Chemostat culturing reduces fecal eukaryotic virus load and delays diarrhea after virome transplantation
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eLife Assessment
In this valuable study, the authors report on an innovative chemostat propagation system to reduce eukaryotic viruses while retaining phages in mixtures used for FVTs (fecal virome transplant). The authors hypothesized that chemostat-propagated viromes could modulate the gut microbiota and reduce necrotizing enterocolitis (NEC) lesions while avoiding potential side effects, such as earlier onset of diarrhea. The study is solid in that it integrates in vitro fermentation, high-resolution metagenomics, immunogenicity assays, and in vivo validation, demonstrating the potential of FVT using eukaryotic-free virome-based therapeutics. However, the study overall has some conceptual and technical limitations.
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Abstract
Abstract
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.
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eLife Assessment
In this valuable study, the authors report on an innovative chemostat propagation system to reduce eukaryotic viruses while retaining phages in mixtures used for FVTs (fecal virome transplant). The authors hypothesized that chemostat-propagated viromes could modulate the gut microbiota and reduce necrotizing enterocolitis (NEC) lesions while avoiding potential side effects, such as earlier onset of diarrhea. The study is solid in that it integrates in vitro fermentation, high-resolution metagenomics, immunogenicity assays, and in vivo validation, demonstrating the potential of FVT using eukaryotic-free virome-based therapeutics. However, the study overall has some conceptual and technical limitations.
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Reviewer #1 (Public review):
Summary:
Fecal virome transfer (FVT) has the potential to take advantage of microbiome-associated phages to treat diseases such as NEC. However, FVT is also associated with toxicity due to the presence of eukaryotic viruses in the mixture, which are difficult to filter out. The authors use a chemostat propagation system to reduce the presence of eukaryotic viruses (these become lost over time during culture). They show in pig models of NEC that chemostat propagation reduces the incidence of diarrhea induced by FVTs.
Strengths:
The authors report an innovative yet simple approach that has the potential to be useful for future applications. Most of the experiments are easy to follow and are performed well.
Weaknesses:
The biggest weakness is that the authors show that their technique addresses safety, but they …
Reviewer #1 (Public review):
Summary:
Fecal virome transfer (FVT) has the potential to take advantage of microbiome-associated phages to treat diseases such as NEC. However, FVT is also associated with toxicity due to the presence of eukaryotic viruses in the mixture, which are difficult to filter out. The authors use a chemostat propagation system to reduce the presence of eukaryotic viruses (these become lost over time during culture). They show in pig models of NEC that chemostat propagation reduces the incidence of diarrhea induced by FVTs.
Strengths:
The authors report an innovative yet simple approach that has the potential to be useful for future applications. Most of the experiments are easy to follow and are performed well.
Weaknesses:
The biggest weakness is that the authors show that their technique addresses safety, but they are unable to demonstrate that they retain efficacy in their NEC model. This could be due to technical issues or perhaps the efficacy of FVT reported in the literature is not robust. If they cannot demonstrate the efficacy of the chemostat-propagated virome mixture, the value of the study is compromised.
The above issue is especially concerning because the chemostat propagation selected for bacteria that may not necessarily be the ones that harbor the beneficial phages. Without an understanding of exactly how FVT works, is it possible to make any conclusion about the usefulness of the chemostat approach?
Finally, can the authors rule out that their observations in THP-1 cells are driven by LPS or some other bacterial product in the media?
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Reviewer #2 (Public review):
The authors hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as the earlier onset of diarrhea. This is interesting.
Major Comments:
(1) As the authors state that the aim of the research is 'We hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as earlier onset of diarrhea'.
a) For the efficacy, in Figure 5, there is no significance in stomach pathology and enterocolitis between groups, even between the control group and experimental groups, is it because of the low incidence of NEC? This may affect the statistical power of the conclusions. Therefore, it is unclear how one can draw the conclusion that chemostat can reduce NEC lesions?
b) …Reviewer #2 (Public review):
The authors hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as the earlier onset of diarrhea. This is interesting.
