Biography

  • Education in clinical medicin (MD), medical microbiology and immunology (MSc) and microbial genetics (PhD)
  • Molecular biology and biotechnology
  • Microbial source tracking (MST) of water fecal contamination
  • Host-specific genetic markers
  • Clinical relevant pathogens present in water, soil and biogas digestate materials
  • Analyses of antibiotic resistance genes (ARGs) in environmental samples
  • Microbial pollution and water quality measures
  • Microbial diversity of water and biogas digestates with special focus on bacteria and archaea
  • High-throughput Next Generation DNA Sequencing (NGS) technology, e.g Illumnia MiSeq platform
  • Bioinformatics analyses on DNA/RNA molecules, quantitative real-time PCR (qPCR) and Illumina sequencing data analysis
  • Functional genetic markers of carbon and nitrogen cycles

 

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Abstract

During June 2019, an outbreak of campylobacteriosis occurred in Askøy, an island northwest of Bergen, Norway. According to the publicly available records, over 2000 residents fell ill and 76 were hospitalised, and two deaths were suspected to be associated with Campylobacter infection. By investigating the epidemic pattern and scope, an old caved drinking water holding pool was identified that had been faecally contaminated as indicated by the presence of Escherichia coli (E. coli). Furthermore, Campylobacter bacteria were found at several points in the water distribution system. In the escalated water health crisis, tracking down the infectious source became pivotal for the local municipality in order to take prompt and appropriate action to control the epidemic. A major task was to identify the primary faecal pollution source, which could further assist in tracking down the epidemic origin. Water from the affected pool was analysed using quantitative microbial source tracking (QMST) applying host-specific Bacteroidales 16S rRNA genetic markers. In addition, Campylobacter jejuni, Enterococcus faecalis, Clostridium perfringens and Shiga toxin-producing E. coli were detected. The QMST outcomes revealed that non-human (zoogenic) sources accounted predominantly for faecal pollution. More precisely, 69% of the faecal water contamination originated from horses.

Abstract

Aquatic microbial diversity, composition, and dynamics play vital roles in sustaining water ecosystem functionality. Yet, there is still limited knowledge on bacterial seasonal dynamics in lotic environments. This study explores a temporal pattern of bacterial community structures in lotic freshwater over a 2-year period. The aquatic bacterial communities were assessed using Illumina MiSeq sequencing of 16S rRNA genes. Overall, the communities were dominated by α-, β-, and γ-Proteobacteria, Bacteroidetes, Flavobacteriia, and Sphingobacteriia. The bacterial compositions varied substantially in response to seasonal changes (cold vs. warm), but they were rather stable within the same season. Furthermore, higher diversity was observed in cold seasons compared to warm periods. The combined seasonal-environmental impact of different physico-chemical parameters was assessed statistically, and temperature, suspended solids, and nitrogen were determined to be the primary abiotic factors shaping the temporal bacterial assemblages. This study enriches particular knowledge on the seasonal succession of the lotic freshwater bacteria.

Abstract

Faecal contamination is one of the major factors affecting biological water quality. In this study, we investigated microbial taxonomic diversity of faecally polluted lotic ecosystems in Norway. These ecosystems comprise tributaries of drinking water reservoirs with moderate and high faecal contamination levels, an urban creek exposed to extremely high faecal pollution and a rural creek that was the least faecally polluted. The faecal water contamination had both anthropogenic and zoogenic origins identified through quantitative microbial source tracking applying host‐specific Bacteroidales 16S rRNA genetic markers. The microbial community composition revealed that Proteobacteria and Bacteroidetes (70–90% relative abundance) were the most dominant bacterial phyla, followed by Firmicutes, especially in waters exposed to anthropogenic faecal contamination. The core archaeal community consisted of Parvarchaeota (mainly in the tributaries of drinking water reservoirs) and Crenarchaeota (in the rural creek). The aquatic microbial diversity was substantially reduced in water with severe faecal contamination. In addition, the community compositions diverge between waters with dominant anthropogenic or zoogenic pollution origins. These findings present novel interpretations of the effect of anthropo‐zoogenic faecal water contamination on microbial diversity in lotic ecosystems.

