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A group of diseases have been shown to correlate with a phenomenon called microbiome dysbiosis, where the bacterial species composition of the gut becomes abnormal. The gut microbiome of an animal is influenced by many factors including diet, exposures to bacteria during post-gestational growth, lifestyle, and disease status. Studies also show that host genetics can affect microbiome composition. We sought to test whether host genetic background is associated with gut microbiome composition in the Norwegian Lundehund dog, a highly inbred breed with an effective population size of 13 individuals. The Lundehund has a high rate of a protein-losing enteropathy in the small intestine that is often reported as Lundehund syndrome, which negatively affects longevity and life-quality. An outcrossing project with the Buhund, Norrbottenspets, and Icelandic sheepdog was recently established to reintroduce genetic diversity to the Lundehund and improve its health. To assess whether there was an association between host genetic diversity and the microbiome composition, we sampled the fecal microbiomes of 75 dogs of the parental (Lundehund), F1 (Lundehund x Buhund), and F2 (F1 x Lundehund) generations. We found significant variation in microbiome composition from the parental Lundehund generation compared to the outcross progeny. The variation observed in purebred Lundehunds corresponded to dysbiosis as seen by a highly variable microbiome composition with an elevated Firmicutes to Bacteroidetes ratio and an increase in the prevalence of Streptococcus bovis/Streptococcus equinus complex, a known pathobiont that can cause several diseases. We tracked several other environmental factors including diet, the presence of a cat in the household, living in a farm and the use of probiotics, but we did not find evidence of an effect of these on microbiome composition and alpha diversity. In conclusion, we found an association between host genetics and gut microbiome composition, which in turn may be associated with the high incidence of Lundehund syndrome in the purebred parental dogs. Gut microbiome dysbiosis is associated with host genetics in the Norwegian Lundehund

Aliivibrio wodanis has been associated with winter ulcer disease outbreaks. It has been reported that A. wodanis may act as a secondary pathogen in disease outbreaks and pathogenesis. The culture supernatant of A. wodanis causes a cytopathogenic effect (CPE) in various salmon cell lines. Moreover, in an Atlantic salmon bath challenge experiment, A. wodanis alone produces clinical symptoms in the fish. However, the contribution of A. wodanis to winter ulcer disease is not clear. Despite the knowledge to date and the research done in the field, there are still unanswered questions and a knowledge gap. Several Vibrio and Aliivibrio species use quorum sensing (QS) to regulate genes connected to host-pathogen interaction, virulence, survival, and adaptation mechanisms. In other aliivibrios, several QS systems, including the ainS/AinR and a transcriptional regulator litR (LuxR homologs), have been reported to regulate various phenotypic traits. Moreover, temperature is an essential factor that governs the prevalence of bacteria in the environment and host and is a potent regulator of pathogenesis in many bacteria. Hence the thesis aimed to understand better the QS`s role and the effect of temperature in A. wodanis. Our study reveals that ainS autoinducer synthase is required to produce the Acyl-Homoserine Lactone (AHL) 3OHC10-HSL in A. wodanis. Furthermore, we found that the 3OHC10-HSL production is cell density and temperature-dependent. The 3OHC10-HSL concentration was higher at 6°C, the temperature below the threshold temperature at which winter ulcer occurs, compared to 12°C. The results also showed that QS and temperature regulate various functions such as AHL production, motility, and production of proteases, hemolysin and siderophores. The cell culture study further revealed that cell density, QS and temperature influence the cytotoxicity in CHSE salmon cell lines. This suggests that A. wodanis produces cytolysins and cytotoxins that are implicated in cytotoxicity. Bacteria use the Type VI secretion system (T6SS) for multiple functions like iron transport, interspecies competition, virulence, and niche adaptations. A. wodanis co-exists with the main pathogen M. viscosa in the infected fish during the winter ulcer disease outbreaks. In addition, the bacterium has been shown to hinder the growth and virulence of M. viscosa. The thesis further investigated the mechanisms by which A. wodanis may survive together with M. viscosa in skin ulcers and during winter ulcer outbreaks. We found that the A. wodanis genome encodes three T6SSs (T6SS1-T6SS3) and auxiliary clusters (Aux1-4); and several potential Type VI secretion system effectors (T6SEs). In addition, the A. wodanis genome is found to contain a type IF CRISPR-Cas system. This suggests that these two mechanisms may play a role during the survival, adaptation, and immune function of A. wodanis in its natural environment, inside the host or during its co-existence with M. viscosa. Next, we analyzed the genome-wide transcriptomics of A. wodanis and QS mutants litR and ainS mutants grown at different temperatures and cell densities. We found that the genes involved in T6SSs, and CRISPR-Cas systems are regulated by cell density, temperature, and QS. The transcriptome analysis showed that the complete T6SS2 apparatus was less expressed in the litR/WT, suggesting litR regulates T6SS2 in A. wodanis. In this study, the transcriptome analysis demonstrated that deletion of litR decreased the hcp1 expression, a gene involved in bacterial competition and virulence in other bacteria. In addition to litR dependent expression, expression value of hcp decreased three times in litR/WT, HCD at 12°C when compared to 6°C and HCD. Our observation suggests that temperature 6°C and litR are crucial for expressing the genes related to virulence. Understanding the strain diversity of the same species is important in exploring strategies to survive in a changing environment. Finally, we wanted to study the genomic similarity and variation between A. wodanis isolates by performing a pan-genome analysis of twenty-two A. wodanis isolates collected from various locations across Norway. The analysis revealed an open pan-genome with a wide inter-species diversity in A. wodanis genomes. We examined the phylogenetic relatedness between the isolates using single nucleotide variants (SNVs) and core genes. The phylogenetic trees were distributed into five groups, where Group 2, 4 and 5 encompassed conserved isolates. The accessory genomes (shell and cloud) accounted for about 73% of the total pan-genome suggesting the genomes have acquired most of the genes through horizontal gene transfer (HGT). Whole and cloud genome functional annotation revealed a larger number of genes related to functional families such as metabolism, signaling and cellular processes and genetic information processing, suggesting they are involved in energy metabolism and environmental interactions. By further analyzing the twenty-two A. wodanis genomes, we identified diverse CRISPR-Cas systems, spacers, and prophages. About 60% of isolates encoded a different CRISPR-Cas system compared to the reference strain. Like the reference strain, the other twenty-one A. wodanis isolates also encode multiple T6SSs where the T6SS2 and Aux-2 are either absent or showed differences in about 80% (18 out of 22) of isolates. In addition to the T6SSs and CRISPR-Cas, other elements relevant for adaptation, virulence, and survival potentials, such as virulence factors (VFs) and biosynthetic gene clusters (BGCs), were explored. We have found that the predicted VFs and BGCs were conserved between the A. wodanis isolates. The results presented in this work yield knowledge about QS regulation of virulence, survival, and adaptation mechanisms in A. wodanis. This aids in understanding the mechanisms connected to co-existence of A. wodanis with M. viscosa and winter ulcer disease development and treatment.

The main aim is to evaluate the content of marine biofilms and different marine bacteria for antimicrobial potential. Hypothesis: antimicrobial compounds can be produced in the presence of other bacterial species; in the context of Winogradsky columns that simulate a naturalistic environment, and in the proximity to other species in the case of marine bacterial isolates. For this main aim and hypothesis, the following subgoals are defined: 1. Document any antimicrobial production by marine bacteria from different marine sources, using a co-culture approach. 2. Document the bacterial content of biofilm in Winogradsky columns (WC) from the seashore, using different techniques. 3. Evaluate the antibacterial production in the bacterial content from Winogradsky biofilms by performing diffusing assays, MIC assays in combinations with chromatographic methods.

Climate change is a major concern in the Arctic region, as large amounts of organic carbon (C) are stored in permafrost soils and sediments. Increasing average temperatures have the potential to release that C and making it available to biologic activity. Carbon-rich, anoxic soils such as peatlands are inhabited by methanogenic archaea that can metabolize by-products of microbial C decomposition and consequently release methane (CH4). Methane oxidizing bacteria (MOB) comprise a major biological filter for CH4 in terrestrial and aquatic ecosystems and thereby regulate CH4 emissions to the atmosphere. The genus Methylobacter has been detected in many CH4 rich ecosystems and several circumpolar locations. Climate change in the Arctic includes changes in both temperature and precipitation as well as ecosystem changes related to plant cover and herbivory. All of these changes have the potential to influence soil structure and soil biological processes. Thus, they are important factors controlling the soil C cycle and eventually the activity of MOB. The aim of this thesis was to investigate the ability of the Arctic biological CH4 filter to adapt to changes in vegetation, CH4 concentrations and temperature. Further, to gain insights in the resilience and resistance of the MOB community to environmental changes. We have shown that herbivory by geese changes the soil structure and thus the vertical distribution of CH4 and oxygen (O2) concentrations in a high Arctic peatland. These differences are accompanied by changes in the potential rates of CH4 oxidation. The highest activity was detected in shallower parts of the peatland in grazed sites compared to sites protected from grazing. Different MOB communities are responsible for the CH4 oxidation, depending on the above ground grazing and these communities are composed of closely related Methylobacter OTUs. Exposing peat soils from both grazed and protected sites to increased CH4 concentrations and temperature revealed that MOB respond strongly to changing CH4 concentrations, but apparently not to temperature. The response to changing CH4 concentrations involved different members of the Methylobacter community depending on the CH4 concentration and their previous exposure to herbivory. Temperature had minor effects on the CH4 oxidation activity and the MOB community pmoA transcription in the soils. However, temperature clearly influences growth, CH4 oxidation and CO2 production in the native peat soil isolate Methylobacter tundripaludum SV96. The temperature adaptation of M. tundripaludum SV96 is modulated by fine tuning transcription and translation to achieve the optimal balance between substrate availability, growth, and energy generation at different temperatures. These findings show that identical CH4 oxidation rates can be produced from very different physiological states. This also explains how the apparent lack of temperature responses in soil MOB communities is a result of physiological acclimation. Our results show that MOB in high Arctic peatlands and particular the genus Methylobacter is both physiologically and ecologically flexible in adapting to changes in plant cover, O2 distribution, CH4 concentrations and temperature. Thus, the MOB community comprises an efficient and resilient CH4 filter in high Arctic peat soils.

The rise in frequency of antibiotic resistant pathogenic bacteria makes the need for new treatment options for previously curable bacterial infections ever more important. In the process of discovering and developing antibacterial agents, one powerful approach has been borrowing wisdom from nature. Antimicrobial peptides (AMPs) are critical components of the innate immune systems found in almost all eukaryotic life forms. Their mode of action includes disruption of the bacterial membrane and to trigger supporting immune responses. Due to these properties, AMPs are considered promising lead structures that can be further developed into commercially available antibiotics to treat or prevent human diseases caused by bacteria. The work conducted in this thesis aims to discover and characterize novel antimicrobial peptides from the Arctic marine ascidian Synoicum turgens by using a marine bioprospecting approach. This includes collection, extraction and fractionation of biomass, antibacterial bioactivity testing and AMP isolation followed by chemical and biological characterization. For one isolated peptide class, truncated versions were prepared, aiming to produce shorter, linear variants with retained antimicrobial activity. In paper I, turgencin A and B and their oxidized derivatives were discovered through bioassay-guided purification. These peptides had an unusual disulfide connectivity, rarely seen in marine AMPs. Both turgencin A and B were potently active against all assayed bacterial strains. Membrane assays showed that the peptides cause bacterial membrane disruption within a few seconds. Turgencin A and B also displayed some cytotoxic activity against two human cell lines. Oxidation of the methionine present in both peptides decreased the bioactivities in all assays. Turgencin A, being the most potent AMP, was subject for sequence analysis and prediction of antimicrobial potential of different regions in paper II. Truncated and modified variants of turgencin A were synthetically produced to make smaller AMPs with the potential of being developed into antimicrobial drug leads. These 10-mer peptides, named StAMP-1–11, were made using an amino acid replacement strategy. Some of the Trp enriched peptides had similar bactericidal activity as the parent peptide turgencin A, and no cytotoxic activity against the mammalian cell lines. During turgencin isolation, a series of other smaller peptides were discovered in the same extracts presumably with antimicrobial activity. These isolated and characterized ~2 kDa, cysteine-rich peptides (CRPs) (described in paper III), were named St-CRP-1 and St-CRP-2 and contained 18-19 amino acids. The St-CRPs shared a disulfide connectivity pattern with alpha-defensins, had a neutral net charge, moderate antibacterial activity and showed no cytotoxicity. In addition, the introduction section provides background information on topics related to the thematic of the articles. This includes an introduction to bacteria, antibiotics and antibiotic resistance, AMPs, ascidians, and the marine environment.

