Publications by authors named "Lars Hestbjerg Hansen"

96 Publications

Engraftment of strictly anaerobic oxygen-sensitive bacteria in irritable bowel syndrome patients following fecal microbiota transplantation does not improve symptoms.

Gut Microbes 2021 Jan-Dec;13(1):1-16

Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.

Dysbiosis of the gut microbiome has been correlated with irritable bowel syndrome (IBS). Fecal microbiota transplantation (FMT) is being explored as a therapeutic option. Little is known of the mechanisms of engraftment of microbes following FMT and whether the engraftment of certain microbes correlate with clinical improvement in IBS. Microbiome data, from a previously reported placebo-controlled trial of treatment of IBS with FMT or placebo capsules, were used to investigate microbial engraftment 15 days, 1, 3 and 6 months after treatment through assessment of gains, losses and changes in abundance of amplicon sequence variants (ASVs) and microbial diversity (CHAO-1 richness) between the FMT group and the placebo group. These data were compared to changes in IBS Symptom Severity Scores (IBS-SSS). Twelve days of treatment with 25 daily multi-donor FMT capsules induced significant short- and long-term changes in the recipients' microbiomes for at least 6 months, with persistent engraftment of a variety of anaerobic bacteria from keystone genera, such as and and increased microbial diversity, particularly in patients with low initial diversity. FMT recipients lost ASVs after treatment, which was seen to a much lesser extent in the placebo group. No ASVs increased to a greater extent between FMT responders and non-responders following treatment. Major long-term changes, lasting for at least 6 months, in the gut microbiomes of IBS patients are seen following treatment with FMT capsules. None of these changes correlated with clinical improvement. The relationship between the microbiome and the etiology of IBS still remains unsolved.
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http://dx.doi.org/10.1080/19490976.2021.1927635DOI Listing
June 2021

Metaviromes Reveal the Dynamics of Host-Specific Phages Cultured and Uncultured by Plaque Assay.

Viruses 2021 May 21;13(6). Epub 2021 May 21.

Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark.

Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or those with the highest ability to make plaques in solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in environmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage community's shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome-host matrix to observe how enrichments in liquid media correspond to the metaviromic data. In this study, we observed a significant shift ( = 0.015) of the 47 identified putative phages with a minimum twofold change above zero in read abundance when adding a   DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the strain, were highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlight how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.
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http://dx.doi.org/10.3390/v13060959DOI Listing
May 2021

The effect of faecal microbiota transplantation on abdominal pain, stool frequency, and stool form in patients with moderate-to-severe irritable bowel syndrome: results from a randomised, double-blind, placebo-controlled study.

Scand J Gastroenterol 2021 May 17:1-9. Epub 2021 May 17.

Gastrounit, Medical Division, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.

Background: Irritable bowel syndrome (IBS) is associated with intestinal dysbiosis. Therefore, faecal microbiota transplantation (FMT) has been hypothesised to have a positive effect in patients with IBS. In this study, we analysed previously unexamined data from our randomised, double-blind, placebo-controlled study (trial registration number NCT02788071). The objective was to evaluate the effect of FMT on abdominal pain, stool frequency, and stool form.

Method: The study included 52 adult patients with moderate-to-severe IBS assigned randomly to treatment with FMT capsules or placebo capsules (1:1) for 12 days. The patients were followed for a total of six months, during which they kept a daily symptom diary tracking their abdominal pain on a scale from 0-10 and their bowel movements using the Bristol Stool Form Scale (BSFS). Diary data were not collected before treatment start.

Results: A statistically significant improvement in stool frequency was found in the FMT group from during treatment to post-treatment and 1 month. No statistically significant differences were found between groups at any time during the study for any of abdominal pain, stool frequency, and stool form (as measured by weighted stool score).

Conclusion: In this analysis of results from a randomised, double-blind, placebo-controlled study, we found no clinically beneficial effect of FMT on abdominal pain, stool frequency, or stool form. However, since the current literature on the potential role of FMT in treating IBS shows conflicting results, further studies are required. To assess treatment efficacy, we recommend future studies to include daily symptom diaries both before and after treatment intervention.
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http://dx.doi.org/10.1080/00365521.2021.1915375DOI Listing
May 2021

An improved direct metamobilome approach increases the detection of larger-sized circular elements across kingdoms.

Plasmid 2021 May 17;115:102576. Epub 2021 Apr 17.

