The Next Generation Fecal Microbiota Transplantation: To Transplant Bacteria or Virome

Abstract Fecal microbiota transplantation (FMT) has emerged as a promising therapeutic approach for dysbiosis‐related diseases. However, the clinical practice of crude fecal transplants presents limitations in terms of acceptability and reproductivity. Consequently, two alternative solutions to FMT are developed: transplanting bacteria communities or virome. Advanced methods for transplanting bacteria mainly include washed microbiota transplantation and bacteria spores treatment. Transplanting the virome is also explored, with the development of fecal virome transplantation, which involves filtering the virome from feces. These approaches provide more palatable options for patients and healthcare providers while minimizing research heterogeneity. In general, the evolution of the next generation of FMT in global trends is fecal microbiota components transplantation which mainly focuses on transplanting bacteria or virome.


Introduction
The human gut microbiome is a complex community consisting of bacteria, archaea, viruses, fungi, eukaryotic parasites, protozoa, and their genomes. [1,2]Researchers are increasingly interested in treatments targeting the gut microbiome that plays a pivotal role in human health. [3]Fecal microbiota transplantation (FMT) is a promising treatment for diseases related to gut dysbiosis, [4,5] as it can help to reconstruct the composition DOI: 10.1002/advs.202301097 and function of imbalanced gut microbiota by transferring fecal preparations from healthy donors. [6]A recent systematic review revealed that FMT has been reported to treat 85 specific diseases in clinical settings globally from 2011 to 2021. [7]In particular, FMT is highly effective in treating recurrent Clostridiodes difficile infection (CDI), with a cure rate of ≈90%. [8][11] The transferred microbiota and metabolites can partly explain the efficacy of FMT.For example, metabolites derived from the Firmicutes phylum, specifically short-chain fatty acids, and secondary bile acids, play multiple beneficial roles in maintaining host homeostasis.These roles include fortifying the gut barrier and mitigating inflammation. [12,13]However, varied FMT methods involving varying doses and delivery routes [14] have caused different clinical responses among different research teams. [15]This heterogeneity presents challenges to research reproducibility.In a parallel progression akin to drug development's evolution from natural formulations to more precise components, FMT is also expected to follow a similar trajectory.Presently, the focus is shifting toward the transplantation of more precise and efficacious microbial components.Both bacteria and the virome, as predominant constituents of the gut microbiota, are believed to play pivotal roles in the effectiveness of FMT. [16,17]20][21] Given the abundance of bacteria in the gut microbiome, bacteria have been the focus of most FMT-related studies. [22][25] The automatic washing process which involves delivering considerations, was coined as washed microbiota transplantation (WMT) [26] and released as consensus by the FMT-standardization Study Group in 2019. [27]The primary objective of WMT is to transplant the enriched bacteria using a washed fecal microbiota preparation involving the elimination of bacterial fragments, metabolites, soluble molecules, proteins, and viruses to the greatest extent possible, while also ensuring the removal of all fungi and parasite eggs (Figure 1). [26]Another method for transplanting bacteria is bacteria spores treatment, which enriches bacteria spores through a preparation that incorporates chemical and physical methods (Figure 1). [25]In recent years, the importance of the virome in gut Figure 1.The conceptual graphs of preparation methodology for fecal transplants.The preparation methods for fecal transplants: fecal microbiota transplantation (rough filtration), washed microbiota transplantation, bacteria spores treatment, and fecal virome transplantation.Additional details can be found in Table 1.
microbiota has also been increasingly recognized. [28,29]However, the methods of WMT and bacteria spores treatment are not actually aimed at transplanting the viral community.[32] Separating the virome for study purposes could offer a solution, enabling the investigation of the mechanism and potential applications of the virome.The gut virome is mainly composed of prokaryotic viruses, [33] with bacteriophages being the dominant type.The total number of gut bacteriophages is estimated to be ≈10 15 . [34]Recently, there has been growing in-terest in fecal virome transplantation (FVT) or fecal filtrate transplantation (FFT), which involve transplanting virome (containing viruses, metabolites, and cellular debris, but not intact bacterial cells) from donor feces to patients, to eliminate adverse events (AE) caused by bacteria (Figure 1). [19,35]In 2017, Ott et al. reported clinical success in treating CDI by FFT, [19] which has spurred further research into the potential benefits of the virome. [22]Of note, to facilitate understanding, we use the term FVT to collectively refer to methods of transplanting fecal virome, which shares similar concepts and methods with FFT.
In the current stage, as the mechanism is partially defined, the next generation of FMT could fulfill the following criteria: uncultured microbes derived from healthy human donors, relative specificity in compositions, higher safety through clinical trials, and gastrointestinal tract as the delivery route.These criteria aim to retain both known and unknown beneficial components in fecal transplants while reducing study heterogeneity through more precise compositions and refined methodologies.In general, the next generation of FMT is progressing towards the direction of transplanting specific microbiota components, namely microbiota components transplantation, mainly targeting bacteria and virome.In this review, the methods involved in transplanting bacteria and viromes meet these criteria.Although their primary components differ, their mechanisms are interconnected and deserve further exploration.It is worth noting that certain promising products with well-defined and reproducible components, including formula (or cocktail) bacteria, [36] bacteriophage cocktail treatment, [37] and bacteriophage consortia treatment, [38] are excluded in this review.
In this review, we discussed milestones of FMT in the medical history, and the safety and regulation concerns associated with FMT.We also displayed the challenges facing FMT and emphasized the importance of improving its methodology.Finally, we discussed cutting-edge research on transplanting bacteria or virome, including their methodology, applications, safety, potential mechanism, research techniques, and prospects.Our goal was to offer guidance for the future development of microbiotabased therapies and enrich the scientific explanations of microbiota medicine [39] as a branch discipline of clinical medicine.

