Shotgun Metagenomics Research Articles

Archaea methanogens are associated with cognitive performance through the shaping of gut microbiota, butyrate and histidine metabolism.

The relationship between bacteria, cognitive function and obesity is well established, yet the role of archaeal species remains underexplored. We used shotgun metagenomics and neuropsychological tests to identify microbial species associated with cognition in a discovery cohort (IRONMET, n = 125). Interestingly, methanogen archaeas exhibited the strongest positive associations with cognition, particularly Methanobrevibacter smithii (M. smithii). Stratifying individuals by median-centered log ratios (CLR) of M. smithii (low and high M. smithii groups: LMs and HMs) revealed that HMs exhibited better cognition and distinct gut bacterial profiles (PERMANOVA p = 0.001), characterized by increased levels of Verrucomicrobia, Synergistetes and Lentisphaerae species and reduced levels of Bacteroidetes and Proteobacteria. Several of these species were linked to the cognitive test scores. These findings were replicated in a large-scale validation cohort (Aging Imageomics, n = 942). Functional analyses revealed an enrichment of energy, butyrate, and bile acid metabolism in HMs in both cohorts. Global plasma metabolomics by CIL LC-MS in IRONMET identified an enrichment of methylhistidine, phenylacetate, alpha-linolenic and linoleic acid, and secondary bile acid metabolism associated with increased levels of 3-methylhistidine, phenylacetylgluamine, adrenic acid, and isolithocholic acid in the HMs group. Phenylacetate and linoleic acid metabolism also emerged in the Aging Imageomics cohort performing untargeted HPLC-ESI-MS/MS metabolic profiling, while a targeted bile acid profiling identified again isolithocholic acid as one of the most significant bile acid increased in the HMs. 3-Methylhistidine levels were also associated with intense physical activity in a second validation cohort (IRONMET-CGM, n = 116). Finally, FMT from HMs donors improved cognitive flexibility, reduced weight, and altered SCFAs, histidine-, linoleic acid- and phenylalanine-related metabolites in the dorsal striatum of recipient mice. M. smithii seems to interact with the bacterial ecosystem affecting butyrate, histidine, phenylalanine, and linoleic acid metabolism with a positive impact on cognition, constituting a promising therapeutic target to enhance cognitive performance, especially in subjects with obesity.

Advances in metagenomic sequencing for infectious disease management: From pathogen profiling to antibiotic resistance

Metagenomics has revolutionized the study of infectious diseases by enabling the analysis of microbial communities without the need for cultivation. This approach, powered by high-throughput sequencing technologies like 16S rRNA sequencing and shotgun metagenomics, allows the identification of both culturable and unculturable microbes, providing a comprehensive view of pathogens. Metagenomics offers critical insights into microbial diversity and interactions, particularly in polymicrobial infections, respiratory and gastrointestinal infections, and bloodstream infections. Advancements in bioinformatics, artificial intelligence (AI), and machine learning have enhanced the accuracy and speed of metagenomic data interpretation, aiding pathogen detection, antibiotic resistance surveillance, and personalized treatment strategies. Despite challenges such as sample complexity, host DNA interference, and data interpretation difficulties, metagenomics continues to evolve as a crucial tool for diagnostics and therapeutic development. Future research aims to address current gaps, including integrating AI into metagenomics for more precise pathogen profiling and developing targeted therapies based on microbial communities. Ultimately, metagenomics holds the potential to transform infectious disease management, offering real-time diagnostics, better global surveillance, and the promise of personalized medicine in the fight against emerging pathogens.