Department of Environmental Science, Aarhus University, Roskilde, Denmark; Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Mobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate genetic diversity. MGEs serve as a vast communal gene pool and include DNA elements such as plasmids and bacteriophages (phages) among others. These mobile DNA elements represent a human health risk as they can introduce new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting environmental circular MGEs, referred to as metamobilomes, may broaden our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomics is affected by a severe bias towards small circular elements, introduced by multiple displacement amplification (MDA). MDA is typically used to overcome limiting DNA quantities after the removal of non-circular DNA during library preparations. By examining the relationship between sequencing coverage and the size of circular MGEs in paired metamobilome datasets with and without MDA, we show that larger circular elements are lost when using MDA. This study is the first to systematically demonstrate that MDA is detrimental to detecting larger-sized plasmids if small plasmids are present. It is also the first to show that MDA can be omitted when using enzyme-based DNA fragmentation and PCR in library preparation kits such as Nextera XT® from Illumina.
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http://dx.doi.org/10.1016/j.plasmid.2021.102576DOI Listing
May 2021

Application of wood ash leads to strong vertical gradients in soil pH changing prokaryotic community structure in forest top soil.

Sci Rep 2021 Jan 12;11(1):742. Epub 2021 Jan 12.

Department of Biology, University of Copenhagen, Copenhagen Ø, Denmark.

Wood ash is alkaline and contains base-cations. Application of wood ash to forests therefore counteracts soil acidification and recycle nutrients removed during harvest. Wood ash application to soil leads to strong vertical gradients in physicochemical parameters. Consequently, we designed an experimental system where small-scale vertical changes in soil properties and prokaryotic community structure could be followed after wood ash application. A mixed fly and bottom ash was applied in dosages of 3 and 9 t ha to the surface of soil mesocosms, simulating a typical coniferous podzol. Soil pH, exchangeable cations and 16S prokaryotic community was subsequently assessed at small depth intervals to 5 cm depth at regular intervals for one year. Wood ash significantly changed the prokaryotic community in the top of the soil column. Also, the largest increases in pH and concentrations of exchangeable cations was found here. The relative abundance of prokaryotic groups directionally changed, suggesting that wood ash favors copiotrophic prokaryotes at the expense of oligotrophic and acidophilic taxa. The effect of wood ash were negligible both in terms of pH- and biological changes in lower soil layers. Consequently, by micro-vertical profiling we showed that wood ash causes a steep gradient of abiotic factors driving biotic changes but only in the top-most soil layers.
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http://dx.doi.org/10.1038/s41598-020-80732-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804945PMC
January 2021

Epigenetic Memories: The Hidden Drivers of Bacterial Persistence?

Trends Microbiol 2021 03 4;29(3):190-194. Epub 2021 Jan 4.

Section of Microbial Ecology and Biotechnology, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.

Epigenetic modifications, including DNA methylation, stably alter gene expression without modifying genomic sequences. Recent evidence suggests that epigenetic regulation coupled with a long-term 'memory' effect plays a major role within bacterial persistence formation. Today, emerging high-resolution, single-molecule sequencing technologies allow an increased focus on DNA modifications as regulatory epigenetic marks, which presents a unique opportunity to identify possible epigenetic drivers of bacterial persistence.
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http://dx.doi.org/10.1016/j.tim.2020.12.005DOI Listing
March 2021

Enterococcus phage Nonaheksakonda infecting clinical isolates of Enterococcus faecalis represents a new lineage in the family Siphoviridae.

Arch Virol 2021 Feb 3;166(2):593-599. Epub 2021 Jan 3.

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark.

Enterococcus phage Nonaheksakonda was isolated from wastewater, using a vancomycin-resistant strain of the opportunistic pathogen Enterococcus faecalis (VRE) as a host. Nonaheksakonda is a lytic phage infecting E. faecalis V583 and clinical isolates with at least four different multi-locus sequence types (MLSTs). The genome is a 41.9-kb double-stranded DNA molecule (34.6% GC) with 74 coding sequences. Comparative analysis revealed only one close relative, Enterococcus phage heks. All other phages had low protein similarity and shared less than 54% nucleotide sequence identity with phage Nonaheksakonda. The most similar phages were all classified and unclassified efquatroviruses. We propose that the phages Nonaheksakonda and heks represent a novel genus within the family Siphoviridae, order Caudovirales, for which we propose the name "Nonaheksakondavirus".
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http://dx.doi.org/10.1007/s00705-020-04905-yDOI Listing
February 2021

Influence of vintage, geographic location and cultivar on the structure of microbial communities associated with the grapevine rhizosphere in vineyards of San Juan Province, Argentina.

PLoS One 2020 14;15(12):e0243848. Epub 2020 Dec 14.

Instituto de Biotecnología y Biología Molecular (IBBM), CCT-La Plata, CONICET, Dto de Cs. Biológicas, Fac. Cs. Exactas, UNLP, La Plata, Argentina.