Infectious diseases have been a problem for humans since the beginning of human existence. The “golden age” of antibiotics started at the end of the 1920s, with the discovery of penicillin by Sir Alexander Fleming. This was followed by the discovery of several life-saving antibiotics. The number of new marketed antibiotics has declined, and most pharmaceutical companies are no longer working in antibiotic development. This in itself would be unproblematic, had it not been for the rapid ability of bacteria to become resistant towards previously debilitating agents. The need for new antibiotics is therefore eminent. Natural products have been important contributors for antibiotic drug discovery and development. Microorganisms have been a particularly proliferative source of antibiotics, providing us with among others the penicillins, aminoglycosides, tetracyclines and polymyxins. Most naturally derived pharmaceuticals, including antibiotics, originate from terrestrial organisms. This is mainly because the terrestrial environment historically has been easier to access compared to the marine environment below the intertidal zone. The marine environment is highly diverse, and there is still a huge biodiversity that is yet to be explored. In this project, Arctic and sub-Arctic marine bacteria and fungi were cultivated and studied for their production of natural products. The cultures were extracted and fractionated, and the fractions were tested for bioactivity, mainly focusing on antibacterial activity. Using bioactivity-guided isolation, compounds were isolated and structurally characterized. Finally, the bioactivity of the isolated compounds was broadly evaluated. In paper I, a known siderophore, serratiochelin A, was isolated from a co-culture of two bacteria, Serratia sp. and Shewanella sp. The compound was not detected in axenic cultures, indicating that cocultivation triggered production. The acid-catalyzed degradation of serratiochelin A into serratiochelin C was also observed. Serratiochelin A had weak activity against Staphylococcus aureus, melanoma cells and non-malignant lung fibroblasts. No activity was observed for the degradation product serratiochelin C, indicating that the oxazoline moiety in the original compound is essential for the bioactivity. In paper II, a marine bacterium Lacinutrix sp. was cultivated and studied for its ability to produce bioactive natural products. Through bioactivity-guided isolation, two new lyso-ornithine lipids were isolated, and their structures elucidated, showing that they only differed by the length of the hydrocarbon tail. Analysis by UHPLC-HR-MS indicate that the purified solutions are mixtures of isomers, but these were not possible to separate by preparative HPLC-MS. The compounds were evaluated for antibacterial activity and antiproliferative activity against human cells. Compound 1 displayed weak activity against Streptococcus agalactiae, while compound 2 had weak activity against melanoma cells. In paper III, a new dimeric naphthopyrone substituted with a sulphate group was isolated in high yields from cultures of an obligate marine fungus in the family Lulworthiaceae. The compound was tested against an extended panel of clinical bacterial isolates and showed potent antibacterial activity against several clinical methicillin-resistant Staphylococcus aureus isolates, with MICs down to 1.56 μg/mL. Acid-catalyzed degradation was also observed. The compound also displayed moderate activities against three human cell lines: melanoma, hepatocellular carcinoma, and non-malignant lung fibroblast. In paper IV, a new chlovalicin variant, chlovalicin B, was isolated from cultures of the obligate marine fungus Digitatispora marina. The fungus has previously been studied for its distribution in the marine environment but has not been extensively studied for its biosynthetic potential. The compound was isolated in low yields, and the structure was elucidated by NMR and HRMS experiments. The compound was assessed for a range of bioactivities and had weak antiproliferative activity against human melanoma cells.

The coastal domain of the Arctic is in rapid change with shifts in discharge phenology and catchment characteristics. Riverine discharge shapes hydrography, under water light climate, and nutrient dynamics during the brief melt season. Nutrients transported from catchment to coast can stimulate primary productivity, yet light attenuation caused by high surface turbidity is considered a limiting factor. This in turn affects the relative role of bacterial growth, with subsequent changes to carbon and metabolic balances. However, the effect of nutrient versus light availability on microbial growth remains understudied. The main goal of this master’s thesis was to assess the impact of riverine inputs on bacterial and primary production in a High Arctic Fjord Estuary. A full melt season study (May to September) was conducted in Adventfjorden, Svalbard, with samples collected across horizontal and vertical salinity and turbidity gradients. Microbial productivity was measured using in vitro incubations for net primary productivity (NPP) and bacterial production (BP), using the 14C-bicarbonate and 3H-methyl-thymidine incorporation essay methods. I paired this with in situ estimations of system metabolic balance (gross community production (GCP) versus community respiration (CR)) over a 24h incubation period. I found that NPP had the potential of exceeding BP by 100 to ~2800 times in freshwater influenced fjord waters, which was up to 3 times higher than the saline fjord max. Light had a strong impact on system metabolic balance, yet the system was net autotrophic even under low light conditions. River influenced areas in Arctic fjords are potential hotspots for high, sustained primary productivity during the melt season, challenging previous consensus. This has implications for our general understanding of nutrient cycling and carbon balances in the Arctic.

The substantial influx of freshwater to High Arctic coastal ecosystems influences nutrient, organic matter, and sediment dynamics, stratification, and light availability throughout the melt season. These changes shape pelagic microbial community composition and functioning, though little is known about impacts on nearshore benthic bacteria. Globally, mudflats are hotspots for biogeochemical cycling, and expected climate change driven increases in terrestrial runoff to coastal areas have highlighted a need to understand the influence these inputs from land might have on Arctic estuarine tidal flat bacteria. In this study, I investigated microbial community composition and function in an estuarine tidal flat through a full melt season, using a combined approach of metabarcoding and carbon-source utilization assays under different salinity treatments. I found that bacterial communities varied through both space and time, as environmental conditions shifted due to riverine inputs and local processes, with salinity as a key structuring gradient. Arctic freshwater bacteria demonstrated higher capacities for degrading a wider range of carbon substrates than Arctic marine microbial communities, indicating higher potential for degradation of complex terrestrially derived organic material in freshwater systems. Terrestrial and riverine taxa were transported with melt water and deposited in sediments, composing up to 60% of sequences in downstream communities. However, their unique functional capabilities appear to be inhibited by the high salinities found in subtidal mudflats, and the highest potential for utilization of terrestrially derived organic matter may be limited to areas where sediments are permeated by freshwater. With anticipated increases in riverine discharge and permafrost thaw in a warming Arctic, tidal flats will likely be more frequently inundated with freshwater and the resident bacteria will have increased access to bioavailable terrestrial organic matter, extending the region where terrestrial organic matter can be readily utilized by microbial communities further towards the sea.

Cold seeps are locations on the seafloor where CH4 migrates from reservoirs below sediments towards the atmosphere, sustaining thereby a high microbial and macrofaunal biomass and a diversity contrasting from the surrounding seafloor. The oxidation of methane and sulphide are typically the main sources of primary productivity of these ecosystems and have therefore gained a particular attention in the global oceans. Yet, despite the ubiquitous presence of these seeping sites and the presence of gas hydrates in the Arctic Ocean and its adjacent shelves, the impact of methane on benthic and pelagic microbial communities in this region have remained limited. Recently, five gas hydrate bearing mounds with ongoing methane seeping activity were discovered south of Svalbard, in the northern Barents Sea. In this PhD project, I studied changes in the structure of microbial communities, including both prokaryotes and eukaryotes, and geochemical profiles at these mounds to highlight key microbial groups and to provide insights on their ecological roles. Different niches were addressed, including: deep anaerobic sediments (Paper I and II); niches at the sediment surface at gas flare locations and within bacterial mats and siboglinid fields (Paper III); and above gas flares in the shallow shelf water column (Paper IV). The microbial biodiversity and the structure of communities were successfully identified for each of the habitats listed above. Our investigations revealed a microbial composition similar to other cold seeps: a predominance of archaeal anaerobic methanotrophs (ANME) and sulphate-reducing bacteria (SRB) in CH4-rich sediments, a higher abundance of methane oxidizing bacteria associated to the Methylococcales in the surface sediments and water column; and a co-occurrence of other commonly found prokaryotic groups. Yet, uncommon biological traits were also uncovered at these methane seeping sites: the anaerobic oxidation of methane was merely only driven by ANME-1 without the co-occurrence of a specific SRB clade; an abundant methanotroph with little genetic similarity in databases was detected; and a strong niche differentiation of sulphide-oxidizing bacteria within the different bacterial mats. This project has thereby extended our knowledge on the microbial biodiversity at Arctic cold seeps and opened further future research perspective toward microbial activity and metabolism at these high latitudes.

