Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of In Vitro Cultivation and Membrane Integrity Methods.

BACKGROUND: Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. METHODS: Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. RESULTS: Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. CONCLUSION: Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

Clinical efficacy and safety of faecal microbiota transplantation in the treatment of irritable bowel syndrome: a systematic review, meta-analysis and trial sequential analysis.

BACKGROUND: The aim of this study is to evaluate the efficacy and safety of faecal microbiota transplantation (FMT) for the treatment of irritable bowel syndrome (IBS). METHODS: We searched four databases for randomised controlled trials (RCTs) that compared FMT with a control intervention in patients with IBS. The revised Cochrane risk-of-bias (RoB) tool was chosen for appraisal. Meta-analysis with trial sequential analysis (TSA) was conducted. Grading of Recommendations Assessment Development and Evaluation (GRADE) methodology was used to assess the certainty of evidence (CoE). RESULTS: We included 12 RCTs with a total of 615 participants. Meta-analyses showed no significant difference between the FMT and control groups in terms of clinical responses (relative risk [RR] = 1.44, 95% confidence interval [CI] 0.88-2.33) and changes in IBS Severity Scoring System (IBS-SSS) scores (standardised mean difference [SMD] = - 0.31, 95% CI - 0.72 to 0.09) and IBS Quality of Life (IBS-QOL) scores (SMD = 0.30, 95% CI - 0.09 to 0.69). Subgroup analysis revealed that in studies with low RoB and using endoscopy, nasojejunal tube and rectal enema delivery, FMT led to a significant improvement in clinical responses and changes in IBS-SSS and IBS-QOL scores. TSA suggested that the current evidence is inconclusive and that the CoE is very low. CONCLUSION: This study suggests that patients with IBS may benefit from FMT especially when it is administered via endoscopy, nasojejunal tube or rectal enema. However, the certainty of evidence is very low. Further research is needed to confirm the efficacy and safety of FMT for IBS treatment. TRIAL REGISTRATION: PROSPERO registration number CRD42020211002.

Reduction in Renal Interstitial Fibrosis in Aged Male Mice by Intestinal Microbiota Rejuvenation.

INTRODUCTION: Renal interstitial fibrosis is an important pathological basis for kidney ageing and the progression of ageing nephropathy. In the present research, we established an aged mouse model of faecal microbiota transplantation (FMT), identified the rejuvenation features of the kidney in aged male mice, and preliminarily analysed the possible mechanism by which the rejuvenation of the intestinal microbiota reduces renal interstitial fibrosis and delays senescence in aged male mice. METHODS: We established an aged male mice model that was treated with FMT (FMT-Old) and a normal aged male mice control group (Old). Differentially expressed cytokines were identified using a cytokine array, and changes in protein expression related to signal transduction pathways in renal tissues were detected using a signalling pathway array. Senescence-associated ß-galactosidase and Masson staining were performed to observe the degrees of renal senescence and tubule interstitial fibrosis. Immunohistochemistry was utilized to detect changes in the expression of the ageing markers p53 and p21 and the inflammation-related protein nuclear factor (NF-κB) subunit (RelA/p65). RESULTS: The pathological features of renal senescence in the FMT-Old group were significantly alleviated, and the levels of the ageing indicators p53 and p21 were decreased (p < 0.05). Ingenuity Pathway Analysis revealed that six differentially expressed cytokines, MIP-3ß (CCL-19), E-selectin (SELE), Fas ligand (Fas L/FASLG), CXCL-11 (I-TAC), CXCL-1 and CCL-3 (MIP-1α) were related to a common upstream regulatory protein, RelA/p65, and the expression of this protein was significantly different between groups according to the signalling pathway array. CONCLUSION: Our findings suggest that the intestinal microbiota regulates the renal microenvironment by reducing immune inflammatory responses through the inhibition of the NF-κB signalling pathway, thereby delaying renal senescence in aged male mice.

Washed Microbiota Transplantation Improves the Sleep Quality in Patients with Inflammatory Bowel Disease.