Soil microbiomes, as a primary reservoir for plant colonizing fungi and bacteria, play a major role in determining plant productivity and preventing invasion by pathogenic microorganisms. The use of 16S rRNA and ITS high-throughput amplicon sequencing for analysis of complex microbial communities have increased dramatically in recent years, establishing links between wine specificity and, environmental and viticultural factors, which are framed into the elusive terroir concept. Given the diverse and complex role these factors play on microbial soil structuring of agricultural crops, the main aim of this study is to evaluate how external factors, such as vintage, vineyard location, cultivar and soil characteristics, may affect the diversity of the microbial communities present. Additionally, we aim to compare the influence these factors have on the structuring of bacterial and fungal populations associated with Malbec grapevine rhizosphere with that of the more widespread Cabernet Sauvignon grapevine cultivar. Samples were taken from Malbec and Cabernet Sauvignon cultivars from two different vineyards in the San Juan Province of Argentina. Total DNA extracts from the rhizosphere soil samples were sequenced using Illumina's Miseq technology, targeting the V3-V4 hypervariable 16S rRNA region in prokaryotes and the ITS1 region in yeasts. The major bacterial taxa identified were Proteobacteria, Bacteroidetes and Firmicutes, while the major fungal taxa were Ascomycetes, Basidiomycetes, Mortierellomycetes and a low percentage of Glomeromycetes. Significant differences in microbial community composition were found between vintages and vineyard locations, whose soils showed variances in pH, organic matter, and content of carbon, nitrogen, and absorbable phosphorus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243848PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735631PMC
February 2021

Potential Rhodopsin- and Bacteriochlorophyll-Based Dual Phototrophy in a High Arctic Glacier.

mBio 2020 11 24;11(6). Epub 2020 Nov 24.

Department of Environmental Science, Aarhus University, Roskilde, Denmark.

Conserving additional energy from sunlight through bacteriochlorophyll (BChl)-based reaction center or proton-pumping rhodopsin is a highly successful life strategy in environmental bacteria. BChl and rhodopsin-based systems display contrasting characteristics in the size of coding operon, cost of biosynthesis, ease of expression control, and efficiency of energy production. This raises an intriguing question of whether a single bacterium has evolved the ability to perform these two types of phototrophy complementarily according to energy needs and environmental conditions. Here, we report four sp. strains () of monophyletic origin isolated from a high Arctic glacier in northeast Greenland (81.566° N, 16.363° W) that are at different evolutionary stages concerning phototrophy. Their >99.8% identical genomes contain footprints of horizontal operon transfer (HOT) of the complete gene clusters encoding BChl- and xanthorhodopsin (XR)-based dual phototrophy. Two strains possess only a complete XR operon, while the other two strains have both a photosynthesis gene cluster and an XR operon in their genomes. All XR operons are heavily surrounded by mobile genetic elements and are located close to a tRNA gene, strongly signaling that a HOT event of the XR operon has occurred recently. Mining public genome databases and our high Arctic glacial and soil metagenomes revealed that phylogenetically diverse bacteria have the metabolic potential of performing BChl- and rhodopsin-based dual phototrophy. Our data provide new insights on how bacteria cope with the harsh and energy-deficient environment in surface glacier, possibly by maximizing the capability of exploiting solar energy. Over the course of evolution for billions of years, bacteria that are capable of light-driven energy production have occupied every corner of surface Earth where sunlight can reach. Only two general biological systems have evolved in bacteria to be capable of net energy conservation via light harvesting: one is based on the pigment of (bacterio-)chlorophyll and the other is based on proton-pumping rhodopsin. There is emerging genomic evidence that these two rather different systems can coexist in a single bacterium to take advantage of their contrasting characteristics in the number of genes involved, biosynthesis cost, ease of expression control, and efficiency of energy production and thus enhance the capability of exploiting solar energy. Our data provide the first clear-cut evidence that such dual phototrophy potentially exists in glacial bacteria. Further public genome mining suggests this understudied dual phototrophic mechanism is possibly more common than our data alone suggested.
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http://dx.doi.org/10.1128/mBio.02641-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7701988PMC
November 2020

Metagenomic analysis of a keratin-degrading bacterial consortium provides insight into the keratinolytic mechanisms.

Sci Total Environ 2021 Mar 29;761:143281. Epub 2020 Oct 29.

Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Keratin is an insoluble fibrous protein from natural environments, which can be recycled to value-added products by keratinolytic microorganisms. A microbial consortium with efficient keratinolytic activity was previously enriched from soil, but the genetic basis behind its remarkable degradation properties was not investigated yet. To identify the metabolic pathways involved in keratinolysis and clarify the observed synergy among community members, shotgun metagenomic sequencing was performed to reconstruct metagenome-assembled genomes. More than 90% genera of the enriched bacterial consortium were affiliated to Chryseobacterium, Stenotrophomonas, and Pseudomonas. Metabolic potential and putative keratinases were predicted from the metagenomic annotation, providing the genetic basis of keratin degradation. Furthermore, metabolic pathways associated with keratinolytic processes such as amino acid metabolism, disulfide reduction and urea cycle were investigated from seven high-quality metagenome-assembled genomes, revealing the potential metabolic cooperation related to keratin degradation. This knowledge deepens the understanding of microbial keratinolytic mechanisms at play in a complex community, pinpointing the significance of synergistic interactions, which could be further used to optimize industrial keratin degradation processes.
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http://dx.doi.org/10.1016/j.scitotenv.2020.143281DOI Listing
March 2021

Bone biodeterioration-The effect of marine and terrestrial depositional environments on early diagenesis and bone bacterial community.

