post-FMT Samples Research Articles

P702 Faecal Microbiota Transplantation Sculpts the faecal and mucosal microbial and metabolomic profiles in patients with Ulcerative Colitis

Abstract Background Gut microbiota modulation via faecal microbiota transplantation (FMT)is known to induce long lasting clinical as well as endoscopic remission in patients with UC. The present study aims to identify microbial and metabolomic changes in the faecal as well mucosal niches, in response to FMT in patients with UC. Methods 28 patients with mild-moderate UC and 16 non-IBD controls were enrolled. Patients were given a weekly infusion of pooled-multidonor-FMT, for 8 weeks, while maintaining a uniform dietary pattern. We collected paired stool and recto-sigmoidal biopsysamples from patients with UC pre-FMT (n=28), post-FMT (n=10) and controls (n=16). 16S (V3-V4) rRNA sequencing and LC-MS based untargeted metabolomics was performed. α and β diversity analysis and differential abundance (DA) analysis of microbiota was performed using DeSeq2 R package. Differential metabolite peaks between the three groups were identified via volcano plot and annotated using LipidMaps, HMDB, Kegg and Metlin databases. (Fig.1) Results α-diversity of patients with UC was not significantly different from that of controls, in both sample matrices. Post-FMT α-diversity of faecal samples differed significantly from the Pre-FMT group, however in mucosal tissue, the difference was non-significant. β-diversity indices were significant in UC pre-FMT vs post-FMT populations for both matrices (Fig.2A and B). DA analysis identified faecal and mucosal controls and UC post-FMT samples to be significantly similar [ANOSIM R=0.07; p=0.1 (faecal) and R=0.02; p=0.315 (mucosal)]. Analysis of core microbiota revealed diminished faecal microbial core in UC, with no major UC-associated loss of mucosal core members, showing relatively conserved nature of mucosal microbiota. FMT restored microbial core along both niches. UC pre-FMT samples displayed increased abundance of Firmicutes (Limosilactobacillus, Lactobacillus, Streptococcus, Veilonella and Sphingomonas) and Proteobacteria (Methylobacterium) in faecal samples, while mucosa-associated microbiota displayed increased abundances of Firmicutes (Streptococcus, Limosilactobacillus, Ligilactobacillus, Eubacterium, Enterococcus, etc), Bacteroidetes (Parabacteroides and Alistipes) and Proteobacteria (Burkholderia)(Fig.3). Distinct functional classes of metabolites, including bacterial cell wall and QS molecules, membrane lipids, metabolism of vitamins, long and medium chain fatty acids, bile acids, and tryptophan etc, were also found to be altered between the three groups in both sample matrices(Fig.4) Conclusion FMT efficiently restores beneficial bacterial populations and related metabolic pathways in faecal as well the relatively reserved mucosal microbial niches, which may pave way for induction of remission in patients with UC.

Faecalibacterium prausnitzii increases following fecal microbiota transplantation in recurrent Clostridioides difficile infection.

ObjectiveFecal microbiota transplantation (FMT) is a highly effective treatment for Clostridioides difficile infection (CDI). However, the fecal transplant’s causal components translating into clearance of the CDI are yet to be identified. The commensal bacteria Faecalibacterium prausnitzii may be of great interest in this context, since it is one of the most common species of the healthy gut microbiota and produces metabolites with anti-inflammatory properties. Although there is mounting evidence that F. prausnitzii is an important regulator of intestinal homeostasis, data about its role in CDI and FMT are relatively scarce.MethodsStool samples from patients with recurrent CDI were collected to investigate the relative abundance of F. prausnitzii before and after FMT. Twenty-one patients provided fecal samples before the FMT procedure, at 2 weeks post-FMT, and at 2–4 months post-FMT. The relative abundance of F. prausnitzii was determined using quantitative polymerase chain reaction.ResultsThe abundance of F. prausnitzii was elevated in samples (N = 9) from donors compared to pre-FMT samples (N = 15) from patients (adjusted P<0.001). No significant difference in the abundance of F. prausnitzii between responders (N = 11) and non-responders (N = 4) was found before FMT (P = 0.85). In patients with CDI, the abundance of F. prausnitzii significantly increased in the 2 weeks post-FMT samples (N = 14) compared to the pre-FMT samples (N = 15, adjusted P<0.001). The increase persisted 2–4 months post-FMT (N = 15) compared to pre-FMT samples (N = 15) (adjusted P<0.001).ConclusionsFMT increases the relative abundance of F. prausnitzii in patients with recurrent CDI, and this microbial shift remains several months later. The baseline abundance of F. prausnitzii in donors or recipients was not associated with future treatment response, although a true predictive capacity cannot be excluded because of the limited sample size. Further studies are needed to discern whether F. prausnitzii plays an active role in the resolution of CDI.

Evolution of intestinal microbiome in a process of faecal microbiota transplantation in a patient with Clostridioides difficile infection: NGS analysis with different bioinformatics software programs

IntroductionClostridioides difficile infection (CDI) has become a global healthcare challenge due to increases in its incidence and mortality rates. Faecal microbiota transfer (FMT) is postulated as a protocol to prevent CDI recurrence. Material and methodsA donor faecal sample and patient faecal samples (pre-FMT and post-FMT) were analysed. The r16S gene was amplified and sequenced by NGS, and its diversity and taxonomy composition were examined. ResultsMicrobial richness increased in post-FMT samples, and the β diversity studies grouped the samples into two clusters. One included the non-pathological samples (donor and pre-FMT samples), and the other included the pathological sample. The results obtained by Qiime2 and Bioconductor were similar. ConclusionThe analysis showed an increase in taxonomic diversity after the FMT, which suggests its usefulness. Moreover, these results showed that standardisation of bioinformatics analysis is key.

Evolución del microbioma intestinal en un proceso de transferencia de microbiota fecal (TMF) en un paciente con infección por Clostridioides difficile: análisis por NGS con diferentes programas bioinformáticos

IntroductionClostridioides difficile infection (CDI) has become a global healthcare challenge due to increases in its incidence and mortality rates. Faecal microbiota transfer (FMT) is postulated as a protocol to prevent CDI recurrence. Material and methodsA donor faecal sample and patient faecal samples (pre-FMT and post-FMT) were analysed. The r16S gene was amplified and sequenced by NGS, and its diversity and taxonomy composition were examined. ResultsMicrobial richness increased in post-FMT samples, and the β diversity studies grouped the samples into two clusters. One included the non-pathological samples (donor and pre-FMT samples), and the other included the pathological sample. The results obtained by Qiime2 and Bioconductor were similar. ConclusionThe analysis showed an increase in taxonomic diversity after the FMT, which suggests its usefulness. Moreover, these results showed that standardisation of bioinformatics analysis is key.

Neuroinflammation in Murine Cirrhosis Is Dependent on the Gut Microbiome and Is Attenuated by Fecal Transplant.

Cirrhosis and hepatic encephalopathy (HE) is associated with an altered gut-liver-brain axis. Fecal microbial transplant (FMT) after antibiotics improves outcomes in HE, but the impact on brain function is unclear. The aim of this study is to determine the effect of colonization using human donors in germ-free (GF) mice on the gut-liver-brain axis. GF and conventional mice were made cirrhotic using carbon tetrachloride and compared with controls in GF and conventional state. Additional GF mice were colonized with stool from controls (Ctrl-Hum) and patients with cirrhosis (Cirr-Hum). Stools from patients with HE cirrhosis after antibiotics were pooled (pre-FMT). Stools from the same patients 15 days after FMT from a healthy donor were also pooled (post-FMT). Sterile supernatants were created from pre-FMT and post-FMT samples. GF mice were colonized using stools/sterile supernatants. For all mice, frontal cortex, liver, and small/large intestines were collected. Cortical inflammation, synaptic plasticity and gamma-aminobutyric acid (GABA) signaling, and liver inflammation and intestinal 16s ribosomal RNA microbiota sequencing were performed. Conventional cirrhotic mice had higher degrees of neuroinflammation, microglial/glial activation, GABA signaling, and intestinal dysbiosis compared with other groups. Cirr-Hum mice had greater neuroinflammation, microglial/glial activation, and GABA signaling and lower synaptic plasticity compared with Ctrl-Hum mice. This was associated with greater dysbiosis but no change in liver histology. Pre-FMT material colonization was associated with neuroinflammation and microglial activation and dysbiosis, which was reduced significantly with post-FMT samples. Sterile pre-FMT and post-FMT supernatants did not affect brain parameters. Liver inflammation was unaffected. Conclusion: Fecal microbial colonization from patients with cirrhosis results in higher degrees of neuroinflammation and activation of GABAergic and neuronal activation in mice regardless of cirrhosis compared with those from healthy humans. Reduction in neuroinflammation by using samples from post-FMT patients to colonize GF mice shows a direct effect of fecal microbiota independent of active liver inflammation or injury.