Abstract

The aquatic microbiota is known to be an important factor in the sustainability of the natural water ecosystems. However, the microbial community also might include pathogens, which result in very serious waterborne diseases in humans and animals. Faecal pollution is the major cause of these diseases. Therefore, it is of immense importance to assess the potential impact of faecal pollution, originating from both anthropogenic and zoogenic sources, on the profile of microbial communities in natural water environments. To this end, the microbial taxonomic diversity of lotic ecosystems in different regions of Norway, representing urban and rural areas, exposed to various levels of faecal pollution, was investigated over the course of a 1-year period. The highest microbial diversity was found in rural water that was the least faecally polluted, while the lowest was found in urban water with the highest faecal contamination. The overall diversity of the aquatic microbial community was significantly reduced in severely polluted water. In addition, the community compositions diverged between waters where the dominant pollution sources were of anthropogenic or zoogenic origin. The results provide new insight into the understanding of how faecal water contamination, specifically that of different origins, influences the microbial diversity of natural waters.

To document

Abstract

Wastewater (WW) has been identified as a major hotspot of microbial emerging contaminants (MECs), such as antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Currently used WW treatment methods cannot efficiently eliminate these pollutants, resulting in passive contamination of adjacent environments receiving undertreated discharge. More effective WW treatment strategies are therefore urgently required. In this study, newly developed and well-characterised semi-interpenetrating polymer network (semi-IPN) hydrogels derived from the valorisation of marine wastes (e.g., shrimp shells) were investigated for their ARG removal potential. The results indicated that multiple ARGs prevalent in WW, such as ermB, qrnS, sul1 and tetO, were removed by up to 100% after being treated by novel hydrogels. In terms of horizontal gene transfer-associated genetic elements, such as integron-1 intl1, transposons tnpA1 (IS4 group) and tnpA2 (IS6 group), substantial reduction approaching 99.9% was also achieved. Moreover, up to 97% of efflux pump-associated qacE∆1 conferring multidrug resistance (MR) was successfully attenuated. To conclude, the semi-INP hydrogels developed exhibited great potential for ARG mitigation towards strengthening WW decontamination, which provides a viable, cost-effective and environmentally friendly novel treatment approach.

To document

Abstract

The present work aims to study the influence of ammonium-quaternary monomers and chitosan, obtained from different sources, upon the effect of semi-interpenetrating polymer network (semi-IPN) hydrogels upon the removal of waterborne pathogens and bacteria from wastewater. To this end, the study was focused on using vinyl benzyl trimethylammonium chloride (VBTAC), a water-soluble monomer with known antibacterial properties, and mineral-enriched chitosan extracted from shrimp shells, to prepare the semi-IPNs. By using chitosan, which still contains the native minerals (mainly calcium carbonate), the study intends to justify that the stability and efficiency of the semi-IPN bactericidal devices can be modified and better improved. The new semi-IPNs were characterized for composition, thermal stability and morphology using well-known methods. Swelling degree (SD%) and the bactericidal effect assessed using molecular methods revealed that hydrogels made of chitosan derived from shrimp shell demonstrated the most competitive and promising potential for wastewater (WW) treatment.

Abstract

Fecal contamination of water constitutes a serious health risk to humans and environmental ecosystems. This is mainly due to the fact that fecal material carries a variety of enteropathogens, which can enter and circulate in water bodies through fecal pollution. In this respect, the prompt identification of the polluting source(s) is pivotal to guiding appropriate target-specific remediation actions. Notably, microbial source tracking (MST) is widely applied to determine the host origin(s) contributing to fecal water pollution through the identification of zoogenic and/or anthropogenic sources of fecal environmental DNA (eDNA). A wide array of host-associated molecular markers have been developed and exploited for polluting source attribution in various aquatic ecosystems. This review is intended to provide the most up-to-date overview of genetic marker-based MST studies carried out in different water types, such as freshwaters (including surface and groundwaters) and seawaters (from coasts, beaches, lagoons, and estuaries), as well as drinking water systems. Focusing on the latest scientific progress/achievements, this work aims to gain updated knowledge on the applicability and robustness of using MST for water quality surveillance. Moreover, it also provides a future perspective on advancing MST applications for environmental research.