PLoS One 2020 15;15(10):e0240512. Epub 2020 Oct 15.

The GLOBE Institute, University of Copenhagen, København, Denmark.

Bacteria play an important role in the degradation of bone material. However, much remains to be learnt about the structure of their communities in degrading bone, and how the depositional environment influences their diversity throughout the exposure period. We genetically profiled the bacterial community in an experimental series of pig bone fragments (femur and humeri) deposited at different well-defined environments in Denmark. The bacterial community in the bone fragments and surrounding depositional environment were studied over one year, and correlated with the bioerosion damage patterns observed microscopically in the bones. We observed that the bacterial communities within the bones were heavily influenced by the local microbial community, and that the general bone microbial diversity increases with time after exposure. We found the presence of several known collagenase producing bacterial groups, and also observed increases in the relative abundance of several of these in bones with tunneling. We anticipate that future analyses using shotgun metagenomics on this and similar datasets will be able to provide insights into mechanisms of microbiome driven bone degradation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561151PMC
December 2020

Fecal Microbiota Transplantation in the Treatment of Chronic Pouchitis: A Systematic Review.

Microorganisms 2020 Sep 18;8(9). Epub 2020 Sep 18.

Department of Plant and Environmental Sciences, Section for Microbial Ecology and Biotechnology, Copenhagen University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

The objective was to evaluate available literature on treatment of chronic pouchitis with fecal microbiota transplantation (FMT) focusing on clinical outcomes, safety, and different approaches to FMT preparation and delivery. A systematic review of electronic databases was conducted using Medline, EMBASE, and the Cochrane Central Register of Controlled Trials Library from inception through April 2020. Human studies of all study types reporting results of FMT to treat chronic pouchitis were included. Nine studies, reporting FMT treatment of 69 patients with chronic pouchitis were found eligible for the review. Most studies were case series and cohort studies rated as having fair to poor quality due to high risk of bias and small sample size. Only one randomized controlled trial was included, finding no beneficial effect of FMT. In total clinical response after FMT was reported in 14 (31.8%) out of 44 evaluated patients at various timepoints after FMT, and clinical remission in ten (22.7%) patients. Only minor self-limiting adverse events were reported. FMT varied greatly regarding preparation, length of treatment, and route of delivery. The effects of FMT on symptoms of chronic pouchitis are not established, though some studies show promising results. Future controlled well-designed studies are warranted.
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http://dx.doi.org/10.3390/microorganisms8091433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565999PMC
September 2020

[Bacteriophage therapy].

Ugeskr Laeger 2020 06;182(27)

Bacteriophages are viruses, which exclusively infect bacteria. Bacteriophage therapy has a great potential in the treatment of pan- or multidrug resistant bacterial infections as argued in this review, and promising results have been published within recent years. The effects of the treatment are, however, still not fully understood and remain to be clarified. Furthermore, the facilities to produce legally approved bacteriophage products for human treatment must be available before the treatment becomes available to Danish patients.
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June 2020

Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function.

Gigascience 2020 06;9(6)

Department of Biological Sciences, University of New Hampshire, 38 Academic Way, Durham, NH 03824, USA.

Background: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape" scale, and few if any have applied the newest nanopore technology.

Results: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers.

Conclusions: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.
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http://dx.doi.org/10.1093/gigascience/giaa053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285869PMC
June 2020

Isolation and characterisation of novel phages infecting Lactobacillus plantarum and proposal of a new genus, "Silenusvirus".

Sci Rep 2020 05 29;10(1):8763. Epub 2020 May 29.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399C, Roskilde, 4000, Denmark.

Bacteria of Lactobacillus sp. are very useful to humans. However, the biology and genomic diversity of their (bacterio)phage enemies remains understudied. Knowledge on Lactobacillus phage diversity should broaden to develop efficient phage control strategies. To this end, organic waste samples were screened for phages against two wine-related Lactobacillus plantarum strains. Isolates were shotgun sequenced and compared against the phage database and each other by phylogenetics and comparative genomics. The new isolates had only three distant relatives from the database, but displayed a high overall degree of genomic similarity amongst them. The latter allowed for the use of one isolate as a representative to conduct transmission electron microscopy and structural protein sequencing, and to study phage adsorption and growth kinetics. The microscopy and proteomics tests confirmed the observed diversity of the new isolates and supported their classification to the family Siphoviridae and the proposal of the new phage genus "Silenusvirus".
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http://dx.doi.org/10.1038/s41598-020-65366-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260188PMC
May 2020

Faecal virome transplantation decreases symptoms of type 2 diabetes and obesity in a murine model.