Purpose: There is scarce evidence to support the effectiveness of faecal microbiota transplantation (FMT) in improving sleep among individuals with inflammatory bowel disease (IBD). Our study aimed to evaluate the effect of washed microbiota transplantation (WMT) (the new method of FMT) on the sleep of patients with IBD in short term. Patients and Methods: This prospective study was conducted as part of two interventional clinical trials (starting on February 2013 and expected to end on December 2025) and placed significant emphasis on evaluating sleep quality in patients with IBD. To measure subjective sleep, we used the Pittsburgh sleep quality index (PSQI). The primary endpoint was the PSQI score one month after WMT. Results: This stage study included 52 eligible patients evaluated by PSQI questionnaire who underwent WMT from January 2020 to March 2021 and 47 patients were enrolled for analysis. The age of the patients ranged from 13 to 60 years, with a mean of 33.4 years, and 57.4% (25/47) of the patients were male. The PSQI scores for all 47 patients one month after undergoing WMT were significantly lower (Cohen d = 0.59, p < 0.001) compared to the baseline. Moreover, baseline PSQI score was correlated with the difference value of the PSQI score before and after WMT (post-PSQI minus pre-PSQI) (r = 0.61, p < 0.05). Conclusion: The study suggests that WMT might be a helpful intervention for improving the sleep quality of patients with IBD, encouraging clinicians to consider its use in clinical practice for addressing poor sleep in IBD patients. Clinical Trial Registration: ClinicalTrials.gov; ID: NCT01793831, NCT01790061.

Personalized Paths: Unlocking Alzheimer's via the Gut-Brain Axis.

Background: Alzheimer's disease (AD) is characterised by abnormal protein aggregates in the brain that lead to cognitive decline. While current therapies only treat symptoms, disease-modifying treatments are urgently needed. Studies suggest that the composition of the microbiota is altered in people with AD, suggesting a link between gut bacteria and AD-related brain changes. Summary: In our narrative review, we explore various microbial interventions, such as faecal microbiota transplantation, probiotics, and diet, as powerful potential treatments. Studies suggest changes in microbiota composition following these interventions, with some beneficial effects on cognitive function. However, the mechanism of action of these microbial interventions is still unknown. Key Message: Our aim was to highlight the importance of personalised approaches, taking into account individual metabolic and microbiome profiles. We try to address gaps in current research and emphasise the need for microbiota analysis at different stages of the disease and its integration with clinical parameters and lifestyle information for a comprehensive understanding of AD progression (summarised in online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000535869).

Efficacy of Donor-Recipient-Matched Faecal Microbiota Transplantation in Patients with IBS-D: A Single-Centre, Randomized, Double-Blind Placebo-Controlled Study.

INTRODUCTION: The imbalance in gut microbiota is contributing to the development and progression of IBS. FMT can improve the gut microbiota, and donor-recipient-matched FMT can help develop individualized treatment plans according to different enterotypes. This study aimed to explore the efficacy of donor-recipient-matched FMT in IBS with predominant diarrhoea (IBS-D) and evaluate its effects on gut microbiota. METHODS: Twenty-seven patients with IBS-D were randomly divided into donor-recipient-matched FMT group (group P), random-donor FMT group (group R), and placebo group (group B). All participants received corresponding FMT treatment after filling in IBS-S, IBS-QoL, GSRS, and HADS questionnaires and having their stool samples collected at 4, 8, and 12 weeks after treatment. The improvement in the symptoms and the changes in the bacterial flora were analysed for three groups. RESULTS: The IBS-SSS, IBS-QoL, GSRS, and anxiety scores of group P were significantly lower after treatment (p < 0.05). The IBS-QoL scores of group R were significantly lower after treatment (p < 0.05). Beta diversity analysis showed that the gut microbiota of group P had an obvious trend of classification after treatment. Seven bacterial genera were related to the differences in the IBS-SSS scores before and after treatment. CONCLUSION: Donor-recipient-matched FMT significantly improved the clinical symptoms, quality of life, and anxiety scores of the patients with IBS-D than random-donor FMT.