The Milestones of FMT in the Medical History
The first milestone of FMT in medical history should be the ancient record in traditional Chinese medicine.The use of FMT for treating human diseases can be traced back to at least the 4 th century, as documented in the Zhou Hou Bei Ji Fang, a Chinese handbook of emergency medicine. [5]According to this ancient medical history, traditional Chinese medicine doctors used oral human fecal suspension to treat patients with food poisoning or severe diarrhea.This historical account meets several criteria that confirm the use of FMT, including the use of human fecal matter as the source material, administration via the digestive tract, efficacy mediated by the microbiota present in fresh fecal water or fermented fecal matter, and clear descriptions of prescriptions, methods, indications, and efficacy in ancient literature. [5]n 1958, FMT through enema was first reported to treat pseudomembranous enterocolitis. [40]In 1983, Schwan et al. reported the first case of recurrent CDI successfully treated with homologous FMT. [41]In 2013, FMT was first time recommended for the treatment of recurrent CDI by the guideline led by Surawicz. [42]his is a milestone for FMT from folk remedies to recommendations by professional teams.In modern medicine, the range of diseases being investigated for treatment with FMT has significantly expanded, but CDI remains the only well-established indication. [4,5]The data as of August 2023 shows that the disease with the highest number of registered clinical trials for FMT treatment on the clinicaltrials.govwebsite is IBD.From 2011 to 2021, a total of 2,400 articles in the English language related to FMT research were published. [43]FMT has also been identified as a potential treatment for several diseases that occur beyond the gut.The concept of specific gut-organ axes has been proposed to reflect the potential correlations in mechanisms of disease development between these organs and the gut.Well-known examples include the gut-brain axis (e.g.autism and depression), [44,45] gut-cardiac axis (e.g.myocarditis and hypertension), [46,47] gutpulmonary axis (e.g.pneumonia), [48] gut-liver axis (e.g.nonalcoholic steatohepatitis), [49,50] gut-immune axis (e.g.systemic lupus erythematosus and graft-versus-host disease, GVHD), [51,52] and gut-skin axis (e.g.atopic dermatitis). [53]Of note, the scope of gut-organ axes is continuously expanding, as researchers are discovering new connections between the gut microbiome and various organs in the body.
Humans must not like feces from other people and have been trying to escape this reality, but have never succeeded.The colonic transendoscopic enteral tubing (TET) was reported as a new delivery of microbiota suspension. [54]It has been used to deliver microbiota for patients with severe intestinal diseases, such as CDI [55] and IBD. [24,56]Therefore, colonic TET represents a milestone in the field of microbiota or drug delivery, [4] offering a promising novel route for repeated administration throughout the colon.This innovative approach not only enhances the safety of treatments but also aligns seamlessly with physiological principles.The widespread adoption of colonic TET in China highlights its clinical versatility and the significant benefits it offers in addressing various challenging scenarios, including in situ sampling in the cecum. [54]A recent animal study unveiled variations in the virome along the gastrointestinal tract, with luminal samples from the large intestine exhibiting the highest loads and diversity of bacteriophages. [57]Similarly, in humans, samples obtained directly from the intestines demonstrated a higher prevalence of prophage induction compared to stool samples. [58]Based on the above, colonic TET can emerge as a promising method for future investigations into the intricate interplay between the virome and hosts.
Another milestone is the drug approvals for FMT.The prepared fecal microbiota has been approved as one of the live biotherapeutic products by Australia and the United States in 2022 and 2023, respectively. [59]The spores collected from donated feces also received final approval from the United States Food and Drug Administration (FDA) in 2023. [21]These approved drugs play a crucial role in advancing the field of microbiota-based therapies by facilitating their utilization in a broader population.

The Safety and Regulation Concerns
A systematic review [60] has summarized the reported FMTrelated AEs in 5688 cases from 2000 to 2020.This review revealed that FMT-related AEs were observed in 19% of FMT procedures, and the most frequently reported FMT-related AEs were diarrhea (10%) and abdominal discomfort/pain/cramping (7%).Significantly, FMT-related serious AEs have been reported in 1.4% of patients.These serious AEs include five FMT-related deaths, attributed to fatal aspiration pneumonia related to the upper gastrointestinal delivery route (two cases had a specific causal relation, and one probably had), [61][62][63] as well as aspiration during sedation for FMT administered via colonoscopy, [64] and transmission of pathogenic bacterial species. [65]A case of transmission of pathogenic bacterial species was reported in 2019, [65] in which a patient developed extended-spectrum beta-lactamase-producing E. coli bacteremia after undergoing FMT.Genomic sequencing confirmed that the causality was linked to the donor.[68] Despite the above FMT-related AEs having been reported, the overall safety profile of FMT is favorable. [69]Although the data on the long-term safety of FMT is insufficient, the immunogenicity of bacteria is stable in human transmission and over time. [70]o further ensure the safety of FMT, it is important to focus on the methodology.1][72][73] In a real-world setting by Chen's group in the United States, patients who underwent FMT at academic centers differed significantly in clinical characteristics from those treated at a private practice. [74]Importantly, the efficacy of FMT can be influenced by various parameters such as the manufacturing process of fecal preparation, doses, and delivery routes. [14,15]The heterogeneity of FMT can also lead to poor reproducibility of the research.By November 2020, a systematic review involving 30 randomized controlled trials (RCT) of FMT suggested that uniform and standardized methods were needed for research. [75]Despite different microbial taxa of donors of responders and non-responders having been observed, the inconsistent results were due to the different investigating criteria.Such inconsistencies might increase the burden of regulating FMT, leading to varied regulations and ethical considerations among countries.
By putting appropriate and effective regulations in place to safeguard patients and donors, the safety and efficacy of FMT can be improved. [76,77]Since 2013, the FDA has permitted the use of FMT under "enforcement discretion" for treating CDI that does not respond to standard therapy. [78]In November 2022, the Australian Therapeutic Goods Administration approved the registration of BIOMICTRA, a donor-derived microbiome-based therapy product, for the treatment of recurrent CDI, and it has been added to the Australian Register of Therapeutic Goods.Beforehand, FMT was regarded as an investigational medicinal product in Australia. [79]FMT was not approved as a drug until 2023 when the FDA granted approval to Rebyota (RBX2660). [59]This microbiota-based live biotherapeutic, prepared from human stool and consisting of a broad consortium of microbes, is indicated for the prevention of recurrence of CDI in individuals 18 years of age and older who have received antibiotic treatment for recurrent CDI.In China, FMT is a permitted medical therapy for CDI and many other diseases under the supervision of the National Health Commission. [18]Significantly, in 2020, the FDA issued a warning regarding the potential risk of transferring SARS-CoV-2 through FMT to patients.As a precautionary measure, they recommended additional safety protocols, including excluding the donor from further donations and refraining from clinical use of any FMT product derived from stool donated by the affected donor within 4 weeks prior to their first positive test.Even though the World Health Organization (WHO) declared the COVID-19 pandemic officially over on May 5, 2023, transitioning into a phase of long-term management, we must remain vigilant regarding the risk of SARS-CoV-2 transmission through fe-cal material.The establishment of stool banks plays a crucial role in standardizing the donor screening process, as well as in monitoring treatment outcomes and potential side effects. [80]Over 20 stool banks have been established worldwide since 2012, such as OpenBiome in America, BiomeBank in Australia, the Netherlands Donor Feces Bank, and the China Microbiota Transplantation System (FmtBank). [7]Fecal microbiota bank centers have partially addressed the accessibility issues of FMT for most hospitals.