Gut 2020 12 12;69(12):2122-2130. Epub 2020 Mar 12.

Food Science, University of Copenhagen, Frederiksberg, Denmark

Objective: Development of obesity and type 2 diabetes (T2D) are associated with gut microbiota (GM) changes. The gut viral community is predominated by bacteriophages (phages), which are viruses that attack bacteria in a host-specific manner. The antagonistic behaviour of phages has the potential to alter the GM. As a proof-of-concept, we demonstrate the efficacy of faecal virome transplantation (FVT) from lean donors for shifting the phenotype of obese mice into closer resemblance of lean mice.

Design: The FVT consisted of viromes with distinct profiles extracted from the caecal content of mice from different vendors that were fed a low-fat (LF) diet for 14 weeks. Male C57BL/6NTac mice were divided into five groups: LF (as diet control), high-fat (HF) diet, HF+ampicillin (Amp), HF+Amp+FVT and HF+FVT. At weeks 6 and 7 of the study, the HF+FVT and HF+Amp+FVT mice were treated with FVT by oral gavage. The Amp groups were treated with Amp 24 hours prior to first FVT treatment.

Results: Six weeks after first FVT, the HF+FVT mice showed a significant decrease in weight gain compared with the HF group. Further, glucose tolerance was comparable between the LF and HF+FVT mice, while the other HF groups all had impaired glucose tolerance. These observations were supported by significant shifts in GM composition, blood plasma metabolome and expression levels of genes associated with obesity and T2D development.

Conclusions: Transfer of caecal viral communities from mice with a lean phenotype into mice with an obese phenotype led to reduced weight gain and normalised blood glucose parameters relative to lean mice. We hypothesise that this effect is mediated via FVT-induced GM changes.
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http://dx.doi.org/10.1136/gutjnl-2019-320005DOI Listing
December 2020

Land use as a driver for protist community structure in soils under agricultural use across Europe.

Sci Total Environ 2020 May 8;717:137228. Epub 2020 Feb 8.

Department of Environmental Science, Aarhus University, Roskilde, Denmark. Electronic address:

Soil biodiversity is threatened by intensification of land use. The consequences of different land use on belowground biodiversity remain insufficiently explored for soil protists. Alongside being abundant and extremely diverse in soil, protists provide many ecosystem services: key players in the microbial loop, turnover of organic matter and stimulation of plant growth-promoting rhizobacteria. However, we lack knowledge of effects of site, land use intensity and management on diversity of soil protists. Here we assessed protist communities in four European arable sites with contrasting land use intensities at each site: Lusignan, France; Moskanjci, Slovenia; Castro Verde, Portugal and Scheyern, Germany as well as two grassland sites: Hainich, Germany and Lancaster, UK. Each site has consistent agricultural management history of low and high land use intensities quantified in terms of land use index (LUI). We employed high-throughput sequencing of environmental DNA, targeting the V4 region of the 18S rRNA gene. By assigning the protist composition to trophic groups, we inspected for effects of management, and other biotic and abiotic variables. While overall protist richness was unaffected by LUI within sites, specific trophic groups such as plant pathogens and saprotrophs were affected. Effects on protist biome across land uses and sites were also observed. LUI sensitive taxa were taxonomically diverse in each plot, and their trophic groups responded in specific patterns to specific practices. The most abundant trophic group was phagotrophs (73%), followed by photoautotrophs (16%), plant pathogens (4%), animal parasites (2%) and saprotrophs (1%). Community compositions and factors affecting the structure of individual trophic groups differed between land uses and management systems. The agricultural management selected for distinct protist populations as well as specific functional traits, and the protist community and diversity were indeed affected by site, LUI and management, which indicates the ecological significance of protists in the soil food web.
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http://dx.doi.org/10.1016/j.scitotenv.2020.137228DOI Listing
May 2020

GC bias affects genomic and metagenomic reconstructions, underrepresenting GC-poor organisms.

Gigascience 2020 02;9(2)

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, 1871, Denmark.

Background: Metagenomic sequencing is a well-established tool in the modern biosciences. While it promises unparalleled insights into the genetic content of the biological samples studied, conclusions drawn are at risk from biases inherent to the DNA sequencing methods, including inaccurate abundance estimates as a function of genomic guanine-cytosine (GC) contents.

Results: We explored such GC biases across many commonly used platforms in experiments sequencing multiple genomes (with mean GC contents ranging from 28.9% to 62.4%) and metagenomes. GC bias profiles varied among different library preparation protocols and sequencing platforms. We found that our workflows using MiSeq and NextSeq were hindered by major GC biases, with problems becoming increasingly severe outside the 45-65% GC range, leading to a falsely low coverage in GC-rich and especially GC-poor sequences, where genomic windows with 30% GC content had >10-fold less coverage than windows close to 50% GC content. We also showed that GC content correlates tightly with coverage biases. The PacBio and HiSeq platforms also evidenced similar profiles of GC biases to each other, which were distinct from those seen in the MiSeq and NextSeq workflows. The Oxford Nanopore workflow was not afflicted by GC bias.