The diatoms make up the largest and most important group of marine microalgae. They are responsible for a quarter of all inorganic carbon fixation in the ocean and are thus of immense importance for global CO2 sequestration. This makes them good candidates for biological carbon capture and utilization (CCU) by mass cultivation using CO2 emissions from industrial point sources. Large scale cultivation of marine diatoms will produce valuable biomass containing marine lipids and proteins with possible commercial application. Commercial production of biomass requires extensive knowledge on the species to be cultivated in terms of optimal cultivation conditions, but also on the bioactivity, cytotoxicity and how the species responds to changing cultivation conditions. Most studies on the bioactivity and biochemistry of marine diatoms have been performed on species cultivated in the laboratory. Cultivation conditions are known to affect the biochemistry of diatoms, and large-scale cultivation will inevitably lead to a change in the environmental conditions. The present study was therefore designed to investigate how Porosira glacialis cultivated in large scale responds to changes in abiotic and biotic cultivation conditions. The first study was designed to investigate whether the direct addition of factory smoke as a source of CO2 would trigger production of bioactive or cytotoxic compounds in P. glacialis. Bioactivity testing revealed activity in anticancer, antibacterial and antibiofilm assays, in addition to cytotoxicity against human lung fibroblasts and effect on the development of sea urchin larvae. Nonetheless, the addition of factory smoke did not increase the cytotoxicity of P. glacialis, nor did it change the beneficial antibacterial and antibiofilm activity. These results are favourable for further development of large-scale production of diatom biomass. P. glacialis was co-cultivated with zooplankton to investigate whether the bioactivity and metabolic expression of the diatom would change when exposed to grazing pressure. The bioactivity testing revealed that the presence of zooplankton increased the cytotoxicity of P. glacialis towards human normal lung fibroblasts. Investigation of active compounds and metabolomic analysis showed that the grazing pressure possibly influence the carotenoid concentration in P. glacialis. It was also found that the bioactivity might be traced to primary metabolites such as chlorophyll derived compounds. The study also showed that the use of OSMAC (one strain many compounds) could be a useful method for further investigation of the bioactivity of diatoms. Investigation of the antibiofilm activity of P. glacialis resulted in isolation of two compounds showing inhibition of biofilm formation by Staphylococcus epidermidis. The compounds isolated were methyl 3-hydroxyoctadecanoate and a pheophorbide-like compound. This is the first time compounds with antibiofilm activity have been isolated from P. glacialis as well as the first evidence of such activity from both the isolated compounds. Kiselalgene utgjør den største og viktigste gruppen av marine mikroalger. De er ansvarlige for en fjerdedel av all uorganisk karbonfiksering i havet og er dermed av enorm betydning for det globale CO2 opptaket. Dette gjør dem til gode kandidater for biologisk karbonfangst og -utnyttelse (CCU) i massedyrking ved bruk av CO2 fra industrielle punktutslipp. Storskala dyrking av marine kiselalger vil produsere verdifull biomasse som inneholder marine lipider og proteiner med mulig kommersiell anvendelse. Kommersiell produksjon av biomasse krever stor kunnskap om arten som skal dyrkes med tanke på best mulige dyrkingsforhold, men også om bioaktivitet, toksisitet og hvordan arten reagerer på endrede dyrkingsforhold. De fleste studier på bioaktivitet og biokjemi av marine kiselalger er utført på arter dyrket i laboratoriet. Det er kjent at dyrkingsforhold påvirker biokjemien til kiselalger, og dyrking i stor skala vil uunngåelig føre til en endring i miljøforholdene. Denne studien ble derfor designet for å undersøke hvordan Porosira glacialis dyrket i stor skala reagerer på endringer i abiotiske og biotiske dyrkingsforhold. Den første studien undersøkte om direkte tilsetning av fabrikkrøyk som kilde til CO2 ville endre produksjon av bioaktive eller toksiske forbindelser i P. glacialis. Bioaktivitetstesting avdekket aktivitet i alle tester: anticancer, antibakteriell og anti-biofilm. I tillegg viste resultatene toksisitet som hemmet utvikling av kråkebolle-larver og humane lungefibroblaster. Likevel økte ikke tilsetting av fabrikkrøyk toksisiteten til P. glacialis, og den hemmet heller ikke den gunstige antibakterielle og anti-biofilm-aktiviteten til algene. Disse resultatene er svært positive for videre utvikling av storskala produksjon av kiselalger. P. glacialis ble dyrket sammen med dyreplankton for å undersøke om bioaktiviteten og utrykket av metabolitter ville endre seg når de ble utsatt for stress i form av beitetrykk. Testing av bioaktivitet avslørte at tilstedeværelsen av zooplankton økte toksisiteten til P. glacialis mot humane normale lungefibroblaster. Undersøkelse av aktive forbindelser og metabolittprofiler viste at beitetrykket muligens påvirker konsentrasjonen av karotenoider i P. glacialis, og at bioaktiviteten muligens kan spores til primære metabolitter som klorofyllderivater. Studien viste også at bruk av OSMAC (one strain many compounds) kunne være en nyttig metode for videre undersøkelse av bioaktiviteten til kiselalger. Undersøkelse av anti-biofilm-aktiviteten til P. glacialis resulterte i isolasjon av to forbindelser som viser inhibering av biofilmdannelse av bakterien Staphylococcus epidermidis. De isolerte forbindelsene var metyl-3-hydroksyoktadekanoat og en pheophorbid-lignende struktur. Dette er første gang en forbindelse med anti-biofilm-aktivitet er blitt isolert fra P. glacialis, og det første beviset på slik aktivitet fra begge de isolerte forbindelsene.

Scientific abstract Antibiotic resistance, especially in Gram-negative pathogens, represents a substantial clinical and financial burden to our society. The presented work investigated mechanisms and evolutionary dynamics that promote the emergence and maintenance of resistance in bacteria. In the first study, conserved collateral susceptibility changes were identified across resistant uropathogenic Escherichia coli, which included also changes towards increased sensitivity of these isolates to certain antibiotics. This so-called collateral sensitivity potentiated the effect of antibiotics and prevented the selection of resistant isolates compared to wildtype strains. The mechanism and fitness cost of resistance were important predictors of collateral responses in resistant bacteria, and their rapid clinical identification could inform future evolution-based infection treatment. The second study demonstrated the potential of a transposable element (Tn1) to spread antibiotic resistance during natural transformation of Acinetobacter baylyi. In the course of this horizontal gene transfer mechanism, Tn1-containing DNA entered the bacterial cell, and specific host, as well as transposon proteins, facilitated Tn1-insertion into the recipient chromosome. A mechanistic model of transposition-mediated natural transformation from a circular, cytoplasmic intermediate is presented. In the third study, uropathogenic E. coli improved its permissiveness towards two unrelated multidrug resistance plasmids while adapting to a new environmental niche. Mutations in the CCR and ArcAB regulatory systems resulted in transcriptional downregulation of plasmid genes and explained plasmid cost reduction. The presented evolutionary dynamics improve our understanding of successful associations between bacterial pathogens and resistance plasmids and provide a novel solution to the so-called ‘plasmid paradox’. In a broader perspective, the findings of this thesis advanced our knowledge on the selection, spread and maintenance of antibiotic resistance in bacteria, which is important to counteract its evolution.

Marine microbes are crucial for the marine food web and carbon cycle. Algae are the main source of organic matter in the oceans with algae blooms triggering reoccurring bacterial succession patterns. Bacteria can recycle nutrients from organic matter coming from land or algae, fueling regenerated primary production. Terrestrial freshwater inputs can have substantial impacts on Arctic marine microbes via import of nutrients, organic matter, and sediments, and via changing the marine hydrography. The aims of this PhD thesis are i) to summarize the current knowledge about microbial ecology in the Arctic seasonal ice zone, ii) to study the effects of terrestrial inputs from rivers and glaciers on the microbial food web over different seasons, and iii) to dive into algae-bacteria interactions with a focus on the importance of regenerated production. We provide evidence that subglacial upwelling is an important process in a tidewater glacier-influenced fjord on Svalbard in spring, a previously understudied season. Subglacial upwelling lead to increased surface water nutrients, a stratified surface layer, and brackish sea ice. At the glacier front, microbial communities were significantly different compared to a sea-ice edge reference site. Phytoplankton primary production was two orders of magnitude higher at the glacier site compared to the reference site, due to upwelling related nutrient inputs and potentially also stratification and a thinner snow cover. During the spring freshet and in summer we investigated the impacts of river runoff on bacterial and archaeal communities with detailed considerations of environmental drivers. We found significant differences between bacterial communities during the spring freshet and late summer, mainly controlled by the concentrations and properties of dissolved organic matter, nutrient concentrations, and fjord hydrography. We recreated major algal spring bloom dynamics in an experimental study with algae-bacteria co-cultures. I used the experimental data to extend a commonly used dynamic algal growth model improving the modeling of multi-nutrient limitations and bacterial remineralisation. The work within this thesis demonstrates that microbial communities and functions are highly regulated by environmental constraints in the coastal seasonal ice zones. Marine mikrober er avgjørende for hele det marine næringsnettet og for karbonsyklusen. Alger er den viktigste kilden til organisk material i havet med algeoppblomstringer som utløser gjentatte suksesjonsmønstre fra forskjellige mikroorganismer. Bakterier kan bryte ned organisk material fra alger eller land og resirkulere næringsstoffer som gir næring til regenerert primærproduksjon. Terrestriske tilførsler av ferskvann, organisk material, næringssalter og sediment har sterk innvirkning på arktiske marine systemer der bakterier og alger er tett forbundet gjennom en rekke interaksjoner. Målet med denne doktorgradsavhandlingen er i) å oppsummere den nåværende kunnskapen om mikrobiell økologi i den arktiske issonen, ii) å studere effekten av terrestriske tilførsler fra elver og isbreer på det mikrobielle næringsnettet over forskjellige årstider, og iii) å utforske interaksjoner mellom alger og bakterier. Vi viser at subglasial oppstrømning er viktig i vinter/vår i et fjordsystem med sjøis på Svalbard, som er en undervurdert årstid. Subglasial oppstrømning har betydelige konsekvenser for mikrobielle samfunn og for karbonsyklusen. Vi fant betydelige forskjeller i mikrobielle samfunn, spesielt i sjøisen ved brefronten. Primærproduksjon av fytoplankton ble betydelig økt ved brefronten på grunn av tilskudd av næringsstoffer, men også på grunn av lagdeling og et tynnere snødekke. I løpet av våren og på sensommeren studerte vi innvirkningene av avrenning fra elven på bakterie- og arkeasamfunn, med et særlig hensyn til miljømessige drivere, som organisk material, tilførsel av næringssalter og fjordhydrografi. Vi fant signifikante forskjeller mellom bakteriesamfunn i løpet av våren og sensommeren, hovedsakelig kontrollert av konsentrasjonene og egenskapene til organisk material, næringssaltkonsentrasjoner og fjordhydrografi. Vi fikk kulturer av våroppblomstrende alger og tilknyttede bakterier fra Van Mijenfjorden og gjenskapte våroppblomstringsdynamikk. Vi brukte dette eksperimentet til å utvide en kjent modell for å representere begrensninger med flere næringsstoffer og bakteriell remineralisering og for å diskutere hvor betydelig det er å inkludere disse. Oppsummert viser arbeidet i denne oppgaven at samfunnet og funksjoner av mikroorganismer er sterk regulert av miljøet som organisk material, næringssalter, lys, og hydrografi.

The Arctic is experiencing dramatic changes including increases in precipitation, glacial melt, and permafrost thaw, resulting in increasing freshwater runoff to coastal waters. During the melt season, terrestrial runoff delivers carbon- and nutrient-rich freshwater to Arctic coastal waters, with unknown consequences for the microbial communities that play a key role in determining the cycling and fate of terrestrial matter at the land-ocean interface. To determine the impacts of runoff on coastal microbial (bacteria and archaea) communities, we investigated changes in pelagic microbial community structure between the early (June) and late (August) melt season in 2018 in the Isfjorden system (Svalbard). Amplicon sequences of the 16S rRNA gene were generated from water column, river and sediment samples collected in Isfjorden along fjord transects from shallow river estuaries and glacier fronts to the outer fjord. Community shifts were investigated in relation to environmental gradients, and compared to river and marine sediment microbial communities. We identified strong temporal and spatial reorganizations in the structure and composition of microbial communities during the summer months in relation to environmental conditions. Microbial diversity patterns highlighted a reorganization from rich communities in June toward more even and less rich communities in August. In June, waters enriched in dissolved organic carbon (DOC) provided a niche for copiotrophic taxa including Sulfitobacter and Octadecabacter. In August, lower DOC concentrations and Atlantic water inflow coincided with a shift toward more cosmopolitan taxa usually associated with summer stratified periods (e.g., SAR11 Clade Ia), and prevalent oligotrophic marine clades (OM60, SAR92). Higher riverine inputs of dissolved inorganic nutrients and suspended particulate matter also contributed to spatial reorganizations of communities in August. Sentinel taxa of this late summer fjord environment included taxa from the class Verrucomicrobiae (Roseibacillus, Luteolibacter), potentially indicative of a higher fraction of particle-attached bacteria. This study highlights the ecological relevance of terrestrial runoff for Arctic coastal microbial communities and how its impacts on biogeochemical conditions may make these communities susceptible to climate change.

Arctic peatlands act as important sources and sinks of carbon. Microbial decomposition takes place in these soils, producing the greenhouse gasses carbon dioxide and methane as end-products. A variety of aerobic and anaerobic microbial pathways are involved in the decomposition of organic material in peat soil. In anoxic soil layers, methane and carbon dioxide is often produced through syntropic partnerships between several fermenters and methanogens. Changes in soil conditions like temperature and substrate availability affect which methanogenic and fermentative pathways are dominant in the soil, thus affecting the final gas emissions. Due to their size and fast metabolism, microorganisms have the potential to respond rapidly to environmental changes like temperature variation and are constantly exposed to short-time temperature changes on daily and hourly basis in many natural and anthropogenic ecosystems. How short-term temperature variation affect soil microbial communities is yet poorly understood. In this master thesis I have investigated microbial responses in Arctic peat soil to temperature changes (heating and cooling). A high resolution 9-week incubation experiment with temperature increase from 2 – 10°C followed by cooling from 10 – 2°C was carried out, thus exposing the peat soil to a temperature range and timeframe similar to Arctic summer season temperature shifts. Gas and metabolite accumulation and microbial community growth and biomass was monitored to establish knowledge about the effects. Methane accumulation was rapidly affected by heating and showed increasing accumulation rates at warmer temperatures. However, exposure to cooling did not immediately reduce the accumulation of methane. This delay might be an effect of established high growth rates at higher temperatures that takes longer time to reverse. A change from no net carbon dioxide emission below 6°C to emission rates increasing rapidly due to heating above 6°C was observed. This change occurred at the same time and temperature as radical changes in concentrations of the fermentative metabolites acetate and propionate and more rapid cell growth. A combination of a change in the ratio between different methanogenic pathways, fermentative pathways and rates of carbon dioxide fixation relative to production are proposed as possible explanations to the shift in carbon dioxide emission seen at 6°C. This master thesis represents a comprehensive study of time-dependent temperature effects on greenhouse gas emissions from anoxic peat soils, an important and understudied topic in literature.

Marine fungi comprise a group of organisms that have been overlooked for a long time. Research interest has increased with the realization of the important ecological role and rich chemistry of marine fungi. Marine fungi have yielded thousands of new natural products the last decade, but many taxa remain unstudied. Marine fungi from the Arctic have not been reported in literature in regard to bioprospecting campaigns and represent a novel source of natural products. The aim of this thesis is to assess the potential of Arctic marine fungi to produce bioactive secondary metabolites by fermentation and genome analysis. This was achieved in three steps. First, fungi were isolated from the Svalbard archipelago. The 20 isolates obtained were characterized based on molecular markers and their antibacterial activity was tested using an agar diffusion assay (Paper 1). Secondly, three distinct marine fungi were whole genome sequenced and characterized. One of the fungi represented a putatively novel species which was circumscribed based on morphology and phylogenetic inference (Paper 2). Finally, a metabolite from one fungus among the 20 obtained around Svalbard was isolated and the bioactivities characterized (Paper 3). In Paper 1, half of the fungal isolates showed activity against pathogenic bacteria and every third isolate represents potentially new species of fungi. Five of these isolates are strictly marine fungi belonging to the order of Lulworthiales. The study showed that the Arctic can yield novel marine fungal diversity that can be utilized in bioprospecting. For Paper 2, three marine fungi were whole genome sequenced and their biosynthetic gene clusters were characterized. Mapping of the biosynthetic gene clusters (BGCs) within the Emericellopsis genome confirmed the detection of the secondary metabolite helvolic acid produced during fermentation. The study revealed numerous unknown biosynthetic gene clusters and a range of carbohydrate active enzymes. Each of the three genomes provides the first genome of their respective taxa and can contribute to understanding their evolutionary adaption to the marine environment. In Paper 3, a novel compound from the fermentation broth of Mytilinidion sp. was isolated and its bioactivity was characterized using seven different bioactivity assays. The compound turned out to be a modified medium component with IC50 of 43 µM in an ACE-inhibitory assay. The compound was novel and this is the first report of its bioactivity. Molecular networking could perhaps have provided early indications that the compound was a modified medium component. Marin sopp er en gruppe av organismer som har vært oversett i lang tid. Interessen for marin sopp har steget i takt med økt forståelse for dens økologi og rike kjemi. Mange tusen nye naturprodukter har blitt beskrevet fra marin sopp de siste tiårene, men det er fremdeles mange grupper som ikke er godt studert. Marin sopp fra Arktis er ikke godt undersøkt i forbindelse med bioprospektering og representerer en ny kilde til naturprodukter. Målet med avhandlingen var å vurdere potensialet til Arktisk marin sopp for å produsere bioaktive metabolitter ved å bruke fermentering og genomanalyse. Dette ble gjennomført i tre steg. Først ble 20 marine sopp isolert i løpet av ett forskningstokt rundt Svalbard. Disse soppene ble karakterisert ved hjelp av molekylære markører og evnen til å produsere antibakterielle forbindelser ble undersøkt ved å bruke agar-diffusjons analyse (Artikkel 1). Det neste som ble gjort var at tre forskjellige marine sopp ble helgenomsekvensert og genomene ble karakterisert. En av soppene er antageligvis ny for vitenskapen og ble beskrevet basert på morfologisk og fylogenetisk analyser (Artikkel 2). Til sist ble ett stoff fra en av soppene fra Artikkel 1 isolert og bioaktiviteten til stoffet ble beskrevet (Artikkel 3). I den første artikkelen viste halvparten av de isolerte soppene antibakteriell aktivitet mot sykdomsfremkallende bakterier. Hvert tredje isolat representerer muligens nye arter basert på tilgjengelige referansesekvenser. Fem av de potensielt nye artene tilhører ordenen Lulworthiales som kun finnes i havet. Studien har vist at det er stort potensiale for å avdekke nye arter og at mange av disse viser evne til å produsere antibakterielle forbindelser. Dette viser at de kan utnyttes i bioprospektering. I Artikkel 2 ble tre ulike marine sopper helgenomsekvenseert og genomet ble karakterisert med tanke på hvilke biosyntetiske genklynger og karbohydrat-aktive enzymer som ble detektert. Karakteriseringen av genklyngene i Emericellopsis bekreftet deteksjonen av metabolitten helvolsyre som soppen produserte under fermentering. Studien avslørte mange ukjente genklynger og en rekke karbohydrat-aktive enzymer. De tre genomene er de første helsekvenserte genomene i deres respektive slekter og kan bidra til å gi ny kunnskap om de evolusjonære tilpasningene de har til det marine miljø. I den siste artikkelen ble ett ukjent stoff fra fermenteringen av Mytilinidion isolert. Bioaktiviteten til stoffet ble karakterisert ved hjelp av syv forskjellige bioaktivitetstester. Stoffet viste seg å være en delvis nedbrutt og modifisert mediekomponent som hadde en IC50 verdi på 42.3 µM i en ACE-inhiberingstest. Analyse ved hjelp av molekylært nettverk kunne kanskje avdekket at stoffet var relatert til dyrkningsmediumet.

Hydrocarbon pollution in marine environments present an acute problem which is aggravated by cold temperatures. This is especially relevant in important environmental and economic northern regions such as the Barents Sea. The Barents Sea region has become the focus of oil industries from Russia and Norway, incrementing the risk of hydrocarbon pollution. Bioremediation is a cost-effective and environmentally sound method to remove hydrocarbon pollution. To study the bioremediation potential of native bacteria from a chronically oil polluted region of the Barents Sea a study was done in the Murmansk seaport (Kola Bay, 68°58′00′′ N, 33°05′00′′ E) analyzing the composition of the bacterial community. This present work aims to do a deeper study of that bacterial community through a metagenomic fosmid library, a selective hydrocarbon-rich medium and bioinformatic analysis. A metagenomic fosmid library was constructed with the environmental DNA, the fosmid clones were transformed in Escherichia coli cells and cultivated in minimal media with concentrations 0.05%, 0.5% and 1% of diesel or crude oil. Ten colonies were selected, sequenced, and subsequently analyzed with the software Geneious 2020.2.2. BLAST searches and other bioinformatic tools were conducted in every Open Reading Frame of the 10 colonies, revealing new links between native bacteria and hydrocarbon degradation as well as promising enzymes for hydrocarbon bioremediation. This work sets the ground for further studies on functional metagenomic analyses with fosmid libraries and further studies with the novel bacterial species and enzymes linked to hydrocarbon biodegradation.

It is difficult to understand plasmid maintenance in the absence of selection and theoretical models predict the conditions for plasmid persistence to be limited. Plasmid-associated fitness costs decrease bacterial competitivity, while imperfect partition allows the emergence of plasmid-free cells during cell division. Although plasmid conjugative transfer allows mobility into plasmid-free cells, the rate of such events is generally not high enough to ensure plasmid persistence. Experimental data suggest several factors that may expand the conditions favorable for plasmid maintenance, such as compensatory mutations and accessory genes that allow positive selection. Most of the previous studies focus on bacteria that carry a single plasmid. However, there is increasing evidence that multiple plasmids inhabit the same bacterial population and that interactions between them affect their transmission and persistence. Here, we adapt previous mathematical models to include multiple plasmids and perform computer simulations to study how interactions among them affect plasmid maintenance. We tested the contribution of different plasmid interaction parameters that impact three biological features: host fitness, conjugative transfer and plasmid loss – which affect plasmid persistence. The interaction affecting conjugation was studied in the contexts of intracellular and intercellular interactions, i.e., the plasmids interact when present in the same cell or when in different cells, respectively. First, we tested the effect of each type of interaction alone and concluded that only interactions affecting fitness (epistasis) prevented plasmid extinction. Although not allowing plasmid maintenance, intracellular interactions increasing conjugative efficiencies had a more determinant impact in delaying extinction than the remaining parameters. Then, we allowed multiple interactions between plasmids and concluded that, in a few cases, a combined effect of (intracellular) interactions increasing conjugation and fitness lead to plasmid maintenance. Our results show a hierarchy among these interaction parameters. Those affecting fitness favor plasmid persistence more than those affecting conjugative transfer and lastly plasmid loss. These results suggest that interactions between different plasmids can favor their persistence in bacterial communities.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited., Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

Staphylococcus haemolyticus is a ubiquitous bacterium recognised as a significant cause of nosocomial infections. Today it represents one of the most clinically relevant staphylococcal species, hallmarked by its extreme genome plasticity and multiresistant phenotype. Despite its relevance, little is known about its virulence repertoire or how it causes disease. The main objective of this PhD thesis was to achieve a better understanding of S. haemolyticus colonization, virulence and hospital adaption by using a combination of comparative genomics and phenotypic assays. By the use of whole genome sequencing and pangenome analysis, we showed a clear phylogenetic separation between clinical and commensal isolates, and a distinct genomic signature of clinical S. haemolyticus isolates. Eighty-eight percent of the clinical isolates were multidrug resistant, compared to only 11 % of the commensal isolates. Clinical isolates typically carried the resistance genes aacA-aphD and mecA, in addition to the associated IS element IS256. These three genes were absent in most of the commensal isolates and were therefore good candidates for markers to differentiate the two groups. Additionally, sraP and the polysaccharide capsule operon, important virulence and immune evasion factors in other staphylococcal species, were more often detected in clinical isolates. We compared the adhesive and biofilm forming properties of clinical and commensal S. haemolyticus isolates, and showed that clinical isolates formed a thicker biofilm. By developing a novel method for investigating surface proteins expressed during human host colonization, we identified several surface proteins, with potential roles in colonization (sdr-like proteins, SceD), biofilm formation (Atl, Ebh) and immune evasion (TirS and SasH-like). In addition, we described a new species of the Staphylococcus genus; Staphylococcus borealis. The novel species was closely related to S. haemolyticus, but compared to S. haemolyticus, S. borealis showed considerable phylogenetic distance, yellow pigmented phenotype and the ability to produce urease. In conclusion, these studies have greatly advanced our knowledge of S. haemolyticus and its potential as a nosocomial pathogen. We have uncovered several potential markers which can distinguish clinical and commensal isolates and potentially be used as diagnostic markers of invasive disease. We also identified several important colonisation, virulence and immune evasion factors in S. haemolyticus, - some of which may be possible future targets for therapy. Finally, we demonstrate the high discriminatory power of whole genome sequencing by identifying a new staphylococcal species, now described as Staphylococcus borealis.