The Methods and Delivery Routes of WMT
Since 2014, microbiota preparations using an automatic purification system (GenFMTer, Nanjing, China) have been used in China. [51,56,81]This is followed by three rounds involving centrifugation and suspension to automatically wash the microbiota from feces (Table 1).The methods of washing the microbiota are different from the traditional crude FMT process that only involves manual suspension and filtration steps to remove larger particles, fibers, and undigested food (Figure 1). [82]In 2018, Zhang et al. [5] proposed that it was time to discontinue manual FMT and promote standardization of the procedure.The improved methodology of FMT based on the automatic washing process and the related delivering consideration was coined as WMT, [26] and released by the FMT-standardization Study Group in 2019. [27]MT not only offers a refined methodology compared to manual FMT but also provides a safe and convenient delivery route.TET is the delivery route to address the current clinical requirements and limitations of manual FMT. [83]TET involves endoscope-assisted implantation of the tube into the deep intestine, followed by fixation.This method has great potential to adapt to the requirements of repeated transplantations. [84]TET is classified into mid-gut TET and colonic TET, depending on the implantation location in the intestine.The colonic TET can reduce the spatial and temporal heterogeneity of different studies because the implantation site can be precisely identified and the sampling time can be standardized. [85]The improved quantitative methods, automatic purification systems, and delivery approach of WMT have led to reduced research heterogeneity and improved safety.

