Biology subjects

Nielsen, D. S.

Publications and source records attributed to Nielsen, D. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Ultra-High Multiplexing Enables Near-Full-Length 16S rRNA Gene Amplicon Sequencing of Over 1,200 Gut Microbiome Samples on a Single Nanopore Flow Cell

Next-generation sequencing (NGS) of the prokaryotic 16S rRNA gene revolutionized gut microbiome research two decades ago. However, short read lengths remain an inherent limitation of platforms such as the widely used Illumina platforms (2 x 150-300 bp). Recent advances in Oxford Nanopore Technologies (ONT) flow cell chemistry (R10.4.1) have substantially improved sequencing accuracy. Combined with a custom multiple-primer strategy that comprehensively targets 16S rRNA gene variants to generate near-full-length amplicons, this approach enables read-by-read taxonomic classification, a feature not feasible with short-read sequencing platforms. Although our multiple-primer strategy could enable parallel sequencing of more than 18,000 samples (192 x 96), current flow cell capacity offers sufficient sequencing depth for approximately 1,000-1,500 samples. To validate the scalability and our per-read classification pipeline, we show that more than a thousand human fecal microbiome samples spiked with two bacterial strains (Imtechella halotolerans and Allobacillus halotolerans), not otherwise present in human fecal samples, can be successfully sequenced on a single flow cell, achieving a per-molecule error rate sufficient for direct per-read classification and at an adequate read depth for downstream analysis. This level of scalability significantly reduces per-sample costs, making the approach more accessible to a broader research community. To embrace these advancements, we have developed RubyRed, a pipeline that processes raw sequencing data and assigns taxonomic classifications on a per-read basis. Using spike-in references (I. halotolerans and A. halotolerans), we demonstrate high mean single-read sequencing accuracy (99% and 98.9%, respectively), with the majority of reads exceeding the canonical threshold required for species-level taxonomic classification based on the 16S rRNA gene.

microbiology↗

bioRxiv2025

A chemically defined medium to support the growth of food-relevant Bacillus species

The Bacillus genus contains many members with food significance, including the food-grade Bacillus subtilis clade often used in fermentations and the pathogenic Bacillus cereus clade. Chemically defined media for Bacillus species are crucial tools to allow detailed investigations of the influence of specific nutrients on growth and also improve reproducibility and consistency of experiments. Previous studies have focused on the development of defined media for single species, while the aim of this study was to develop a chemically defined medium that supports the growth of multiple food relevant Bacillus species. The new medium, Pafoba, was tested using two pathogenic strains of the Bacillus cereus clade and eleven strains of the Bacillus subtilis clade representing seven different clade members. All thirteen Bacillus strains were able to grow on Pafoba, of which ten displayed a similar or higher maximum OD600 on Pafoba medium compared to rich medium (Brain Heart Infusion broth). Detailed analysis revealed a biotin requirement for Bacillus subtilis strain PRO64, and the necessity of including essential amino acids for Bacillus weihenstephanensis and Bacillus cereus strains. In conclusion, the chemically defined Pafoba medium provides a controlled and reproducible growth environment for fundamental studies and is suitable for detection and enumeration of a broad range of Bacillus spp. related to food processing and safety. ImportanceBacillus species are important in both food fermentation and food safety. While members of the Bacillus subtilis clade are used in the production of fermented foods, those in the Bacillus cereus group are associated with foodborne illness. This study presents a chemically defined medium that supports the growth of multiple food-relevant Bacillus species, enabling precise control over nutrient composition. Knowledge of Bacillus cereus metabolism under defined conditions is essential to support efforts in food safety, risk assessment, and the development of targeted intervention strategies. Likewise, an understanding of Bacillus subtilis metabolism under defined conditions is essential to optimize fermentation processes, improve product consistency, and enhance functional food development. By providing a standardized and reproducible growth environment, the medium developed in this study will facilitate research that advances both microbial food safety and the controlled use of beneficial Bacillus strains in food production.