The gut microbiota and diabetes: research, translation, and clinical applications - 2023 Diabetes, Diabetes Care, and Diabetologia Expert Forum.

This article summarises the state of the science on the role of the gut microbiota (GM) in diabetes from a recent international expert forum organised by Diabetes, Diabetes Care, and Diabetologia, which was held at the European Association for the Study of Diabetes 2023 Annual Meeting in Hamburg, Germany. Forum participants included clinicians and basic scientists who are leading investigators in the field of the intestinal microbiome and metabolism. Their conclusions were as follows: (1) the GM may be involved in the pathophysiology of type 2 diabetes, as microbially produced metabolites associate both positively and negatively with the disease, and mechanistic links of GM functions (e.g. genes for butyrate production) with glucose metabolism have recently emerged through the use of Mendelian randomisation in humans; (2) the highly individualised nature of the GM poses a major research obstacle, and large cohorts and a deep-sequencing metagenomic approach are required for robust assessments of associations and causation; (3) because single time point sampling misses intraindividual GM dynamics, future studies with repeated measures within individuals are needed; and (4) much future research will be required to determine the applicability of this expanding knowledge to diabetes diagnosis and treatment, and novel technologies and improved computational tools will be important to achieve this goal.

Long-term efficacy of washed microbiota transplantation in overweight patients.

BACKGROUND: Faecal microbiota transplantation holds promise in mitigating fat accumulation and improving obesity. This study aimed to evaluate the long-term efficacy of washed microbiota transplantation (WMT) among overweight patients. METHODS: The clinical data pertaining to the treatment of patients with WMT were collected retrospectively. Compared alterations in body mass index (BMI), blood glucose, blood lipids and blood pressure prior to and following WMT treatment. Comprehensive efficacy evaluation and atherosclerosis cardiovascular disease (ASCVD) grading evaluation were carried out, with an analysis of gut microbiota composition before and after WMT. RESULTS: A total of 186 patients were included (80 overweight, 106 normal weight). WMT not only had the effect of improving overweight patients to the normal weight patients (p < .001), but also could significantly reduce BMI in the long term by restoring gut microbiota homeostasis (p < .001). In addition, the BMI improvement value of multi course was more significant than that of single course or double course. WMT had a significant ASCVD downgrade effect on the high-risk and medium-risk groups outside 1 year, while it did not increase the risk of upgrading ASCVD for low-risk group. CONCLUSIONS: WMT could significantly reduce the BMI of overweight patients and still had an improvement effect in the long term.

Exploring the Gut Microbiome's Role in Inflammatory Bowel Disease: Insights and Interventions.

Inflammatory Bowel Disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), is a chronic and relapsing inflammatory condition of the intestine that significantly impairs quality of life and imposes a heavy burden on healthcare systems globally. While the exact etiology of IBD is unclear, it is influenced by genetic, environmental, immunological, and microbial factors. Recent advances highlight the gut microbiome's pivotal role in IBD pathogenesis. The microbial dysbiosis characteristic of IBD, marked by a decline in beneficial bacteria and an increase in pathogenic microbes, suggests a profound connection between microbial imbalance and disease mechanisms. This review explores diagnostic approaches to IBD that integrate clinical assessment with advanced microbiological analyses, highlighting the potential of microbiome profiling as a non-invasive diagnostic tool. In addition, it evaluates conventional and emerging treatments and discusses microbiome-targeted intervention prospects, such as probiotics, symbiotics, and faecal microbiota transplantation. The necessity for future research to establish their efficacy and safety is emphasised.

The effect of non-pooled multi-donor faecal microbiota transplantation for inducing clinical remission in patients with chronic pouchitis: Results from a multicentre randomised double-blinded placebo-controlled trial (MicroPouch).