Conclusions: These findings indicate potential sources of difficulty, arising from GC biases, in genome sequencing that could be pre-emptively addressed with methodological optimizations provided that the GC biases inherent to the relevant workflow are understood. Furthermore, it is recommended that a more critical approach be taken in quantitative abundance estimates in metagenomic studies. In the future, metagenomic studies should take steps to account for the effects of GC bias before drawing conclusions, or they should use a demonstrably unbiased workflow.
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http://dx.doi.org/10.1093/gigascience/giaa008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016772PMC
February 2020

Seasonal epiphytic microbial dynamics on grapevine leaves under biocontrol and copper fungicide treatments.

Sci Rep 2020 01 20;10(1):681. Epub 2020 Jan 20.

Environmental Microbial Genomics (EMG), Plant and Environmental Microbiology, Copenhagen University, Copenhagen, DK, Denmark.

Winemakers have long used copper as a conventional fungicide treatment on grapevine and only recently, the use of biocontrol agents depicted a promising alternative. Most of the studies that investigate the impact of fungicide treatments, focus on specific pathogenic microbes. In the present work instead, a field experiment conducted in South Africa shows the seasonal microbial change occurring on grapevine leaves, periodically treated with two different fungicide treatments: copper sulphate and Lactobacillus plantarum MW-1. In this work, NGS data were combined with strain-specific and community qPCRs to reveal the shift of the microbial communities throughout the growing season and highlight the impact of fungicides on the microbiota. Only the family of Lactobacillaceae systematically changed between treatments, while the bacterial community remained relatively stable over time. MW-1 was exclusively detected on biocontrol-sprayed leaves. Conversely, the fungal community was largely shaped by sampling time, underlining the succession of different dominant taxa over the months. Between treatments, only a few fungal taxa appeared to change significantly and the fungal load was also comparable. Monitoring the dynamics of the microbial community under different fungicide treatments may advise the best timing to apply treatments to the plants, toward the realization of more sustainable agricultural practices.
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http://dx.doi.org/10.1038/s41598-019-56741-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971271PMC
January 2020

Microbiota in foods from Inuit traditional hunting.

PLoS One 2020 14;15(1):e0227819. Epub 2020 Jan 14.

Department of Food Science, The University of Copenhagen, Frederiksberg, Denmark.

The foods we eat contain microorganisms that we ingest alongside the food. Industrialized food systems offer great advantages from a safety point of view, but have also been accused of depleting the diversity of the human microbiota with negative implications for human health. In contrast, artisanal traditional foods are potential sources of a diverse food microbiota. Traditional foods of the Greenlandic Inuit are comprised of animal-sourced foods prepared in the natural environment and are often consumed raw. These foods, some of which are on the verge of extinction, have not previously been microbiologically characterized. We mapped the microbiota of foods stemming from traditional Inuit land-based hunting activities. The foods included in the current study are dried muskox and caribou meat, caribou rumen and intestinal content as well as larval parasites from caribou hides, all traditional Inuit foods. This study shows that traditional drying methods are efficient for limiting microbial growth through desiccation. The results also show the rumen content of the caribou to be a highly diverse source of microbes with potential for degradation of plants. Finally, a number of parasites were shown to be included in the biodiversity of the assessed traditional foods. Taken together, the results map out a diverse source of ingested microbes and parasites that originate from the natural environment. These results have implications for understanding the nature-sourced traditional Inuit diet, which is in contrast to current day diet recommendations as well as modern industrialized food systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959823PMC
April 2020

Complete Genome and Plasmid Sequences of Salmonella enterica subsp. Serovar Enteritidis PT1, Obtained from the Reference Laboratory at Public Health England, Colindale, United Kingdom.

Microbiol Resour Announc 2020 Jan 9;9(2). Epub 2020 Jan 9.

Department of Environmental Science, Aarhus University RISØ Campus, Roskilde, Denmark.

Hybrid assembly of Illumina and Oxford Nanopore sequencing was used here to produce the complete circular genome and plasmid sequences of subsp. serovar Enteritidis PT1 (phage type 1). The organism was obtained from the Reference Laboratory at Public Health England, Colindale, UK.
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http://dx.doi.org/10.1128/MRA.01064-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952643PMC
January 2020

Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina.

Syst Appl Microbiol 2020 Jan 22;43(1):126044. Epub 2019 Nov 22.