microbiology↗

bioRxiv2025

Stool Dynamics and the Developing Gut Microbiome During Infancy

The infant gut microbiome is a dynamic ecosystem that plays a crucial role in early development, influences immune system maturation, and overall health. Recent insights reveal that the gut microbiota undergoes changes across the 24-h day, raising the possibility that it may act as "zeitgeber", helping to regulate the hosts sleep-wake organisation. Despite its importance, timing factors influencing microbiome composition are poorly understood, limiting its use as a health predictor. This study investigates the relationship between stool dynamics (interval since last defecation, collection time of the sample), sleep pressure (interval since last sleep), meal timing and the composition of the gut microbiome. Stool samples from 198 healthy infants, aged 3 to 31 months, were analysed to assess microbial diversity, richness evenness, and abundance. Our findings reveal that longer intervals since the last bowel movement are associated with increased microbial diversity, evenness, and richness. Stool timing is associated with shifts in microbial composition, especially in younger infants, indicating that diurnal microbial fluctuations may become more stable as infants mature. We found that longer periods of wakefulness were associated with increased microbial diversity in early infancy, although this effect appeared to diminish with age. Feeding schedules appeared to have a limited effect on the gut microbiome. Longer fasting before sampling showed no significant associations with most microbial parameters, except for a positive association with microbial richness. At the phylum level, results indicate that infant gut microbial composition is influenced by behaviour and physiology. Longer intervals between bowel movements were associated with shifts in bacterial abundance, with Proteobacteria decreasing and Actinobacteria increasing. Stool timing and meal timing also affected microbial diversity: later sampling times showed higher Actinobacteria levels, and longer fasting was associated with reduced Bacteroidetes. Sleep pressure showed a trend effect with Firmicutes displaying a slight decrease in infants who had been awake longer. Our findings underscore the importance of time-based factors on infant gut microbiome composition.

systems biology↗

bioRxiv2025

Characterization of a virulent bacteriophage consortium targeting Enterobacteriaceae from inflamed preterm gut mucosa

Preterm infants have a high risk of intestinal inflammation which can progress to necrotizing enterocolitis (NEC). The gut microbial colonization commencing at birth is essential for proper intestinal maturation, but this process is often disrupted in preterm infants, leading to dysbiosis and increased risk of developing NEC. Bacteriophages (phages), viruses that specifically infect bacteria, are an important constituent of the gut microbiome and protects the gut epithelium against invading bacteria. This study aimed to isolate and characterize phages for use as a preventive measure against NEC-associated bacteria. We initially cultured Enterobacteriaceae from ileal mucosa of preterm piglets that exhibited severe NEC-like pathology. We then screened 23 donor fecal samples for inhibition of bacterial growth and isolated a collection of unique phages to use further. The phages were characterized by whole genome sequencing, host receptor binding determination, and immune cell activation in vitro. The final phage collection consisted of ten virulent phages within five genera, representing myovirus, podovirus and siphovirus morphologies. All phages in the collection induced expression of both pro-and anti-inflammatory genes in co-culture with macrophage-like THP-1 cells, but to different extents than Escherichia coli. Ultimately, we selected one phage for a high-dose oral administration to newborn piglets and assessed its infectivity and presence in different gut segments. This intervention did not result in any direct side effects, while both infective phages and signatures of phage DNA were detected in the intestinal content and mucosa. Having characterized a set of rationally selected virulent phages, we support the advancement of phage therapy as a potential protection against NEC.

microbiology↗

bioRxiv2025

Schistosoma mansoni-induced host immunopathology is gut microbiota dependent

Despite progress in schistosomiasis control during recent decades in endemic areas, this parasitic blood-fluke infection continues to pose a substantial public health burden. In depth understanding of the mechanisms behind the complex immunopathology and drivers of morbidity in schistosomiasis is urgently needed. Chronic infection with the parasitic blood fluke Schistosoma mansoni manifests the most severe pathology in relation to egg-induced host immune responses and fibrosis development. Host immune homeostasis is influenced by the commensal gut microbiota, which may therefore also affect systemic immunopathology induced by S. mansoni. Both humans and experimental animal models show metabolomic changes related to gut microbial and liver metabolism during S. mansoni infection, further supporting a link between gut microbiota and regulation of S. mansoni pathology. To investigate whether a radically changed gut microbiota composition would result in changed S. mansoni egg-induced pathology, a mouse model combining antibiotic treatment and infection was established. In the model, the commensal gut microbial composition of female C57BL/6-NTac mice was altered by oral administration of a broad-spectrum ampicillin-vancomycin cocktail prior to infection with S. mansoni and throughout the experiment. An ecosystem view of the host immune milieu was collated by measuring a wide range of parameters: gut microbiome characterisation, immune profiling on the transcriptional level (ileum and liver) supported by flow cytometry of cells (spleen and mesenteric lymph nodes) and bead-based measurements of immune parameters in sera, short-chain fatty acid measurements in sera and caecal content, intestinal permeability assessment, liver enzyme levels in sera, and histology and stereology to characterise immunopathology (ileum and liver). The application of stereological principles to address the non-spherical nature of egg-induced liver granulomas and ileum inflammation supported by liver enzyme levels, collagen deposition, and immune parameters, enabled demonstration of significantly less granuloma formation in livers from antibiotics treated mice compared to controls. The observed difference in the degree of egg-induced inflammatory response in livers from infected mice mediated by antibiotics treatment, was not observed in ileum tissue and could not be explained by infection burden. Our mouse model results demonstrate a role for the gut microbiota and intestinal immune milieu in regulation of systemic S. mansoni infection-driven liver pathology and, hence, potential morbidity. In translational context, these findings warrant that drivers of gut microbial changes and intestinal immune milieu must be considered when pursuing novel holistic interventions for treatment or alleviation of persistent schistosomiasis.

immunology↗

bioRxiv2025

Rhythms of Early Life: Gut Microbiota Rhythmicity and Circadian Maturation in infants

BackgroundThe human gut microbiota undergoes daily fluctuations, yet its interaction with sleep-wake patterns during infancy remains largely uncharted. This study aims to elucidate the relationship between gut microbiota rhythmicity and the development of sleep patterns in infants over the first year of life. We continuously monitored 162 healthy infants across multiple days at 3, 6, and 12 months of age using ankle actigraphy and 24-hour diaries. The Circadian Function Index (CFI) was computed as a proxy for sleep-wake rhythm maturation. Stool samples were collected to profile gut microbiota taxa composition via 16S rRNA gene amplicon sequencing, and microbial oscillations were assessed through sine and cosine fitting to detect 24-hour patterns. ResultsOur findings revealed that the relative abundance of bacterial taxa exhibited rhythmic patterns, with 26 zOTUs (1.74%) following a sine pattern and 100 zOTUs (6.69%) displaying cosine rhythmicity. Cosine rhythmicity became more pronounced with age, showing strong maturation: 7 zOTUs at 3 months, 2 zOTUs at 6 months, and 86 zOTUs at 12 months. Notably, 105 zOTUs (7.02%) were associated with CFI, demonstrating a significant relationship between gut microbiota rhythms and sleep development. Among these, 27 zOTUs with sine dynamics and 96 zOTUs with cosine dynamics were linked to CFI, with this association strengthening as infants aged. ConclusionsThese results highlight the increasing synchronization between gut microbiota rhythmicity and sleep-wake cycles during infancy, pointing to a critical window for potential health interventions. This novel observation, previously reported in rodents and adults, underscores the role of gut microbiota in early human development, offering new avenues for enhancing developmental outcomes through targeted interventions.

developmental biology↗

bioRxiv2024

Chemostat culturing reduces fecal eukaryotic virus load and delays diarrhea after virome transplantation