Global warming has especially detrimental effects on Arctic regions. One major issue is permafrost thaw and sub-permafrost methane escape via surface seeps. While the mitigation potential of methane-consuming bacteria on marine pingo-like methane seeps is well described, terrestrial methane seeps are still understudied. Recently, open system pingos have gained attention as terrestrial methane seeps. Lagoon Pingo (N 78°14'22'', E15°45'16''), a near-shore open system pingo in the Adventdalen valley, Svalbard, was chosen as a study site to investigate the potential impact of methane-oxidizing bacteria on methane evasion from open system pingos. During a fieldwork campaign in August 2019, methane fluxes were measured across the entire site and compared to the distribution of the site’s methane oxidation potentials. The centre of evasion was found at the groundwater-discharging source. The discharged waters were carried away by an associated stream, which gradually emitted methane to a distance of up to 80 meters from the source. While waters from the site were not shown to possess the ability to oxidize methane aerobically, an abundance of methane oxidizing bacteria was found in sediments that were covered with methane emitting waters, creating the potential to oxidize methane. Furthermore, using Lagoon Pingo sediments as inoculum, enrichments have brought a methane oxidizing bacterial strain in culture. This novel Methylobacter sp. seems to produce exospores, a feature not previously described for cultured Methylobacter species. The new knowledge provided by this thesis is a fundament for evaluating the bacterial impact on methane evasion from open system pingos. This in turn could be used to improve predictions of the contribution of open system pingos to the global methane budget.

Microbial communities have inherently high levels of metabolic flexibility and functional redundancy, yet the structure of microbial communities can change rapidly with environmental perturbation. To understand whether such changes observed at the taxonomic level translate into differences at the functional level, we analyzed the structure of taxonomic and functional gene distribution across Arctic and Antarctic locations. Taxonomic diversity (in terms of alpha diversity and species richness) differed significantly with location. However, we found that functional genes distributed evenly across bacterial networks and that this functional distribution was also even across different geographic locations. For example, on average 15% of the functional genes were related to carbon cycling across all bacterial networks, slightly over 21% of the genes were stress-related and only 0.5% of the genes were linked to carbon degradation functions. In such a distribution, each bacterial network includes all of the functional groups distributed following the same proportions. However, the total number of functional genes that is included in each bacterial network differs, with some clusters including many more genes than others. We found that the proportion of times a specific gene must occur to be linked to a specific cluster is 8%, meaning the relationship between the total number of genes in the cluster and the number of genes per function follows a linear pattern: smaller clusters require a gene to appear less frequently to get fixed within the cluster, while larger clusters require higher gene frequencies. We suggest that this mechanism of functional association between equally rare or equally abundant genes could have implications for ecological resilience, as non-dominant genes also associate in fully functioning ecological networks, potentially suggesting that there are always pre-existing functional networks available to exploit new ecological niches (where they can become dominant) as they emerge, for example, in the case of rapid or sudden environmental change. Furthermore, this pattern did not correlate with taxonomic distribution, suggesting that bacteria associate based on functionality and this is independent of its taxonomic position. Our analyses based on ecological networks also showed no clear evidence of recent environmental impact on polar marine microbial communities at the functional level, unless all communities analyzed have changed exactly in the same direction and intensity, which is unlikely given we are comparing areas changing at different rates.

Beta diversity is a key component of biodiversity with implications ranging from species dynamics to ecosystem functioning. However, β-diversity and its drivers have received little attention, especially for micro-eukaryotes which play key roles in soil functioning. We studied the diversity of soil micro-eukaryotes in a Swiss lowland floodplain using high-throughput Illumina sequencing of soil DNA. We determined the temporal vs. spatial patterns of soil micro-eukaryotic α- and β-diversity in six contrasted habitats sampled over one year. We identified the drivers of these patterns among soil conditions and functions and identified indicator taxa of habitats in each season. We found higher spatial than temporal variability and a strong space-time interaction in soil micro-eukaryotic diversity patterns as well as in their edaphic drivers, which contrasts with previous observation of bacterial diversity patterns. Our results show that, although soil micro-eukaryotic diversity indeed varies seasonally, it is correlated most strongly with edaphic variables and vegetation but the strength of correlations with individual drivers varied seasonally. Microbial diversity patterns and their drivers can thus differ quite substantially among seasons and taxa. Despite the dominance of spatial patterns, the temporal component of microbial diversity should not be ignored to accurately estimate the diversity and the complexity of soil microbial community assembly processes. Given the importance of soil microbial diversity for ecosystem functioning such knowledge is relevant for land management.

Graphical abstract, Transposon insertion sequencing methods such as Tn-seq revolutionized microbiology by allowing the identification of genomic loci that are critical for viability in a specific environment on a genome-wide scale. While powerful, transposon insertion sequencing suffers from limited reproducibility when different analysis methods are compared. From the perspective of population biology, this may be explained by changes in mutant frequency due to chance (drift) rather than differential fitness (selection). Here, we develop a mathematical model of the population biology of transposon insertion sequencing experiments, i.e. the changes in size and composition of the transposon-mutagenized population during the experiment. We use this model to investigate mutagenesis, the growth of the mutant library, and its passage through bottlenecks. Specifically, we study how these processes can lead to extinction of individual mutants depending on their fitness and the distribution of fitness effects (DFE) of the entire mutant population. We find that in typical in vitro experiments few mutants with high fitness go extinct. However, bottlenecks of a size that is common in animal infection models lead to so much random extinction that a large number of viable mutants would be misclassified. While mutants with low fitness are more likely to be lost during the experiment, mutants with intermediate fitness are expected to be much more abundant and can constitute a large proportion of detected hits, i.e. false positives. Thus, incorporating the DFEs of randomly generated mutations in the analysis may improve the reproducibility of transposon insertion experiments, especially when strong bottlenecks are encountered.

Microbial eukaryotes, including protists and fungi, play diverse functions in virtually all ecosystems. In the High Arctic, their high biomass and diversity reflects crucial ecological importance and the performance of key ecological processes. Protists are the main primary producers in arctic seas, whereas fungi are an important group of decomposers and symbiotic partners of plants in terrestrial habitats. During the last decade, along with the development of new high-throughput sequencing methods, our knowledge regarding arctic microbial eukaryotes has expanded. Previous studies have identified the major groups of microbial eukaryotes present in Svalbard and how their richness and abundance may vary along various temporal and spatial scales. Those studies used high-throughput sequencing to reveal the dynamics, biodiversity patterns and community composition of diverse microbial eukaryotes such as marine protists, soil and root-associated fungi. However, altogether these studies have just scratched the surface of disentangling the biodiversity and its drivers. Basic questions regarding taxonomic diversity, community composition and their drivers are addressed in a limited manner, often leaving most of the observed variation unexplained. Regarding functionality of these organisms, even less is known. At the same time, these findings have also increased the amount of questions about microbial eukaryotes, their life histories, strategies, seasonality, sensitivity to changes in environmental conditions, as well as functional importance of these organisms at different scales. Previously unexplained variation and other emerging knowledge gaps regarding microbial eukaryotes formed a backstage for this thesis. The main focus was to look at these organisms from a functional angle regarding variation related to methodology, seasonality and biotic factors through case studies addressing the following knowledge gaps. Firstly, to understand the functionality of biodiversity in a temporal and spatial context of cold soils, we need to determine if our methods estimate biodiversity of the active community of microbial eukaryotes. In other words, does DNA-based detection of species provide good enough approximation to continue or is a different methodology needed? We found that the choice of marker gene template influenced diversity measures and read numbers in abundant fungal groups such as Helotiales and Agaricales. However, it did not impact the community structure. Secondly, the aim was to understand the role of biodiversity and functionality of plant root-associated fungi in relation to host plant performance. We explored putative effects of fungal diversity on plant morphology and the interplay between functional diversity and abiotic factors in a spatial context. Our results revealed the importance of fungal richness and functional diversity, but no impact of community structure on plant morphometrics. Moreover, we showed that temperature affects fungal richness, below- and aboveground parts of the plant in different ways, making it difficult to predict its impact on the biological outcomes in natural systems. The third aim was to address the lack of polar night investigations of microbial eukaryotes in general, especially with a strong focus on their functions. Here, we investigated the impact of strong seasonality on functions of microbial eukaryotes in the marine environment. Community-level gene expression was driven primarily by seasonal patterns of light availability. Among the most expressed transcripts, nearly ⅔ transcripts were not functionally annotated, providing further evidence for distinct genetic makeup of the Arctic Ocean. Through these three case studies, this thesis contributed some building blocks to close important knowledge gaps, but also revealed that there are more unknowns to be addressed. This thesis aimed to increase awareness of the importance of functional understanding of the roles of microbial eukaryotes in the High Arctic ecosystems. Finally, it highlights further possibilities and developments that could improve the understanding of possible future responses of these organisms and processes that they control.

Arctic soil microbiomes may have to face drastic climate changes in the coming century. Currently, the arctic tundra act as a carbon sink due to slow decomposition rates of soil organic carbon, which partly owes to low temperatures and poor water drainage. However, with elevated temperatures, large, latent carbon pools stored in arctic permafrost are exposed to mineralization by the active layer microbiota. This could cause increased emissions of potent climate gases, such as CH4 and CO2 to the atmosphere. Potentially changing the status of the arctic tundra into a net carbon source and further result in a positive feedback-loop to the climate system. Along with the climatic changes, altered precipitation regimes are predicted to cause higher water contents in some areas, while others are predicted to become drier. In turn, these changes are likely to trigger a response in the diversity and functioning of the soil microbial communities, which again might have an impact on biogeochemical cycles. Methane oxidizing bacteria (MOB) are bacteria that works as a filter for CH4, mainly produced by anaerobic methanogenic archaea. Some MOB also possesses the ability to consume CH4 from the atmosphere (atmMOB). Studies have shown that high water saturation might impede O2 availability, which is demonstrated to lower CH4 oxidation rates. Here we investigate how the bacterial biodiversity, and, in more depth, the MOB community changes along a moisture gradient in high-Arctic tundra, Svalbard. We have used next generation sequencing of the 16S rRNA gene, and the MOB functional gene, pmoA, to infer differences in community composition along the gradient. Statistical analyses were used to deduce the effect of environmental variables on the bacterial- and MOB community structure. Both moisture and pH were shown to have significant effects on the bacterial community composition. Proteobacteria, Actinobacteria and Acidobacteria were overall the most abundant phyla. Cyanobacteria had a high abundance in the top layer of wet soil, while Chloroflexi were abundant in the deep layers. Most interestingly, we found that the MOB community was dominated by members of the upland soil cluster  (USC), a group of atmMOB not previously found in Svalbard. Our results demonstrate that dry, neutral to slightly alkalic upland cryosols could be a potential significant previously unrecognized CH4 sink. An analysis of variance showed that in addition to pH, moisture had a significant effect on the MOB community, which also was shown by a lack pmoA product in the wet sites. This implies that the MOB communities in these soils are vulnerable to alterations in water saturation in future climate change scenarios.