The Efficacy and Safety of Washed Microbiota Transplantation
Two previous studies [24,56] have reported that WMT is an independent factor that reduces AEs in patients with ulcerative colitis (UC) and Crohn's disease (CD).The latest study [18] updated the safety data of WMT and compared it with manual FMT.The incidence of AEs decreased from 35.5% (11/31) of manual FMT to 7.2% (65/902) of WMT in patients with UC, and from 21.7% (15/69) of FMT to 4% (35/882) of WMT in patients with CD.An animal study [26] explained the improved safety of WMT could be attributed to the repeated washing process, which helps to Items WMT [5,18] Bacteria spores treatment [86] FVT [19] Conditions GMP GMP Biosafety cabinet Fecal weight ≥50 g ≥50 g ≥50 g Origin Allogenic Allogenic Allogenic Equipment GenFMTer Undefined Blender, and custom-built air pressure filtration system Processes 1) Suspension: at the ratio of 500 mL of sterile saline per 100 g of feces.2) Microfiltration: multi-level automatic filtration within a disposable sterile closed-loop system (GenFMTer).3) Centrifugation and washing. 1) Suspension: normal saline with feces.
1) Suspension: at the ratio of 500 mL of sterile saline per 50 g of feces.
2) Centrifugation and collection of the supernatant.3) Filtration with different pore sizes.GMP, good manufacturing practice.
remove a growing number of pro-inflammatory metabolites (such as leukotriene B4, corticosterone, and prostaglandin G2), and viruses.
Intriguingly, fresh WMT achieved the same duration of remission in a patient with refractory UC with less frequent treatments compared to fresh FMT (Table 2). [23]In this study, the patient achieved the same duration of eight-month remission with only two courses of WMT via colonic TET, while the same duration was achieved with 60 times of FMT via a percutaneous endoscopic cecostomy tube.In clinical practice, reducing both the frequency of treatment and the length of hospital stays can contribute to increasing patients' compliance.Although the biomarkers of responsiveness to WMT for IBD are unclear, decreasing Candida abundance has been positively correlated with the amelioration of disease severity. [87]Patient with refractory UC suffering from recurrent fungal infection has also been successfully cured by WMT (Table 2). [88]Within one week after the initial WMT, the C. glabrata infection in fecal fungal culture turned negative.
In metabolic diseases, the latest studies have demonstrated the efficacy and safety of WMT in improving diabetes. [89,90]The animal study has observed that fecal transplantation from patients with diabetes complicating distal symmetric polyneuropathy to mice could lead to the transfer of diabetes phenotypes.This transfer may be attributed to several potential mechanisms, including a compromised gut barrier, elevated antigen load, and systemic inflammation.Specifically, lipopolysaccharide (LPS) was able to breach the compromised gut barrier and enter the bloodstream.Subsequently, LPS induced the production of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and TNF- in a Toll-like receptor 4-dependent manner, thereby promoting inflammation and exacerbating damage to islet  cells, consequently accelerating the progression of diabetes. [91]In the RCT segment of the same study, it was demonstrated that frozen WMT could improve neuropathic pain in patients.The WMT group showed a significant improvement in neuropathic pain compared to the placebo group (WMT group: 53.3% vs. placebo group: 14.29%). [90]This improvement following WMT could potentially be attributed to an increase in the gut microbiota's butyric acid production capacity and a decrease in LPS production.Another study reported that 72.22% of patients with high blood glucose restored their fasting blood glucose to normal within 1 month after fresh WMT. [89]In addition, long-term (≈6 months) effects on lowering blood pressure after WMT have also been shown in patients with high blood glucose (Table 2). [89]Previous studies in animal models have suggested that gut microbiota play a role in regulating blood pressure. [92,93]For example, hypertensive models such as SHRs and Ang II mice have fewer SCFA-producing bacteria than normotensive mice. [94]Decreased SCFA could cause neuroinflammation, further affecting blood pressure. [95]In clinical practice, a recent study indicated that fresh WMT had a short-term antihypertensive effect on patients with hypertension (Table 2). [96]Compared with baseline, patients with hypertension after WMT had an increased abundance of Senegalimassilia species and a decreased abundance of Parasutterella and Solobacterium species.Among these species, Solobacterium species has been proven to have an association with atherosclerotic cardiovascular disease. [97]Another study involving patients with dyslipidemia showed that 35.14% of patients with hyperlipidemia returned to normal blood lipid levels one month after fresh WMT. [98]In the hypolipidemia group, [98] 36.36% and 47.06% became normal in one and two months after WMT, respectively (Table 2).
A recent case report presented clinical evidence of the effectiveness of WMT in halting the progression of amyotrophic lateral sclerosis (ALS), and trends in the microbiome and metabolome were consistent with improvements in disease status (Table 2). [99]imilarly, in individuals with ASD, alterations in the composition of the microbiota have been recognized by researchers as a key feature of this pervasive developmental disorder. [100]A retrospective study of children with ASD has demonstrated that symptoms of ASD, gastrointestinal issues, sleep disorders, and systemic inflammation can be improved with fresh WMT (Table 2). [101]The potential mechanism might be attributed to that WMT could mitigate the production of p-cresol sulfate and 5-hydroxytryptamine, which are associated with microbiota dysbiosis. [102,103]he methodology of WMT has been widely used in China since 2014, as described in the studies on the treatment of antibiotic-associated diarrhea, [104] radiation enteritis, [105] and GVHD (Table 2). [51]As mentioned above, WMT has shown its efficacy and safety in treating numerous diseases (Figure 2), providing researchers with confidence in its potential.Case report [23] One patient with refractory UC

Healthy individuals
Colonic TET; gastroscopy The patient underwent 60 FMTs via percutaneous endoscopic cecostomy tube in two months and achieved clinical remission for eight months.Then the patient underwent seven courses of WMT (a total 10 WMTs, 8 by colonic TET).After two courses of WMT, the patient achieved clinical remission for eight months.The total remission time induced and maintained by the last two courses of WMT added up to 45 months.
Case report [88] One patient with refractory UC complicated with recurrent

High blood glucose
Real-world study [89] 195 patients (20  Animal study plus a randomized, double-blind, and placebo-controlled trial [90] Animal study one: mice (M)-normal glucose (NG) group RCT: After 84 days, several positive outcomes were observed in the group that underwent WMT compared to the placebo group.These included improvements in neuropathic symptoms, neuropathic pain, anxiety levels, sleep quality, and electrophysiological functions of peripheral nerves.Notably, the relief of moderate to severe neuropathic pain showed a significant difference between the two groups, with 53.3% experiencing relief in the WMT group compared to only 14.29% in the placebo group.Additionally, sensory nerve conduction velocities were significantly increased in the WMT group.

Healthy individuals
Nasojejunal tube; Colonic TET After WMT, the blood pressure at hospital discharge was significantly lower than that at hospital admission (change in systolic blood pressure: −5.09 ±

ALS
Case report [99] One patient

Healthy individuals
Colonic TET; mid-gut TET Constipation and ALS symptoms such as impaired balance and gait were well controlled since the first course of WMT.Although the patient was administered antibiotics after several months because of an accidental fall and then her muscle tone returned to her worst status, the rescue WMT successfully stopped the progression of the disease again with quick improvement.Microbial analysis indicated that the diversity and composition of the gut microbiota from the patient after WMT treatment were closer to healthy donors.

Healthy individuals
Colonic TET Aberrant Behavior Checklist, Childhood Autism Rating Scale, and Sleep Disturbance Scale for Children scores, the proportion of children with constipation and abnormal fecal forms, and WBC and globulin levels were all significantly lower in ASD children after WMT.

Healthy individuals
Mid-gut TET; gastroscopy 3/5 patients responded to WMT which was defined as a 8 patients

Healthy individuals
Nasoduodenal tube All the patients achieved clinical symptomatic remission after the first WMT.Among patients with diarrhea, their stool volumes and frequencies were reduced to 3-4 times/day after WMT.In the 2-week follow-up after the first WMT, two patients' diarrhea and all the other five patients' abdominal pain disappeared (three patients died within this period unrelated to WMT).Compared to those who did not receive WMT, these eight patients achieved a higher progression-free survival.

Healthy individuals nasojejunal tube
In all 5 patients, FVT restored normal stool habits and eliminated symptoms of CDI for a minimum period of 6 months.Bacterial phylogeny and virome profile analyses of fecal samples from recipients indicated longitudinal changes in microbial and viral community structures after FVT.