Major Comments:
(1) As the authors state that the aim of the research is 'We hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as earlier onset of diarrhea'.
a) For the efficacy, in Figure 5, there is no significance in stomach pathology and enterocolitis between groups, even between the control group and experimental groups, is it because of the low incidence of NEC? This may affect the statistical power of the conclusions. Therefore, it is unclear how one can draw the conclusion that chemostat can reduce NEC lesions?
b) Convincing pathology images would be helpful.
c) For the safety, such as body weight development, FVT had no statistical significance difference from control, CVT, and CVT-MO. So how can the authors draw the conclusion that chemostat can avoid potential side effects?
d) There is a lack of evidence to convince the reader that there is a decrease in eukaryotic viruses. More quantitative data here would be useful.(2) Questions regarding Figure 3F:
a) How can the medium have 'the baseline viral content'?
b) What is the statistical significance of the relative abundance of specific eukaryotic viruses?
c) The hosts for some of the listed eukaryotic viruses are neither pigs or humans, as such, the significance of a decrease in these viruses to humans is unclear.(3) In this study, pH 6.5 was selected as the pH value for chemostat cultivation, but considering the different adaptability of different bacteria to pH, it is recommended to further explore the effect of pH on bacteria and virus groups. In particular, it was optimized to maintain the growth of beneficial bacteria such as Lactobacillaceae and Bacteroides in order to improve the effect of chemostat cultivation.
(4) Please improve the quality of the images, charts, error bars, and statistical significance markers throughout and mark the n's. used in each experiment.
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Reviewer #3 (Public review):
This study investigated the in vitro amplification of donor fecal virus using chemostat culturing technology, aiming to reduce eukaryotic virus load while preserving bacteriophage community diversity, thereby optimizing the safety and efficacy of FVT. The research employed a preterm pig model to evaluate the effects of chemostat-propagated viromes (CVT) in preventing necrotizing enterocolitis (NEC) and mitigating adverse effects such as diarrhea.
Strengths:
(1) Enhanced Safety Profile:
Chemostat cultivation effectively reduced eukaryotic virus load, thereby minimizing the potential infection risks associated with virome transplantation and offering a safer virome preparation method for clinical applications.(2) Process Reproducibility:
The chemostat system achieved stable amplification of bacteriophage …Reviewer #3 (Public review):
This study investigated the in vitro amplification of donor fecal virus using chemostat culturing technology, aiming to reduce eukaryotic virus load while preserving bacteriophage community diversity, thereby optimizing the safety and efficacy of FVT. The research employed a preterm pig model to evaluate the effects of chemostat-propagated viromes (CVT) in preventing necrotizing enterocolitis (NEC) and mitigating adverse effects such as diarrhea.
Strengths:
(1) Enhanced Safety Profile:
Chemostat cultivation effectively reduced eukaryotic virus load, thereby minimizing the potential infection risks associated with virome transplantation and offering a safer virome preparation method for clinical applications.(2) Process Reproducibility:
The chemostat system achieved stable amplification of bacteriophage communities (Bray-Curtis similarity >70%), mitigating the impact of donor fecal variability on therapeutic efficacy.Weaknesses:
(1) Loss of Phage Functionality:
The chemostat cultivation resulted in a reduction in phage diversity (e.g., the loss of Lactobacillaceae phages), which may compromise their protective effects against NEC (potentially linked to the immunomodulatory functions of Lactobacilli). The authors should explicitly address this limitation in the discussion section, particularly if additional experiments cannot be conducted to resolve it within the current study.(2) Limitations in Experimental Design:
The low incidence of NEC lesions in the control group reduced the statistical power of the study. This limitation undermines the ability to conclusively evaluate the efficacy and safety of the chemostat-propagated virome as a novel intervention for NEC. Future studies should optimize experimental conditions (e.g., using a more NEC-susceptible model or diet) to ensure adequate disease incidence for robust statistical comparisons. -
