Introduction

The gut microbiome plays a significant role in the development, progression, and treatment of various gastrointestinal (GI) diseases. However, analyzing the microbiome remains a time-consuming challenge, mainly due to the lack of validated methodologies that can extract DNA and RNA simultaneously. This study aimed to determine the impact of storage conditions and nucleic acid extraction protocols on the recovery and integrity of microbial genetic material from stool samples.

Aims & Methods

Stool samples from seven healthy individuals were stored under different conditions: in conventional stool collection tubes at room temperature (condition A); or in collection tubes with DNA stabilizer (INVITEK) at room temperature (condition B), at -20ºC (condition C), or at -80ºC (condition D). For DNA and RNA extraction, the Chemagic DNA Stool Extraction kit (Perkin Elmer) was used. Although this kit was only validated for DNA extraction, modifications to the standard protocol were made to achieve RNA extraction. The yield and purity of both DNA and RNA were evaluated using the Nanodrop2000. The applicability of the kit for RNA extraction was ultimately assessed using 16s rRNA sequencing and whole viral genome sequencing (Illumina MiSeq).

Results

Collecting stool samples in tubes containing DNA stabilizer and keeping them frozen allowed for higher yields (mean DNA and RNA concentrations of 70±52 and 51±42ng/µl, respectively, for samples at -80ºC vs. 61±41 and 50±45ng/µl at -20ºC), with good purity. Conversely, significantly lower concentrations were obtained for samples at room temperature, particularly if tubes without stabilizer were used. Perkin Elmer's extraction kit successfully extracted DNA and RNA independently of RNase treatment, although higher yields were obtained without RNase (mean RNA concentrations of 150±123 vs. 51±42ng/µl). Whole genome sequencing confirmed the ability to extract and categorize viral material.

Table 1. DNA and RNA concentrations in samples stored under different conditions.

Conclusion

The simultaneous extraction of DNA and RNA using Perkin Elmer's kit is feasible and accurate, optimizing time and resources. The application of these protocols to samples from patients with GI conditions, particularly those with inflammatory bowel disease, is underway.

Introduction

Understanding the intra- and inter-individual variability of gut microbiota is crucial for advancing microbiota research and its clinical applications. Gut microbial markers play essential roles in maintaining intestinal health, with functional groups including opportunistic, proteolytic, equilibrium, immunoprotective, mucosal homeostasis, proinflammatory, and methanogenic microbes.

Aims & Methods

This study aimed to evaluate the temporal stability of 15 microbial markers and identify potential transient disruptions associated with lifestyle and dietary factors.
A longitudinal observational study was conducted with 6 participants, including 4 healthy individuals and 2 with mild IBS, each providing 7 fecal samples (42 samples total) over a one-month period on randomly selected days. Microbial DNA was extracted from fecal samples and analyzed using a qPCR kit of a specific microbiota test, which includes a panel of 15 microbial markers. Participants completed questionnaires on each sampling day, documenting dietary habits, lifestyle factors, and digestive health. To assess the stability of the microbial markers in relation to differences among individuals, the Intraclass Correlation Coefficient (ICC) was calculated after adjusting a mixed effects model for each marker. These metrics provide numerical insights into the consistency and variability of the microbial populations over time. However, it is essential to contrast these numbers with the nature or behaviour of each marker, allowing for a more accurate and contextualized interpretation of the results, as the observed variability and stability may be influenced by intrinsic factors to each type of microbial marker.

Results

Distinct individual-specific microbiota patterns were observed, with clear inter-individual variability in baseline distributions. Transient alterations were observed in some cases, with many of them associated with dietary factors, such as specific food types or eating habits reported prior to sample collection. Notably, indicative markers of intestinal health, such as Faecalibacterium prausnitzii, Roseburia, and Ruminococcus, remained stable. In contrast, modifications in Akkermansia muciniphila and Methanobrevibacter smithii levels were observed coincident with variations in dietary intake suggesting that these could be an influent factor. Particularly, these disruptions were generally short-lived, with markers returning to baseline levels within a few days. The ICC has demonstrated its effectiveness in refining results and quantifying the relationship among intra- and inter-personal variability of markers. For example, Faecalibacterium prausnitzii exhibited a low ICC (0.112), indicating minimal differences among participants, whereas Methanobrevibacter smithii had a high ICC (0.921), reflecting significant variability among participants.

