate the possibilities to establish profiles from minute amounts of extracted DNA. The main methods used during this thesis were polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE). These methods are well-established tools both in biomedical science and at The Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine. Different factors were optimized and the acquired knowledge resulted in application of DNA on FTA® Micro Cards. The cards are used in the daily routines and are easy to use. Several experiments were then performed on peripheral lymphocytes based on the knowledge acquired during the process. Applying a low amount of lymphocytes on FTA cards proved to be very successful and the method generates DNA profiles at a single cell level. The method is applicable for approximately 5-10 cells.

Handhygien är en grundläggande princip som förebygger, kontrollerar och reducerar vårdrelaterade infektioner och är ensam den mest effektiva metoden för att bryta en smittspridning. Syfte: att belysa vårdpersonalens följsamhet till de basala rutinerna gällande handhygien. Metod: Författarna gjorde en litteratursökning och granskning av vetenskapliga artiklar inom området. Resultat: Vårdpersonalens följsamhet till handhygienrutinerna visade sig ligga mellan 16,5%-70% och var större efter än före patientvård. Det fanns faktorer som gynnade och minskade följsamheten. Diskussion: Författarna fann stöd i sin teori om att vårdpersonalen skyddar sig i första hand själv och utför därför handhygienen i större grad än efter patientkontakt. Då det visade sig att kunskap leder till ökad följsamhet ansåg författarna att en årlig genomgång av handhygienrutinerna är lika viktigt som att repetera hjärt- och lungräddning.

ABSTRACT Nontypable Haemophilus influenzae is a common cause of otitis, sinusitis and conjunctivitis. It is the most common bacterial pathogen associated with chronic obstructive pulmonary disease (COPD). Studies have shown that nonpathogenic Haemophilus haemolyticus are often mistaken for Haemophilus influenzae due to an absent hemolytic reaction on blood agar. Distinguishing H. haemolyticus from H. influenzae is important to prevent unnecessary antibiotic use, and to understand the role of H. influenzae in clinical infections. In this study, PCR-primers for amplifying 16S rDNA sequences were used to set up a method for distinguishing H. haemolyticus from H. influenzae. The aim was to use the method for analyzing apparent H. influenzae strains, to investigate if some strains were in fact H. haemolyticus. However, because of problems with unspecific primerannealing,no conclusions could be drawn regarding misclassification of H. haemolyticus. Moraxella catarrhalis is the second most common bacterial pathogen associated with COPD. It also causes otitis and sinusitis. An important virulence factor of M. catarrhalis is the outer membrane protein Moraxella catarrhalis IgD-binding protein (MID). One part of the protein; MID764-913 , has been shown to function as an adhesin, and this part has been fragmented to further investigate its adhesive properties. The aim of this second, independent study, was to express some of these proteinfragments by cloning in E. coli. The time spent on this project was too short, and no proteins could be expressed duing this period.

The purpose of the study was to set up a method for cultivation of C. pneumoniae from infected mouse tissue in Hep2 cells. We also evaluated a new reagent, Chlamatis, which is considered to increase the detection sensitivity of the bacterium with both PCR and cultivation. All 10 serum samples treated with Chlamatis were negative for C. pneumoniae in PCR. The cultivation of tissue was found to work without problems. The yield of the bacteria was highest at the speed of 30 Hz using homogenization with TissueLyser. Mice infected with C. pneumoniae were killed at days 4, 8, 20 and 40. The highest yields of C. pneumoniae were found at days 4 and 8 using PCR with all infected mice. The results obtained with PCR and cultivation confirmed each other to a large extent. For heart tissue, PCR positive mice were found only at days 4 and 8. The number of PCR positive lung samples confirmed to a large extent the number found by cultivation, except for mice killed after 4 days and 40 days where the results differed slightly. This indicated that the optimization of the cultivation method was successful

Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis. Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus. Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined. Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested. Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.