BACKGROUND AND AIMS: To investigate if treatment with non-pooled multi-donor faecal microbiota transplantation (FMT) for four weeks was superior to placebo to induce clinical remission in patients with chronic pouchitis. METHODS: The study was a randomised double-blinded placebo-controlled study with a 4-week intervention period and 12-month follow-up. Eligible patients with chronic pouchitis were recruited from five Danish hospitals. Participants were randomised to non-pooled multi-donor FMT derived from four faecal donors, or placebo. Treatment was delivered daily by enema for two weeks followed by every second day for two weeks. Disease severity was accessed at inclusion and 30-day follow-up, using the Pouchitis Disease Activity Index (PDAI); PDAI <7 was considered equivalent to clinical remission. Faecal samples from participants and donors were analysed by shotgun metagenomic sequencing. RESULTS: Inclusion was stopped after inclusion of 30 participants who were randomised 1:1 for treatment with FMT or placebo. There was no difference in participants achieving clinical remission between the two groups at 30-day follow-up, relative risk 1.0 (95%CI(0.55;1.81)). Treatment with FMT resulted in a clinically relevant increase in adverse events compared to placebo, incidence rate ratio 1.67 (95%CI(1.10;2.52)); no serious adverse events within either group. Faecal microbiota transplantation statistically significantly increased the similarity of participant faecal microbiome to the faecal donor microbiome at 30-days follow-up (p=0.01), which was not seen after placebo. CONCLUSIONS: Non-pooled multi-donor FMT was comparable to placebo in inducing clinical remission in patients with chronic pouchitis but showed a clinically relevant increase in adverse events compared to placebo.

Faecal Microbiota Transplantation Engraftment After Budesonide or Placebo in Patients With Active Ulcerative Colitis Using Pre-selected Donors: A Randomized Pilot Study.

BACKGROUND: Faecal microbiota transplantation [FMT] shows some efficacy in treating patients with ulcerative colitis [UC], although variability has been observed among donors and treatment regimens. We investigated the effect of FMT using rationally selected donors after pretreatment with budesonide or placebo in active UC. METHODS: Patients ≥18 years old with mild to moderate active UC were randomly assigned to 3 weeks of budesonide [9 mg] or placebo followed by 4-weekly infusions of a donor faeces suspension. Two donors were selected based on microbiota composition, regulatory T cell induction and short-chain fatty acid production in mice. The primary endpoint was engraftment of donor microbiota after FMT. In addition, clinical efficacy was assessed. RESULTS: In total, 24 patients were enrolled. Pretreatment with budesonide did not increase donor microbiota engraftment [p = 0.56] nor clinical response, and engraftment was not associated with clinical response. At week 14, 10/24 [42%] patients achieved [partial] remission. Remarkably, patients treated with FMT suspensions from one donor were associated with clinical response [80% of responders, p < 0.05] but had lower overall engraftment of donor microbiota. Furthermore, differences in the taxonomic composition of the donors and the engraftment of certain taxa were associated with clinical response. CONCLUSION: In this small study, pretreatment with budesonide did not significantly influence engraftment or clinical response after FMT. However, clinical response appeared to be donor-dependent. Response to FMT may be related to transfer of specific strains instead of overall engraftment, demonstrating the need to characterize mechanisms of actions of strains that maximize therapeutic benefit in UC.

Safety and efficacy of faecal microbiota transplantation in patients with mild to moderate Parkinson's disease (GUT-PARFECT): a double-blind, placebo-controlled, randomised, phase 2 trial.