Abstract

Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health problems, such as waterborne outbreaks, which have caused huge economic and social losses. In this context, the prompt detection of microbial contamination becomes essential to enable early warning and timely reaction with proper interventions. Recently, molecular diagnostics have been increasingly employed for the rapid and robust assessment of microbial water quality implicated by various microbial pollutants, e.g., waterborne pathogens and antibiotic-resistance genes (ARGs), imposing the most critical health threats to humans and the environment. Continuous technological advances have led to constant improvements and expansions of molecular methods, such as conventional end-point PCR, DNA microarray, real-time quantitative PCR (qPCR), multiplex qPCR (mqPCR), loop-mediated isothermal amplification (LAMP), digital droplet PCR (ddPCR), and high-throughput next-generation DNA sequencing (HT-NGS). These state-of-the-art molecular approaches largely facilitate the surveillance of microbial water quality in diverse aquatic systems and wastewater. This review provides an up-to-date overview of the advancement of the key molecular tools frequently employed for microbial water quality assessment, with future perspectives on their applications.

Abstract

Zoogenic faecal contamination of the environment is one of the indices included in the evaluation of ecological threats, health hazards and adverse impacts on various ecosystems. The risks and environmental concerns are associated with the fact that faeces of wild and domesticated animals constitute the largest source of environmental loading of enteropathogens associated with transmission of zoonotic diseases (enteric zoonoses). Although sick animals are more likely to transmit pathogens, healthy ones can also be the carriers and defecate them into the environment. This is of particular importance given the close human-animal interactions and health effects resulting from human and ecological exposures to faecal hazards from companion and farm animals. We have therefore set out to investigate whether healthy equines can carry and defecate human infectious pathogens. For this purpose, we set up a pilot study to examine the faecal DNA of horses using culture-independent molecular diagnostics – fluorescent probe-based quantitative real-time PCR. Our results revealed that among a total of 23 horses, 6 were found to carry Campylobacter jejuni (C. jejuni), and 5 had Salmonella enterica serovar Typhimurium (S. Typhimurium). Moreover, Enterococcus faecalis (E. faecalis) was found in 14 horses, while 19 were positive for Clostridium perfringens (C. perfringens). Furthermore, the frequently reported protozoan parasites in livestock, Cryptosporidium parvum (C. parvum) and Giardia lamblia (G. lamblia), were discovered in 8 and 7 samples, respectively. This pilot study shed new light on the phenomenon of healthy horses carrying C. jejuni and other human-health-related enteropathogens.

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Abstract

Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a “green enzyme,” laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda’s gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7–8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.

Abstract

This study describes microbial and chemical source tracking approaches for water pollution in rural and urban catchments. Culturable faecal indicator bacteria, represented by Escherichia coli, were quantified. Microbial source tracking (MST) using host-specific DNA markers was applied to identify the origins of faecal contamination. Chemical source tracking (CST) was conducted to determine contaminants of emerging concern (CEC) of human/anthropogenic origin, including pharmaceuticals and personal care products (PPCPs) and endocrine-disrupting chemicals (EDCs). In addition, the eutrophication-causing macronutrients nitrogen and phosphorus were studied. MST tests revealed both anthropogenic and zoogenic faecal origins, with a dominance of human sources in the urban stream; non-human/environmental sources were prevalent in the rural creek. CST analyses revealed a higher number of CECs in the urban stream than in the rural watercourse. Positive correlations between PPCPs and both E. coli and the human DNA marker were uncovered in the urban stream, while in the rural creek, PPCPs were only highly correlated with the anthropogenic marker. Interestingly, macronutrients were strongly associated with primary faecal pollution origins in both watercourses. This correlation pattern determines the main pollutant contributors (anthropogenic or zoogenic) to eutrophication.