The marine pathogen Aliivibrio salmonicida is the causative agent of cold-water vibriosis, affecting mainly farmed salmonid fish when water temperatures are below 10°C. Even though cold-water vibriosis is no longer threatening Norwegian aquaculture, the reemergence of the disease is still a possibility. Therefore, it is crucial to gain knowledge and understanding of the pathogenicity of A. salmonicida. Quorum sensing (QS) is one of the communication systems used by bacteria to regulate gene expression in a synchronized way in response to cell density by secreting and sensing extracellular signals called autoinducers (AIs). QS system controls various physiological processes, particularly virulence system and biofilm formation in many pathogenic bacteria. With the increased emergence of antibiotic-resistant in recent years, understanding and targeting QS system is expected to bring potential new breakthroughs for the prevention and treatment of Vibrio infections. The present work was initiated to increase the knowledge on the QS system and its regulation on phenotypic traits that may be important for survival and host-pathogen interaction in A. salmonicida. Alternative sigma factors such as RpoS provide the main line of responses to changes in the environment by altering gene transcription. In several vibrios, RpoS has been shown to be connected to QS system. The obtained results in this thesis, clearly indicate that an RpoS-like sigma factor, RpoQ (VSAL_II0319) is a component of the QS system and involved in regulating colony rugosity, biofilm formation, and motility in a cell density dependent manner. The transcriptomics analysis further revealed that RpoQ is involved in influencing expression of a large panel of genes including the syp operon involved in polysaccharide production. This suggests that the downregulation of biofilm development and wrinkled colony phenotype were due to RpoQ-dependent repression on polysaccharide biosynthesis genes (syp genes) at high cell density. In addition to cell density dependent control on biofilm formation and colony rugosity through QS, temperature was shown to influence the regulation of RpoQ on these phenotypes, linking this environmental factor to the development of cold-water vibriosis in seawater at low temperatures. Previous reports have shown that A. salmonicida possesses two functional autoinducer synthases, the LuxI and AinS, which are responsible for the production of eight acyl homoserine lactones (AHLs). In this thesis, the inactivation of luxI, but not ainS, led to the formation of wrinkled colonies similar to those formed by the ΔrpoQ mutant. The transcriptome analysis showed that LuxI is required for repression of syp expression, where repression of syp is likely operated through the RpoQ sigma factor. When both systems were inactivated simultaneously, strains (ΔainSluxI−) with wrinkled colonies and mushroom structured biofilm were formed. Furthermore, the exogenous addition of either LuxI, N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or AinS, N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), to the ΔainSluxI- double mutant, inhibited biofilm development. This suggested that the downregulation of biofilm formation is operated through a common pathway when the AHL concentrations are high. The results presented in this work, add new knowledge about the nature of the QS mechanism of A. salmonicida and elucidate some aspects of the complex mechanism of biofilm formation, contributing to advancement of research in this field.

Sedimentary biofilms comprising microbial communities mediating the anaerobic oxidation of methane are rare. Here, we describe two biofilm communities discovered in sediment cores recovered from Arctic cold seep sites (gas hydrate pingos) in the north-western Barents Sea, characterized by steady methane fluxes. We found macroscopically visible biofilms in pockets in the sediment matrix at the depth of the sulphate-methane-transition zone. 16S rRNA gene surveys revealed that the microbial community in one of the two biofilms comprised exclusively of putative anaerobic methanotrophic archaea of which ANME-1 was the sole archaeal taxon. The bacterial community consisted of relatives of sulphate-reducing bacteria (SRB) belonging to uncultured Desulfobacteraceae clustering into SEEP-SRB1 (i.e. the typical SRB associated to ANME-1), and members of the atribacterial JS1 clade. Confocal laser scanning microscopy demonstrates that this biofilm is composed of multicellular strands and patches of ANME-1 that are loosely associated with SRB cells, but not tightly connected in aggregates. Our discovery of methanotrophic biofilms in sediment pockets closely associated with methane seeps constitutes a hitherto overlooked and potentially widespread sink for methane and sulphate in marine sediments.

The recent introduction of novel β-lactam/β-lactamase inhibitor combinations like ceftazidime-avibactam has increased our ability to treat infections caused by multidrug-resistant Gram-negative bacteria, including carbapenemase-producing Enterobacterales. However, the increasing number of cases of reported resistance to ceftazidime-avibactam is a concern. OXA-48 is a carbapenemase that has no significant effect on ceftazidime, but is inhibited by avibactam. Since isolates with OXA-48 frequently harbor extended-spectrum β-lactamases that are inhibited by avibactam, it is likely that ceftazidime-avibactam will be used to treat infections caused by OXA-48-producing Enterobacterales. Our data show that exposure to ceftazidime-avibactam can lead to changes in OXA-48, resulting in increased ability to hydrolyze ceftazidime and withstand the inhibitory effect of avibactam. Thus, resistance toward ceftazidime-avibactam among OXA-48-producing Enterobacterales should be monitored. Interestingly, the compromising effect of the amino acid substitutions in OXA-48 on other β-lactams and the effect of ceftazidime-avibactam exposure on the epidemic OXA-48 plasmid indicate that the evolution of ceftazidime-avibactam resistance comes with collateral effects., Infections due to carbapenemase-producing Gram-negative pathogens are associated with limited treatment options and consequently lead to increased mortality and morbidity. In response, combinations of existing β-lactams and novel β-lactamase inhibitors, such as ceftazidime-avibactam (CAZ-AVI), have been developed as alternative treatment options. To understand the development of resistance and evolutionary trajectories under CAZ-AVI exposure, we studied the effects of ceftazidime (CAZ) and CAZ-AVI on the carbapenemase OXA-48 and the epidemic OXA-48 plasmid in Escherichia coli. Exposure of CAZ and CAZ-AVI resulted in single (P68A) and double (P68A,Y211S) amino acid substitutions in OXA-48, respectively. The antimicrobial susceptibility data and enzyme kinetics showed that the P68A substitution was responsible for an increased activity toward CAZ, whereas P68A,Y211S led to a decrease in the inhibitory activity of AVI. X-ray crystallography and molecular modeling of the mutants demonstrated increased flexibility within the active site, which could explain the elevated CAZ hydrolysis and reduced inhibitory activity of AVI. Interestingly, these substitutions resulted in collateral effects compromising the activity of OXA-48 toward carbapenems and penicillins. Moreover, exposure to CAZ-AVI selected for mutations within the OXA-48-encoding plasmid that severely reduced fitness in the absence of antimicrobial selection. These evolutionary trade-offs may contribute to limit the evolution of OXA-48-mediated CAZ and CAZ-AVI resistance, as well as potentially resensitize isolates toward other therapeutic alternatives. IMPORTANCE The recent introduction of novel β-lactam/β-lactamase inhibitor combinations like ceftazidime-avibactam has increased our ability to treat infections caused by multidrug-resistant Gram-negative bacteria, including carbapenemase-producing Enterobacterales. However, the increasing number of cases of reported resistance to ceftazidime-avibactam is a concern. OXA-48 is a carbapenemase that has no significant effect on ceftazidime, but is inhibited by avibactam. Since isolates with OXA-48 frequently harbor extended-spectrum β-lactamases that are inhibited by avibactam, it is likely that ceftazidime-avibactam will be used to treat infections caused by OXA-48-producing Enterobacterales. Our data show that exposure to ceftazidime-avibactam can lead to changes in OXA-48, resulting in increased ability to hydrolyze ceftazidime and withstand the inhibitory effect of avibactam. Thus, resistance toward ceftazidime-avibactam among OXA-48-producing Enterobacterales should be monitored. Interestingly, the compromising effect of the amino acid substitutions in OXA-48 on other β-lactams and the effect of ceftazidime-avibactam exposure on the epidemic OXA-48 plasmid indicate that the evolution of ceftazidime-avibactam resistance comes with collateral effects.

It was once a long-held view that the Antarctic was a pristine environment with low biomass, low biodiversity and low rates of microbial activity. However, as the intensity of scientific investigation has increased, so these views have started to change. In particular, the role and impact of human activity toward indigenous microbial communities has started to come under more intense scrutiny. During the Subglacial Lake Ellsworth exploration campaign in December 2012, a microbiological survey was conducted to determine the extent and likelihood of exogenous input into the subglacial lake system during the hot-water drilling process. Snow was collected from the surface to represent that used for melt water production for hot-water drilling. The results of this study showed that snow used to provide melt water differed in its microbiological composition from that of the surrounding area and raised the question of how the biogeography of snow-borne microorganisms might influence the potential outcome of scientific analyses. In this study, we investigated the biogeography of microorganisms in snow around a series of Antarctic logistic hubs, where human activity was clearly apparent, and from which scientific investigations have been undertaken. A change in microbial community structure with geographical location was apparent and, notably, a decrease in alpha diversity at more remote southern latitudes. Soil-related microorganisms dominated microbial assemblages suggesting terrestrial input, most likely from long-range aeolian transport into continental Antarctica. We also observed that relic DNA was not a major issue when assessing snow samples. Overall, our observations might have profound implications for future scientific activities in Antarctica, such as the need to establish “no-go” protected areas, the need for better characterization of field sites and improved protocols for sterilization and verification of ice drilling equipment.

This is a pre-print of an article published in the ISME Journal. The final authenticated version is available online at: https://doi.org/10.1038/s41396-019-0396-x. Microbial community succession is a fundamental process that affects underlying functions of almost all ecosystems; yet the roles and fates of the most abundant colonizers are often poorly understood. Does early abundance spur long term persistence? How do deterministic and stochastic processes influence the ecological contribution of colonizers? We performed a succession experiment within a hypersaline ecosystem to investigate how different processes contributed to the turnover of founder species. Bacterial and eukaryotic colonizers were identified during primary succession and tracked through a defined, 79-day biofilm maturation period using 16S and 18S rRNA gene sequencing in combination with high resolution imaging that utilized stable isotope tracers to evaluate successional patterns of primary producers and nitrogen fixers. The majority of the founder species did not maintain high abundance throughout succession. Species replacement (versus loss) was the dominant process shaping community succession. We also asked if different ecological processes acted on bacteria versus Eukaryotes during succession and found deterministic and stochastic forces corresponded more with microeukaryote and bacterial colonization, respectively. Our results show that taxa and functions belonging to different kingdoms, which share habitat in the tight spatial confines of a biofilm, were influenced by different ecological processes and time scales of succession.

Describing dynamics of belowground organisms, such as fungi, can be challenging. Results of studies based on environmental DNA (eDNA) may be biased as the template does not discriminate between metabolically active cells and dead biomass. We analyzed ribosomal DNA (rDNA) and ribosomal RNA (rRNA) coextracted from 48 soil samples collected from a manipulated snow depth experiment in two distinct vegetation types in Svalbard, in the High Arctic. Our main goal was to compare if the rDNA and rRNA metabarcoding templates produced congruent results that would lead to consistent ecological interpretation. Data derived from both rDNA and rRNA clustered according to vegetation types. Different sets of environmental variables explained the community composition based on the metabarcoding template. rDNA and rRNA-derived community composition of symbiotrophs and saprotrophs, unlike pathotrophs, clustered together in a similar way as when the community composition was analyzed using all OTUs in the study. Mean OTU richness was higher for rRNA, especially in symbiotrophs. The metabarcoding template was more important than vegetation type in explaining differences in richness. The proportion of symbiotrophic, saprotrophic and functionally unassigned reads differed between rDNA and rRNA, but showed similar trends. There was no evidence for increased snow depth influence on fungal community composition or richness. Our findings suggest that template choice may be especially important for estimating biodiversity, such as richness and relative abundances, especially in Helotiales and Agaricales, but not for inferring community composition. Differences in study results originating from rDNA or rRNA may directly impact the ecological conclusions of one’s study, which could potentially lead to false conclusions on the dynamics of microbial communities in a rapidly changing Arctic.