Background: Dysregulation of the gut microbiome has been implicated in Parkinson's disease (PD). This study aimed to evaluate the clinical effects and safety of a single faecal microbiota transplantation (FMT) in patients with early-stage PD. Methods: The GUT-PARFECT trial, a single-centre randomised, double-blind, placebo-controlled trial was conducted at Ghent University Hospital between December 01, 2020 and December 12, 2022. Participants (aged 50-65 years, Hoehn and Yahr stage 2) were randomly assigned to receive nasojejunal FMT with either healthy donor stool or their own stool. Computer-generated randomisation was done in a 1:1 ratio through permutated-block scheduling. Treatment allocation was concealed for participants and investigators. The primary outcome measure at 12 months was the change in the Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score obtained during off-medication evaluations. Intention-to-treat analysis was performed using a mixed model for repeated measures analysis. This completed trial is registered on ClinicalTrials.gov (NCT03808389). Findings: Between December 2020 and December 2021, FMT procedures were conducted on 46 patients with PD: 22 in the healthy donor group and 24 in the placebo group. Clinical evaluations were performed at baseline, 3, 6, and 12 months post-FMT. Full data analysis was possible for 21 participants in the healthy donor group and 22 in the placebo group. After 12 months, the MDS-UPDRS motor score significantly improved by a mean of 5.8 points (95% CI -11.4 to -0.2) in the healthy donor group and by 2.7 points (-8.3 to 2.9) in the placebo group (p = 0.0235). Adverse events were limited to temporary abdominal discomfort. Interpretation: Our findings suggested a single FMT induced mild, but long-lasting beneficial effects on motor symptoms in patients with early-stage PD. These findings highlight the potential of modulating the gut microbiome as a therapeutic approach and warrant a further exploration of FMT in larger cohorts of patients with PD in various disease stages. Funding: Flemish PD patient organizations (VPL and Parkili), Research Foundation Flanders (FWO), Biocodex Microbiota Foundation.

Assessment of Faecal Microbiota Transplant Stability in Deep-Freeze Conditions: A 12-Month Ex Vivo Viability Analysis.

BACKGROUND: Faecal microbiota transplantation (FMT) is an established treatment for Clostridioides difficile infection and is under investigation for other conditions. The availability of suitable donors and the logistics of fresh stool preparation present challenges, making frozen, biobanked stools an attractive alternative. AIMS: This study aimed to evaluate the long-term viability of bacterial populations in faecal samples stored at -80°C for up to 12 months, supporting the feasibility of using frozen grafts for FMT. METHODS: Fifteen faecal samples from nine healthy donors were processed, mixed with cryoprotectants and stored at -80°C. Samples were assessed at baseline and after 3, 6 and 12 months using quantitative culturing methods to determine the concentration of live bacteria. RESULTS: Quantitative analysis showed no significant decrease in bacterial viability over the 12-month period for both aerobic and anaerobic cultures (p = 0.09). At all timepoints, the coefficients of variability in colony-forming unit (CFU) counts were greater between samples (102 ± 21% and 100 ± 13% for aerobic and anaerobic cultures, respectively) than the variability between measurements of the same sample (30 ± 22% and 30 ± 19%). CONCLUSIONS: The study confirmed that faecal microbiota can be preserved with high viability in deep-freeze storage for up to a year, making allogenic FMT from biobanked samples a viable and safer option for patients. However, a multidonor approach may be beneficial to mitigate the risk of viability loss in any single donor sample.

Role of bacteria and microbial metabolites in immune modulation during early life.

Host-microbiome interplay from birth is essential for immune imprinting and tuning. Live gut microbes and microbial-derived metabolites regulate the development and modulation of the immune system, but whether microbial metabolites solely are sufficient to induce immune maturation remains unclear. Sterile faecal filtrates (FFT) were generated from murine gut contents. Newborn germ-free (GF) mice were treated twice daily with FFT (GF-FFT) or saline (GF-NaCl) from post-natal day 5 until 4 weeks of age. A third group of GF neonates were conventionalized by the transfer of caecal microbiota with live gut microbes. Host immune compartments were comprehensively immunophenotyped and systemically analysed in all available immune-related organs using flow cytometry. Oral FFT was associated with reduced survival among neonates (n = 7/19; 36.8% mortality), while saline treatment was well tolerated (n = 1/17, 5.9% mortality). Four-week-old FFT-treated pups were comparable in body weight to GF-NaCl, and the major B-cell, conventional T-cell and unconventional T-cell subsets were unchanged from saline-treated mice. Live bacteria administered during early life induced clear changes in proportions of B cells, T cells and T-cell subsets in all mucosal tissues and secondary lymphoid organs compared to GF-FFT, including restoration of intestinal natural killer T (NKT) cells with characteristics similar to conventional pups. Our findings show that oral administration of a FFT made of microbial metabolites, antigens and bacteriophages alone is insufficient to induce normal immune development elicited by the presence of live bacteria. Reduced survival during neonatal FFT treatment suggests a potential bioactive attribute of sterile faecal filtrates.