Fecal virome transfer (FVT) shows promise in reducing necrotizing enterocolitis (NEC), likely due to donor bacteriophages preventing the gut dysbiosis preceding disease. However, concurrent transfer of eukaryotic viruses may carry a risk of infection for the recipient. To increase safety, we investigated chemostat propagation as a method to eliminate eukaryotic viruses from donor feces while maintaining a diverse and reproducible bacteriophage community. Donor feces was collected from healthy suckling piglets and inoculated into a fermenter containing growth media supplemented with lactose and milk oligosaccharides (MOs). During continuous medium exchange (20% volume/h), dilution significantly reduced eukaryotic viruses. Viral richness was concurrently reduced although still preserving a stable community of 200-250 bacteriophages. Inclusion of MOs in the medium ensured higher bacterial richness and a bacterial community closer resembling donor feces. Fecal Lactobacillaceae bacteria were lost during cultivation but partially replaced by members of the Bacteroidota phylum in MO-supplemented cultures, accompanied by phages predicted to have Parabacteroides as host. After cultivation, virus-like particles (VLPs) were isolated, and their ability to reduce NEC incidence tested in vivo. Preterm piglets were delivered by cesarean section and received either the lactose- or MO-propagated viromes by oral route (n = 14-15/group). These were compared with groups receiving the same dose of donor fecal virome (1010 VLPs/kg) or vehicle control. The piglets were subsequently fed infant formula for 96 hours followed by euthanasia and tissue sampling. Both chemostat-propagated viromes effectively mitigated diarrhea compared to the donor virome. The donor virome partially engrafted in recipients and led to higher levels of Lactobacillaceae bacteria and Lactobacillaceae targeting phages. However, these signatures were lost in recipients of chemostat-propagated viromes, and only minor microbiome effects and no NEC prevention were observed. To conclude, we provide in vivo proof-of-concept for chemostat propagation of fecal viruses as a means to deplete eukaryotic viruses and in turn reduce side effects in newborn virome recipients. However, chemostat culture conditions need further optimization to preserve the donor phageome.

microbiology↗

bioRxiv2024

Protection against necrotizing enterocolitis by fecal filtrate transfer requires an active donor virome

Necrotizing enterocolitis (NEC) remains a frequent catastrophic disease in preterm infants, but fecal filtrate transfer (FFT) has been identified as a promising prophylactic therapy in preclinical studies. This study examined the importance of the FFT virome viability on gut colonization and NEC occurrence. We established an ultraviolet irradiation-based viral inactivation protocol and demonstrated total loss of infectivity of a viral mock community. Using this protocol, we inactivated an aliquot of sterile-filtered donor feces and compared the response in preterm piglets subjected to experimental NEC induction. Gut pathology and barrier properties were assessed, and bacterial and viral compositions were determined by 16S rRNA amplicon and viral metagenomics sequencing, respectively. Native FFT decreased NEC severity and proinflammatory cytokines, but inactivated FFT (iFFT) completely abolished these effects. Mild side effects in the form of diarrhea manifested earlier in recipients of native FFT than iFFT or controls. A distinct gut colonization pattern of increased viral heterogeneity increased bacterial homogeneity and reduction in pathobionts like Clostridium perfringens and Escherichia was observed in the group receiving native FFT, but not in the iFFT group. The present study uncovered a clear distinction between active and inactivated transferred viromes in the ability to modulate gut colonization after preterm birth and decrease NEC. FFT efficacy is potentially driven by active bacteriophages targeting pathogenic bacteria.

microbiology↗

bioRxiv2024

The effect of microbial metabolites from colonic protein fermentation on bacteria-induced cytokine production in dendritic cells

ScopeCompared to the well-defined immune-modulating effect of butyrate, the knowledge of other metabolites from colonic protein fermentation is limited. Methods and resultsThe effect of protein-derived metabolites (valerate, branched-chain fatty acids, ammonium, phenol, p-Cresol, indole, and H2S) on cytokine production in murine bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, Lactobacillus acidophilus NCFM, or Staphylococcus aureus USA300 was investigated. The metabolites modulated the cytokine profile differently and depended on the specific microbial stimulus with short-chain fatty acids (SCFAs) exhibiting the strongest effects and no toxicity. After short-term treatment, SCFAs affected the cytokine profile similar to but weaker than butyrate, reflected by inhibition of IL-12p70 and IL-10 but enhanced IL-23 (LPS and S. aureus USA300) and IL-1{beta} production. Compared to valerate, butyrate exhibited a stronger and more prompt effect on cytokine gene expression without influencing reactive oxygen species (ROS) formation. Oppositely, long-term treatment with the two SCFAs resulted in similar anti-inflammatory effects, i.e. abrogation of LPS-induced IL-12 and enhancement of IL-10 and the expression of aryl hydrocarbon receptor (Ahr) and LPS-stimulated dual specificity phosphatase 1 (Dusp1). ConclusionOur data reveals immune-modulating effects of various protein fermentation metabolites, and valerate in specific holds activities resembling but not identical to butyrate.