High throughput single cell metagenomic sequencing with semi-permeable capsules: unraveling microbial diversity at the single-cell level in sewage and fecal microbiomes

Single-cell sequencing may serve as a powerful complementary technique to shotgun metagenomics to study microbiomes. This emerging technology allows the separation of complex microbial communities into individual bacterial cells, enabling high-throughput sequencing of genetic material from thousands of singular bacterial cells in parallel. Here, we validated the use of microfluidics and semi-permeable capsules (SPCs) technology (Atrandi) to isolate individual bacterial cells from sewage and pig fecal samples. Our method involves extracting and amplifying single bacterial DNA within individual SPCs, followed by combinatorial split-and-pool single-amplified genome (SAG) barcoding and short-read sequencing. We tested two different sequencing approaches with different numbers of SPCs from the same sample for each sequencing run. Using a deep sequencing approach, we detected 1,796 and 1,220 SAGs, of which 576 and 599 were used for further analysis from one sewage and one fecal sample, respectively. In shallow sequencing data, we aimed for 10-times more cells and detected 12,731 and 17,909 SAGs, of which we used 2,456 and 1,599 for further analysis for sewage and fecal samples, respectively. Additionally, we identified the top 10 antimicrobial resistance genes (ARGs) in both sewage and feces samples and linked them to their individual host bacterial species.

Persistent functional and taxonomic groups dominate an 8,000-year sedimentary sequence from Lake Cadagno, Switzerland

Most of our knowledge of deep sedimentary life comes from marine environments; however, despite their relatively small volume, lacustrine sediments constitute one of the largest global carbon sinks and their deep sediments are largely unexplored. Here, we reconstruct the microbial functional and taxonomic composition of an 8,000-year Holocene sedimentary succession from meromictic Lake Cadagno (Switzerland) using shotgun metagenomics and 16S rRNA gene amplicon sequencing. While younger sediments (<1,000 years) are dominated by typical anaerobic surface sedimentary bacterial taxa (Deltaproteobacteria, Acidobacteria, and Firmicutes), older layers with lower organic matter concentrations and reduced terminal electron acceptor availability are dominated by taxa previously identified as “persistent populations” within deep anoxic marine sediments (Candidatus Bathyarchaeia, Chloroflexi, and Atribacteria). Despite these dramatic changes in taxonomic community composition and sediment geochemistry throughout the sediment core, higher-order functional categories and metabolic marker gene abundances remain relatively consistent and indicate a microbial community capable of carbon fixation, fermentation, dissimilatory sulfate reduction and dissimilatory nitrate reduction to ammonium. As the conservation of these metabolic pathways through changes in microbial community compositions helps preserve the metabolic pathway connectivity required for nutrient cycling, we hypothesize that the persistence of these functional groups helps enable the Lake Cadagno sedimentary communities persist amidst changing environmental conditions.

Prevalence of HSV Genomic Signatures Among Acanthamoeba Hosts and Contaminated Lens Cases: A Molecular and Clinical Study.

To document the presence of herpes simplex virus (HSV) genomic signatures among Acanthamoeba hosts recovered from patients with Acanthamoeba keratitis (AK) and in contaminated lens cases. We used a combination of PCR sequencing and shotgun metagenomics to detect and confirm the presence of HSV genomic signatures in Acanthamoeba hosts and lens cases. Clinical outcomes were correlated with the prevalence of host HSV signatures. HSV genomic signatures were detected in 26% (n = 6) of Acanthamoeba hosts recovered from patients with culture confirmed AK. HSV-1 and HSV-2 or both were identified in 33%, 50%, and 17% of isolates, respectively. Fifty-two percent of patients (12/23) were misdiagnosed initially as presenting with HSV keratitis. Patients with HSV-positive Acanthamoeba isolates had a mean best-corrected visual acuity of 1.43 LogMAR at diagnosis and 0.53 LogMAR at follow-up, compared with 1.85 and 0.92 LogMAR, respectively, in HSV-negative cases. Contact lens use was identified as a risk factor in 83% of 18 patients. We detected 46,597 viral signatures in 5 of 14 contaminated lens cases (35.7%). Distribution included 33% bacteriophages, 8.2% giant viruses, 4.1% nonhuman Herpesviridae, and 0.39% human Herpesviridae. Among the 184 human Herpesviridae genomic signatures, HSV types 1 or 2 or both were documented in 5.7%, VZV in 39.7%, HHV7 in 38.6%, HHV6 in 15.0%, and Epstein-Barr virus in 0.5%. This study is the first to identify HSV-positive genomic signatures in clinical AK isolates and/or contact lens cases. Taken together, the high prevalence of HSV genomic signatures in both amebic hosts and lens cases, might signal an unrecognized Acanthamoeba-HSV association and the need for reassessing current management.

