Multi-omics Dissection of Gut Microbiome Engraftment During FMT

Gut microbiota regulates key functions in humans, i.e. immunity and metabolism, and is a pathogenic pathway of many disorders, including recurrent C. difficile infection (rCDI), ulcerative colitis (UC), and metabolic syndrome (MetS). Microbiota therapeutics (MT) have raised high expectations without comparable results. Among MT, fecal microbiota transplantation (FMT), the transfer of healthy donor feces to a recipient with a microbiome-associated disease, has achieved high (nearly 90%) cure rates of rCDI but lower and less consistent results in chronic disorders, i.e. UC or MetS. Clinical and microbial features seem to be related with FMT outcomes, but consistent predictors are not available.

Donor-recipient microbiome engraftment may be critical for the clinical success of FMT. In a pooled meta-analysis of 20 FMT studies in different diseases by our group and the Segata Lab , donor recipient microbiome engraftment was associated with clinical response regardless of disease, differed among bacterial taxa, and was predicted by machine learning (ML). Other factors could influence engraftment, but evidence is unclear. Virome and fungome, have been linked to FMT success, but their engraftment kinetics is unknown. Host factors, i.e. genetics, gut immunity and microbial metabolites are supposed to play a role in engraftment, but supporting data are still absent.

Crucial issues of the engraftment dynamics remain still unsolved, including 1) which are the interactions among host factors, microbial strains, and products during FMT; 2)whether and how they influence engraftment and 3) clinical outcomes.Our aim is to disentangle the dynamics of microbiome engraftment and correlate them to clinical outcomes.

OBJECTIVES

Primary objectives - To assess the longitudinal multidomain interactions of host and microbiome variables and their influence on microbial engraftment

Secondary Objectives

\- To assess the longitudinal multidomain interactions of host and microbiome variables and their influence on clinical outcomes

Endpoints

Primary - The longitudinal evaluation of multidomain interactions of host and microbiome variables throughout a multi-omics approach at 90 days after the last FMT

Secondary

\- The longitudinal evaluation of multidomain interactions of host and microbiome variables throughout a multi-omics approach at 7,30,180,360 days after the last FMT

Procedures:

Baseline assessment

At baseline enrolled patients will be evaluated by the gastroenterology staff and endocrine and metabolic Unit staff of the Fondazione Policlinico Universitario A. Gemelli IRCCS and their demographic, clinical characteristics and laboratory data will be recorded, specifically:

* Disease clinical and endoscopic activity for UC patients, expressed using Mayo score * Insulin sensitivity, assessed by Matsuda index and OGIS index after an oral glucose tolerance test (OGTT), for MetS patients. * Clinical characteristic, the occurrence of diarrhea and fecal C. difficile toxin, in patients affected by recurrent CDI

In addition, the following data for patients in all cohorts will be collected:

* Erythrocyte Sedimentation Rate (ESR) and C Reactive Protein (CRP) serum levels. * White Blood Count, Red Blood Count, serum creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), urine analysis. At baseline (pre - FMT), a blood and stool sample, and gut (colonic) biopsy will be collected.

After the baseline assessment, all patients will undergo to the FMT combined (colonoscopy + capsules) procedure.

Follow-up visits

All patients will undergo follow-up visits at day 7, 30, 90, 180, 360 after the last FMT.

At each time point, the gastroenterology staff and endocrine and metabolic unit staff of the Fondazione Policlinico Universitario A. Gemelli IRCCS, will assess the same items assessed at baseline (demographic, clinical characteristics including disease activity, and laboratory data) and the same blood and stool sample will be collected. Moreover, a stool sample will be collected after the pre-conditioning with nonabsorbable antibiotics.

Gut biopsies will be collected at day 30,180 and 360 after the last FMT.

Treatment

Patients will receive a first donor FMT by colonoscopy, after a pre-conditioning with vancomycin and neomycin + bacitracin for 3 days, because data from our group show that pre-FMT antibiotics are associated with higher rates of microbial engraftment. Then they will receive two cycles, respectively after 3 and 7 days after colonoscopy - FMT, of frozen donor FMT capsules (15 capsules b.i.d. per 3 days). Patients will always receive feces from the same donor.

