Biology subjects

Rasmussen, T. S.

Publications and source records attributed to Rasmussen, T. S..

11 recordsLinked to original sources

bioRxiv2026

Fecal virome addition promotes biofilm biomass and produces distinct bacterial responses in biofilm and planktonic communities in an in vitro colon model

The gut microbiota is spatially organized, but microorganisms at the intestinal mucus surface remain understudied in virome microbiota research. Here, we investigated how fecal virome addition affects biofilm biomass and bacterial composition in surface-attached and planktonic communities using a biofilm-adapted in vitro colon model, CoMiniGut. Fecal samples were collected from three healthy donors at two time points approximately one year apart. Fecal communities from both collection time points were fermented for 48 h in CoMiniGut reactors containing mucin-coated glass beads. Each community was exposed separately to virome fractions from all three donors, testing autologous and heterologous combinations alongside matched microbiome-only controls. Two parallel CoMiniGut systems provided two reactor replicates for each donor treatment combination at each collection time point. Biofilm biomass was quantified by crystal violet staining, and biofilm-associated and planktonic communities from the same reactors were characterized by 16S rRNA gene sequencing. Across 36 reactor-level observations representing combinations of bacterial donor, virome donor, fecal collection time point, and CoMiniGut system, biomass was classified as increased in 18, neutral in 11, and decreased in 7. Each observation represented the mean of 5 - 6 beads normalized to the matched microbiome-only control. Among non-neutral observations, increases were more frequent than decreases (18 versus 7; two-sided exact binomial test, P = 0.043), whereas the magnitude of the relative change did not differ significantly between Increase and Decrease observations (P = 0.220). Virome addition also explained a small but significant proportion of variation in bacterial community composition (R2 = 0.024, P = 0.029), without substantially altering alpha diversity. No interaction between virome treatment and growth mode was detected for the overall community-level response, while separate differential-abundance analyses identified different sets of virome-associated taxa in biofilm-associated and planktonic samples. Thus, fecal virome addition was associated with a directional bias towards increased biofilm biomass and with distinct taxonomic patterns in biofilm-associated and planktonic communities, without extensive community restructuring.

microbiology↗

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↗

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

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↗

bioRxiv2022

Fecal virome transfer improves proliferation of commensal gut Akkermansia muciniphila and unexpectedly enhances the fertility rate in laboratory mice

Probiotics have been suggested as nutritional supplements to improve gastrointestinal health. However, the probiotics marketed today only colonize the densely populated gut to a limited extent. Bacteriophages comprise the majority of viruses in the human gut virome and there are strong indications that they play important roles in shaping the gut microbiome (GM). Here we investigate the use of fecal virome transplantation (FVT) as a mean to alter GM composition to lead the way for persistent colonization of two types of probiotics: Lacticaseibacillus rhamnosus GG (LGG) representing a well-established probiotic and Akkermansia muciniphila (AKM) representing a putative next-generation probiotic. Male and female C57BL/6NTac mice were cohoused in pairs at 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT originated from donor mice with high relative abundance of A. muciniphila. All animals were terminated at age 9 weeks. The FVT treatment did not increase the relative abundance of the administered LGG or AKM in the recipient mice. Instead FVT significantly (p<0.05) increased the abundance of naturally occurring A. muciniphila compared to the control. This highlights the potential of stimulating the commensal "probiotics" that already are permanent members of the gut. Being co-housed male and female, a fraction of the female mice became pregnant. Unexpectedly, the FVT treated mice were found to have a significantly (p<0.05) higher fertility rate independent of probiotic administration. These preliminary observations urge for follow-up studies investigating GM/fertility interactions.

microbiology↗

bioRxiv2022

CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice

Many prokaryotes harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, very few of these systems have been experimentally confirmed to be active in gut bacteria. Here, we experimentally demonstrate that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage-assays in vitro but also in vivo using gnotobiotic (GB) mice. An increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Interestingly, much less new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.

microbiology↗

bioRxiv2019

Sporaefaciens musculi gen. nov., sp. nov., a novel bacterium isolated from the caecum of an obese mouse