T2D and obesity
Animal study [20] 40 mice were divided into 5 groups including a low-fat diet (as lean control), high-fat (HF) diet, HF+ampicillin, HF+ ampicillin +FVT, and HF+FVT C57BL/6N mice fed LF diet for 14 weeks oral gavage Six weeks after the first FVT from lean donors, A decreased weight gain and a normalized blood glucose tolerance in a diet-induced obesity mouse model were observed.FVT significantly changed the bacterial and viral gut microbiota component, as well as the plasma metabolome and the expression profiles of obesity and T2D-associated genes.

SIBO
Animal study [ 106] 36 mice divided into two dietary groups (n = 18 each): standard diet (SD) or high-fat diet (HFD) 18 mice fed HFD for 30 days oral gavage In both recipient groups, FVT produced similar results to the whole FMT.FVT did not significantly alter the population density of resident small intestinal bacterial community composition in SD recipients.However, it did induce a transition in the small intestinal bacterial composition, making it resemble that of an HFD microbiome.Following FVT, the bacterial density in the ileum of recipients on an HFD was reduced.
16 mice before antibiotic treatment oral gavage An autochthonous virome transfer reshaped the bacterial communities of mice following antibiotic treatment, resulting in a microbiota profile that closely resembled the pre-antibiotic state.This effect was not observed in mice that received non-viable phages.Metagenomic sequencing of the virome showed that the abundance and diversity of fecal bacteriophages differed over time between the FVT group and the control group.Notably, the phages introduced through FVT persisted in the mice that received them.

16) healthy suckling piglets
Enema; oral gavage Oro-gastric FVT completely prevented NEC, which was confirmed by microscopy, whereas FMT did not perform better than the control.Oro-gastric FVT increased viral diversity and reduced Proteobacteria relative abundance in the ileal mucosa relative to control.Induction of mucosal immunity was observed in response to FMT but not FVT.Oro-gastric FVT also performed better than FMT in a series of safety parameters, including body growth rate, the relative weight of the small intestine, small intestinal permeability, and mucosal integrity.

The Methodology and Delivery Routes of Bacteria Spores Treatment
Another method to enrich bacteria for transplanting is by purifying bacteria spores from fecal matter. [25]Bacteria spores are a means of survival in hostile environments, such as those created by antibiotic treatments.This presents a significant challenge for clinicians, as antibiotics cannot kill spores. [109]However, it has been observed that utilizing nontoxigenic bacteria spores that compete with toxigenic spore-forming bacteria has the potential to solve this challenge. [21,110]The methods of purifying nontoxigenic bacteria spores (Figure 2) involve solvent treatments (a combination of freeze-thaw in 50% ethanol followed by 2 h in 70% ethanol at room temperature) and purification steps (pelleting by centrifugation, washing with saline to remove ethanol, resuspending with sterile glycerol) to clear vegetative bacteria, fungi, parasites, and viruses (Table 1). [25,86]Following these processes, patients are given capsules containing purified bacteria spores through oral administration. [25,86]

The Efficacy and Safety of Bacteria Spores Treatment
Purifying bacteria spores has been primarily used in the treatment and prevention of recurrent CDI (Figure 2). [21,110]C. difficile is a Gram-positive, spore-forming, toxin-producing, obligate anaerobic bacterium.The spore coat is a physical barrier that protects C. difficile against chemical insults from hosts and natural environments.C. difficile spores are resistant to antibiotics and can rapidly germinate into vegetative bacteria after antibiotic treatment is stopped. [109]In 2015, Gerding et al. [110] reported that administration of spores from nontoxigenic C. difficile strain M3 could prevent recurrent CDI.They hypothesized that nontoxigenic C. difficile strain M3 could compete for the same metabolic or adherence niche in the gastrointestinal tract with toxigenic C. difficile.Recently, an investigational oral microbiome therapeutic called SER-109, [21,86] composed of donor's live purified Firmicutes bacterial spores from ≈ 34 different genera, has been developed to compete metabolically with C. difficile for essential nutrients and modulate bile-acid profiles to reestablish resistance to colonization by C. difficile.Purified Firmicutes bacterial spores have shown high efficacy and safety in treating CDI (Table 2). [111]n April 2023, SER-109 (tradename: Vowst) received FDA approval as an oral microbiota drug, with the indication for the prevention of recurrent CDI.Because SER-109 is considered to restore the balance of gut microbiota rather than killing C. difficile.
Although the compositions of practical spores may vary among donors, the improved methodology involving solvent treatments and purification steps can increase safety without relying solely on donor screening. [21]Besides, oral administration and capsule preparation facilitate quantification of the therapeutic dose, [25] which can help reduce research heterogeneity.

The Future of Transplanting Fecal Bacteria
Although studies have shown that enriching the bacteria from feces did not reduce the efficacy of WMT, [24,56] it is worth noting that some substances, such as viruses, microbial secretions, and metabolites, have been mostly washed out.Exploring more precise bacteria for transplanting is an emerging trend in the development of microbiota-based therapies.Strain level diversity and complementarity are considered to be the strongest determinants of FMT results, [16] and more accurate sequencing strategies would contribute to evaluating the efficacy or selecting the most suitable enriched bacteria for patients. [112,113]Live biotherapeutic products consisting of defined bacterial consortia, such as VE303, have also demonstrated stable and effective results. [36,114]owever, it may eliminate certain fecal components whose functions are not yet fully understood but are believed to play crucial roles.Although existing sequencing techniques are adequate for identifying the organisms present in the gut microbiota and inferring their functional potential, they fall short in pinpointing which species are more active and their specific functional contributions within the human body.To address this limitation, mature multiomics sequencing strategies, such as the introduction of metatranscriptome analysis, can be employed. [115]Besides, the human gut microbiome is a highly complex and heterogeneous ecosystem, with significant variations even within the same individual's microbiota. [116]Careful attention to experimental design, including refined sample collection processes with reduced variation, and enhanced data analysis techniques such as absolute microbiome measurements, can also contribute to overcoming the challenges associated with certifying the efficacy of defined bacterial consortia across a large and diverse population. [117] The Development of Transplanting Fecal Virome