Donors Recruitment

Potential donor candidates will be evaluated by the gastroenterology staff of the Fondazione Policlinico Universitario A. Gemelli IRCCS, following protocols recommended by international guidelines and according to the recent recommendations imposed by the reorganization of faecal microbiota transplant during the COVID-19 pandemic.

In addition, in relation to the national and international spread of human cases of monkey pox, according to the reports of the European Centre for Disease Prevention and Control (ECDC) and the FDA and as indicated in the circulars of the General Directorate for Health Prevention of the National Ministry of Health of 25/5/2022 (Prot. DGPREV 0026837), of 02/08/2022 (Prot. DGPREV 0034905) and in The National Transplants Center note of 07/06/2022 (Prot. ISS 0021745).

Collection and storage of stool and blood samples

Stool samples will be collected in donors and in patients at baseline and at each follow-up visit, using a Zymo buffer, to preserve feces at room temperature for up to 48 hours. Fecal samples will be stored at -80°C and assigned de-identified IDs. Blood samples will be collected in donors and in patients at each timepoint (1 mL of whole blood per sample), centrifuged at 2000 rpm for 15 minutes for serum collection, stored at a temperature of -80°C. Both stool and blood samples will be stored until the end of the clinical study (after the end of the follow-up of the last enrolled patient). Then, blood samples will be used for the analysis of human genes, associated with microbiome and beta diversity, while stool samples will be used for microbiome analysis.

RNA extraction

Total RNA will be prepared by the RNeasy kit (Qiagen) based on manufacturer's instructions. Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. 100 ng/μl of RNA will be analyzed for RNA integrity and then interrogated by microarray using Affymetrix technology. Fold-change data will be calculated for each gene from the microarray analysis, comparing expression among different groups of samples. Data set will be trimmed to include only those genes showing at least a ±1.5-fold change relative to the C. difficile infection cohort, which will be then submitted to Ingenuity Pathway Analysis (IPA, Qiagen) for unsupervised clustering analysis. Potential gene networks will be identified by the Global Molecular Network algorithm.

Genome extraction and shotgun metagenomic sequencing

DNA extraction will be performed by using the Dneasy PowerSoil Pro Kit (QIAGEN, Germany) according to the manufacturer's procedures. DNA concentration will be measured with Qubit (Thermo Fisher Scientific, USA), and DNA will be then stored at - 20°C. Sequencing libraries will be prepared using the Illumina® DNA Prep (M) Tagmentation kit (Illumina, California, USA) following the manufacturer's guidelines.

Shotgun metagenomic sequencing will be performed on the Illumina NovaSeq platform.

A \>7.5Gb/sample of 150nt paired end reads (insert size \~150nt) will be generate. This will be sufficient to cover at 2x a 4Mb bacterial, fungal, and viral genome present at an abundance of 0.1% after accounting for human DNA reads removal, and to detect with our markerbased strategy organisms at abundances as low as 0.01%. All the above procedures have been extensively validated. As gut mycobiome is hardly detected by WGS, it will be assessed also by sequencing the ITS2 region and the 18SrRNA gene.

Metagenome quality control and pre-processing

Newly generated shotgun metagenomic sequences will be pre-processed and quality controlled using the pipeline available at https://github.com/SegataLab/preprocessing.

Reads will be quality-controlled and those of low quality (quality score 2 ambiguous nucleotides were removed with Trim Galore. Contaminant and host DNA will be identified with Bowtie2 using the parameter -sensitive-local, allowing confident removal of the phiX 174 Illumina spike-in and human reads (hg19 human genome release). Remaining high-quality reads will be sorted and split to create forward reverse and unpaired reads output files for each metagenome.

Microbiome taxonomic profiling

Microbiome taxonomic profiling will be performed following the general guidelines and relying on the bioBakery computational environment. The taxonomic profiling and quantification of organisms' relative abundances of all metagenomic samples will be quantified using MetaPhlAn 3.0 (species-level profiling) and StrainPhlAn 3 (strain-level profiling).

• Longitudinal Analysis of Host-Microbiome Interactions and Their Impact on Clinical Outcomes [Time frame: 60 months]