Conclusion

This study provides preliminary insights into the stability and individual variability of gut microbiota markers analyzed using the qPCR kit of this microbiota test. These findings highlight the importance of establishing personal baselines for accurate diagnostic interpretation and underscore the resilience of the gut microbiota in response to daily lifestyle fluctuations Moreover, the stability of intestinal health indicators markers supports their potential as reliable indicators of gut health. These results provide valuable data for a deeper understanding of gut health dynamics and underscore the importance of personalized approaches in microbiota diagnostics and management.

Disclosure

This work has been previously presented at an international meeting.
Authors: Lia Oliver, Marta Malagón, Mariona Serra-Pagès, and Daniel Rius are employees of GoodGut SLU. Xavier Aldeguer has also collaborated with GoodGut as a medical advisor.
Funding: This project has been partially funded by the AGAUR grant for Industrial Doctorates.

Introduction

Faecal microbiota transplantation (FMT) involves the transfer of microbiota from healthy donors to patients with the aim of correcting dysbiosis and restoring intestinal diversity. FMT has been shown to be successful in the treatment of recurrent or refractory Clostridioides difficile infection. The main limiting factor to carry out FMT is access to treatments due to the absence of intestinal microbiota banks (IMB), a process that requires a complex structure.

Aims & Methods

• Define and standardize procedures for the creation of an IMB.
• Fabrication of viable FMT samples.

The project was carried out at the Hospital Clinic of Barcelona. Processes were defined for recruitment donors, collection and processing samples for their later use in clinical practice. A multidisciplinary team was made up with gastroenterologists, microbiologists, infectologists and two clinical nurses.

Results

From May to December 2021, through a questionnaire distributed to the donation unit of the Blood and Tissue Bank (BTB) of Hospital Clinic, 207 potential donors were identified. 52.7% (109/207) of them were considered suitable, but after the a first screening including clinical visit and microbiological analytical studies carried out, only 18.3% (20/109) of them were able to start the donation process. During this period, 79 donations were collected which enabled the processing of 155 stool samples for FMT. After 2 months of quarantine and the second screening, only 73% of the samples (114/155) were ready to be released for clinical use. Two donors were responsible for 40.5% of the donations, while 30% (6/20) did not contribute any samples during the donation period. If we consider suitable donors those who did not present exclusion criteria in any phase of the established screening, the final number was only 9.1% (10/109).

Conclusion

1. Correct organization of all processes is essential for the establishment of a IMB.
2. Loyalty of committed donors is essential.
3. This was the first step of a collaborative project currently ongoing at the Clinic Hospital and the Bellvitge Hospital, supported by the Department of Health in Catalonia.

Introduction

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection. A pool of healthy stool donors is essential for any FMT program. However, donor
recruitment is not easy due to screening failures and the commitment requested.

Aims & Methods

We aimed to report data from the first two years of stool donor screening, recruitment, and stool banking from the first public Italian FMT center. We prospectively collected data from donor screening questionnaires, laboratory evaluations, and donations. Donor screening has been performed according to the Italian Health Ministry regulation and the FMT European consensus FMT (1): potential donors, all aged below 50 years old, were initially screened for the presence of concomitant gastrointestinal and systemic diseases or for the anamnestic drugs intake or recent antibiotic intake. The subsequent clinical evaluation aimed at excluding at-risk conditions for infectious diseases, while the microbiological screening evaluated the colonization by potential pathogens. Bacterial counts in terms of aerobes and anaerobes concentration [colony forming unit (CFU)/mL] from aliquots of each frozen fecal material after aerobic samples preparation were assessed after 1 week and subsequently monthly until 18 months from the donation.