Heart failure-induced microbial dysbiosis contributes to colonic tumour formation in mice.

AIMS: Heart failure (HF) and cancer are the leading causes of death worldwide. Epidemiological studies revealed that HF patients are prone to develop cancer. Preclinical studies provided some insights into this connection, but the exact mechanisms remain elusive. In colorectal cancer (CRC), gut microbial dysbiosis is linked to cancer progression and recent studies have shown that HF patients display microbial dysbiosis. This current study focussed on the effects of HF-induced microbial dysbiosis on colonic tumour formation. METHODS AND RESULTS: C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16 s rRNA sequencing, and pooled for faecal microbiota transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane/Dextran sodium sulphate. The CRC mice were transplanted with faeces from MI or sham mice. MI-induced HF resulted in microbial dysbiosis, characterized by a decreased α-diversity and microbial alterations on the genus level, several of which have been associated with CRC. We then performed faecal microbiota transplantation with faeces from HF mice in CRC mice, which resulted in a higher endoscopic disease score and an increase in the number of tumours in CRC mice. CONCLUSION: We demonstrated that MI-induced HF contributes to colonic tumour formation by altering the gut microbiota composition, providing a mechanistic explanation for the observed association between HF and increased risk for cancer. Targeting the microbiome may present as a tool to mitigate HF-associated co-morbidities, especially cancer.

Long-term beneficial effect of faecal microbiota transplantation on colonisation of multidrug-resistant bacteria and resistome abundance in patients with recurrent Clostridioides difficile infection.

BACKGROUND: Multidrug-resistant (MDR) bacteria are a growing global threat, especially in healthcare facilities. Faecal microbiota transplantation (FMT) is an effective prevention strategy for recurrences of Clostridioides difficile infections and can also be useful for other microbiota-related diseases. METHODS: We study the effect of FMT in patients with multiple recurrent C. difficile infections on colonisation with MDR bacteria and antibiotic resistance genes (ARG) on the short (3 weeks) and long term (1-3 years), combining culture methods and faecal metagenomics. RESULTS: Based on MDR culture (n = 87 patients), we notice a decrease of 11.5% in the colonisation rate of MDR bacteria after FMT (20/87 before FMT = 23%, 10/87 3 weeks after FMT). Metagenomic sequencing of patient stool samples (n = 63) shows a reduction in relative abundances of ARGs in faeces, while the number of different resistance genes in patients remained higher compared to stools of their corresponding healthy donors (n = 11). Furthermore, plasmid predictions in metagenomic data indicate that patients harboured increased levels of resistance plasmids, which appear unaffected by FMT. In the long term (n = 22 patients), the recipients' resistomes are still donor-like, suggesting the effect of FMT may last for years. CONCLUSIONS: Taken together, we hypothesise that FMT restores the gut microbiota to a composition that is closer to the composition of healthy donors, and potential pathogens are either lost or decreased to very low abundances. This process, however, does not end in the days following FMT. It may take months for the gut microbiome to re-establish a balanced state. Even though a reservoir of resistance genes remains, a notable part of which on plasmids, FMT decreases the total load of resistance genes.

Faecal microbiota transplantation in children: A systematic review.