To document

Abstract

Wastewater (WW) has been widely recognized as the major sink of a variety of emerging pathogens (EPs), antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which may disseminate and impact wider environments. Improving and maximizing WW treatment efficiency to remove these microbial hazards is fundamentally imperative. Despite a variety of physical, biological and chemical treatment technologies, the efficiency of ARG removal is still far from satisfactory. Within our recently accomplished M-ERA.NET project, novel functionalized nanomaterials, i.e., molecularly imprinted polymer (MIP) films and quaternary ammonium salt (QAS) modified kaolin microparticles, were developed and demonstrated to have significant EP removal effectiveness on both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB) from WW. As a continuation of this project, we took the further step of exploring their ARG mitigation potential. Strikingly, by applying MIP and QAS functionalized kaolin microparticles in tandem, the ARGs prevalent in wastewater treatment plants (WWTPs), e.g., blaCTXM, ermB and qnrS, can be drastically reduced by 2.7, 3.9 and 4.9 log (copies/100 mL), respectively, whereas sul1, tetO and mecA can be eliminated below their detection limits. In terms of class I integron-integrase I (intI1), a mobile genetic element (MGE) for horizontal gene transfer (HGT), 4.3 log (copies/100 mL) reduction was achieved. Overall, the novel nanomaterials exhibit outstanding performance on attenuating ARGs in WW, being superior to their control references. This finding provides additional merit to the application of developed nanomaterials for WW purification towards ARG elimination, in addition to the proven bactericidal effect.

Abstract

During June 2019, an outbreak of campylobacteriosis occurred in Askøy, an island northwest of Bergen, Norway. According to the publicly available records, over 2000 residents fell ill and 76 were hospitalised, and two deaths were suspected to be associated with Campylobacter infection. By investigating the epidemic pattern and scope, an old caved drinking water holding pool was identified that had been faecally contaminated as indicated by the presence of Escherichia coli (E. coli). Furthermore, Campylobacter bacteria were found at several points in the water distribution system. In the escalated water health crisis, tracking down the infectious source became pivotal for the local municipality in order to take prompt and appropriate action to control the epidemic. A major task was to identify the primary faecal pollution source, which could further assist in tracking down the epidemic origin. Water from the affected pool was analysed using quantitative microbial source tracking (QMST) applying host-specific Bacteroidales 16S rRNA genetic markers. In addition, Campylobacter jejuni, Enterococcus faecalis, Clostridium perfringens and Shiga toxin-producing E. coli were detected. The QMST outcomes revealed that non-human (zoogenic) sources accounted predominantly for faecal pollution. More precisely, 69% of the faecal water contamination originated from horses.

To document

Abstract

Despite major efforts to combat pollution, the presence of pathogenic bacteria is still detected in surface water, soil and even crops due to poor purification of domestic and industrial wastewaters. Therefore, we have designed molecularly imprinted polymer films and quaternary ammonium-functionalized- kaolin microparticles to target specifically Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB) in wastewaters and ensure a higher purification rate by working in tandem. According to the bacteriological indicators, a reduction by 90 % was registered for GNB (total coliforms and Escherichia coli O157) and by 77 % for GPB (Clostridium perfringens) in wastewaters. The reduction rates were confirmed when using pathogen genetic markers to quantify particular types of GNB and GPB, like Salmonella typhimurium (reduction up to 100 %),Campylobacter jejuni (reduction up to 70 %), Enterococcus faecalis (reduction up to 81 %), Clostridium perfringens (reduction up to 97 %) and Shiga toxin-producing Escherichia coli (reduction up to 64 %). In order to understand the bactericidal activity of prepared films and microparticles, we have performed several key analyses such as Cryo-TEM, to highlight the auto-assembly mechanism of components during the films formation, and 29 Si/13 C CP/MAS NMR, to reveal the way quaternary ammonium groups are grafted on the surface of kaolin microparticles.

Abstract

Aquatic microbial diversity, composition, and dynamics play vital roles in sustaining water ecosystem functionality. Yet, there is still limited knowledge on bacterial seasonal dynamics in lotic environments. This study explores a temporal pattern of bacterial community structures in lotic freshwater over a 2-year period. The aquatic bacterial communities were assessed using Illumina MiSeq sequencing of 16S rRNA genes. Overall, the communities were dominated by α-, β-, and γ-Proteobacteria, Bacteroidetes, Flavobacteriia, and Sphingobacteriia. The bacterial compositions varied substantially in response to seasonal changes (cold vs. warm), but they were rather stable within the same season. Furthermore, higher diversity was observed in cold seasons compared to warm periods. The combined seasonal-environmental impact of different physico-chemical parameters was assessed statistically, and temperature, suspended solids, and nitrogen were determined to be the primary abiotic factors shaping the temporal bacterial assemblages. This study enriches particular knowledge on the seasonal succession of the lotic freshwater bacteria.

To document

Abstract

To meet increasing demand for animal protein, swine have been raised in large Chinese farms widely, using antibiotics as growth promoter. However, improper use of antibiotics has caused serious environmental and health risks, in particular Antimicrobial resistance (AMR). This paper reviews the consumption of antibiotics in swine production as well as AMR and the development of novel antibiotics or alternatives in China. The estimated application of antibiotics in animal production in China accounted for about 84240 tons in 2013. Overuse and abuse of antibiotics pose a great health risk to people through food-borne antibiotic residues and selection for antibiotic resistance. China unveiled a national plan to tackle antibiotic resistance in August 2016, but more support is needed for the development of new antibiotics or alternatives like plant extracts. Antibiotic resistance has been a major global challenge, so international collaboration between China and Europe is needed.

Abstract

Faecal contamination is one of the major factors affecting biological water quality. In this study, we investigated microbial taxonomic diversity of faecally polluted lotic ecosystems in Norway. These ecosystems comprise tributaries of drinking water reservoirs with moderate and high faecal contamination levels, an urban creek exposed to extremely high faecal pollution and a rural creek that was the least faecally polluted. The faecal water contamination had both anthropogenic and zoogenic origins identified through quantitative microbial source tracking applying host‐specific Bacteroidales 16S rRNA genetic markers. The microbial community composition revealed that Proteobacteria and Bacteroidetes (70–90% relative abundance) were the most dominant bacterial phyla, followed by Firmicutes, especially in waters exposed to anthropogenic faecal contamination. The core archaeal community consisted of Parvarchaeota (mainly in the tributaries of drinking water reservoirs) and Crenarchaeota (in the rural creek). The aquatic microbial diversity was substantially reduced in water with severe faecal contamination. In addition, the community compositions diverge between waters with dominant anthropogenic or zoogenic pollution origins. These findings present novel interpretations of the effect of anthropo‐zoogenic faecal water contamination on microbial diversity in lotic ecosystems.

Abstract

The aquatic microbiota is known to be an important factor in the sustainability of the natural water ecosystems. However, the microbial community also might include pathogens, which result in very serious waterborne diseases in humans and animals. Faecal pollution is the major cause of these diseases. Therefore, it is of immense importance to assess the potential impact of faecal pollution, originating from both anthropogenic and zoogenic sources, on the profile of microbial communities in natural water environments. To this end, the microbial taxonomic diversity of lotic ecosystems in different regions of Norway, representing urban and rural areas, exposed to various levels of faecal pollution, was investigated over the course of a 1-year period. The highest microbial diversity was found in rural water that was the least faecally polluted, while the lowest was found in urban water with the highest faecal contamination. The overall diversity of the aquatic microbial community was significantly reduced in severely polluted water. In addition, the community compositions diverged between waters where the dominant pollution sources were of anthropogenic or zoogenic origin. The results provide new insight into the understanding of how faecal water contamination, specifically that of different origins, influences the microbial diversity of natural waters.

To document

Abstract

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

Abstract

Faecal contamination of water has both anthropogenic and zoogenic origins that can shade various point and nonpoint/diffuse sources of pollution. Due to the dual origin and number of sources of faecal contamination, there are immense challenges in the implementation of effective measures to protect water bodies from pollution that poses threats to human and environmental health. The main health threats refer to infections, illnesses and deaths caused by enteric pathogenicmicrobes, in particular those responsible for waterborne zoonoses. To detect and identify the origins and sources of faecal pollution simultaneously, various methods and indicators have been compiled into a comprehensivemeasuring toolbox. Molecular diagnostics using genetic markers derived from Bacteroidales 16S rRNA gene sequences are quite prevalent in the current methodological implementation for the identification of faecal contamination sources in water. For instance, a culture- and library-independent microbial source tracking toolbox combining micro- and molecular biology tests run as a three-step procedure has been implemented in Norway. Outcomes from the Norwegian studies have identified two general trends in dominance of contributors to faecal water contamination in urban environments. Firstly, there is a tendency of higher contributions from anthropogenic sources during the cold season. Secondly, the identification of the dominance of zoogenic sources in faecalwater contamination during warm periods of the year.

