Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.

BACKGROUND: Fecal 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 hinder their wider clinical application in treating less acute diseases. METHODS: To overcome these challenges, we developed methods to broaden FVT's 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. RESULTS: FVT-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 FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight 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. CONCLUSIONS: This proof-of-concept study shows that specific modifications of 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 of donor phages emerging as promising approaches. Video Abstract.

Transfer of modified gut viromes improves symptoms associated with metabolic syndrome in obese male mice.

Metabolic syndrome encompasses amongst other conditions like obesity and type-2 diabetes and is associated with gut microbiome (GM) dysbiosis. Fecal microbiota transplantation (FMT) has been explored to treat metabolic syndrome by restoring the GM; however, concerns on accidentally transferring pathogenic microbes remain. As a safer alternative, fecal virome transplantation (FVT, sterile-filtrated feces) has the advantage over FMT in that mainly bacteriophages are transferred. FVT from lean male donors have shown promise in alleviating the metabolic effects of high-fat diet in a preclinical mouse study. However, FVT still carries the risk of eukaryotic viral infections. To address this, recently developed methods are applied for removing or inactivating eukaryotic viruses in the viral component of FVT. Modified FVTs are compared with unmodified FVT and saline in a diet-induced obesity model on male C57BL/6 N mice. Contrasted with obese control, mice administered a modified FVT (nearly depleted for eukaryotic viruses) exhibits enhanced blood glucose clearance but not weight loss. The unmodified FVT improves liver pathology and reduces the proportions of immune cells in the adipose tissue with a non-uniform response. GM analysis suggests that bacteriophage-mediated GM modulation influences outcomes. Optimizing these approaches could lead to the development of safe bacteriophage-based therapies targeting metabolic syndrome through GM restoration.

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 the 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 and how the choice of ultrafilter might influence the extracted viral community. Clinical samples (feces, vaginal swabs, and tracheal suction samples) were spiked with a mock community of known phages (T4, c2, Φ6, Φ29, Φx174, and Φ2972), filtered, and quantified using spot and plaque assays to estimate the loss in recovery. The enveloped Φ6 phage is especially severely affected by the choice of filter, but also tailed phages such as T4 and c2 have a reduced infectivity after ultrafiltration. We conclude that the pore size of ultrafilters may affect the recovery of phages in a strain- and sample-dependent manner, suggesting the need for greater thought when selecting filters for virus extraction.

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

Probiotics are intended to improve gastrointestinal health when consumed. 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. Here, we investigate the use of fecal virome transplantation (FVT, sterile filtrated feces) as a mean to alter the gut microbiome 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 from 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, LGG+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT donor material originated from 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 propagating the existing commensal "probiotics" that have already permanently colonized 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 interactions between the gut microbiome and fertility.

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

Many bacteria and archaea 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, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally 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. Both high phage titer and 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. Fewer 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 to 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.

Inter-vendor variance of enteric eukaryotic DNA viruses in specific pathogen free C57BL/6N mice.

The laboratory mouse strain C57BL/6 is widely used as an animal model for various applications. It is becoming increasingly clear that the bacterial enteric community highly influences the phenotype. Eukaryotic viruses represent a sparsely investigated member of the enteric microbiome that might also affect the phenotype. We here investigated the presence of enteric eukaryotic DNA viruses (EDVs) in specific pathogen-free (SPF) C57BL/6N mice purchased from three vendors upon arrival and after being fed a low-fat diet (LFD) or high-fat diet (HFD). We detected genetic fragments of EDVs belonging to the viral families of Herpes-, Mimi-, Baculo- and Phycodnaviridae represented by two genera; Chlorovirus and Prasinovirus. The EDVs were detected in the mice upon arrival and persisted for 13 weeks. However, these signals of EDVs were only detected at notable levels in mice fed LFD from 2 out of 3 vendors, which suggested that the enteric composition of these EDVs were affected by both vendor (p < 0.003) and different dietary regimes (p < 0.013). This highlights the need of additional studies assessing the potential function of these EDVs that may influence the mouse phenotype and the reproducibility of animal studies using this C57BL/6N substrain.

Bacteriophage-mediated manipulation of the gut microbiome - promises and presents limitations.

Gut microbiome (GM) composition and function are linked to human health and disease, and routes for manipulating the GM have become an area of intense research. Due to its high treatment efficacy, the use of fecal microbiota transplantation (FMT) is generally accepted as a promising experimental treatment for patients suffering from GM imbalances (dysbiosis), e.g. caused by recurrent Clostridioides difficile infections (rCDI). Mounting evidence suggests that bacteriophages (phages) play a key role in successful FMT treatment by restoring the dysbiotic bacterial GM. As a refinement to FMT, removing the bacterial component of donor feces by sterile filtration, also referred to as fecal virome transplantation (FVT), decreases the risk of invasive infections caused by bacteria. However, eukaryotic viruses and prophage-encoded virulence factors remain a safety issue. Recent in vivo studies show how cascading effects are initiated when phage communities are transferred to the gut by e.g. FVT, which leads to changes in the GM composition, host metabolome, and improve host health such as alleviating symptoms of obesity and type-2-diabetes (T2D). In this review, we discuss the promises and limitations of FVT along with the perspectives of using FVT to treat various diseases associated with GM dysbiosis.