AIM: Novel technologies offer insights into the potential role of the intestinal microbiota in human health and disease. Dysbiosis has been associated with several diseases, and it is thought to play a role in the pathogenesis of different gastrointestinal diseases. Faecal microbiota transplantation (FMT) is emerging as a method to modulate the gastrointestinal microbial ecosystem. While recurrent Clostridioides difficile infection is the recognised FMT indication, exploration of other therapeutic uses is ongoing. METHODS: Following PRISMA guidelines, we conducted a systematic review, extracting 583 articles from Embase and PubMed (index date to October 2022). RESULTS: The search yielded 58 studies for full review, with 50 included in the systematic review. Articles were categorised by FMT indication, study design, efficacy, adverse events, donor selection and administration route. FMT appears safe and effective for recurrent Clostridioides difficile infection, although severe adverse events are reported in children. However, there are currently insufficient data to support the use of FMT for other potential therapeutic indications (such as irritable or inflammatory bowel disease or obesity), beside the potential to decolonise multi-drug resistant organisms. CONCLUSION: This underscores the need for randomised, controlled, prospective cohort studies in children to assess FMT effectiveness in diverse conditions and counteract publication bias.

Horizontal gene transfer after faecal microbiota transplantation in adolescents with obesity.

BACKGROUND: Horizontal gene transfer (HGT) describes the transmission of DNA outside of direct ancestral lineages. The process is best characterised within the bacterial kingdom and can enable the acquisition of genetic traits that support bacterial adaptation to novel niches. The adaptation of bacteria to novel niches has particular relevance for faecal microbiota transplantation (FMT), a therapeutic procedure which aims to resolve gut-related health conditions of individuals, through transplanted gut microbiota from healthy donors. RESULTS: Three hundred eighty-one stool metagenomic samples from a placebo-controlled FMT trial for obese adolescents (the Gut Bugs Trial) were analysed for HGT, using two complementary methodologies. First, all putative HGT events, including historical HGT signatures, were quantified using the bioinformatics application WAAFLE. Second, metagenomic assembly and gene clustering were used to assess and quantify donor-specific genes transferred to recipients following the intervention. Both methodologies found no difference between the level of putative HGT events in the gut microbiomes of FMT and placebo recipients, post-intervention. HGT events facilitated by engrafted donor species in the FMT recipient gut at 6 weeks post-intervention were identified and characterised. Bacterial strains contributing to this subset of HGT events predominantly belonged to the phylum Bacteroidetes. Engraftment-dependent horizontally transferred genes were retained within recipient microbiomes at 12 and 26 weeks post-intervention. CONCLUSION: Our study suggests that novel microorganisms introduced into the recipient gut following FMT have no impact on the basal rate of HGT within the human gut microbiome. Analyses of further FMT studies are required to assess the generalisability of this conclusion across different FMT study designs and for the treatment of different gut-related conditions. Video Abstract.

Faecal Microbiota Transplantation Alleviates Ferroptosis after Ischaemic Stroke.

Ischaemic stroke can induce changes in the abundance of gut microbiota constituents, and the outcome of stroke may also be influenced by the gut microbiota. This study aimed to determine whether gut microbiota transplantation could rescue changes in the gut microbiota and reduce ferroptosis after stroke in rats. Male Sprague-Dawley rats (6 weeks of age) were subjected to ischaemic stroke by middle cerebral artery occlusion (MCAO). Fecal samples were collected for 16S ribosomal RNA (rRNA) sequencing to analyze the effects of FMT on the gut microbiota. Neurological deficits were evaluated using the Longa score. triphenyl tetrazolium chloride (TTC) staining was performed in the brain, and kits were used to measure malondialdehyde (MDA), iron, and glutathione (GSH) levels in the ipsilateral brain of rats. Western blotting was used to detect the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and the transferrin receptor 2 (TFR2) in the ipsilateral brain of rats. Stroke induced significant changes in the gut microbiota, and FMT ameliorated these changes. TTC staining results showed that FMT reduced cerebral infarct volume. In addition, FMT diminished MDA and iron levels and elevated GSH levels in the ipsilateral brain. Western blot analysis showed that FMT increased GPX4 and SLC7A11 protein expression and decreased TFR2 protein expression in the ipsilateral brain after stroke. FMT can reverse gut microbiota dysbiosis, reduce cerebellar infarct volume, and decrease ferroptosis after stroke.