To document

Abstract

In anaerobic digestion, studies of feeding frequency have produced conflicting results. Hence, the effect of feeding frequency on process variables and microbial community structure was investigated by comparing a laboratory-scale digester fed steam exploded food waste 10 times daily vs. one fed an equivalent amount once daily. The Frequently Fed Digester (FFD) produced on average 20% more methane and had lower effluent concentrations of long-chain fatty acids. Greater daily fluctuations in acetate, pH and biogas production rate could explain the lower specific methane yield and β-oxidation. Feeding frequency also influenced the microbial community whereby Tenericutes (42%) dominated in FFD but Firmicutes (31%) was most abundant in the Daily Fed Digester (DFD). Feeding frequency effects are therefore postulated to occur more often in digesters fed labile feedstocks at high organic loading rates.

To document

Abstract

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S)envelope protein are currently used in universal vaccination programs and achieve protective immuneresponse in more than 90% of recipients. However, new vaccination strategies are necessary for successfulimmunization of the remaining non- or low-responders. We have previously characterized a novel HBVchimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L)envelope protein (genotype D). The S/preS121–47chimera produced in mammalian cells and Nicotianabenthamiana plants, induced a significantly stronger immune response in parenterally vaccinated micethan the S protein. Here we describe the transient expression of the S/preS121–47antigen in an edibleplant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral admin-istration of adjuvant-free Lactuca sativa expressing the S/preS121–47antigen, three times, at 1lg/dose,was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies wereable to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) moreefficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results sup-port the S/preS121–47antigen as a promising candidate for future development as an edible HBV vaccine.

To document

Abstract

Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most costeffective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.

To document

Abstract

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases ( e.g . cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca s ativa ) using Agrobacterium - mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2 Δ N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2 Δ N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.

Abstract

This study describes the first Norwegian microbial source tracking (MST) approach for water quality control and pollution removal from catchment run-off in a nature-based treatment system (NBTS) with a constructed wetland. The applied MST tools combined microbial analyses and molecular tests to detect and define the source(s) and dominant origin(s) of faecal water contamination. Faecal indicator bacteria Escherichia coli and host-specific Bacteroidales 16 s rRNA gene markers have been employed. The study revealed that the newly developed contribution profiling of faecal origin derived from the Bacteroidales DNA could quantitatively distinguish between human and non-human pollution origins. Further, the outcomes of the MST test have been compared with the results of both physicochemical analyses and tests of pharmaceutical and personal care products (PPCPs). A strong positive correlation was discovered between the human marker and PPCPs. Gabapentin was the most frequently detected compound and it showed the uppermost positive correlation with the human marker. The study demonstrated that the NBTS performs satisfactorily with the removal of E. coli but not PPCPs. Interestingly, the presence of PPCPs in the water samples was not correlated with high concentrations of E. coli. Neither has the latter an apparent correlation with the human marker.

To document

Abstract

Biochar and its properties can be significantly altered according to how it is produced, and this has ramifications towards how biochar behaves once added to soil. We produced biochars from corncob and miscanthus straw via different methods (slow pyrolysis, hydrothermal and flash carbonization) and temperatures to assess how carbon cycling and soil microbial communities were affected. Mineralization of biochar, its parent feedstock, and native soil organic matter were monitored using 13C natural abundance during a 1-year lab incubation. Bacterial and fungal community compositions were studied using T-RFLP and ARISA, respectively. We found that persistent biochar-C with a half-life 60 times higher than the parent feedstock can be achieved at pyrolysis temperatures of as low as 370 °C, with no further gains to be made at higher temperatures. Biochar re-applied to soil previously incubated with our highest temperature biochar mineralized faster than when applied to unamended soil. Positive priming of native SOC was observed for all amendments but subsided by the end of the incubation. Fungal and bacterial community composition of the soil-biochar mixture changed increasingly with the application of biochars produced at higher temperatures as compared to unamended soil. Those changes were significantly (P < 0.005) related to biochar properties (mainly pH and O/C) and thus were correlated to pyrolysis temperature. In conclusion, our results suggest that biochar produced at temperatures as low as 370 °C can be utilized to sequester C in soil for more than 100 years while having less impact on soil microbial activities than high-temperature biochars.