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

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.

Mouse Vendor Influence on the Bacterial and Viral Gut Composition Exceeds the Effect of Diet.

Often physiological studies 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 not only on 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.

The Gut Microbiome on a Periodized Low-Protein Diet Is Associated With Improved Metabolic Health.

A periodized (14 days on/14 days off) 5% low protein-high carbohydrate (pLPHC) diet protects against weight gain, improves glucose tolerance in mice and interacts with concurrent voluntary activity wheel training on several parameters including weight maintenance and liver FGF21 secretion. The gut microbiome (GM) responds to both diet and exercise and may influence host metabolism. This study compared the cecal GM after a 13.5-week intervention study in mice on a variety of dietary interventions ± concurrent voluntary exercise training in activity wheels. The diets included chronic chow diet, LPHC diet, 40 E% high protein-low carbohydrate (HPLC) diet, an obesigenic chronic high-fat diet (HFD) and the pLPHC diet. Our hypothesis was that the GM changes with pLPHC diet would generally reflect the improved metabolic health of the host and interact with concurrent exercise training. The GM analyses revealed greater abundance phylum Bacteroidetes and the genus Akkermansia on chronic and periodized LPHC and higher abundance of Oscillospira and Oscillibacter on HFD. The differences in diet-induced GM correlated strongly with the differences in a range of host metabolic health-measures. In contrast, no significant effect of concurrent exercise training was observed. In conclusion, pLPHC diet elicits substantial changes in the GM. In contrast, only subtle and non-significant effects of concurrent activity wheel exercise were observed. The pLPHC-associated microbiome may contribute to the healthier host phenotype observed in these mice.

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

A bacterial strain, designated WCA-9-b2T, was isolated from the caecal content of an 18-week-old obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate was rod-shaped, strictly anaerobic, spore-forming, non-motile and Gram-stain-positive, under the conditions tested. Colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the order Clostridiales with Dorea longicatena ATCC 27755T (94.9 % sequence identity), Ruminococcus gnavus ATCC 29149T (94.8%) and Clostridium scindens ATCC 35704T (94.3%) being the closest relatives. Whole genome sequencing showed an average nucleotide identity <74.23 %, average amino acid identity <64.52-74.67 % and percentage of conserved proteins values <50 % against the nine closest relatives (D. longicatena, Ruminococcus gnavus, C. scindens, Dorea formicigenerans, Ruminococcus lactaris, Clostridium hylemonae, Merdimonas faecis, Faecalicatena contorta and Faecalicatena fissicatena). The genome-based G+C content of genomic DNA was 44.4 mol%. The major cellular fatty acids were C16 : 0 (24.5%), C18 : 1 cis9 (19.8 %), C16 : 0 DMA (11.7%), C18 : 0 (8.4%) and C14 : 0 (6.6%). Respiratory quinones were not detected. The predominant metabolic end products of glucose fermentation were acetate and succinate. Production of CO2 and H2 were detected. Based on these data, we propose that strain WCA-9-b2T represents a novel species within a novel genus, for which the name Sporofaciens musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T=CECT 30156T).

Effect of fecal microbiota transplantation route of administration on gut colonization and host response in preterm pigs.

This study examined gut colonization patterns and host responses to fecal microbiota transplantation (FMT) by different administration routes after preterm birth. In two separate experiments, cesarean-delivered, preterm pigs were administered combined oral + rectal, or exclusively rectal donor feces, and compared with saline controls. After 5 days, stomach and colon bacterial compositions were determined by 16S rRNA gene amplicon sequencing, and organic acid metabolites measured. Further, gut pathology, mucosa bacterial adherence, and goblet cell density were assessed. FMT increased the relative abundance of obligate anaerobes in the colon without affecting total bacterial load. Bacteroides colonized recipients despite low abundance in the donor feces, whereas highly abundant Prevotella and Ruminococcaceae did not. Further, FMT changed carbohydrate metabolism from lactate to propionate production thereby increasing colonic pH. Besides, FMT preserved goblet cell mucin stores and reduced necrotizing enterocolitis incidence. Only rectal FMT increased the stomach-to-colon pH gradient and resistance to mucosa bacterial adhesion. Conversely, oral + rectal FMT increased bacterial adhesion, internal organ colonization, and overall mortality. Our results uncovered distinctions in bacterial colonization patterns along the gastrointestinal tract, as well as host tolerability between oral and rectal FMT administration in preterm newborns. Besides, FMT showed the potential to prevent necrotizing enterocolitis.

Genetic manipulation of structural color in bacterial colonies.

Naturally occurring photonic structures are responsible for the bright and vivid coloration in a large variety of living organisms. Despite efforts to understand their biological functions, development, and complex optical response, little is known of the underlying genes involved in the development of these nanostructures in any domain of life. Here, we used Flavobacterium colonies as a model system to demonstrate that genes responsible for gliding motility, cell shape, the stringent response, and tRNA modification contribute to the optical appearance of the colony. By structural and optical analysis, we obtained a detailed correlation of how genetic modifications alter structural color in bacterial colonies. Understanding of genotype and phenotype relations in this system opens the way to genetic engineering of on-demand living optical materials, for use as paints and living sensors.