A bacterial strain, designated WCA-9-b2, was isolated from the caecal content of an 18-week-old obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate is rod-shaped, Gram-positive, strictly anaerobic, spore-forming and non-motile under the conditions tested. Bacterial colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene indicated that the isolate belonged to the family Lachnospiraceae with Clostridium scindens ATTC 35704 (94.9% sequence identity) and Dorea formicigenerans ATCC 27755 (94.8%) being the closest relatives. Whole genome sequencing showed average nucleotide identity (ANI) ranging from 69.80-74.23% and percentage of conserved proteins (POCP) values < 50% against the nine closest relatives. The genome-based G+C content of genomic DNA was 44.4%. The predominant metabolic end products of glucose fermentation were acetate and succinate. Based on these data, we propose that strain WCA-9-b2 represents a novel species within a novel genus, for which the name Sporaefaciens musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T = CCUG pending IDT). RepositoriesThe GenBank accession number for the 16S rRNA gene sequence of strain WCA-9-b2T is MN756014, and the accession number for the genome assembly is PRJNA592877. Raw sequencing Illumina NextSeq (PRJEB35655) and ONT MinION (PRJEB35656) data can be accessed at EMBL-EBI.

microbiology↗

bioRxiv2019

Faecal virome transplantation decrease symptoms of type-2-diabetes and obesity in a murine model

ObjectiveDevelopment 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. 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.\n\nDesignThe FVT consisted of viromes extracted from the caecal content of mice fed a low-fat (LF) diet for fourteen weeks. Male C57BL/6NTac mice were divided into five groups: LF (as control), high-fat diet (HF), HF+Ampicillin (Amp), HF+Amp+FVT and HF+FVT. At week six and seven of the study the HF+FVT and HF+Amp+FVT mice were treated with FVT by oral gavage. The Amp groups were treated with ampicillin 24 h prior to first FVT treatment.\n\nResultsSix weeks after first FVT the HF+FVT mice showed a significant decrease in weight gain compared to the HF group. Further, glucose tolerance was comparable between the lean 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 involved in obesity and T2D development.\n\nConclusionsTransfer of gut viral communities from mice with a lean phenotype into those with an obese phenotype reduce weight gain and normalise blood glucose parameters relative to lean mice. We hypothesise that this effect is mediated via FVT-induced GM changes.\n\nSignificance of this studyWhat is already known about this subject?\n\nO_LIObesity and type-2-diabetes (T2D) are associated with gut microbiota (GM) dysbiosis.\nC_LIO_LIFaecal microbiota transplant from lean donors has previously shown potential in treating obesity and T2D.\nC_LIO_LIPatients suffering from recurrent Clostridium difficile infections (rCDI) have been cured with sterile filtered donor faeces (containing enteric viruses and no bacteria), here defined as faecal virome transplantation (FVT).\nC_LI\n\nWhat are the new findings?\n\nO_LIFVT from lean donors lead to decreased weight gain and normalised blood sugar tolerance in a diet-induced obesity (DIO) mouse model.\nC_LIO_LIFVT significantly changed the bacterial and viral GM component, as well as the plasma metabolome and the expression profiles of obesity and T2D associated genes.\nC_LIO_LIInitial treatment with ampicillin prior FVT seems to counteract the beneficial effects associated with FVT.\nC_LI\n\nHow might it impact on clinical practice in the foreseeable future?\n\nO_LIWe here show a proof-of-concept, providing a solid base for designing a clinical study of FVT targeting obesity and T2D in humans. This is further augmented by the increased safety related to FVT, since bacteria and other microorganisms are removed from the donor faeces, and therefore minimises the risk of disease transmission.\nC_LIO_LIThese findings highlight the potential application of FVT treatment of various diseases related to GM dysbiosis and further support the vital role of the viral community in maintaining and shaping the GM.\nC_LI

microbiology↗

bioRxiv2019

Mouse vendor influence on the bacterial and viral gut composition exceeds the effect of diet

Often physiological studiess using mice from one vendor show different outcome when being reproduced using mice from another vendor. These divergent phenotypes between similar mouse strains from different vendors have been assigned to differences in the gut microbiome. During recent years, evidence has mounted that the gut viral community plays a key role in shaping the gut microbiome and may thus also influence mouse phenotype. However, to date inter-vendor variation in the murine gut virome has not been studied. Using a metavirome approach, combined with 16S rRNA gene sequencing, we here compare the composition of the viral and bacterial gut community of C57BL/6N mice from three different vendors exposed to either a chow-based low-fat diet or high-fat diet. Interestingly, both the bacterial and the viral component of the gut community differed significantly between vendors. The different diets also strongly influenced both the viral and bacterial gut community, but surprisingly the effect of vendor exceeded the effect of diet. In conclusion, the vendor effect is substantial on not only the gut bacterial community, but also strongly influences viral community composition. Given the effect of GM on mice phenotype this is essential to consider, for increasing reproducibility of mouse studies.

microbiology↗