INTECH (Instituto Tecnológico Chascomús), CCT-La Plata, CONICET - Universidad Nacional de San Martín, Avenida Intendente Marino, Km 8.2, 7130 Chascomús, Argentina. Electronic address:

Three symbiotic nitrogen-fixing bacteria (BD68, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099, M. erdmanii USDA 3471, M. carmichaelinearum ICMP 18942, M. opportunistum WSM 2975 and M. jarvisii ATCC 33699, with sequence identities of 99.72%-100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68 was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68 and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied. Based on these results, BD68, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68 (=CECT 9304=LMG 30179).
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http://dx.doi.org/10.1016/j.syapm.2019.126044DOI Listing
January 2020

A novel hybrid concept for implementation in drinking water treatment targets micropollutant removal by combining membrane filtration with biodegradation.

Sci Total Environ 2019 Dec 1;694:133710. Epub 2019 Aug 1.

Aarhus University, Dept. Environmental Science, Section for Environmental Microbiology & Biotechnology, Roskilde, Denmark; University of Copenhagen, Dept. of Plant- and Environmental Science, Section for Microbial Ecology and Biotechnology, Copenhagen, Denmark. Electronic address:

Groundwater extracted for drinking water production is commonly treated by aeration and sand filtration. However, this simple treatment is typically unable to remove pesticide residues. As a solution, bioaugmentation of sand filter units (i.e., the addition of specific degrader strains) has been proposed as an alternative "green" technology for targeted pesticide removal. However, the introduced degraders are challenged by (i) micropollutant levels of target residue, (ii) the oligotrophic environment and (iii) competition and predation by the native microorganisms, leading to loss of population and degradation potential. To overcome these challenges, we propose the introduction of a novel hybrid treatment step to the overall treatment process in which reverse osmosis filtration and biodegradation are combined to remove a target micropollutant. Here, the reverse osmosis produces a concentrated retentate that will act as a feed to a dedicated biofilter unit, intended to promote biodegradation potential and stability of an introduced degrader. Subsequently, the purified retentate will be re-mixed with the permeate from reverse osmosis, for re-mineralization and downstream consumption. In our study, we investigated the effect of reverse osmosis retentates on the degradation potential of an introduced degrader. This paper provides the first promising results of this hybrid concept using the 2,6-dichlorobenzamide (BAM)-degrading bacteria Aminobacter sp. MSH1 in batch experiments, spiked with radiolabeled BAM. The results showed an increased degradation potential of MSH1 in retentate waters versus untreated water. Colony-forming units and qPCR showed a stable MSH1 population, despite higher concentrations of salts and metals, and increased growth of native bacteria.
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http://dx.doi.org/10.1016/j.scitotenv.2019.133710DOI Listing
December 2019

Fungicides and the Grapevine Wood Mycobiome: A Case Study on Tracheomycotic Ascomycete Reveals Potential for Two Novel Control Strategies.

Front Plant Sci 2019 31;10:1405. Epub 2019 Oct 31.

LEAF-Linking Landscape Environment Agriculture and Food, Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal.

is a tracheomycotic fungus that colonizes the xylem of grapevines ( L.), causing wood discoloration, brown wood streaking, gummosis, and wood necrosis, which negatively affect the overall health, productivity, and life span of vines. Current control strategies to prevent or cope with infections are frequently ineffective. Moreover, it is unclear how fungicides commonly applied in vineyards against downy and powdery mildew agents affect the wood mycobiome, including wood pathogens such as . In this study, we used next-generation sequencing to assess the effects of foliar spray of grapevines with inorganic (copper oxychloride and sulfur), synthetic (penconazole and fosetyl-aluminum), and natural (Blad) fungicides currently used against the downy and powdery mildews. The subjects of our investigation were (i) the resident wood mycobiome, (ii) the early colonization by a consortium of fungal wood endophytes (ACEA1), (iii) the wood colonization success of , and (iv) the interaction between and ACEA1, under greenhouse conditions, in rooted grapevine cuttings of cv. Cabernet Sauvignon. The data obtained suggest that the resident mycobiome is affected by different fungicide treatments. In addition, the early colonization success of the endophytes composing ACEA1 varied in response to fungicides, with relative abundances of some taxa being overrepresented or underrepresented when compared with the control. The wood colonization by comported significant changes in the mycobiome composition, and in addition, it was greatly affected by the foliar spray with Blad, which decreased the relative abundance of this pathogen 12-fold (4.9%) when compared with the control (60.7%) and other treatments. The presence of the pathogen also decreased considerably when co-inoculated into the plant with ACEA1, reaching relative abundances between 13.9% and 2.0%, depending on the fungicide treatment applied. This study shows that fungicides sprayed to prevent infections of powdery and downy mildews have an effect on non-target fungi that colonize the endosphere of grapevines. We suggest two potential control strategies to fight , namely, the foliar spray with Blad and the use of ACEA1. Further studies to confirm these results are required.
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http://dx.doi.org/10.3389/fpls.2019.01405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836639PMC
October 2019

Unravelling plasmidome distribution and interaction with its hosting microbiome.