The Methodology and Delivery Routes of FVT
Viral therapy can date back to at least the 20th century, as bacteriophages were discovered in 1915 [118] and their use in treating dysentery was reported shortly after. [119]Among human gut bacteriophages, the tailed double-stranded DNA Caudovirales order and the single-stranded DNA Microviridae family are the most common.As the main vector of horizontal gene transfer, bacteriophages can strongly influence bacterial evolution, diversity, and metabolism. [120]Therefore, bacteriophages play a key role in shaping the community structure and stability of human gut microbiota. [35]In recent years, researchers have found that the gut virome is associated with several diseases, including recurrent CDI, [121] IBD, [122,123] metabolic syndrome, [124] GVHD, [125] and even cancer. [126]One angle of research supporting the role of the virome is the association between efficacy and overlapped viral communities.Studies [17,121,127] of recurrent CDI indicated that recipients who benefited from FMT had an increased relative abundance of Microviridae and a decreased abundance of Caudovirales.These results suggested that the Microviridae family and Caudovirales order potentially played a pivotal role in the efficacy of FMT.Moreover, Broecker et al. [128] observed that stable bacteriophages remained present for at least 4.5 years following FMT and were better correlated with successful FMT than bacterial communities.The negative correlation between recovery of the virome and CDI recurrence can further certify the vital role of the virome in FMT.For example, one study [121] reported that CDI patients who only had restored bacterial communities suffered from disease recurrence.In subjects with metabolic syndrome who did not respond to FMT, the differences between the viral communities shared by the recipient and the respective donor were greater. [124]nother angle that demonstrates the potential of the virome is the difference in virome characteristics between disease and health.Several studies have shown an increasing richness of Caudovirales in patients with IBD compared to healthy participants. [123,129,130]Furthermore, a recent study [131] indicated that transplanting the virome of UC patients into mice could exacerbate the severity of DSS colitis, leading to shortening colon length and increasing pro-inflammatory cytokine production such as TNF- and IL-1.This study excluded the interference of endotoxin in the virome preparations, such as lipopolysaccharide, because mice given heat-killed virome displayed reduced colitis severity compared to those given intact virome of UC patients. [131]Gut virome also relates to environmental factors, such as diet [132] and antibiotic exposure, [133] which are known as risk factors for IBD.138] GVHD is a serious complication that is one of the most common causes of death arising from allogeneic hematopoietic stem cell transplant. [139]Immunosuppressants are typically used to treat GVHD, and a study found a higher presence of eukaryotic viruses relative to bacteriophages in the GVHD patient before FMT, [125] which may be connected to immunosuppressant use. [140]Compared to individuals without GVHD after allogeneic hematopoietic stem cell transplantation, enteric GVHD patients have exhibited a decrease in phage richness and a continuous increase in the number and detection rate of DNA viruses (Anelloviridae, Herpesviridae, Papillomaviridae, and Polyomaviridae). [141]In the latest meta-analysis, viruses mainly associated with butyrate-producing bacteria were found to be reduced in patients with colorectal cancer. [126]Butyrate-producing bacteria, such as F. prausnitzii, have anti-tumorigenic properties and might contribute to preventing colorectal cancer development. [142]Hence, differences in the virome may serve as cancer-associated viral markers to guide future practice.
FVT has emerged as a potential alternative to FMT.Unlike transplanting the entire fecal microbiome, FVT focuses on transplanting the virome in the feces of donors.To prepare the fecal virome, bacteria are removed from the donor feces through a sterile preparation process that employs filters with pore sizes smaller than bacteria (Table 1). [19,35]This allows for the retention of the virome while removing potentially harmful bacteria.Accordingly, FVT has the potential to decrease the risk of invasive bacterial infections. [17,143]Of note, the delivery route of FVT varied in different studies, such as nasojejunal tube, [19] orogastric, [131] and enema (Table 2). [144]This variability could be attributed to the fact that FVT is still in the exploratory stage and has yet to be widely adopted in clinical practice.

The Efficacy and Safety of FVT
In 2017, a preliminary investigation mentioned that the fresh sterile fecal filtrates (with fecal virome) could alleviate symptoms of patients with CDI (Table 2). [19]After FVT, they restored normal stool habits and experienced the elimination of CDI symptoms for at least 6 months.Although this study had a small sample size and was non-randomized, the significant benefits to patients gave researchers confidence to study FVT further.
In metabolic diseases, a mice study [20] demonstrated that FVT induced the reduction of symptoms of type 2 diabetes (T2D) and obesity.They observed that the reduced Shannon Diversity Index of the bacterial community in mice that were fed an HFD could be restored to normal levels after receiving FVT from lean donors.Besides, they found increasing gene expressions such as Lepr, Klb, Ppargc1a, and Igfbp2 in ileum tissue (Table 2).Importantly, previous studies [145][146][147][148] have proved a positive correlation between all of the above genes with protection against T2D or lower body weight.A recent study yielded similar results, showing that virome-induced perturbations could modify the fecal microbiome in vivo, and this alone was adequate to induce both lean and obese phenotypes in mice. [149]Moreover, a high-fat diet not only increases the risk of obesity and T2D but also disrupts the composition and function of the gut microbiota, leading to a range of gastrointestinal disorders including small intestinal bacterial overgrowth (SIBO).In a study conducted on mice, [150] it was observed that after being on a high-fat diet for 30 days, the intervillous zone of the ileum was occupied by a dense microbiota, which is a characteristic of SIBO. [151]Another study [106] created a mice model of SIBO by feeding them a high-fat diet.These mice became donors and provided fecal transplants for mice on a standard diet or high-fat diet.The researchers found that both FMT and FVT could convert the gut microbiota of healthy mice into high-fat diet-related components.Intriguingly, although FMT and FVT had similar effects on the recipients, mice fed with different diets had different results.The bacterial density of recipients on a high-fat diet reduced, while those on a standard diet did not (Table 2).This result suggests that both FMT and FVT could have similar effects on normalizing the mice with SIBO.SIBO has been identified in up to 78% of IBS patients. [152,153]Antibiotics are typically used as the firstline treatment for SIBO.However, a study showed that 43.7% (35/80) of SIBO patients experienced a relapse 9 months after completing antibiotic treatment. [154]Therefore, there is a need for alternative therapies, and FVT may have the potential to be one such therapy.Although other studies did not directly involve the treatment of certain diseases, they demonstrated the potential of FVT in replicating the effects of certain diets.One such study explored the effects of FMT or FVT, prepared from mice fed with green tea polyphenol, on mice with DSS-induced colitis. [155]he study revealed that both FMT and FVT could ameliorate DSS-induced colitis, as indicated by decreased inflammation factors in the plasma and improved symptoms.However, the degree of improvement observed after FMT was better than that of FVT.In another study, it was found that weight gain in mice, induced by feeding risperidone, could be achieved by FVT prepared from mice fed with risperidone. [156]Furthermore, a recent study demonstrated that FVT derived from mice with a high relative abundance of A. muciniphila could enhance the proliferation of commensal gut A. muciniphila in the recipients.Surprisingly, this also resulted in an improvement in fertility rates. [157]However, the underlying mechanism behind this effect warrants further investigation.
Strategies are also needed to address gut microbiota dysbiosis caused by broad-spectrum antibiotics. [158]An animal study [107] showed that autochthonous FVT (collected and frozen before antibiotic treatment) can reshape the gut microbiota after antibiotic treatment (Table 2).After 15 days of initial gavage, the mice that received FVT showed less difference in abundant taxa (14% of fecal OTUs and 36% of cecally derived) when compared to the initial microbiota, whereas the difference was 87% and 50% in mice treated with heat-or nuclease-treated FVT, respectively.This study suggested the potential of FVT in preventing gut microbiota dysbiosis following the use of antibiotics.
FVT might be effective for preventing necrotizing enterocolitis (NEC) in preterm infants. [108]NEC is a tough challenge to clinicians because of its high frequency, lethality, and unpreventable characteristics in preterm infants. [159]A study conducted on preterm piglets showed that collecting fecal materials from healthy suckling piglets and using FVT with frozen virome to prevent NEC in preterm piglets was effective. [108]After stomatogastric FVT, the macroscopic and microscopic incidence of NEC reduced to 0% and lower than 20%, respectively.However, rectally administered FMT failed to reduce the severity and incidence of NEC compared to control groups (Table 2).The recognized microbiota feature of NEC in neonatal units is increased Proteobacteria and reduced Bacteroidetes relative abundances. [144]Interestingly, stomatogastric FVT increased viral diversity and decreased Proteobacteria relative abundance in the ileal mucosa, highlighting the potential of FVT as a promising intervention for NEC prevention in preterm infants. [108]Importantly, in this study, there was no significant alteration observed in gene expressions, suggesting that the virome did not have a substantial impact on host immune cells to induce mucosal immunity.This finding contrasts with the previously discussed study that FVT could improve T2D by altering tissue gene expression. [20]

The Future of Transplanting Fecal Virome
Although transplanting the fecal virome has shown significant potential in treating some diseases (Figure 2), the concept is still in its early stages.Bacteriophages, which are predominant in gut virome, may be sufficient for contributing to the efficacy of transplanting virome, [160,161] but the contribution of metabolites, proteins, and bacterial fragments in the sterile filtration cannot be excluded. [162,163]There are two possible explanations for the effectiveness.One is that bacterial cell wall components or DNA fragments make efforts by stimulating host response. [162,163]Another is the interaction of bacteriophages and gut microbiota, resulting in the resolution of initial gut dysbiosis. [160,161]Of note, bacteriophages rely on bacteria as hosts to function. [164,165]They can either destroy the bacteria (virulent or lysogenic phages) or modify their behavior (lysogenic phages), including drug resistance and virulence.These phages show promise in combating pathogen-led diseases like CDI and antibiotic-resistant bacterial infections. [166,167]They can also help regulate the intestinal environment without the need for excessive microbiota supplementation. [168]However, their effectiveness may be limited in diseases caused by microbiota dysbiosis.Moreover, there is a risk of exacerbating gut microbiota disorders by introducing inappropriate viral groups.Many pathogenic eukaryotic viruses exist in the human gut, including papillomaviruses, herpes viruses, hepatitis viruses, bocaviruses, enteroviruses, rotaviruses, and sapoviruses. [169]Norovirus transmission to patients with CDI via FMT has been reported because these patients developed post-FMT norovirus gastroenteritis. [170]Besides, some unwanted phage-encoded genes may be transferred by bacteriophages, [128] which can induce antibiotic resistance or bacterial virulence.For example, Bacteroidetes, Firmicutes, Verrucomicrobia, and Fusobacteria gained antimicrobial peptide resistance amiABC after FMT by inserting these lysogenic phage genomes into their genome. [17]he characteristics of phages suggest that the development of the transplanted virome is closely intertwined with transplanted bacteria.This implies that the ongoing research directions focused on transplanted bacteria have the potential to shed light on the role of the virome as well.However, there is still a need for significant advancements in terms of efficacy, mechanisms, sequencing identification, and other technical aspects related to virome.Microbiota medicine, as an emerging clinical medicine discipline, [39] can integrate various microbiome-related technologies with FMT as the core, and employ education as the core force to mitigate the challenges faced by FVT.