Plasmids are genetic elements that play a role in bacterial evolution by providing new genes that promote adaptation to diverse conditions. Plasmids are also known to reduce bacterial competitiveness in the absence of selection for plasmid-encoded traits. It is easier to understand plasmid persistence when considering the evidence that plasmid maintenance can improve during co-evolution with the bacterial host, i.e. the chromosome. However, bacteria isolated from nature often harbor diverse mobile elements: phages, transposons, genomic islands and even other plasmids. Recent interest has emerged on the role such elements play on the persistence and evolution of plasmids. Here, we mainly review interactions between different plasmids, but also discuss their interactions with other genetic elements. We focus on interactions that impact fundamental plasmid traits, such as the fitness effect imposed on their hosts and the transfer efficiency into new host cells. We illustrate these phenomena with examples concerning clinically relevant organisms and the spread of plasmids carrying antibiotic resistance genes and virulence factors.

Enterococcus faecium is a ubiquitous bacterium that only recently emerged as a nosocomial pathogen. Infections mostly affect immunocompromised patients and multi-resistance of E. faecium often hampers treatment. This thesis focuses on determinants, which give pathogenic potential to E. faecium. In the first paper, two TirE proteins are described as novel virulence factors of E. faecium. The tirE encoding genes are exclusive to nosocomial E. faecium strains and localized on a putative mobile genetic element of phage origin. Comparison of the wild-type and its isogenic mutant which lacks the tirE locus showed that the tirE locus promotes bacterial proliferation in human blood. Both TirE proteins were detected in bacterial supernatant and one of them was associated with membrane vesicles. Membrane vesicles of E. faecium are described for the first time in the second paper. An isolation protocol using ultracentrifugation and purification of vesicles over a density gradient was established. Using a proteomic approach the proteinaceous content of vesicles of nosocomial strains under varying culture conditions was described. In addition to virulence factors, the vesicles contain vaccine candidate proteins and antimicrobial resistance related proteins. In the third paper, a megaplasmid from a blood culture isolate encoding high-level gentamicin resistance was investigated for its putative virulence determinants. The megaplasmid was transferred to a commensal strain, where it enhanced proliferation in blood. Plasmid-encoded blood proliferation-enhancing factors are predicted to be involved in primary metabolic processes and membrane transport. Genes encoding these factors are enriched among clinical strains. Megaplasmids are therefore likely to serve as vessels for various genes, including those needed for niche adaption in infection. Overall, the findings of this thesis advanced the field of E. faecium’s virulence in different aspects and will hopefully inspire novel therapy development.

Source at https://doi.org/10.1186/s40168-019-0681-y. Background - The population of Atlantic cod (Gadus morhua), also known as Northeast Arctic cod, migrating Atlantic cod, or simply “skrei,” lives mainly in the Barents Sea and Svalbard waters and migrates in annual cycles to the Norwegian coast in order to spawn eggs during late winter. It is the world’s largest population of Atlantic cod, and the population is distinct from the Norwegian coastal cod (or “fjord” cod). Despite the biological, economic, and cultural importance of migrating Atlantic cod, current knowledge on the associated microbiota is very limited. Using shotgun metagenomics and metaproteomics approaches, we present here the gut microbiota, metagenome-assembled genomes (MAGs) of the most abundant bacterial species, DNA-based functional profile, and the metaproteome of Atlantic cod specimens caught at a spawning area in an open ocean outside of Tromsø, Norway. Results - Our analyses identified 268 bacterial families in DNA isolated from feces of 6 individual migrating Atlantic cod. The most abundant family was Vibrionaceae (52%; 83% if unclassified reads are excluded), with Photobacterium (genus) representing the vast majority. The recovery of metagenome-assembled genomes provided further details and suggests that several closely related Photobacterium strains from the Photobacterium phosphoreum clade are the most abundant. A genomic-based functional profiling showed that the most abundant functional subsystems are “Carbohydrates”; “Amino Acids and Derivatives”; “Protein Metabolism”; “Cofactors, Vitamins, Prosthetic, Groups, and Pigments”; and “DNA Metabolism,” which is in agreement with other studies of gut microbiomes of marine organisms. Finally, the MS-based metaproteomic dataset revealed that the functional category “Protein Metabolism” is highly overrepresented (3×) when compared to the genome-based functional profile, which shows that ribosomal proteins are rich in the bacterial cytosol. Conclusion - We present here the first study of bacterial diversity of the gut of migrating Atlantic cod using shotgun sequencing and metagenome-assembled genomes (MAGs). The most abundant bacteria belong to the Photobacterium genus (Vibrionaceae family). We also constructed functional profiles of the gut microbiome. These may be used in future studies as a platform for mining of commercially interesting cold-active enzymes.

While the sources and fates of plastic pollution are receiving growing attention, major knowledge gaps exist. Among these, microbial degradation (aka biodegradation) of plastics remains poorly investigated. The process of biodegradation begins with the formation of biofilm on the polymer surface; our study aimed to investigate microbial colonization of polymer surfaces in the Arctic marine environment around Tromsø, Norway. An immersion experiment was designed to assess microbiome community composition on four different types of pre-production microplastic (

Source at https://doi.org/10.1186/s12917-018-1338-y . Background Infectious keratoconjunctivitis (IKC) is one of the most common ocular diseases in ruminants worldwide. In addition to keratitis and conjunctivitis, animals with IKC can develop uveitis, corneal ulcer, and in severe cases, blindness. The bacteria Moraxella spp. has been described as the primary causative agent of infectious bovine keratoconjunctivitis (IBK) in cattle (Bos taurus), while Chlamydia spp. and Mycoplasma conjunctivae are considered the main causative agents of IKC in sheep (Ovis aries). Previous studies indicated cervid herpesvirus 2 (CvHV2) as the primary causative agent of IKC in semi-domesticated reindeer (Rangifer tarandus tarandus). The aim of the study was to investigate the presence and prevalence of potential pathogens for IKC in reindeer, and compare the ocular microbiota of animals with IKC, with apparently healthy animals. Results Semi-domesticated reindeer (n = 341), with (n = 108) or without (n = 113) ocular clinical signs, or with no information on clinical status (n = 120), were sampled in Norway, Sweden and Finland in 2010–2014. Seroprevalence was 37.4% for alphaherpesvirus (95/254), 3.8% for gammaherpesvirus (8/211) and 7.1% for pestivirus (15/211) (ELISA). PCR analyses of conjunctival swab samples revealed a prevalence of 28.5% for CvHV2 (57/200), 11.9% for Chlamydiaceae (16/135) and 1.0% for M. conjunctivae (2/197). Bacteriological cultivation of 202 conjunctival swab samples revealed bacterial growth from 75.2% of the samples, with Moraxella spp. being isolated from 21.6% (11/51) of the animals with and 5.6% (5/84) without ocular clinical signs. A significant association (p Conclusions These results support the hypothesis that CvHV2 is the primary agent of IKC in semi-domesticated reindeer in Fennoscandia, with Moraxella bovoculi being a secondary candidate, since it was isolated in two different outbreaks of IKC. Further studies should be carried out to better understand the infection biology and the pathogenesis of IKC in reindeer.

Source at https://doi.org/10.1038/s41396-018-0107-z. The Kryos Basin is a deep-sea hypersaline anoxic basin (DHAB) located in the Eastern Mediterranean Sea (34.98°N 22.04°E). It is filled with brine of re-dissolved Messinian evaporites and is nearly saturated with MgCl2-equivalents, which makes this habitat extremely challenging for life. The strong density difference between the anoxic brine and the overlying oxic Mediterranean seawater impedes mixing, giving rise to a narrow chemocline. Here, we investigate the microbial community structure and activities across the seawater–brine interface using a combined biogeochemical, next-generation sequencing, and lipid biomarker approach. Within the interface, we detected fatty acids that were distinctly 13C-enriched when compared to other fatty acids. These likely originated from sulfide-oxidizing bacteria that fix carbon via the reverse tricarboxylic acid cycle. In the lower part of the interface, we also measured elevated rates of methane oxidation, probably mediated by aerobic methanotrophs under micro-oxic conditions. Sulfate reduction rates increased across the interface and were highest within the brine, providing first evidence that sulfate reducers (likely Desulfovermiculus and Desulfobacula) thrive in the Kryos Basin at a water activity of only ~0.4 Aw. Our results demonstrate that a highly specialized microbial community in the Kryos Basin has adapted to the poly-extreme conditions of a DHAB with nearly saturated MgCl2 brine, extending the known environmental range where microbial life can persist.

Carbapenem-resistant Pseudomonas aeruginosa is defined as a “critical” priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-β-lactamase-expressing isolate belonging to the high-risk sequence type 233.

Cervid herpesvirus 2 (CvHV2) is an alphaherpesvirus found in Rangifer subspecies throughout most of the circumpolar Arctic and the causative agent of infectious keratoconjunctivitis (IKC) in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus). IKC occurs as regular outbreaks, affecting dozens of reindeer in a herd, and is most common and severe among calves and young animals. IKC often appears as mild clinical signs from which the animals often recover, but the disease can progress to more advanced stages where the eye is severely damaged, resulting in blindness or death. Development of an antiviral therapy for CvHV2 could improve animal welfare conditions and reduce economic losses within reindeer herding industry in Fennoscandia. To our knowledge, only one pilot study has previously tested the effectiveness of antiviral drugs against CvHV2 and indicated that the nucleoside analog drug Acyclovir, commonly used as an antiherpetic treatment in humans and other species, was not successful inhibiting viral replication. This master’s study aimed to further investigate the effect of antiviral drugs against CvHV2 by testing Ganciclovir and Cidofovir, and to evaluate their potential use as part of a treatment for IKC in semi-domesticated reindeer in Fennoscandia. An in vitro experiment which used Madin-Darby bovine kidney (MDBK) cell cultures was used as a preliminary model for natural CvHV2 infection in reindeer. A negative dose-response relationship was found for both Ganciclovir and Cidofovir, however, neither drug was able to completely inhibit the viral replication even at the highest drug concentrations and lowest viral titers tested. These findings indicate that both drugs are tentative candidates for the development of an antiviral treatment for CvHV2, but further studies to attempt to increase the therapeutic index of either drug should be strongly considered before testing in live animals.

Paper III of this thesis is not available in Munin. Paper III: Salgado‐Flores, A., Tveit, A. T., Wright, A. D., Pope, P. B., Sundset, M. A.: “Characterization of the cecum microbiome from wild and captive rock ptarmigans from Svalbard and northern Norway in relation to diet composition”. (Manuscript). Enteric methane (CH4) from gut microbial fermentation of complex organic polymers in ruminants comprises an important source of the anthropogenic CH4 emissions and may also represent a significant loss of metabolic energy to the host animal depending on diet. CH4 is produced by specialized microbes called methanogens. Extensive research exists on enteric CH4 production and methanogens in domestic ruminants, but little is known about arctic herbivores. Reindeer (Rangifer tarandus tarandus), muskox (Ovibos moschatus), and rock ptarmigan (Lagopus muta), typically consume plants rich in toxic plant secondary metabolites (PSMs). PSMs may depress enteric CH4 production in high concentrations. The objective of this PhD project was to characterize the gut microbiome from these animals, with special emphasis on CH4 metabolism and related to diets high in PSMs. Investigating the microbiology of methanogenesis would help better understand their digestive physiology. Molecular biology techniques were used for these studies: quantitative real-time PCR, amplicon sequencing, and shotgun metagenomics. Reindeer fed lichens (high in PSMs) and muskoxen feeding on an autumn pasture presented increased proportions of methanogens (Methanobrevibacter ruminantium and Methanobrevibacter olleyae) that may be related to the low CH4 output previously described for these ruminants. Housing methanogens with expected low CH4 potentials might result in reduced energy losses in reindeer. Other factors apart from high PSMs contents may account for the presence of these methanogens. Rock ptarmigans mostly contained methanol-utilizing methanogens, potentially related to methanol production from pectin degradation. This thesis presents the first molecular study of the gut microbiota in muskoxen and rock ptarmigans. Muskoxen possessed bacteria mediating fiber degradation, which would allow them live off fibrous plants like graminoids. Rock ptarmigans presented a diverse microbiota with bacteria involved in PSMs degradation and a variable range of polysaccharides (e.g. hemicellulose, starch). In both cases, their gut microbiota would allow them utilizing the available food.

Source at https://doi.org/10.1371/journal.pone.0186576 Extended spectrum β-lactamase producing Escherichia coli (ESBL-EC) are excreted via effluents and sewage into the environment where they can re-contaminate humans and animals. The aim of this observational study was to detect and quantify ESBL-EC in recreational water and wastewater, and perform a genetic and phenotypic comparative analysis of the environmental strains with geographically associated human urinary ESBL-EC. Recreational fresh- and saltwater samples from four different beaches and wastewater samples from a nearby sewage plant were filtered and cultured on differential and ESBL-selective media. After antimicrobial susceptibility testing and multi-locus variable number of tandem repeats assay (MLVA), selected ESBL-EC strains from recreational water were characterized by whole genome sequencing (WGS) and compared to wastewater and human urine isolates from people living in the same area. We detected ESBL-EC in recreational water samples on 8/20 occasions (40%), representing all sites. The ratio of ESBL-EC to total number of E. coli colony forming units varied from 0 to 3.8%. ESBL-EC were present in all wastewater samples in ratios of 0.56–0.75%. ST131 was most prevalent in urine and wastewater samples, while ST10 dominated in water samples. Eight STs and identical ESBL-EC MLVA-types were detected in all compartments. Clinical ESBL-EC isolates were more likely to be multidrug-resistant (p

Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo , which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo , since fewer S. aureus subsp. aureus NCTC8325-4 Δ sdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood.

Background Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells. Results We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation. Conclusions Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-0953-2) contains supplementary material, which is available to authorized users.

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background: Largemouth bronze gudgeon (Coreius guichenoti) is of economic importance in China, distributed in upstream regions of the Yangtze River in China. But it has recently dramatically declined and is close to elimination. However, there is little knowing about the character of its intestinal microbiota. This study was conducted to elucidate the intestinal microbiota of wild largemouth bronze gudgeon with different body weight and gender. Results: Thirty wild largemouth bronze gudgeon were measured for body length and body weight, and identified for male and female according to gonadal development, and thereafter the intestinal microbiota’s were assessed by MiSeq sequencing of 16S rRNA genes. The results revealed that phyla Proteobacteria and Tenericutes were dominant in wild largemouth bronze gudgeon intestine independent of the body weight. Shannon’s and Inverse Simpson’s diversity indexes were significant (P < 0.05) different between male and female fish. The phylum profile in the intestine of male fish revealed that phylum Proteobacteria was dominant, in contrast to female fish where five phyla Tenericutes, Proteobacteria, Firmicutes, Fusobacteria and Spirochaetes were dominant. The genus profile revealed that genera Shewanella and Unclassified bacteria were dominant in male fish, while genus Mycoplasma was dominant in female fish. Conclusions: Our results revealed that the intestinal microbial community of wild largemouth bronze gudgeon was dominated by the phyla Proteobacteria and Tenericutes regardless of the different body weight, but the communities are significant different between male and female fish. These results provide a theoretical basis to understand the biological mechanisms relevant to the protection of the endangered fish species.

The increase in antimicrobial resistance in bacteria is a serious problem in public heath limiting the efficacy of available antimicrobials for treatment of infections. E. coli is the most frequent cause of uncomplicated urinary tract infections. Ciprofloxacin is an important antimicrobial for treatment of uncomplicated urinary tract infections. Unfortunately, resistance to ciprofloxacin is commonly found in the clinics and confers cross-resistance to other antimicrobials. Recent studies on a previously discovered phenomenon known as collateral sensitivity, the opposite of cross-resistance, where resistance to one antimicrobial confers increased sensitivity to others. Recently, some have suggested that using collateral sensitivity to choose the order of antimicrobial used can beneficially slow the progress of antimicrobial resistance during drug cycling. Therefore, we aim to study the collateral sensitivity and cross-resistance profiles across different clinical isolates of E. coli from urinary tract infections. In this study we generated 10 ciprofloxacin resistant mutants and tested their susceptibility to 8 different antimicrobials. Our results show that E. coli resistant to ciprofloxacin above clinical breakpoints are cross-resistant to many other clinically-relevant antimicrobials, such as mecillinam, trimethoprim, nitrofurantoin, chloramphenicol, and ceftazidime. Our results also showed that ciprofloxacin resistant mutants are collaterally sensitive to gentamicin. This study provides important data on cross-resistance and collateral sensitivity in a collection of clinical E. coli isolates resistant to ciprofloxacin. Since it is important to confirm previous findings on laboratory strains with clinical isolates, hopefully these findings will add to growing data on collateral sensitivity and inform future drug cycling treatment strategies to combat antimicrobial resistance.

Publisher's version, source: http://dx.doi.org/10.1371/journal.pone.0155213 . Reindeer (Rangifer tarandus tarandus) are large Holarctic herbivores whose heterogeneous diet has led to the development of a unique gastrointestinal microbiota, essential for the digestion of arctic flora, which may include a large proportion of lichens during winter. Lichens are rich in plant secondary metabolites, which may affect members of the gut microbial consortium, such as the methane-producing methanogenic archaea. Little is known about the effect of lichen consumption on the rumen and cecum microbiotas and how this may affect methanogenesis in reindeer. Here, we examined the effects of dietary lichens on the reindeer gut microbiota, especially methanogens. Samples from the rumen and cecum were collected from two groups of reindeer, fed either lichens (Ld: n = 4), or a standard pelleted feed (Pd: n = 3). Microbial densities (methanogens, bacteria and protozoa) were quantified using quantitative real-time PCR and methanogen and bacterial diversities were determined by 454 pyrosequencing of the 16S rRNA genes. In general, the density of methanogens were not significantly affected (p>0.05) by the intake of lichens. Methanobrevibacter constituted the main archaeal genus (>95% of reads), with Mbr. thaueri CW as the dominant species in both groups of reindeer. Bacteria belonging to the uncharacterized Ruminococcaceae and the genus Prevotella were the dominant phylotypes in the rumen and cecum, in both diets (ranging between 16–38% total sequences). Bacteria belonging to the genus Ruminococcus (3.5% to 0.6%; p = 0.001) and uncharacterized phylotypes within the order Bacteroidales (8.4% to 1.3%; p = 0.027), were significantly decreased in the rumen of lichen-fed reindeer, but not in the cecum (p = 0.2 and p = 0.087, respectively). UniFrac-based analyses showed archaeal and bacterial libraries were significantly different between diets, in both the cecum and the rumen (vegan::Adonis: pseudo-F

In 2015, The Norwegian Environment Agency requested the Norwegian Scientific Committee for Food Safety (VKM) to provide a scientific assessment of the information requirements laid down in the declaration form for the Regulation on microbial products and its guidelines, if these are sufficient to conduct a health and environmental risk assessment of the use of microbial cleaning products in Norway. VKM appointed a working group consisting of members of the Panel on Microbial Ecology. The Panel on Microbial Ecology has reviewed and revised the draft prepared by the working group, and the assessment has been adopted. Vitenskapskomiteen for mattrygghet (VKM) har på oppdrag fra Miljødirektoratet vurdert om dagens krav til informasjon om rengjøringsprodukter som inneholder mikroorganismer gir et godt nok grunnlag til å foreta helse- og miljørisikovurderinger av produktene. Den som importerer, produserer eller omsetter mikrobiologiske produkter i Norge er pålagt å merke og deklarere produktene i Produktregisteret i henhold til et eget deklarasjonsskjema. Miljødirektoratet har bedt om en faglig vurdering av hvilken informasjon direktoratet skal etterspørre når et produkt skal vurderes med hensyn til helse- og miljørisiko i Norge. Hvis dagens krav ikke er gode nok, ble VKM bedt om å vurdere hvilke krav som bør stilles.

Publisher's version, source: http://dx.doi.org/10.1371/journal.pone.0147150. INTRODUCTION Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus. METHODS Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on “Rodent-Borne Pathogens”) in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. KR732654 and KR732653). RESULTS The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal “hot spot” for foreign DNA integration and exchange. CONCLUSION Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.

Infectious keratoconjunctivitis (IKC) is one of the most common ocular diseases in ruminants worldwide. Animals affected by this disease can show a wide variety of clinical symptoms, including keratitis, uveitis, corneal ulcer, conjunctivitis and in severe cases, blindness. Moraxella spp., Chlamydia spp. and Mycoplasma conjunctivae have been described as primary causative agents in ruminant species such as cattle (Bos taurus), sheep (Ovis aries) or alpine ibex (Capra ibex), but previous studies indicated that the reindeer alphaherpesvirus (cervid herpesvirus 2; CvHV2) could be associated with IKC as a primary agent in reindeer (Rangifer tarandus spp.). To address this further, 344 semi-domesticated reindeer (R. t. tarandus), with (n = 127) or without (n = 126) clinical symptoms of IKC, or with no details on clinical symptoms provided (n = 91), were sampled in Norway, Sweden and Finland between 2010 and 2014. Serum was tested for antibodies against CvHV2 (enzyme-linked immunosorbent assay; ELISA) and swab samples obtained from conjunctiva were subjected to bacteriological cultivation and also tested (polymerase chain reaction; PCR) for the presence of Chlammydiaceae, M. conjunctivae and CvHV2 specific deoxyribonucleic acid (DNA). This master project summarizes the data obtained from the sampled reindeer populations and evaluates the assumed link between the presence of CvHV2 and clinical symptoms of IKC. The significant association detected in the present study between clinical symptoms of IKC and the presence of CvHV2 in affected eyes, which is not present for any of the other microorganisms studied, leads to the hypothesis that CvHV2 is the primary agent of the IKC in semi-domesticated reindeer in Fennoscandia.

Published version. Also available at http://dx.doi.org/10.3389/fmicb.2015.00356 The response of methanogens to thawing permafrost is an important factor for the global greenhouse gas budget. We tracked methanogenic community structure, activity, and abundance along the degradation of sub-Arctic palsa peatland permafrost. We observed the development of pronounced methane production, release, and abundance of functional (mcrA) methanogenic gene numbers following the transitions from permafrost (palsa) to thaw pond structures. This was associated with the establishment of a methanogenic community consisting both of hydrogenotrophic (Methanobacterium, Methanocellales), and potential acetoclastic (Methanosarcina) members and their activity. While peat bog development was not reflected in significant changes of mcrA copy numbers, potential methane production, and rates of methane release decreased. This was primarily linked to a decline of potential acetoclastic in favor of hydrogenotrophic methanogens. Although palsa peatland succession offers similarities with typical transitions from fen to bog ecosystems, the observed dynamics in methane fluxes and methanogenic communities are primarily attributed to changes within the dominant Bryophyta and Cyperaceae taxa rather than to changes in peat moss and sedge coverage, pH and nutrient regime. Overall, the palsa peatland methanogenic community was characterized by a few dominant operational taxonomic units (OTUs). These OTUs seem to be indicative for methanogenic species that thrive in terrestrial organic rich environments. In summary, our study shows that after an initial stage of high methane emissions following permafrost thaw, methane fluxes, and methanogenic communities establish that are typical for northern peat bogs.