Environ Microbiol 2020 01 15;22(1):32-44. Epub 2019 Dec 15.

Faculty of Natural Sciences, Department of Life Sciences, The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O.B. 653 Beer-Sheva, Building 41, Room 228, Beer-Sheva, Israel.

Horizontal gene transfer via plasmids plays a pivotal role in microbial evolution. The forces that shape plasmidomes functionality and distribution in natural environments are insufficiently understood. Here, we present a comparative study of plasmidomes across adjacent microbial environments present in different individual rumen microbiomes. Our findings show that the rumen plasmidome displays enormous unknown functional potential currently unannotated in available databases. Nevertheless, this unknown functionality is conserved and shared with published rat gut plasmidome data. Moreover, the rumen plasmidome is highly diverse compared with the microbiome that hosts these plasmids, across both similar and different rumen habitats. Our analysis demonstrates that its structure is shaped more by stochasticity than selection. Nevertheless, the plasmidome is an active partner in its intricate relationship with the host microbiome with both interacting with and responding to their environment.
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http://dx.doi.org/10.1111/1462-2920.14813DOI Listing
January 2020

The Mobilome: Metagenomic Analysis of Circular Plasmids, Viruses, and Other Extrachromosomal Elements.

Methods Mol Biol 2020 ;2075:253-264

Department of Environmental Science, Aarhus University, Roskilde, Denmark.

Isolation, sequencing, and analysis of circular genetic elements bring new insights to mobile genetic elements related to microbial ecology. One method used to study circular plasmids, viruses, and other elements is called the mobilome method. The mobilome method presented here is an unamplified mobilome approach allowing fast isolation of circular DNA elements from a variety of samples followed by directly building unamplified Illumina-compatible sequencing libraries using enzymatic tagging and fragmentation. Several methods for bioinformatic analysis of mobilome data are also suggested.
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http://dx.doi.org/10.1007/978-1-4939-9877-7_18DOI Listing
January 2021

Complete Genome Sequences of Highly Arsenite-Resistant Bacteria sp. Strain CS2 and Micrococcus luteus AS2.

Microbiol Resour Announc 2019 Aug 1;8(31). Epub 2019 Aug 1.

Department of Environmental Science, Aarhus University, Roskilde, Denmark.

The complete genome sequences of two highly arsenite-resistant isolates are presented. Both genomes are G+C rich and consist of a single chromosome containing homologs of known arsenite resistance genes.
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http://dx.doi.org/10.1128/MRA.00531-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675986PMC
August 2019

Characterization of the Wood Mycobiome of in a Vineyard Affected by Esca. Spatial Distribution of Fungal Communities and Their Putative Relation With Leaf Symptoms.

Front Plant Sci 2019 12;10:910. Epub 2019 Jul 12.

LEAF - Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal.

Esca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (, , , , and ) and numerous hitherto unreported species. Esca-associated fungi and sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., spp., ). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting 'tiger stripes' symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of esca.
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http://dx.doi.org/10.3389/fpls.2019.00910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640213PMC
July 2019

Expanding the Diversity of Phages Infecting -A Novel Lineage of Phages Comprising Five New Members.

Viruses 2019 07 4;11(7). Epub 2019 Jul 4.

Department of Environmental Science, Aarhus University, Frederiksborgvej, 399, 4000 Roskilde, Denmark.

is a bacterium with probiotic properties and promising applications in the food industry and agriculture. So far, bacteriophages of this bacterium have been moderately addressed. We examined the diversity of five new phages via whole genome shotgun sequencing and in silico protein predictions. Moreover, we looked into their phylogeny and their potential genomic similarities to other complete phage genome records through extensive nucleotide and protein comparisons. These analyses revealed a high degree of similarity among the five phages, which extended to the vast majority of predicted virion-associated proteins. Based on these, we selected one of the phages as a representative and performed transmission electron microscopy and structural protein sequencing tests. Overall, the results suggested that the five phages belong to the family , they have a long genome of 137,973-141,344 bp, a G/C content of 36.3-36.6% that is quite distinct from their host's, and surprisingly, 7 to 15 tRNAs. Only an average 41/174 of their predicted genes were assigned a function. The comparative analyses unraveled considerable genetic diversity for the five phages in this study. Hence, the new genus "Semelevirus" was proposed, comprising exclusively of the five phages. This novel lineage of phages provides further insight into the genetic heterogeneity of phages infecting sp. The five new phages have potential value for the development of more robust starters through, for example, the selection of mutants insensitive to phage infections. The five phages could also form part of phage cocktails, which producers would apply in different stages of fermentations in order to create a range of organoleptic outputs.
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http://dx.doi.org/10.3390/v11070611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669764PMC
July 2019