Abstract

Water quality problems in Norway are caused mainly by high phosphorus (P) inputs from catchment areas. Multiple pollution sources contributes to P inputs into watercourses, and the two main sources in rural areas are agricultural runoff and discharge from on-site wastewater treatment systems (OWTSs). To reduce these inputs, Constructed wetlands (CWs) treating catchment runoff have been implemented in Norway since early 1990s. These CWs have been proven effective as supplements to agricultural best management practices for water quality improvements and therefore there are more than 1000 CWs established in Norway at present. This study aims to present some overall data on the present status of CWs treating catchment runoff in Norway, and in particular recent results of source tracking and retention of sediments and total phosphorus (TP) in a model, full-scale, long-term operated CW, which in practice treats runoff from a typical rural catchment with pollution from both point and diffuse sources. Nutrient contributions from agricultural runoff and OWTSs have been quantified in eight catchments, while the source tracking and retention of sediments and P has been studied in the model CW. P runoff in the catchments was largely affected by precipitation and runoff situation, and varied both throughout the year (every single year) and from one year to another. Annual TP contribution that origins from OWTSs was in general limited, and only 1 % in the catchment of the model CW. Monthly contribution, however, was higher than 30 % during warm/dry season, and cold months with frost season. For the purpose of source tracking study, faecal indicator bacteria (reported in terms of Escherichia coli - E. coli) and host-specific 16S rRNA gene markers Bacteroidales have been applied. High E.coli concentrations were well associated with high TP inputs into waterbodies during dry or/and cold season with little or no agriculture runoff, and further microbial source tracking (MST) tests proved human contribution. There are considerable variations in retention of sediments and TP in the CW between the years, and the annual yearly retention was about 38 % and 16 %, respectively. During the study period, the average monthly retention of sediments and TP was 54 % and 32 %, respectively. E. coli concentrations were also reduced in water passing the CW. The study confirmed that runoff from agricultural areas is the main P source in watercourses, however, discharges from OWTS can also be of great importance for the water quality, especially during warm/dry- and cold/frosty periods. Small CWs treating catchment runoff contribute substantially to the reduction of sediments, TP and faecal indicator bacteria transport into water recipients.

Abstract

Elevated nutrient concentrations in streams in the Norwegian agricultural landscape may occur due to faecal contamination. Escherichia coli (E. coli) has been used conventionally as an indicator of this contamination; however, it does not indicate the source of faecal origin. This work describes a study undertaken for the first time in Norway on an application of specific host-associated markers for faecal source tracking of water contamination. Real-time quantitative polymerase chain reaction (qPCR) on Bacteroidales host-specific markers was employed for microbial source tracking (MST) to determine the origin(s) of faecal water contamination. Four genetic markers were used: the universal AllBac (Bacteroidales) and the individual specific markers BacH (humans), BacR (ruminants) and Hor-Bac (horses). In addition, a pathogenicity test was carried out to detect the top seven Shiga toxin-producing E. coli (STEC) serogroups. The ratio between each individual marker and the universal one was used to: (1) normalise the markers to the level of AllBac in faeces, (2) determine the relative abundance of each specific marker, (3) develop a contribution profile for faecal water contamination and (4) elucidate the sources of contamination by highlighting the dominant origin(s). The results of the qPCR MST analyses indicated the actual contributions of humans and animals to faecal water contamination. The pathogenicity test revealed that water samples were STEC positive at a low level, which was in proportion to the concentration of the ruminant marker. The outcomes were verified statistically by coupling the findings of major contamination sources with observations in the field regarding local land use (residential or agricultural). Furthermore, clear correlations between the human marker and E. coli counts as well as the ruminant marker and STEC quantity in faecally contaminated water were observed. The results of this study have the potential to help identify sources of pollution for targeted mitigation of nutrient losses.