Microbial bile salt hydrolases mediate the efficacy of faecal microbiota transplant in the treatment of recurrent Clostridioides difficile infection

ObjectiveFaecal microbiota transplant (FMT) effectively treats recurrent Clostridioides difficile infection (rCDI), but its mechanisms of action remain poorly defined. Certain bile acids affect C. difficile germination or vegetative growth. We...

618. Do Clinical Factors Affect Microbial Engraftment After Fecal Microbiota Transplantation in Recurrent Clostridium difficile Infection?

BackgroundFecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (rCDI). Few studies have evaluated clinical factors associated with microbial engraftment. We describe microbial changes post-FMT and clinical factors impacting engraftment.MethodsPatients undergoing FMT for rCDI via colonoscopy were enrolled. Clinical data and stool were collected pre- and 8 weeks post-FMT. Microbial profiles were assessed by 16S rRNA sequencing. Difference in microbial alpha and β-diversity between groups was determined. Significance testing was assessed using Mann–Whitney–Wilcoxon and PERMANOVA tests. The Jensen Shannon divergence (JSD) between donor and their recipient post-FMT was used as a measure of engraftment. The association of clinical factors on engraftment was evaluated by linear regression.ResultsA total of 12 patients received an FMT from 12 unique donors. The efficacy rate was 92%. Mean recipient age was 60 years (range: 33–87) with more females (7/12).Recipients pre-FMT alpha diversity was significantly lower compare to donors (P = 0.04, Figure 1a). This difference dissipated post-FMT (P = 0.67). On β-diversity analysis, the recipients pre-FMT samples clustered separately from their post-FMT samples (P = 0.01, Figure 1b), with the post-FMT samples shifting closer to the donor samples. Proteobacteria was dominant in patients’ pre-FMT samples and were substantially reduced post-FMT, combined with an expansion in Bacteroidetes (Figure 2).On linear regression analysis, clinical factors (age, sex, previous recurrent CDI episodes, inflammatory bowel disease, proton pump inhibitor, immunosuppression, previous anti-CDI antibiotic courses, probiotics) were not significantly associated with engraftment outcomes.ConclusionThere is a significant and durable shift in recipients’ microbial profile to resemble their donor post-FMT. Recipients’ pre-FMT clinical factors did not significantly affect microbial engraftment. Future metagenomic studies may help elucidate whether clinical factors impact engraftment.Figure 1: (A) Alpha diversity. (B) Bray–Curtis principle component plot.Figure 2: Relative abundance of taxa at Phylum level.Disclosures All authors: No reported disclosures.

Engraftment and Augmentation of Microbiome Following Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection

BackgroundRecurrent Clostridum diffcile infection (rCDI) poses major challenges to healthcare providers and patients. Fecal Microbiota Transplantation (FMT) is an effective therapy for rCDI, but the exact mechanism of its efficacy is unknown. Current metagenomics literature indicates that abundance of Bacteroidetes and Firmicutes may protect against CD proliferation and recurrence. However, this is too broad to be useful for developing refined and targeted microbial-specific therapy for rCDI, because the long-term safety of FMT remains unknown. We examined the phylogeny of bacteria pre- and post- FMT to determine the key organisms associated with successful FMT to the genera level.MethodsA subset of patient stool samples (n = 35) from a phase 2 study comparing fresh vs. frozen FMT for rCDI was sequenced at four time points: pre-FMT; at day 10; at week 5; and at week 13, following the last FMT. The matching donor stool was sequenced simultaneously with the corresponding patients’ pre- and post-FMT samples.Using the binary outcome to a single FMT as the response, we have developed an in-house machine learning algorithm, Φ-LASSO, to isolate key genera using the bacterial phylogenetic structure.Engraftment was defined as: newly detected operational taxonomic unit (OTUs) in the patient post-FMT, which were present in the donor but undetected in the patient pre-FMT. Augmentation was defined as: non-donor OTUs whose levels substantially increased post-FMT. Figure 1 (below) displays the distribution of engrafted and augmented OTUs at varying thresholds. We observed increases over time points within each threshold level.Results Akkermansia, Blautia and Roseburiaappear to be key genera for successful FMT. The Φ-LASSO fits with consistently positive coefficients, see Figure 2.ConclusionIn this preliminary study, using Φ-LASSO, we have shown that specific microbes to the genera level are uniformly present in successful FMT. This information may lead to developing refined and targeted microbial-therapy for rCDI.Figure 1Observed (a) engraftment of distinct donor OTUs on patients and (b) augmentation of distinct OTUs in patients for day 10 (D10), week 5 (W5), and week 13 (W13) post-treatment.Figure 2Fitted coefficients for donor OTUs selected by Φ-LASSO.Disclosures All authors: No reported disclosures.