immunology↗

bioRxiv2024

A single strand-based library preparation method for unbiased virome characterization

The gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent limitations and biases associated with current sequencing library preparation methods. To overcome these problems, we repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA+Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches.

microbiology↗

bioRxiv2024

Gut microbial-mediated polyphenol metabolism is restrained by parasitic whipworm infection and associated with altered immune function in mice

Polyphenols are phytochemicals commonly found in plant-based diets which have demonstrated immunomodulatory and anti-inflammatory properties. However, the interplay between polyphenols and pathogens at mucosal barrier surfaces has not yet been elucidated in detail. Here, we show that proanthocyanidin (PAC) polyphenols interact with gut parasites to influence immune function and gut microbial-derived metabolites in mice. PAC intake inhibited mastocytosis during infection with the small intestinal roundworm Heligmosomoides polygyrus, and induced a type-1, interferon-driven mucosal immune response during infection with the large intestinal whipworm Trichuris muris. PAC also induced alterations in mesenteric lymph node T-cell populations that were dependent on infection model, with a Th2/Treg bias during H. polygyrus infection, and a Th1 bias during T. muris infection. In the absence of infection, PAC intake promoted the expansion of Turicibacter sp. within the gut microbiota, increased faecal short chain fatty acids, and enriched phenolic metabolites such as phenyl-{gamma}-valerolactones in the caecum. However, these putatively beneficial effects were reduced in PAC-fed mice infected with T. muris, suggesting concomitant parasite infection can attenuate gut microbial-mediated PAC catabolism. Collectively, our results suggest an inter-relationship between a phytonutrient and infection, whereby PAC may augment parasite-induced inflammation (most prominently with the caecum dwelling T. muris), and infection may abrogate the beneficial effects of health-promoting phytochemicals.

microbiology↗

bioRxiv2023

De novo clustering of long-read amplicons improves phylogenetic insight into microbiome data

Long-read amplicon profiling through read classification limits phylogenetic analysis of amplicons while community analysis of multicopy genes, relying on unique molecular identifier (UMI) corrections, often demands deep sequencing. To address this, we present a long amplicon consensus analysis (LACA) workflow employing multiple de novo clustering approaches based on sequence dissimilarity. LACA controls the average error rate of corrected sequences below 1% for the Oxford Nanopore Technologies (ONT) R9.4.1 and ONT R10.3 data, 0.2% for ONT R10.4.1, and 0.1% for high-accuracy ONT Duplex and Pacific Biosciences (PacBio) circular consensus sequencing (CCS) data in both simulated 16S rRNA and real 16-23S rRNA amplicon datasets. In high-accuracy PacBio CCS data, the clustering-based correction matched UMI correction, while outperforming 4xUMI correction in noisy ONT R10.3 and R9.4.1 data. Notably, LACA preserved phylogenetic fidelity in long operational taxonomic units and enhanced microbiome-wide phenotype characterization for synthetic mock communities and human vaginal samples.