Alpha synuclein overexpression can drive microbiome dysbiosis in mice

Growing evidence indicates that persons with Parkinson disease (PD), have a unique composition of indigenous gut microbes. Given the long prodromal or pre-diagnosed period, longitudinal studies of the human and rodent gut microbiome before symptomatic onset and for the duration of the disease are currently lacking. PD is partially characterized by the accumulation of the protein α-synuclein (α-syn) into insoluble aggregates, in both the central and enteric nervous systems. As such, several experimental rodent and non-human primate models of α-syn overexpression recapitulate some of the hallmark pathophysiologies of PD. These animal models provide an opportunity to assess how the gut microbiome changes with age under disease-relevant conditions. Here, we used a transgenic mouse strain, which overexpress wild-type human α-syn to test how the gut microbiome composition responds in this model of PD pathology during aging. Using shotgun metagenomics, we find significant, age and genotype-dependent bacterial taxa whose abundance becomes altered with age. We reveal that α-syn overexpression can drive alterations to the gut microbiome composition and suggest that it limits diversity through age. Taxa that were most affected by genotype-age interaction were Lactobacillus and Bifidobacteria. In a mouse model, we showed direct link between alpha synuclein geneotype (hallmark of PD), a dysbiotic and low-diversity gut microbiome, and dysbiotic levels of Bifidobacteria and Lactobacillus (most robust features of PD microbiome). Given emerging data on the potential contributions of the gut microbiome to PD pathologies, our data provide an experimental foundation to understand how the PD-associated microbiome may arise as a trigger or co-pathology to disease.

Alterations in gut microbiome in age- and diet-induced colorectal cancer (CRC) progression in a preclinical model.

223 Background: CRC is the third leading cause of cancer related deaths worldwide with age and diet among the strongest risk factors. Emerging evidence suggests gut microbiome plays important role in CRC and is highly impacted by the exposome primarily the food intake. Since diet and age plays an important role in gut microbiome diversity, the present study aimed to investigate the alterations in the gut microbiome in young versus old mice harboring CRC allografts and fed with different diets. Methods: Two cohorts of C57BL/6 including young (n = 9, age = 6 weeks) and old (n = 9, age = 20-24 months) mice were implanted with 1x10 6 MSI (microsatellite instable) CRC MC38 tumor cells. Mice in both cohorts were randomized into three different diet groups: normal diet (ND; standard chow), high-fat (HF) and calorie-restricted (CR: 30% reduction in total calories). Mice were housed individually under standard laboratory conditions. Mice fecal samples were collected and subjected to DNA isolation (ZymoBIOMICS-96 MagBead DNA Kit), followed by metagenomic shotgun and whole genome sequencing (ZymoBIOMICSe and Illumina NovaSeq, respectively). P ≤ 0.05 were considered as statistically significant. Results: Fecal microbiome analysis showed that old microbiome has higher alpha diversity compared to the young mice. No statistically significant differences in were found in microbiome diversities between the diet groups. Pairwise permanova results showed statistically significant differences between microbiomes of old vs. young groups ( P : 0.001), CR vs. HF groups ( P : 0.012) and HF vs. ND groups ( P : 0.021). ANCOM-BC analysis determined the differentiating features and microbial functional pathways in the young mice compared to the old mice group and in different diet groups with relative abundance differences of larger than log 10 2. Bacteroides thetaiotaomicron ( P : 1.43E-39) and Parabacteroides goldsteinii ( P : 2.20E-23) were enriched in young and old groups, respectively. Akkermansia muciniphila ( P : 0.008) was significantly enriched in ND compared to CR group. Lactococcus lactis ( P : 9.94E-160) and Lachnospiraceae bacterium A4 ( P : 4.95E-10) were significantly enriched in HF compared to ND diet groups and vice-versa, respectively. Among the functional pathways, CMP-legionaminate biosynthesis I ( P : 2.38E-13) and L-arginine biosynthesis IV (archaebacteria) ( P : 4.77E-11) were the most significantly enriched in young and old groups, respectively. Conclusions: Our findings highlight the differential diversity and microbial features of gut microbiome in age- and diet-induced CRC progression. Collectively, alteration in diet and age of the host lead to changes in gut microbiota which has a direct impact on activation of specific signaling pathways, metabolism and local and systemic immune responses, which may in turn affect chemosensitivity and outcome in CRC

Bifidobacterium adolescentis-derived nicotinic acid improves host skeletal muscle mitochondrial function to ameliorate sarcopenia.

Sarcopenia significantly diminishes quality of life and increases mortality risk in older adults. While the connection between the gut microbiome and muscle health is recognized, the underlying mechanisms are poorly understood. In this study, shotgun metagenomics revealed that Bifidobacterium adolescentis is notably depleted in individuals with sarcopenia, correlating with reduced muscle mass and function. This finding was validated in aged mice. Metabolomics analysis identified nicotinic acid as a key metabolite produced by B.adolescentis, linked to improvements in muscle mass and functionality in individuals with sarcopenia. Mechanistically, nicotinic acid restores nicotinamide adenine dinucleotide (NAD+) levels in muscle, inhibits the FoxO3/Atrogin-1/Murf-1 axis, and promotes satellite cell proliferation, reducing muscle atrophy. Additionally, NAD+ activation enhances the silent-information-regulator 1 (SIRT1)/peroxisome-proliferator-activated-receptor-γ-coactivator 1-alpha (PGC-1α) axis, stimulating mitochondrial biogenesis and promoting oxidative metabolism in slow-twitch fibers, ultimately improving muscle function. Our findings suggest that B.adolescentis-derived nicotinic acid could be a promising therapeutic strategy for individuals with sarcopenia.

Prevalence and Molecular Characterization of Porcine Parvovirus 2 in Southwest China During 2020–2023

Porcine parvovirus (PPV) is a non-enveloped, single-stranded linear DNA virus that induces reproductive disorders in sows, particularly abortions in primiparous sows. This study investigated the prevalence of PPV in the southwestern region and conducted molecular characterization of PPV strains. An epidemiological survey was conducted on 1534 aborted fetuses from the southwestern region between 2020 and 2023, revealing an abortion rate of 3.00% due to PPV2, with the highest rate of 3.77% in Sichuan. Additionally, 2973 blood samples from sows were tested using ELISA, showing a PPV2 antibody positivity rate of 73.03% to 90%. Through shotgun metagenomics, PPV2 SC2020 was identified in aborted fetal samples from a pig farm in Pengzhou, Sichuan. PCR sequencing analysis yielded seven PPV2 genomic sequences, and the phylogenetic analysis of eight PPV2 strains with thirty reference strains showed distinct evolutionary branches. The virus was successfully isolated from PPV2-positive samples, and the phylogenetic analysis of PPV2 SC2020 revealed ORF1 gene homology of 94.9% to 99.3% and the ORF2 gene homology of 93.1% to 98.0%, with 34 reference strains. Homologous recombination analysis indicated that SC2020 is a recombinant strain of HeB03 and S1.

Contribution of shotgun metagenomics in the diagnosis of a subcutaneous phaeohyphomycosis caused by Parathyridaria percutanea.

Contribution of shotgun metagenomics in the diagnosis of a subcutaneous phaeohyphomycosis caused by Parathyridaria percutanea.

Motility genes are associated with the occurrence of Drosophila melanogaster-associated gut microbes

Abstract Recent work highlighted the role of motility genes in dispersing fly-associated microbes and their spread between hosts. We investigated whether bacterial genes encoding motility are associated with the occurrence of bacteria above passive dispersal levels in the gut of wild Drosophila melanogaster. We revisited 16S amplicon and shotgun metagenome data of wild flies and correlated four genera of bacteria (Commensalibacter, Gluconobacter, Lactobacillus, and Tatumella) with motility genes. We plotted the microbes against neutral models of ecological drift and passive dispersal. Microbes with positive correlations to motility were exclusively found at or above neutral model predictions, suggesting motility genes are crucial for fly microbiota spread and colonization. This information is crucial for understanding how specific gene functions contribute to microbial community dispersal and colonization within the fly host. Moreover, this study’s findings serve as a proof of concept for using the neutral model to predict microbial functions essential for survival and dissemination in diverse hosts.

Antibiotic exposure is associated with minimal gut microbiome perturbations in healthy term infants

BackgroundThe evolving infant gut microbiome influences host immune development and later health outcomes. Early antibiotic exposure could impact microbiome development and contribute to poor outcomes. Here, we use a prospective longitudinal birth cohort of n = 323 healthy term African American children to determine the association between antibiotic exposure and the gut microbiome through shotgun metagenomics sequencing as well as bile acid profiles through liquid chromatography-mass spectrometry.ResultsStool samples were collected at ages 4, 12, and 24 months for antibiotic-exposed (n = 170) and unexposed (n = 153) participants. A short-term substudy (n = 39) collected stool samples at first exposure, and over 3 weeks following antibiotics initiation. Antibiotic exposure (predominantly amoxicillin) was associated with minimal microbiome differences, whereas all tested taxa were modified by breastfeeding. In the short-term substudy, we observed microbiome differences only in the first 2 weeks following antibiotics initiation, mainly a decrease in Bifidobacterium bifidum. The differences did not persist a month after antibiotic exposure. Four species were associated with infant age. Antibiotic exposure was not associated with an increase in antibiotic resistance gene abundance or with differences in microbiome-derived fecal bile acid composition.ConclusionsShort-term and long-term gut microbiome perturbations by antibiotic exposure were detectable but substantially smaller than those associated with breastfeeding and infant age.

Seed microbiomes promote Astragalus mongholicus seed germination through pathogen suppression and cellulose degradation

BackgroundSeed-associated microorganisms play crucial roles in maintaining plant health by providing nutrients and resistance to biotic and abiotic stresses. However, their functions in seed germination and disease resistance remain poorly understood. In this study, we investigated the microbial community assembly features and functional profiles of the spermosphere and endosphere microbiomes related to germinated and ungerminated seeds of Astragalus mongholicus by using amplicon and shotgun metagenome sequencing techniques. Additionally, we aimed to elucidate the relationship between beneficial microorganisms and seed germination through both in vitro and in vivo pot experiments.ResultsOur findings revealed that germination significantly enhances the diversity of microbial communities associated with seeds. This increase in diversity is driven through environmental ecological niche differentiation, leading to the enrichment of potentially beneficial probiotic bacteria such as Pseudomonas and Pantoea. Conversely, Fusarium was consistently enriched in ungerminated seeds. The co-occurrence network patterns revealed that the microbial communities within germinated and ungerminated seeds presented distinct structures. Notably, germinated seeds exhibit more complex and interconnected networks, particularly for bacterial communities and their interactions with fungi. Metagenome analysis showed that germinated seed spermosphere soil had more functions related to pathogen inhibition and cellulose degradation. Through a combination of culture-dependent and germination experiments, we identified Fusarium solani as the pathogen. Consistent with the metagenome analysis, germination experiments further demonstrated that bacteria associated with pathogen inhibition and cellulose degradation could promote seed germination and vigor. Specifically, Paenibacillus sp. significantly enhanced A. mongholicus seed germination and plant growth.ConclusionsOur study revealed the dynamics of seed-associated microorganisms during seed germination and confirmed their ecological role in promoting A. mongholicus seed germination by suppressing pathogens and degrading cellulose. This study offers a mechanistic understanding of how seed microorganisms facilitate successful seed germination, highlighting the potential for leveraging these microbial communities to increase plant health.9Vhf17h9wMs7kvS_yZTv5-Video

Exploring animal food microbiomes and resistomes via 16S rRNA gene amplicon sequencing and shotgun metagenomics.

As a diverse and complex food matrix, the animal food microbiota and repertoire of antimicrobial resistance (AMR) genes remain to be better understood. In this study, 16S rRNA gene amplicon sequencing and shotgun metagenomics were applied to three types of animal food samples (cattle feed, dry dog food, and poultry feed). ZymoBIOMICS mock microbial community was used for workflow optimization including DNA extraction kits and bead-beating conditions. The four DNA extraction kits (AllPrep PowerViral DNA/RNA Kit, DNeasy Blood & Tissue Kit, DNeasy PowerSoil Kit, and ZymoBIOMICS DNA Miniprep Kit) were compared in animal food as well as the use of peptide nucleic acid blockers for 16S rRNA gene amplicon sequencing. Distinct microbial community profiles were generated, which varied by animal food type and DNA extraction kit. Employing peptide nucleic acid blockers prior to 16S rRNA gene amplicon sequencing was comparable with post-sequencing in silico filtering at removing chloroplast and mitochondrial sequences. There was a good agreement between 16S rRNA gene amplicon sequencing and shotgun metagenomics on community profiles in animal feed data sets; however, they differed in taxonomic resolution, with the latter superior at resolving at the species level. Although the overall prevalence of AMR genes was low, resistome analysis of animal feed data sets by shotgun metagenomics revealed 10 AMR gene/protein families, including beta-lactamases, erythromycin/lincomycin/pristinamycin/tylosin, fosfomycin, phenicol, and quinolone. Future expansion of microbiome and resistome profiling in animal food will help better understand the bacterial and AMR gene diversity in these commodities and help guide pathogen control and AMR prevention efforts.IMPORTANCEWith the growing interest and application of metagenomics in understanding the structure/composition and function of diverse microbial communities along the One Health continuum, this study represents one of the first attempts to employ these advanced sequencing technologies to characterize the microbiota and AMR genes in animal food. We unraveled the effects of DNA extraction kits on sample analysis by 16S rRNA gene amplicon sequencing and showed similar efficacies of two strategies at removing chloroplast and mitochondrial reads. The in-depth analysis using shotgun metagenomics shed light on the community compositions and the presence of an array of AMR genes in animal food. This exploration of microbiomes and resistomes in representative animal food samples by both sequencing approaches laid important groundwork for future metagenomic investigations to gain a better understanding of the baseline/core microbiomes and associated AMR functions in these diverse commodities and help guide pathogen control and AMR prevention efforts.

P0060 GPR15L counteracts chronic colitis by shaping the intestinal microbiota

Abstract Background The G protein-coupled receptor 15 (GPR15) is expressed on gut-homing T cells and the interaction of the GPR15 ligand GPR15L with GPR15 has recently been shown to crucially control integrin-dependent adhesion to the endothelium. However, the impact of GPR15L, which is predominantly expressed by colonic epithelial cells, on experimental colitis and inflammatory bowel disease (IBD) is currently not clear. Methods We studied GPR15L in experimental colitis with Gpr15l-deficient and -overexpressing mice as well as with rectal application of recombinant GPR15L. We performed phenotypical and molecular disease characterization using endoscopy, IVIS in vivo imaging, histology, immunofluorescence, FISH, qPCR, and multiplex ELISA. We also performed bulk RNA and metagenomic sequencing. We analyzed the expression of GPR15L in samples from IBD patients and non-IBD controls using qPCR and immunofluorescence and correlated it with clinical information. We explored the function of GPR15L in vitro using radial diffusion assays and antimicrobial assays. Results Acute DSS colitis was aggravated in Gpr15l-deficient mice compared to Gpr15l-proficient mice, while it was attenuated in Gpr15l-transgenic mice. This was T cell-independent, since the phenotype was conserved in Rag-/-Gpr15l-/- mice. Moreover, it was independent of Gpr15 in T cell transfer colitis with Gpr15-deficient and -proficient donor cells. RNA sequencing of colon tissue from Gpr15l-/- and Gpr15l+/+ mice with DSS colitis showed differential regulation in genes related to mucosal barrier function and host-microbiota crosstalk. Accordingly, 16S rRNA sequencing revealed a substantial difference in the intestinal microbial diversity of Gpr15l-/- and Gpr15l+/+ mice. Shotgun metagenomics and radial diffusion assays revealed that GPR15L acts as a selective anti-microbial peptide on community and single strain level, respectively, that predominantly restrains the growth of pathogenic bacteria. Rectal supplementation of recombinant GPR15L in Gpr15l-/- mice rescued increased disease severity in acute DSS colitis and reduced bacterial abundance and translocation. Patients with IBD showed reduced expression of GPR15L compared to controls. Furthermore, the expression of GPR15L was lower in patients with high disease severity compared to those with low severity. Finally, flare-free survival in patients with high GPR15L expression was substantially longer than in patients with low GPR15L expression. Conclusion GPR15L counteracts experimental colitis and seems beneficial in human IBD. Mechanically, it acts as an anti-microbial peptide to promote the homeostasis of the intestinal microbiota. Thus, GPR15L emerges as a potential future therapeutic approach for IBD.

Gut Microbiota Is Not Significantly Altered by Radioiodine Therapy

Purpose: Radiotherapy treatments are known to alter the gut microbiota. However, little is known regarding the effect of nuclear medicine treatments on gut microbiota, and it is established that nuclear medicine is inherently different from radiotherapy. To address this knowledge gap, we conducted a prospective study to identify changes in the gut microbiota of patients treated with [131I]NaI by comparing fecal samples before and after RAIT. Methods: Fecal samples of 64 patients (37 with thyroid cancer and 27 with hyperthyroidism) with indication for RAIT were collected 2 to 3 days before treatment and 8 to 10 days post-treatment. After DNA extraction, the gut microbiota’s richness, diversity, and composition were analyzed by shotgun metagenomics. In addition, LEfSe was performed to compare compositional changes in specific bacteria. Results: Gut microbiome richness and diversity remained unchanged after RAIT, with few changes in its composition identified, especially in patients with hyperthyroidism. Conclusions: This study provides a conceptual and analytical basis for increasing our understanding of the effects of radiopharmaceuticals on gut microbiota. Our preliminary results indicate that RAIT, contrary to radiotherapy, does not cause major disruptions to the human gut microbiota.

Metagenomic investigation of viruses in green sea turtles (Chelonia mydas).

Green sea turtles are listed on the International Union for Conservation of Nature's Red List of Threatened Species. Thus, conservation efforts, including investigation of factors affecting the health of green sea turtles, are critical. Viral communities play vital roles in maintaining animal health. In the present study, shotgun metagenomics was used for the first time to survey viruses in the feces of green sea turtles. Most viral contigs were DNA viruses that mainly belonged to Caudoviricetes, followed by Crassvirales. Additionally, most of the viral contigs were not assigned to any known family or genus, implying a large knowledge gap in the taxonomy of green sea turtle gut viruses. Host prediction showed that most viruses were connected to two phyla: Bacteroidetes and Firmicutes. Furthermore, KEGG enrichment analysis showed that the viral genes were mainly involved in phage-associated and metabolic pathways. Phylogenetic tree reconstruction of Caudovirales terminase large-subunit (TerL) protein showed that most of the sequences were phylogenetically distant. This study expands our understanding of the viral diversity in green sea turtles. In particular, analysis of the virome RNA fraction is exceedingly important for investigating intestinal viromes; therefore, future studies could use metatranscriptomics to study RNA viruses.

Integrating Metagenomic and Culture-Based Techniques to Detect Foodborne Pathogens and Antimicrobial Resistance Genes in Malaysian Produce

Foodborne illnesses pose a significant global health threat, often caused by pathogens like Escherichia coli, Listeria monocytogenes, and Salmonella spp. The emergence of antibiotic-resistant strains further exacerbates food safety challenges. This study combines shotgun metagenomics and culture-based approaches to detect foodborne pathogens and antimicrobial resistance genes (ARGs) in Malaysian produce and meats from the Kinta Valley region. A total of 27 samples comprising vegetables, meats, and fruits were analyzed. Metagenomics provided comprehensive microbial profiles, revealing diverse bacterial communities with species-level taxonomic resolution. Culture-based methods complemented these findings by identifying viable pathogens. Key foodborne pathogens were detected, with Listeria monocytogenes identified in meats and vegetables and Shigella flexneri detected inconsistently between the methods. ARGs analysis highlighted significant resistance to cephalosporins and penams, particularly in raw chicken and vegetable samples, underscoring the potential public health risks. While deli meats and fruits exhibited a lower antimicrobial resistance prevalence, resistant genes linked to E. coli and Salmonella strains were identified. