Methodological insights into fecal microbiota transplantation: Dissecting key approaches for success

Abstract

Fecal microbiota transplantation (FMT) has emerged as a revolutionary treatment strategy for restoring gut microbiota in recurrent Clostridioides difficile infection and has also been explored across a broader range of dysbiosis-related diseases such as inflammatory bowel disease where it has demonstrated promising results and potential therapeutic benefits. The success of FMT largely depends on the careful implementation of best practices, which include selecting appropriate donors, preparing the stool properly, and choosing the right delivery methods. This mini-review explores the evolution of FMT methodologies, including donor screening protocols, advances in stool preparation, and innovations in administration routes. We also discuss emerging approaches, such as synthetic microbiota and microbiome engineering, alongside the challenges and future directions for standardizing FMT. These methodological advancements aim to enhance safety, efficacy, and accessibility of FMT, establishing it as a key player in microbiome-based therapies.

Key Words: Fecal microbiota transplantation; Microbiome; Fecal microbiota transplantation methodology; Fecal microbiota transplantation challenges; Standardization of fecal microbiota transplantation

Core Tip: Fecal microbiota transplantation (FMT) is a promising therapy for restoring gut microbiota, with established success in recurrent Clostridioides difficile infection and emerging potential in dysbiosis-related diseases. Key methodological advancements, including improved donor screening, stool processing, and novel delivery methods like capsule-based formulations and live biotherapeutic products, have improved FMT’s safety and accessibility. Despite ongoing concerns about long-term safety and efficacy, future directions such as personalized FMT, synthetic microbiota, and microbiome engineering hold significant promise.

INTRODUCTION

Gut microbiota plays an integral part in maintaining human health, influencing digestion, immunity, metabolism, and even brain function (gut-brain axis)[1]. Maintaining a balanced and diverse microbiota is key to preventing disease and supporting overall health. Alterations in this microbial community, known as dysbiosis, is implicated in a wide range of conditions like Clostridiodes difficile infection (CDI), inflammatory bowel disease (IBD), obesity, diabetes, autoimmune diseases, neuropsychiatric disorders, cardiovascular diseases, liver diseases, cancer, irritable bowel syndrome and many others[1-3].

One of the defining features of dysbiosis in chronic inflammatory intestinal diseases is a marked decrease in microbial alpha diversity, particularly involving the loss of key beneficial commensal organisms such as Bacteroides, Firmicutes, Clostridia, Ruminococcaceae, Bifidobacterium, Lactobacillus, and Faecalibacterium prausnitzii, the latter recognized for producing anti-inflammatory metabolites. This microbial depletion is accompanied by an increased abundance of potentially pathogenic organisms, notably those of the Proteobacteria class such as adherent-invasive Escherichia coli, as well as Fusobacterium, both of which are associated with mucosal inflammation and disease exacerbation[4]. Functionally, these microbial shifts result in diminished synthesis of short-chain fatty acids (SCFAs), such as butyrate and propionate, which are essential for preserving epithelial barrier integrity and regulating host immune responses. The microbial capacity for amino acid biosynthesis is diminished. Instead, there is a rise in auxotrophy and an upregulation of amino acid transport systems, making a shift towards microbial communities that are less self-sufficient and more dependent on host-derived nutrients. Additionally, an increase in sulfate transport, increased oxidative stress and upregulation of type II secretion systems, which facilitate toxin secretion occur[4]. Collectively, these changes underscore a transition from a symbiotic to a pathogenic microbiome, which can perpetuate inflammation and tissue damage in the gastrointestinal (GI) tract.

Fecal microbiota transplantation (FMT) is the process of transferring processed and well-screened stool from a healthy donor into the GI tract of a recipient to restore microbial diversity and functionality and cure specific disease conditions[5].

The mechanism of FMT is complex and can be divided into two primary categories[6]: (1) Direct interactions between donor and recipient microbiota, leading to competitive exclusion, where beneficial bacteria from the donor outcompete harmful pathogens in the recipient’s gut; and (2) Indirect alterations to the recipient's physiology mediated by the donor microbiota, such as restoration of SCFA-producing species, leading to attenuation of inflammation and restoration of gut barrier integrity; restoration of bile acid metabolism pathways leading to increased secondary bile acids necessary for colonization resistance against pathogens; and immune-modulatory effects by improving T cell function, particularly in situations where immune exhaustion or resistance occurs. Additionally, FMT has been associated with reduced oxidative stress in the intestinal environment, likely through rebalancing redox-active microbial species and reducing host-derived reactive oxygen species (ROS), which collectively support mucosal healing and immune equilibrium.

FMT is the most effective therapy recommended by guidelines for recurrent CDI (rCDI) in order to prevent further relapses and can be used to treat severe or fulminant cases of CDI that do not respond to antibiotics[7]. FMT is typically considered for patients who have experienced a second CDI recurrence or a third CDI episode, or in select patients at high risk of either rCDI or a morbid CDI recurrence. Select use includes patients who have recovered from severe, fulminant, or particularly treatment-refractory CDI and patients with significant comorbidities[7]. The recurrence rates of CDI are significantly lower after FMT (5%-15%) compared to those following antibiotic treatments with vancomycin (35%-65%) or fidaxomicin (25%)[8]. FMT is currently indicated primarily for rCDI by guidelines, nevertheless, increasing evidence suggests that FMT may be effective in treating disorders beyond CDI, including ulcerative colitis (UC) and other dysbiosis-related disorders, though further research is needed for many of these conditions.

The success of FMT is highly dependent on well-defined methodologies. This mini-review discusses the methodological advancements in FMT, including donor selection, stool preparation, administration techniques, regulatory considerations, and emerging technologies aimed at optimizing efficacy and safety.

HISTORY AND MODERN RESURGENCE OF FMT

FMT has historical roots dating back to 4^th century China, where it was described as “yellow soup” for treating severe diarrhea[9]. In modern medicine, FMT first gained attention in 1958, when Dr. Ben Eiseman successfully treated pseudomembranous colitis, later recognized as CDI, using fecal enemas[10]. FMT’s resurgence began in the early 2000s, as recurrent CDI emerged as a major healthcare challenge due to the indiscriminate use of antibiotics. The landmark 2013 randomized controlled trial (RCT) by van Nood et al[11] demonstrated FMT’s superior efficacy over antibiotics in recurrent CDI, cementing its role in clinical practice. Since then, numerous trials have confirmed FMT’s effectiveness in rCDI, while others have explored its potential in conditions beyond CDI, yielding promising results. Additionally, the first standardized FMT-based product, RBX2660 (Rebyota)[12], received FDA approval in 2022, followed by SER-109 (Vowst)[13] in 2023, both for rCDI. These approvals signal a pivotal shift towards live biotherapeutic products (LBPs), which are microbiome-based therapies that offer safer, more consistent, and standardized alternatives to traditional FMT. As research in next-generation microbiome-based therapies, synthetic consortia, and precision-microbiome engineering continues to progress, these innovations are poised to further transform the field.

METHODOLOGY

Methodology involves the comprehensive process of donor selection and screening, stool processing and administration to ensure the safety, efficacy and quality of FMT.

Donor selection and screening

Selecting an appropriate donor is critical to ensuring the safety and efficacy of FMT. The ideal donor should meet stringent health criteria to minimize the risk of transmitting infections or causing adverse effects in the recipient.

The ideal donor age range is typically between 18 and 50, as this age range is associated with optimal overall health and a well-balanced, diverse gut microbiota[14,15]. Health care workers are usually excluded due to the higher risk of colonization with Clostridiodes difficile.

An appropriate informed consent is obtained from all potential donors. The screening process begins with a thorough evaluation of the candidate’s medical history, including a clinical questionnaire (Table 1) that screens for infectious diseases, chronic illnesses, high-risk behavior, recent travel, treatments, and other factors that may compromise gut microbiota or pose risks to recipients[5,15]. Individuals with any of these conditions or risk factors are not considered eligible donors. If the recipient has any known food or medication allergy, the donor must not ingest the allergen for several days before donation[5].

Table 1 Clinical questionnaire to select potential donors.

	Question
Medical history	Any history of
	Concurrent acute, medical illness?
	Any symptoms pertaining to gastrointestinal disease (nausea/ vomiting/ pain abdomen/ diarrhea/ blood in stool)?
	Chronic gastrointestinal (GI) disease (personal or family history), including functional GI disorders, inflammatory bowel disease, celiac disease or other chronic GI disease
	Any history of chronic illness (such as diabetes/ hypertension/ heart disease/ kidney disease/ liver disease/ HIV/malignancy)?
	Acute diarrhea (in the prospective donor or his/her contacts) in the past four weeks
	Typhus or other salmonellosis
	Tuberculosis (acute or past)
	Systemic autoimmune diseases, e.g., multiple sclerosis, connective tissue disorders
	Atopic diseases
	Neurological or psychiatric diseases [depression, chronic pain (fibromyalgia, chronic fatigue syndrome, etc.)]
	Obesity (body mass index > 30)
	Known food or medication allergy
	Cancer, including GI cancer or polyposis syndrome
Travel history	Any history of high-risk travel in the past six months
Social history	Any history of
	High risk sexual behavior
	Imprisonment
	Illicit drug use
	Body modifications such as tattooing, piercing, etc., in the past six months
	Occupational activity in a hospital or other health-care institution with patient contact; occupational activity in agriculture
Family history	Any history of premature colon cancer or early onset polyposis syndromes in first-degree family members
Treatment history	Any history of
	Recent hospitalization/discharge from long-term care facilities (past 6 months)
	Antibiotic treatment in the past three months
	Blood transfusions in the past five years
	Long-term drug treatment (e.g., with antibiotics, immunosuppressants, proton pump inhibitors, chemotherapy)
	Tissue/organ transplant
	Having been a participant in any clinical trial in the past six months
	Bowel resection or gastric reduction surgery

All candidate donors who have passed the medical interview must undergo blood and stool testing to exclude potentially transmittable diseases (Table 2)[15].

Table 2 Donor blood and stool testing.

Blood testing
Complete blood count with differential
Blood sugar, renal and liver function tests
Hepatitis A, hepatitis B, hepatitis C and hepatitis E viruses
HIV-1 and HIV-2
Cytomegalovirus and Epstein-Barr Virus serology
Treponema pallidum (syphilis)
Nematodes (Strongyloides stercoralis)
Stool testing
Clostridioides difficile
Common enteric pathogens, including Salmonella, Shigella, Campylobacter, Shiga toxin-producing Escherichia coli, Yersinia and Vibrio cholerae
Antimicrobial-resistant bacteria and antimicrobial resistance gene testing
Norovirus, rotavirus, adenovirus
Giardia lamblia, Cryptosporidium spp., Isospora, Cyclospora and Microsporidia
Protozoa and helminths/ova and parasites (including Entamoeba histolytica, Blastocystis hominis and Dientamoeba fragilis)
Helicobacter pylori faecal antigen (for upper route of FMT delivery)

FMT: Fecal microbiota transplantation.

On the day of donation, donors must complete a questionnaire to identify any recent health changes, including new signs and/or symptoms (diarrhea, nausea, vomiting, abdominal pain, jaundice, fever or swollen lymph nodes), medication use, high-risk behavior, or travel that may impact donor suitability[15]. Donors who provide stool samples repeatedly undergo clinical assessment and a complete panel of laboratory testing every 8-12 weeks[15].

Stool collection, processing and storage

Donors should collect the stool sample in a clean, wide-mouthed, sterile and leak-proof container, avoiding contamination with urine or water. Thorough hand hygiene must be observed before and after the collection process. Ideally, the sample should be freshly passed and processed within 6 hours of collection to preserve microbial viability and composition (six-hour FMT protocol). If immediate processing is not possible, the stool can be temporarily stored at 4 °C (refrigeration) for short durations or frozen at -80 °C for long-term storage, especially for preparation of capsule-based formulation. Each sample should be appropriately labelled and documented, including donor identification, date, and time of collection[14,15]. Table 3 highlights different steps in stool collection, processing and storage for FMT[5,14-23].

Table 3 Stool collection, processing and storage.

Processing step	Description	Common variations/methods
Stool collection	Freshly passed stool is collected from a healthy, screened donor in a sterile container[17]	Anaerobic conditions: Certain protocols recommend oxygen-free collection and processing methods to preserve strict anaerobes[14,18,19]
		Pre-collection diet: Some studies suggest a high-fiber diet prior to donation to enhance microbial diversity[20,21]
Stool quantity	The amount of stool collected varies based on the protocol	Typically, 50-60 g of stool added to 250-300 mL of diluent[5,17]. A minimum of 25 g of feces is recommended for lower GI delivery, and 12.5 g for upper GI delivery[14,15]
Diluents/buffer solutions	Stool is diluted with a buffered solution to create a homogeneous slurry, and to a consistency that can be injected through the biopsy channel of scope	Commonly used diluents[5,22]:
		Normal saline (0.9% NaCl)-Most commonly used
		Sterile water (less preferred due to hypotonicity)
		Phosphate buffered saline
		Non-bacteriostatic saline (used to avoid antimicrobial preservatives)
		Milk or milk-based solutions (rarely)
Homogenization	Stool sample is mixed with the diluent to create a uniform suspension, ensuring even distribution of microbiota and removal of large particulate matter before filtration	Done by manual stirring with sterile spatula; laboratory blender. Performed under aerobic or anaerobic conditions[18,19]
Filtration	Large particles (undigested fiber, debris) are removed	Using[5]:
		Gauze filtration
		Stainless steel sieve (250-500 μm)
		Sterile mesh filters
Centrifugation	Further purification or concentration of microbiota; retain microbial rich supernatant	Low (3000-4000 × g), medium (5000-6000 × g) or high (up to 10000 × g) speed centrifugation; relative centrifugal force = 6000 × g for 15 minutes[23]. May be followed by re-suspension in diluent
Washing (optional step)	Component of microfiltration plus centrifugation, called washed microbiota transplantation	Integration of multiple filtration and washing steps, often performed using automated purification systems[16]; automated systems (e.g., GenFMTer) reduces host cells, debris, endotoxins (one-hour FMT protocol)[16]
Storage and handling	Stool preparation is handled under strict sterile conditions to prevent contamination. Can be used fresh or stored	Fresh use: Stored at 4 °C and used within 6 hours
		Frozen use: Stored at -80 °C with glycerol (10%) for long-term use (up to 2 years)

GI: Gastrointestinal; FMT: Fecal microbiota transplantation.

Prior to use, donor stool undergoes rigorous laboratory processing, typically classified into three main methods: (1) Rough filtration; (2) Filtration plus centrifugation, and (3) Microfiltration plus centrifugation (MPC). These methods involve increasing levels of purification and standardization[16]. Table 4 shows a comparative analysis of different stool processing methods.

Table 4 Comparative analysis of different stool processing methods.

Method	Pros	Cons	Recommended setting
Rough filtration	Simple, low cost	Low microbial purity, higher adverse events	Suitable for basic setups
Filtration plus centrifugation	Improved bacterial concentration	Moderate technical demands	Used in standard protocols
Microfiltration plus centrifugation	High microbial purity, fewer toxic reactions, delivers a precise dose of the enriched microbiota instead of using the weight of stool	Expensive, automated system e.g., GenFMTer needed	Ideal for advanced centers

All handling should follow standard biosafety procedures to prevent exposure or cross-contamination. The production of capsulized FMT for oral delivery also may involve the addition of a lipid emulsion so that capsules remain stable for ingestion[17].

Washed microbiota transplantation

Washed microbiota transplantation (WMT) utilizes a technique known as MPC, which involves fine filtration followed by centrifugation to concentrate and purify the microbiota. This method, often carried out using automated systems such as GenFMTer, includes multiple washing steps to effectively remove fecal residues, host cells, pro-inflammatory metabolites, soluble molecules and other impurities thereby improving the consistency and safety of the final transplant material. Compared to traditional FMT, WMT has shown promise in achieving better clinical outcomes with fewer side effects, particularly in patients with rCDI and other microbiota-related disorders[16,24].

Fresh vs frozen FMT

Frozen FMT involves processing the stool and storing it at -80 °C for future use. After stool collection and filtration to remove large particulate matter, the suspension is mixed with a cryoprotectant solution, commonly 10% glycerol, to preserve microbial viability during freezing[15]. The processed suspension is then aliquoted into sterile containers or syringes in standardized volumes. These are rapidly frozen and stored at -80 °C, allowing for long-term use.

On the day of fecal infusion, the frozen sample is thawed in a warm water bath at 37 °C and infused within 6 hours from thawing. If required, sterile saline can be added to obtain a desired suspension volume. It is critical to avoid repetitive freezing and thawing, as this can significantly reduce bacterial viability and compromise treatment efficacy[15].

A pivotal randomized, double-blind, non-inferiority trial conducted by Lee et al[25] enrolled 232 adults with recurrent or refractory CDI, comparing frozen (n = 114) and fresh (n = 118) FMT via enema. Clinical resolution at 13 weeks was 83.5% for frozen and 85.1% for fresh FMT, demonstrating non-inferiority (P < 0.001) and confirming that frozen FMT is equally effective as fresh FMT.

A network meta-analysis involving eight studies in rCDI demonstrated comparable effectiveness between fresh and frozen FMT with success rates of 93% and 88% respectively (P = 0.18)[26]. Similarly, a meta-analysis of 22 studies in UC showed no significant difference in clinical efficacy between fresh and frozen preparations, with remission rates of 34.4% and 46.8%, respectively[27].

Pre-FMT recipient conditioning

Pre-FMT recipient conditioning typically includes the discontinuation of antibiotics 24-48 hours prior to the procedure, known as the washout period[7]. This interval helps reduce residual antimicrobial effects that could hinder engraftment of the donor microbiota. In most protocols, a bowel lavage is performed during this interval, especially for lower GI delivery, to eliminate residual stool and antibiotic remnants, further facilitating microbiota colonization. However, the necessity of bowel preparation remains uncertain, as large RCTs supporting the efficacy of FMT for rCDI have been conducted both with and without prior bowel lavage[17]. Proton pump inhibitors (PPIs) may be administered before upper GI FMT routes to reduce gastric acid exposure[7]. Screening for GI obstruction or motility disorders is an important step before FMT, especially when using upper GI routes, to reduce the risk of complications such as aspiration or perforation. Additionally, obtaining informed consent is essential to ensure patient understanding of the procedure, risks, benefits, and alternatives.

Modes of administration

FMT can be administered through various routes, each with distinct advantages, limitations, and efficacy profiles. Table 5 summarizes the key features of different modes of FMT administration, including stool dose, suspension volume, and route-specific considerations[6,28].

Table 5 Comparison of common methods of fecal microbiota transplantation administration.

	Enema	Capsule	Colonoscopy	Nasogastric, nasoduodenal or nasojejunal tube
Delivery	Distal colon	Small intestine/colon	Right colon/terminal ileum	Upper GI tract to stomach/duodenum/jejunum
Starting amount of feces	25-200 g	80-100 g	25-200 g	12.5-150 g
Final delivery volume per dose	300-500 mL	30-40 capsules	30-500 mL	30-500 mL
Pros	Well tolerated, no sedation, can be done at home	Non-invasive, faster, well-tolerated	Higher efficacy	Minimally invasive, avoids sedation risks
Cons	Retention difficulty, lower efficacy	Requires multiple doses, risk of gastric acid degradation; avoid FMT capsules if the patient has dysphagia, difficulty swallowing pills or gastroparesis	Invasive, requires sedation, risk of perforation	Risk of aspiration, patient discomfort

GI: Gastrointestinal; FMT: Fecal microbiota transplantation.

A systematic review and meta-analysis[29] of 15 studies involving 1150 patients evaluated the efficacy of various FMT protocols for CDI. Multiple infusions significantly improved efficacy (76% vs 93%), regardless of delivery route. Colonoscopic delivery showed higher efficacy (P = 0.006), while duodenal delivery and fecal amounts ≤ 50 g were linked to lower success rates (P = 0.039 and P = 0.006, respectively). Importantly, the use of fresh or frozen stool did not impact outcomes.

Lower GI delivery method, such as colonoscopy has generally shown 5%-10% higher cure rates compared to upper GI routes[30,31]. However, clinical response to FMT can vary, and some patients refractory to lower GI delivery have shown improvement with oral capsule administration[32]. Therefore, while lower GI routes are generally preferred for higher efficacy, individual patient factors, tolerance, and feasibility should guide the choice of delivery method. More robust, head-to-head studies are needed to establish the optimal route for FMT delivery.

FMT administration technique

In colonoscopy, the fecal suspension is infused into the cecum or terminal ileum over 2-3 minutes using syringes connected to suction tubing which in turn is connected to the accessory channel of the colonoscope; if full insertion is not feasible, infusion may be done in the proximal or distal colon. The endoscope is then withdrawn carefully, aspirating air only from the distal colon for patient comfort. For upper GI delivery, a nasogastric, nasoduodenal or nasojejunal tube is used, with radiologic confirmation of placement, followed by slow infusion of desired volume to reduce nausea and aspiration risk. Enemas may be self-administered using squeeze bottles (50-60 mL) or gravity-fed enema bags (up to 300 mL). Patients should retain the enema for at least 4 hours, ideally overnight, to maximize microbiota engraftment and clinical efficacy[5].

Patient position post FMT

Following colonoscopy or enema, patients are generally advised to remain in a supine or lateral position for 30-60 minutes to promote retention of the transplanted material and reduce early expulsion. Alternating between right and left lateral positions may further aid in even distribution of microbiota throughout the colon. For upper GI FMT delivery (e.g., via nasogastric or nasoduodenal tube), placing the patient in a reverse Trendelenburg position (head elevated) can help minimize aspiration risk and facilitate gravitational transit of the fecal suspension through the GI tract[14].

FACTORS AFFECTING FMT SUCCESS

The success of FMT depends on donor, recipient, and procedural factors. Donor microbial diversity and composition, overall health, and lack of recent antibiotic use are crucial for effective engraftment. A high-fiber diet in donors may further enhance microbiota richness. In recipients, immune status, underlying conditions, and concurrent medications can influence outcomes. Those with profound dysbiosis might exhibit better microbial engraftment. Procedural elements such as the delivery route, stool preparation (fresh, frozen, or lyophilized) and pre-treatment with antibiotics or bowel cleansing also affect treatment outcomes. Additionally, multiple FMT sessions may be needed for sustained efficacy in chronic or relapsing cases. Optimizing these variables collectively is key to improving therapeutic results.

Figure 1 summarizes the key factors influencing FMT success, highlighting donor characteristics, recipient conditions, procedural variables, and environmental aspects that can impact microbial engraftment and clinical outcomes[33].

Figure 1 Factors affecting fecal microbiota transplantation success. FMT: Fecal microbiota transplantation.

SAFETY

FMT is generally considered safe, with most adverse effects being mild and self-limiting, such as abdominal discomfort, bloating, flatulence, low-grade fever, or transient diarrhea. Rare but more serious complications include infections, aspiration pneumonia (with upper GI delivery), and procedural risks like bleeding or perforation during colonoscopy[34,35]. Transmission of multidrug-resistant organisms has also been reported in isolated cases[36], emphasizing the need for stringent donor screening. Emerging concerns about potential long-term adverse effects, such as metabolic disturbances, altered immune responses, and theoretical risks of cancer or disease transmission, remain under investigation, though current evidence is limited[35,37]. Therefore, long-term follow-up after FMT is essential. Patients should be monitored over several years, with regular assessment of clinical symptoms and relevant laboratory parameters. Establishment of patient registries and implementation of large-scale, longitudinal observational studies will be critical to comprehensively evaluate the long-term safety profile of FMT.

EVALUATING CLINICAL EFFICACY OF FMT AND ENGRAFTMENT

Efficacy endpoints vary by indication. In rCDI, symptom resolution and recurrence rates are typically used, while in IBD, symptom resolution, changes in clinical scores (e.g., Mayo score) and mucosal healing are more common. Engraftment refers to the successful colonization of donor microbial strains in the recipient’s gut and is considered a key determinant of FMT efficacy[33]. Strain engraftment serves as a proxy for FMT success, yet its assessment lacks standardization due to challenges in defining microbial strains and limitations in sequencing depth. The persistence of engrafted strains over time and its impact on long-term clinical outcomes remain open questions, necessitating further research with well-defined clinical endpoints and longitudinal sampling[33].

THE 5D FRAMEWORK FOR FMT

The 5 Ds of FMT provide a structured approach to the FMT process[38].

Decision: Identifying appropriate indication for FMT.

Donor: Screening and selecting a suitable stool donor.

Discussion: Informed consent and patient counselling regarding risks, benefits, and alternatives to FMT.

Delivery: Choosing the optimal route of FMT administration.

Discharge: Post-procedure care, monitoring and follow-up.

STEP UP FMT STRATEGY

The step-up FMT strategy enhances treatment efficacy through escalating interventions.

Step 1: Single FMT.

Step 2: Multiple FMTs.

Step 3: FMT combined with conventional therapies, such as steroids or immunosuppressants.

Each step builds on the previous one, with medications introduced at Step 3 as the reconstructed gut microbiota may influence the host’s immune response, intestinal barrier integrity, and responsiveness to treatment. This strategy is particularly effective in managing refractory IBD, immune-related diseases, and severe or complicated CDI, especially when standard therapies fail[39,40].

INNOVATIONS IN FMT METHODOLOGY

Capsule FMT

Capsule-based FMT involves processing donor stool into a concentrated microbial slurry, which is either frozen with cryoprotectants (such as glycerol or trehalose) or lyophilized into a dry powder form. The material is then encapsulated in acid-resistant capsules for targeted intestinal delivery. Frozen capsules are stored at -80 °C and slightly thawed before use, while lyophilized capsules, containing freeze-dried stool powder, offer improved shelf-life and can be stored at room temperature or in a refrigerator, providing a more convenient, stable alternative[16,41,42].

LBP

LBP are an advanced alternative to traditional FMT, using defined microbial consortia instead of whole fecal material. LBPs enhance safety, standardization, and regulatory compliance by minimizing pathogen transmission while ensuring targeted microbial restoration.

Key LBPs in clinical use: (1) Rebyota™ (RBL): The first FDA-approved LBP, containing a broad microbial consortium with a defined Bacteroides threshold. In the PUNCH CD3[12] trial, a single rectal dose achieved a 70.6% success rate vs 57.5% with placebo in rCDI; and (2) Vowst™ (VOS): A capsule-based, Firmicutes spore-only LBP purified via ethanol processing. The ECOSPOR III[13] trial showed 88% sustained response vs 60% with placebo at 8 weeks (P < 0.0001).

LBPs represent a breakthrough in microbiome-based therapies, offering safer, standardized, and effective alternatives to traditional FMT. Their success in rCDI paves the way for broader applications in gut and systemic diseases.

Advanced microbiota delivery strategies

In a dextran sulfate sodium-induced murine colitis model, Liu et al[43] enhanced the probiotic Escherichia coli Nissle 1917 (EcN) using a double-layer coating of tannic acid (TA) and enteric L100. TA protected EcN from harsh GI conditions, while L100 improved mucoadhesion and colonic retention, boosting both prophylactic and therapeutic effects. In a follow-up study[44], EcN was further modified with ROS-scavenging nanoparticles, significantly enhancing its efficacy in inflamed colonic tissue. These novel material-based enhancements of probiotic delivery open exciting possibilities for next-generation FMT capsules, where such protective and targeted delivery systems could improve microbial viability, site-specific colonization, and therapeutic impact.

CLINICAL EVIDENCE SUPPORTING FMT

Currently, the only established indication of FMT recommended by guidelines is rCDI. It is primarily recommended to prevent further relapses in patients with multiple recurrences, typically after a second recurrence or third overall episode. In select high-risk individuals-such as those with severe, fulminant, or treatment-refractory CDI, or those with significant comorbidities, FMT may also be considered[7].

FMT in IBD

A systematic review and meta-analysis of six double-blind RCT involving 324 patients demonstrated that compared with placebo, FMT had significant benefit in inducing combined clinical and endoscopic remission (odds ratio, 4.11; 95%CI: 2.19-7.72; P < 0.0001)[45]. A recent network meta-analysis comparing therapies for induction of remission in UC found that FMT had a pooled odds ratio of 2.8 (95%CI: 1.4-5.8) vs placebo, which was comparable to biologics[46]. In terms of efficacy, infliximab ranked highest, followed by tofacitinib, ustekinumab, FMT, with vedolizumab and adalimumab ranking lower. A systematic review and meta-analysis of eleven cohort studies and one RCT involving 228 patients with Crohn’s disease showed that 57% (95%CI: 49%-64%) of patients achieved clinical remission 2 to 4 weeks after FMT[47]. While these studies indicate promise, heterogeneity in trial design, patient selection, delivery methods and endpoints make comparison difficult.

FMT in other dysbiosis-associated diseases

FMT is being increasingly explored as a therapeutic option for various dysbiosis-associated diseases. While it has demonstrated promising benefit in some conditions, ongoing research and clinical trials are investigating its potential in several others. Table 6 summarizes diseases where FMT has shown promise or is currently being studied[7,11-14,45,47-75]. It is, however, important to note that most non-rCDI applications remain investigational, with small sample sizes with heterogeneity limiting definitive conclusions.

Table 6 Fecal microbiota transplantation: Current uses and future horizons.

Established indication
Clostridioides difficile infection[7,11-14,48]
Investigational/emerging indications
Ulcerative colitis[45,49]	Primary sclerosing cholangitis[62,63]
Crohn’s Disease[47,50]	Intestinal pseudoobstruction[64]
Pouchitis[51]	Neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, multiple sclerosis)[65]
Irritable Bowel Syndrome[52]	Graft vs host disease[66]
Alcoholic hepatitis[53]	Human immunodeficiency virus[67]
Cirrhosis or hepatic encephalopathy[54]	Methicillin-resistant Staphylococcus aureus enterocolitis[68]
Obesity/metabolic syndrome[55]	Multidrug-resistant organisms[69]
Diabetes mellitus[56]	Post-stem cell transplant[70]
NAFLD/NASH[57]	Autologous FMT (preventive)[71,72]
Hepatitis B[58,59]	Autism spectrum disorders[73]
Pancreatitis[60]	Cancer[74]
Immune checkpoint inhibitor resistance[61]	Fecal incontinence[75]

FMT: Fecal microbiota transplantation; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis.

CONTRAINDICATIONS OF FMT

FMT is generally not recommended in the following clinical scenarios[76]: (1) Active systemic infection or sepsis; (2) Fulminant colitis or toxic megacolon; (3) Ongoing, significant GI hemorrhage; (4) GI perforation; (5) Significant luminal narrowing due to fibrotic strictures; (6) High output intestinal fistula; (7) Severe immunosuppression, whether congenital, acquired (e.g., HIV/AIDS), or iatrogenic (e.g., due to chemotherapy or immunosuppressive drugs); and (8) Pregnancy and lactation.

However, it is important to note that FMT has been explored in select cases of immunocompromised patients, including those with HIV/AIDS or other immunodeficiency states[34], with encouraging safety outcomes in certain contexts. Therefore, immunosuppression should not be considered an absolute contraindication. A careful risk-benefit assessment on a case-by-case basis is essential before proceeding with FMT in such populations.

REGULATORY VARIATIONS

FMT regulation varies globally, reflecting differences in risk perception, classification frameworks, and clinical use, as depicted in Table 7[33,77,78].

Table 7 Regulatory variations in fecal microbiota transplantation.

Country	FMT classification
United States	Restricted use in CDI in line with FDA enforcement discretion policy[78]; investigational new drug approval used in context of clinical trials for other diseases
Belgium, Netherlands, Italy	Regulated as a tissue transplant, under European Union Tissue and Cells Directive
Australia	Regulated as a biological drug
United Kingdom, Germany, France, Ireland, Switzerland	Regulated as a medicinal drug -flexible use allowed

FMT: Fecal microbiota transplantation; CDI: Clostridioides difficile infection.

CHALLENGES AND LIMITATIONS

Despite its therapeutic promise, FMT faces several challenges that limit its broader clinical application: (1) Variability in donor screening, stool processing, and delivery methods affects standardization and clinical outcomes; (2) Risk of transmitting undetected or drug-resistant pathogens despite screening; (3) Limited long-term safety data and unclear potential risks; (4) Regulatory ambiguity and classification inconsistencies (investigational drug vs biological product) hinder broader application; (5) Limited availability of eligible donors due to strict screening protocols; (6) Psychological discomfort or aversion to the idea of using fecal matter reduces patient acceptance and compliance; (7) Insufficient high-quality evidence for conditions beyond CDI; and (8) Storage, cold chain requirements and specialized facilities increase cost and complexity.

PERSONALIZED FMT AND PREDICTIVE MODELING

As our understanding of gut microbiota deepens, the traditional “one stool fits all” approach in FMT is being increasingly challenged, especially for conditions beyond rCDI. Treatment success in these cases depends heavily on strain-level microbial engraftment, highlighting the need for personalized FMT, where donor selection is matched to the recipient’s microbiome profile. While “super-donors” with high microbial diversity may offer benefits, optimal donor profiles are often patient-specific[33,79]. To aid this personalization, machine learning models, particularly random forest algorithms, are being used to predict donor-recipient compatibility and treatment outcomes by analyzing microbiome and host data[33,80]. As microbiome datasets expand, such tools will enable precision, data-driven FMT strategies to improve clinical effectiveness.

FMT-UNANSWERED QUESTIONS AND FUTURE IMPLICATIONS

Despite the growing success of FMT, several critical questions remain, guiding ongoing research toward its future applications. Figure 2 highlights the key unanswered questions and future directions of FMT.

Figure 2 Unanswered questions and future implications of fecal microbiota transplantation. FMT: Fecal microbiota transplantation.

CONCLUSION

FMT has emerged as a powerful tool in restoring gut microbial balance, with proven efficacy in rCDI and growing promise across a spectrum of dysbiosis-associated diseases. Methodological advancements, from donor screening and stool processing to novel delivery routes and innovations like washed microbiota, capsule-based formulations, and LBPs, have enhanced its safety, standardization, and accessibility. However, concerns regarding long-term safety, regulatory challenges, and variable efficacy still persist. Future directions, including personalized FMT, synthetic microbiota, and microbiome engineering, offer exciting potential to revolutionize microbiome-based therapies. Continued research, standardized protocols, and robust clinical trials will be key to fully unlocking the therapeutic scope of FMT in modern medicine.