Identification of p‐cresol sulfate and secondary bile salts in human urine as sensitive biomarkers of fecal microbiota transplantation in R‐CDI patients

Fecal microbiota transplantation (FMT) has become an effective therapy for recurrent Clostridium difficile infection (R‐CDI). Besides reestablishing a normal intestinal microbiome that offers better resistance to R‐CDI, the introduction of microflora from healthy donors through FMT is also expected to change the metabolomes in the intestine and the whole body due to diverse metabolic activities of transplanted microbiota. Therefore, monitoring the metabolic changes in biofluids and excretes provides the opportunities for identifying noninvasive biomarkers of microbial recolonization and understanding the metabolic influences of FMT on host and microflora. In this study, FMT‐induced metabolic changes were examined by the metabolomic comparison of pre‐FMT and post‐FMT urine samples from 18 R‐CDI patients who had symptoms rapidly resolved after receiving FMT from two donors. After the liquid chromatography‐mass spectrometry (LC‐MS) analysis of urine samples from the patients and the donors, the LC‐MS data were processed by partial‐least squares‐discriminant analysis (PLS‐DA), leading to the establishment of a multivariate model revealing the separation between pre‐ and post‐FMT urine samples as well as the similarity between post‐FMT and donor samples. Among a group of urinary metabolites affected by the FMT, the most prominent biomarkers are p‐cresol sulfate and secondary bile acid conjugates, which were absent in most pre‐FMT samples, but dramatically increased in the post‐FMT samples. The presence of p‐cresol sulfate and secondary bile acid conjugates in urine after FMT reflected the recovery of microfloral metabolism on tyrosine and primary bile acids in R‐CDI patients. Their levels in urine may function as effective and sensitive therapeutic indicators of the readiness of CDI patient for FMT as well as the recolonization of healthy microfloral colonies after FMT.Support or Funding InformationNIAID grant R21 AI114722 and NIFA project MIN‐18‐092

Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection in the Elderly: Long-Term Outcomes and Microbiota Changes.

Fecal microbiota transplantation (FMT) has become the cornerstone in management of recurrent Clostridium difficile infection (RCDI) in adults. However, data on efficacy, safety, long-term outcomes, and microbiota alterations are limited in elderly patients (>65years). Twenty-nine consecutive elderly patients with RCDI underwent FMT with combined jejunal and colonic method and monitored for long-term outcomes. Fecal samples from five elderly RCDI patients (G65) were subjected to genomic analysis before and after FMT, and microbiota changes were compared with matched RCDI patients below 65years (L65). FMT resulted in marked improvement in all clinical parameters, including abdominal pain, bloating, and diarrhea in all elderly RCDI patients. Fecal C. difficile toxin was positive in all 29 patients and turned negative in all 27 patients, who agreed to undergo this test after FMT. Statistically significant improvement in leukocytosis was noted (p<0.05). Only adverse events reported were transient mild fever (2/29) and bloating (3/29). Long-term follow-up over 25.4±12.8months did not reveal any additional adverse events or RCDI recurrence. Genomic analysis suggested that overall microbiota diversity increased post-FMT in elderly RCDI patients. However, this response was less robust than the younger group. While Firmicutes did not change markedly, Proteobacteria decreased significantly in post-FMT samples in elderly RCDI patients. These observations suggest that FMT in elderly patients with RCDI appears to be highly efficacious with no recurrence of infection over long-term follow-up. Alterations in microbiota in this group of patients are characterized by less robust increase in microbial diversity and marked reduction in phylum Proteobacteria.