bioinformatics↗

bioRxiv2023

Choice of Ultrafilter affects Recovery Rate of Bacteriophages

Studies into the viral fraction of complex microbial communities like in the mammalian gut have recently garnered much interest. Yet there is still no standardized protocol for extracting viruses from such samples, and the protocols that exist employ procedures that skew the viral community of the sample one way or another. The first step of the extraction pipeline often consists of basic filtering of macromolecules and bacteria, yet even this affects the viruses in a strain-specific manner. In this study we investigate a protocol for viral extraction based on ultrafiltration and6 how the choice of ultrafilter might influence the viral community. Clinical samples (feces, vaginal7 swabs, and tracheal suction samples) were spiked with a mock community of known phages (T4,8 c2, {Phi}6, {Phi}29, {Phi}x174, and {Phi}2972), filtered, and quantified by spot and plaque assays to estimate the9 loss in recovery. Especially the enveloped {Phi}6 phage is severely affected by choice of filter, but also10 tailed phages such as T4 and c2 have a reduced infectivity after ultrafiltration. We conclude that11 the pore size of ultrafilters may affect the recovery of phages in a strain- and sample dependent12 manner, suggesting the need for greater thought when selecting filters for virus extraction.

microbiology↗

bioRxiv2023

The effect of colonic pH on microbial activity and metabolite production using common prebiotics as substrates: an in vitro study

The interplay between gut microbiota (GM) and host via degradation of dietary components leading to the production of metabolites such as short-chain fatty acids (SCFAs) is affected by a range of factors including colonic pH and carbohydrate source. Here we investigate how differences in colonic pH influence GM composition and metabolite production using different substrates including inulin, lactose, Galactooligosaccharides (GOS), and Fructooligosaccharide (FOS) in an in vitro colon setup. We investigated 3 different pH regimes (low, 5.2 increasing to 6.4; medium, 5.6 increasing to 6.8 and high, 6.0 increasing to 7.2) and found that Bacteroides spp. decreased but Bifidobacterium spp. became abundant under low pH regimes, suggesting complex interactions of the bacterial community in the face of pH fluctuations in the colon. The butyrate producers Butyricimonas and Christensenella were enriched at higher pH conditions, where also butyrate production was increased using inulin as substrate. The relative abundance of Phascolarctobacterium, Bacteroides, and Rikenellaceae was also increased at higher colonic pH, which was accompanied by increased production of propionate using GOS and FOS as substrate. The gastrointestinal factors are linked in a complex network, where microbial activity leads to the production of SCFAs and other compounds that influence pH, which in turn seems to influence microbial activity. Taken together, our results show that dynamic changes in colonic pH under in vitro simulated conditions have a strong effect on gut microbial activity with SCFA production being higher at colonic pH conditions close to neutral.

microbiology↗

bioRxiv2023

A reproducible enteric phage community improves blood glucose regulation in an obesity mouse model

Metabolic syndrome encompasses amongst other conditions like obesity, type-2 diabetes, and metabolic dysfunction associated fatty liver disease (MAFLD), which are all associated with gut microbiome (GM) dysbiosis. Fecal microbiota transplantation (FMT) has been explored to treat metabolic syndrome by restoring the GM. FMT is generally safe, but motivated by case reports, accidental transfer of pathogenic bacteria remains a concern. As a safer alternative, fecal virome transplantation (FVT, sterile-filtrated feces) has the advantage over FMT in that mainly bacteriophages are transferred and FVT from lean male donors has shown promise in alleviating the metabolic effects of a high-fat diet in a preclinical mouse study. However, FVT still carries the risk of eukaryotic viral infections. To address this, we here apply recently developed modification methodologies to inactivate or remove the eukaryotic viral component of FVT while maintaining an active enteric bacteriophage community. Modified FVTs were compared with unmodified FVT and saline in an animal model of diet-induced obesity using male C57BL/6N mice. In contrast to the obese control group, mice administered a modified FVT, nearly depleted from eukaryotic viruses (0.1%), exhibited enhanced blood glucose clearance, although without a concurrent reduction in weight gain. The unmodified FVT improved liver pathology and reduced the proportions of immune cells in the adipose tissue with a non-uniform response. GM analysis suggested that bacteriophage-mediated GM modulation had influenced these outcomes. When optimized, this may pave the way for developing safe bacteriophage-based therapies targeting metabolic syndrome through GM restoration.

microbiology↗

bioRxiv2023

Development of safe and effective bacteriophage-mediated therapies against C. difficile infections a proof-of-concept preclinical study

BackgroundFecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinders their wider clinical application in treating less acute diseases. MethodsTo overcome these challenges, we developed methods to broaden FVTs clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: 1) Chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), 2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and 3) pyronin-Y treatment to inhibit RNA-virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. ResultsFVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to the FMT, FVT-PyT, and saline control (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and toxin A/B levels. There was a potential elimination of C. difficile colonization, with 7 out of 8 mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. ConclusionsThis proof-of-concept study show that specific modifications to FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat-propagation emerging as promising approaches.

microbiology↗

bioRxiv2023

Reproducible chemostat cultures to eliminate eukaryotic viruses from fecal transplant material

The effect of fecal microbiota transplantation (FMT) on various gut-related diseases is intensively investigated in clinical trials. In addition to bacteria, the gut microbiome also contains eukaryotic, archaeal, and bacterial viruses (bacteriophages, in short phages), which collectively is referred to as the gut virome. Application of FMT in clinical settings is associated with a potential risk for the recipient of transferring infectious eukaryotic viruses or bacteria, despite strict screening procedures for the donor material. A safer and more targeted method to modulate the gut microbiota is therefore needed to extend the application width of FMT. Emerging evidence suggests that gut phages play a key role in maintaining a balanced gut microbiome as well as in FMT efficacy. Thus, a phageome from a cultured fecal donor microbiome may be a more efficient alternative to modulate the gut bacteriome than FMT. Here, we analyzed the dynamic changes of the viromes of mice cecal and human fecal matter inoculated chemostat cultures. Sequencing results showed that the relative abundance of eukaryotic viruses remarkably decreased during continuous cultivation, likely due to the lack of eukaryotic hosts. The corresponding phageome profiles showed dilution rate dependency, a reproducibility between biological replicates, and maintained high diversity of phages although being different from the inoculum phageome. This proof-of-concept study may constitute the first step of developing therapeutic tools to target a broad spectrum of gut-related diseases and thereby replacing FMT with a safer phage-mediated therapy.

microbiology↗

bioRxiv2023

The influence of early life exposures on the infant gut virome

Large cohort studies have contributed significantly to our understanding of the factors that influence the development of the bacterial component of the gut microbiome (GM) during the first years of life. However, the factors that shape the colonization by other important GM members such as the viral fraction remain more elusive. Most gut viruses are bacteriophages (phages), i.e., viruses attacking bacteria in a host specific manner, and to a lesser extent, but also widely present, eukaryotic viruses, including viruses attacking human cells. Here, we utilize the deeply phenotyped COPSAC2010 birth cohort consisting of 700 infants to investigate how social, pre-, peri- and postnatal factors may influence the gut virome composition at one year of age, where fecal virome data was available from 645 infants. Among the different exposures studied, having older siblings and living in an urban vs. rural area had the strongest impact on gut virome composition. Differential abundance analysis from a total of 16,118 viral operational taxonomic units (vOTUs) (mainly phages, but also 6.1% eukaryotic viruses) identified 2,105 vOTUs varying with environmental exposures, of which 5.9% were eukaryotic viruses and the rest was phages. Bacterial hosts for these phages were mainly predicted to be within the Bacteroidaceae, Prevotellaceae, and Ruminococcaceae families, as determined by CRISPR spacer matches. Spearman correlation coefficients indicated strong co-abundance trends of vOTUs and their targeted bacterial host, which underlined the predicted phage-host connections. Further, our findings show that some gut viruses encode important metabolic functions and how the abundance of genes encoding these functions is influenced by environmental exposures. Genes that were significantly associated with early life exposures were found in a total of 42 vOTUs. 18 of these vOTUs had their life styles predicted, with 17 of them having a temperate lifestyle. These 42 vOTUs carried genes coding for enzymes involved in alanine, aspartate and glutamate metabolism, glycolysis-gluconeogenesis, as well as fatty acid biosynthesis. The latter implies that these phages could be involved in the utilization and degradation of major dietary components and affect infant health by influencing the metabolic capacity of their bacterial host. Given the importance of the GM in early life for maturation of the immune system and maintenance of metabolic health, these findings provide a valuable source of information for understanding early life factors that predispose for autoimmune and metabolic disorders.

microbiology↗