The Sequencing and Big-Data Analysis Technology for Microbiome
Microbiome research has rapidly advanced due to the widespread application of next-generation sequencing (NGS) technology, which enables the discovery and characterization of microbes at a large scale.16S rRNA sequencing, [171] shotgun metagenomic sequencing (for all the DNA), [172] and RNA sequencing [173] can be used to study the mechanism of transplanting bacteria.Importantly, 16S rRNA sequencing cannot detect strain-level changes while shotgun metagenomic sequencing and RNA sequencing can. [174][177] Compared with bacteriome, virome requires a higher standard of sequencing technologies because of the amplified background noise interference and lower annotated rate of virome. [178]Shotgun metagenomic sequencing and RNA sequencing can study the mechanism of transplanting the virome (DNA and RNA virus, respectively). [172,173]However, DNA or RNA virus alone cannot represent the whole virome.Besides, the characteristics of NGS, such as short read length and long sequencing time, [179] would provide both biased and fragmentary knowledge of virome.Oxford Nanopore Technology (ONT) is one of the single-molecule real-time (SMRT) sequencing technologies. [180]The improved sequencing can provide genomelength reads that cover the entire mutation within a single virus particle, [181] and information on epigenetic modifications. [182]A complete working protocol has been established to profile gut virome using physical enrichment, reverse transcription, random amplification, and eventually the SMRT platform of ONT. [181]esides, hybrid and ultra-deep metagenomic sequencing represent crucial sequencing strategies for providing a comprehensive understanding of the genomic and functional aspects of the virome. [183,184]As sequencing technology continues to advance, it will lead to an exponential increase in data, commonly referred to as big data, making it imperative to develop robust methods for processing and analyzing this wealth of information.These analytical strategies for handling big data have the potential to greatly enhance our exploration of the intricate interactions between the gut microbiome and the human body. [185]Moreover, they hold the promise of enabling precision medicine through the microbiome, encompassing precise drug selection, [186] and tailored approaches to cancer treatment, [187] among other medical applications.

Allogenic or Autologous Transplantation
Almost all bacteria and viromes used for transplantation are collected from allogenic donated feces (Table 2).The recent approval of Rebyota as a new drug by the FDA was based on the allogenic healthy fecal matter. [188]An intriguing idea proposed by Ke et al. suggests the creation of a personal microbial Noah's ark using stool banks to collect hosts' stool samples at optimal health and cryopreserve them for future autologous transplantation. [189]This approach may increase the safety of transplanting bacteria or the virome in future practice.[192] Several studies involving at least 792 healthy adults in China have shown significant variation in virome, mycobiome, and archaeome between residents from rural and urban regions, [190][191][192] with geography, urbanization, and diet have a strong impact on the microbiome.

Conclusions
Increasingly studies have shown that FMT has great potential in clinical practice for many diseases.The next generation of FMT for dysbiosis-related diseases, including CDI, antibioticsassociated dysbiosis, metabolic diseases, and others, will likely involve transplanting bacteria (including WMT and purified bacteria spores) or the virome (including FVT).While most current studies use allogenic donated feces for preparing transplants, autologous transplantation may also be an option in the future.All developed microbiota transplantation or microbiota-based drugs can be delivered through colonic TET according to physicians' decision.The new sequencing technology for providing genomelength reads containing all mutations will help improve the development of FMT.In summary, the next generation of FMT is mainly moving forward for transplanting the enriched bacteria or the virome from healthy donors.
Recurrence rates of CDI were significantly lower in the SER-109 group (12%) compared to the placebo group (40%).Most AEs were mild to moderate and similar in both groups.mid-gut TET 74.3% (81/109) and 51.4% (56/109) of patients achieved clinical response at 1 month and 3 months after WMT, respectively; and 25.7% (28/109) and 20.2% (22/109) of patients achieved clinical remission at 1 month and 3 months after WMT, respectively.During the follow-up period of one-five years, 17.4% of AEs occurred.No AE beyond one month was observed.
patient was diagnosed with UC (E3, severe activity, Mayo score = 11), C. glabrata infection, adrenocortical insufficiency, osteoporosis, femoral head necrosis, psoriasis, Hashimoto thyroiditis, and moderate malnutrition.After five times of WMTs and then plus cyclosporine, the stool frequency decreased from 10-15 times per day to twice per day 2 weeks later.The fecal fungus culture remained negative 3 and 6 months after the first WMT.
individuals Mid-gut TET During one month after WMT, 13.6% of mild AEs occurred.No AE beyond one month was observed.The rate of clinical response and clinical remission was 45% (9/20) and 20% (4/20) in the patients with AE, and 75.6% (90/119) and 63.0% (75/119) in the group without AE.
the hyperlipidemia group, hyper-blood lipid decreased to normal within 33-47 days (35.14%; p <0.001), and LDL-C changed to normal within 34-63 days (33.33%; p = 0.013) after WMT.In the hypolipidemia group, 36.36% and 47.06% changed to normal within 33-47 days (p = 0.006) and 34-63 days (p = 0.005) after WMT.In the normal blood lipid group and the low-risk group of atherosclerotic cardiovascular disease, the change was not statistically significant after WMT.

Figure 2 .
Figure 2. The applications of transplanting bacteria or virome.Applications that have clinical or preclinical evidence of transplanting bacteria or virome are listed on the corresponding side.If an application has evidence on both sides, it is highlighted in red.

Table 1 .
The preparation methodology for fecal transplants.

Table 2 .
Summary of clinical and animal studies into transplanting bacteria or virome.

Table 2 .
1-grade reduction in Radiation Therapy Oncology Group (RTOG/EORTC) late toxicity grade from baseline by 8 weeks after WMT.No WMT-induced death and infectious complications were observed.One mild AE occurred in case 4, who experienced transient nausea immediately after WMT and self-resolved within 24 hours.