The Lyme disease causing spirochaete Borrelia burgdorferi sensu lato is transmitted by ticks within the genus Ixodes. These ticks are liberal host seekers and parasitise mammals, birds and reptiles. Prior to this study, the distribution of I. ricinus ticks and Lyme disease was thought to be restricted to the southern half of Sweden. On the island Norrbyskär, located in the Bothnian Gulf, there were reports of a high incidence of tick infestation on humans. To investigate the occurrence of B. burgdorferi s.l. in these ticks and to characterise presumptive isolates at the molecular level we sampled a number of I. ricinus ticks. Three different isolates were obtained from two different ticks, NBS16 from a nymph and NBS23a and NBS23b from an adult female tick. The seabird associated tick I. uriae is circumpolar distributed in both hemispheres. On the island Bonden, which house one of the largest seabird colonies in the Baltic Sea, I. uriae were collected and surveyed for spirochaetes. One isolate of B. burgdorferi s.l. was obtained. This B. burgdorferi s.l. isolate is identical to the Lyme disease Borrelia strain NBS16 isolated from Norrbyskär. To investigate the role of seabirds in the epidemiology of B. burgdorferi s.l., I. uriae were collected from seabird colonies in the southern and northern hemispheres. Borrelia DNA was extracted from the ticks and from cultured spirochaetes. Sequence analysis of the flagellin gene revealed that the DNA obtained was from B. garinii, regardless of the geographical origin of the sample. Identical fla gene fragments in ticks collected in both hemispheres indicate a transhemispheric exchange of B. garinii. A marine ecological niche and epidemiological route for Lyme disease Borrelia are proposed. The prevalence of B. burgdorferi s.l. infected ticks on migrating passerine birds was studied. A total of 22, 998 birds were caught and examined for ticks. The presence of spirochaetes in the 967 collected ticks was determined by DNA amplification by PCR on all ticks. To determine which B. burgdorferi s.l. species were present, classification was performed by DNA amplification using species-specific 16S rDNA primers and by DNA sequencing. Flagellin gene sequences of all species of B. burgdorferi s.l. previously recorded in Europe were found. B. garinii was the most prevalent. These data support the notion that passerine birds are at least partly responsible for the distribution of Lyme disease Borrelia spirochaetes in Europe. To elucidate the distribution of B. burgdorferi s.l. in subarctic regions, strains isolated from I. ricinus and I. uriae ticks found on islands in the northern Atlantic and Baltic Sea were characterised molecularly. All isolates were verified as B. garinii by 16S-rRNA gene analysis and immunoblotting using monoclonal antibodies specific for the outer surface proteins A and C. Three ribotypes (RT's) of B. garinii were found. The I. ricinus associated RT1 is phenotypically the most heterogeneous. RT2 is restricted to the islands in the northern Baltic Sea, whereas RT3 was also recovered from ticks found on islands in the North Atlantic. The heterogeneity of the B. garinii population in the Baltic Sea might be influenced by two geographically opposite directions, North Atlantic (RT3) and Euroasia (RT1). / digitalisering@umu.se

Giardia intestinalis is a two-staged flagellated protozoan parasite which infects the lumen of the small intestine causing the gastrointestinal disease called giardiasis. This disease infects millions of people per year, commonly caused by inadequate water treatment and proximity to livestock. The main line of treatment against this disease has been metronidazole, a member of the nitroimidazole family of prodrugs, which when activated cause non-specific damage to the proteins and genetic material within anaerobic cells. Until recently, G. intestinalis has shown little resistance to metronidazole, but as resistance climbs it has become clear that genetic regulation is key. This study attempts to investigate a select number of key genes through the use of modified transfected plasmids for overexpression and with knockdown using morpholino oligos. Wild type (WB) G. intestinalis cells, transfected with plasmids modified to both overexpress specific genes and incorporate the human influenza hemagglutinin tag (HA-tag) were seen to change in survival rate when exposed to metronidazole. Localization of a select group of these genes was also found using the HA-tag. Two of these genes (quinone oxidoreductase and nitroreductase 1) had protein folding simulations run in order to be visualizee and compare their structures to closely matching equivalents. Finally, morpholino oligo nucleotides were used to knockdown expression in these two genes, leading to a significant decrease in survival for those targeted against quinone oxidoreductase when exposed to metronidazole.