Results

Two hundred and 12 patients completed screening questionnaires. Of these, 29 (13.7%) were excluded for concurrent gastrointestinal symptoms (altered bowel habits) or diseases, 50 (23.6%) for concomitant systemic diseases, and 8 (3.8%) for antibiotic intake in the previous 6 months. Finally, of the 120 donors clinically evaluated, 79 (37.3%) were excluded for at-risk conditions for infectious disease (e.g., use of illegal drugs, risky sexual behavior, recent body tattoo, piercing, travel in tropical countries, healthcare workers, etc.). Among the remaining 41 donors microbiologically screened, 19 (9%) were excluded for pathogens or potential pathogens presence (6 pathogenic Escherichia coli, 1 Vancomycin-resistant Enterococci, 2 Helicobacter pylori, 1 Rotavirus, 1 Giardia, and 8 for other reasons). Finally, 22 subjects (10.4%) were suitable for donation. Among the 10 healthy donors actively donating stools (50% females), 6 were excluded for pathogens infections diagnosed during donation screenings after a median of 3 donations (3 pathogenic Escherichia coli, 1 Salmonella enterica, 1 Campylobacter jejuni, 1 Cryptosporidium). Among the remaining 4 subjects, 2 discontinued the donation program due to the commitment requested. Finally, 22 subjects (10.4%) were suitable for donation. Ten healthy donors actively donated stools (50% females), but 6 were excluded for pathogens/potentially pathogenic bacteria diagnosed during donation screenings after a median of 3 donations. Among the remaining 4 subjects, 2 discontinued the donation program due to the commitment requested.
Aliquots evaluation showed no differences in aerobic counts until 18 months, while significant differences were found for anaerobic counts already after 1 month (1.33x10^10 CFU/mL vs 7.6x10^9 CFU/mL at donation and after 1 month, respectively, p=0.028).

Conclusion

Only one in ten potential healthy donors is finally eligible for stool donation. Frozen stool samples showed a significant decrease in anaerobic concentration already after 1 month from storage, compared to fresh material.

References

1. Cammarota G., et al. Gut. 2017 Apr;66(4):569-580.

Disclosure

Lecture grants from EG Pharma, AlfaSigma, Bromatech, Echosens, Ferring, Mayoly Spindler and Schwabe Pharma.

Introduction

Fecal microbiota transplantation (FMT) is a highly effective treatment for recurrent Clostridioides difficile infection (CDI), but its safety is jeopardized by the potential risk of transmitting microbial pathogens. Stool biobanks have established to provide both a widespread and equitable access to FMT and high safety, quality, and traceable workflows. To guarantee the safety of each donor stool batch, international guidelines recommend either a quarantine or a direct stool testing with a rapid molecular assay for gut pathogens. While reports of the quarantine-based approach are emerging, data on the direct testing-based approach are still limited

Aims & Methods

Our aim is to report outcomes of a donor screening program for FMT including a direct stool testing-based approach based on a rapid molecular assay for gut pathogens.We prospectively evaluated all candidates enrolled in our FMT donor program since the introduction of a direct stool testing with a rapid molecular assay for gut pathogens, in 2019. Following international guidelines, donor screening consisted of a five-step framework, including: 1) collection of clinical history; 2) blood and stool testing; 3) a further questionnaire the day of each donation; 4) nasopharyngeal swab and stool RT-PCR assay for SARS-CoV-2 the day of each donation (after March 2020); 5) a direct stool testing with a rapid molecular assay for gut pathogens and cultures for MDRO evaluation. Specifically, the RT-PCR Allplex™ Gastrointestinal Panel Assay (Seegene, Korea) was used for a comprehensive identification of common gastrointestinal pathogens. The QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) was used for nucleic acid extraction. We evaluated rates of donations per year, rates and reasons of candidates’ exclusion at each step of the donor screening, as well as the rate of unsuitable fecal donations and the intestinal pathogens that caused their rejection. Finally, we recorded the number of total fecal aliquots provided by eligible donors, and related safety outcomes at 12 weeks after their use within FMT.

Results

From January 2019 to December 2022, 227 candidates were assessed for eligibility. Of them, 122 were excluded at different steps of the screening process. Finally, we enrolled 69 eligible donors (37 females, 53,6%; mean age 37, 30% of the total pool). Thirty-seven fecal aliquots were collected in 2019 (13 donors), 39 in 2020 (20 donors); 51 in 2021 (24 donors), and 104 in 2022 (43 donors). Overall, we collected 231 fecal donations, of which 136 (58.8%) were considered eligible and stored at -80°C for FMT. Eight donations were discarded for insufficient amount of feces (<50 g - 3%), and 87 (37.6%) for positivity to intestinal pathogens, including Enteropathogenic E. coli (EPEC) (n=31 samples – 13.4%), ESBL (extended-spectrum beta-lactamase) E. coli (n=15 samples, 6.5%), Blastocystis hominis (n=17 samples, 7.3%).Notably, we found a seasonal variation among pathogen positivity, as most pathogens were observed in Autumn (from September to December: n=48/87, 55.8%).All donations have been used, and no serious adverse events or infections have been observed at 12 weeks after FMT.

Conclusion

Our screening of donor feces based on direct stool testing guarantees high safety of FMT, although with high rates of donor exclusion, with the need of a well-established framework to guarantee a continuous availability of eligible donors to avoid shortages of fecal aliquots.

Disclosure

G.I. has received personal fees for acting as speaker for Biocodex, Danone, Sofar, Malesci, Metagenics and Tillotts Pharma, and for acting as consultant and/or advisor for Ferring Therapeutics, Giuliani, Malesci and Tillotts Pharma. A.G. reports personal fees for consultancy from Eisai Srl, 3PSolutions, Real Time Meeting, Fondazione Istituto Danone, SinergieSrl, Board MRGE and Sanofi SpA personal fees for acting as a speaker for Takeda SpA, AbbVie and Sandoz SpA and personal fees for acting on advisory boards for VSL3 and Eisai. G.C. has received personal fees for acting as advisor for Ferring Therapeutics. All other authors have no conflicts of interest to disclose.

Introduction

The microbiota of human gastrointestinal tract is a complex and dynamic population of microorganisms and plays a major role in the maintenance of gut health and disease conditions. The microbial population, structure and function vary considerably throughout the upper and lower gut, but specific population and its diversity for each segment of GI tract is still not well described.

Aims & Methods

Aim: The retrospective evaluation of microorganism population and its diversity separately for upper and lower GI tract by cultivation of tissue samples that were obtained at routine endoscopy of 271 patients indicated to examination for different reasons.
Methods: Biopsies were taken using sterilized standard endoscopic biopsy forceps. Two pieces of tissue were obtained without the removal of forceps from the duodenum, from the antrum of the stomach and in same way from different segments of large intestine. Samples were placed into a 10% glucose solution for microbial cultivation.

Results

Overall, 271 biopsy specimens from the upper and lower GI tract were cultivated, specifically in 235 patients samples were obtained from antrum of stomach and duodenum, in 127 patients from large intestine and in 36 patients from both parts of GI tract. The presence of bacteria in upper GI tract was found in 70,21% and was negative in 29,79% of cases, while in lower GI tract bacteria was found in 93,70% and was negative 6,30% of cases. Tissue samples from both parts of GI tract was obtained from 36 patients and the same genera was found in 44,44%. In upper GI tract E. coli was present in 15,74%, followed by Enterobacter cloacae in 10,64%, Pseudomonas aeruginosa in 8,51%, Enterococcus species in 7,66%, Klebsiella pneumonie subs pneumoniae in 5,11%, Candida albicans in 4,26%, Helicobacter pylori and Klebsiella pneumoniae in 2,98%, Proteus mirabilis in 2,55%, Stenotrophomonas maltofilia in 1,70%, Neisseria subflava and E. coli ESBL positive in 0,85% and 0,43% belongs to Klebsiella pneumoniae ESBL positive, Streptococcus salivarius, Acinetobacter baumani, Staphylococcus warneri, Enterococcus faecalis, Streptococcus maltofilia, Enterococcus hirae and Candida complex. In lower GI tracts E. coli was present in 64,57%, followed by Klebsiella pneumoniae in 8,66%, Candida albicans, Proteus mirabilis and Enterobacter cloacae in 3,15%, Enterococcus species and Klebsiella pneumoniae subs pneumoniae in 2,36%, E. coli ESBL positive, Enterococcus faecalis and Pseudomonas aeruginosa in 1,57%, Candida non albicans and Citrobacter koseri in 0,79%.
The diversity of upper and lower GI tract was measured by Shannon diversity index, and it was 2.48 for upper GI tract and 1.29 for lower GI tract.

Conclusion

Most of studies which reveals fundamental differences between microbiota of upper and lower GI tract is based on examination of luminal content. We are presenting our results of the diversity of microbiota based on cultivation of biopsy samples.

Introduction

The composition of the gut microbiota differs between individuals with (advanced) adenomas (AA) and colorectal cancer (CRC) compared to controls, suggesting its importance in the adenoma–carcinoma sequence and highlighting its potential as a non-invasive biomarker. While 16S ribosomal RNA sequencing (16S rRNA) is a commonly used method for microbiome research, metaproteomic (MP) analysis of faecal samples is an emerging alternative. MP is generally used to study human and bacterial proteomes, but additionally allows for microbiota profiling. However, a direct comparison between MP and 16S is lacking.

Aims & Methods

This study aims to evaluate the comparability between faecal microbiota composition profiles derived from bacterial MP and 16S rRNA gene sequencing in patients with colorectal neoplasia.
Subjects scheduled for colonoscopy, regardless of indication, were eligible to provide a faecal sample prior to bowel cleansing. Based on endoscopic and histological findings, patients were divided into subgroups. Both 16S rRNA sequencing and MP analysis by liquid chromatography–tandem mass spectrometry were performed on all faecal samples. Only proteins with a unique peptide were quantified and taxonomically annotated using the Human Microbiome NIH project. For 16S, the SILVA 132 99% OTUs database was used.

Results

Faecal samples from 12 CRC, 10 AA, 11 non-advanced adenoma patients, and 20 controls were included in this study. No significant differences in alpha or beta diversity were observed for either technique. After taxonomic annotation, 225 operational taxonomic units (OTUs) were obtained for 16S consisting of 10 different phyla, 53 families, 180 genera, 14 species and no subspecies. Taxonomic annotation of the bacterial proteins resulted in 354 OTUs across 7 different phyla, 45 families, 81 genera, 261 species and 250 subspecies. At phylum level, Bacteroidetes and Firmicutes accounted for the majority of the relative abundance in both analytical strategies. In total, 5 phyla, 27 families, 44 genera and 4 species were detected with both techniques, including CRC-associated genera such as Bacteroides, Parvimonas, and Prevotella. However, CRC-associated Fusobacterium was detected exclusively with MP, and Peptostreptococcus with 16S. Among CRC-associated species, Ruminococcus torques was detected with both methods, while Fusobacterium nucleatum, Parvimonas micra, and Bacteroides fragilis were detected exclusively with MP. The top 12 most abundant families and genera account for over half of the relative abundance in both methods. For these most abundant taxa, overlap was found for 9 families and 5 genera. The distribution among individuals in the various study groups was subsequently examined at family and genus level. Notably, 16S exhibited a higher inter-individual variability and a higher number of outliers compared to MP.

Conclusion

Metaproteomics had a more limited range for detecting bacterial diversity at genus level but offered higher resolution for identifying bacteria at the (sub)species level. In contrast, the highest resolution for 16S rRNA sequencing is typically achieved up to genus-level, resulting in undetected species. There was considerable overlap between 16S and MP analyses at family level regarding the top 12 most prevalent bacteria, but less overlap at genus level between the two analytical methods. This underscores the value of combining both methods for a more complete analysis of bacterial diversity.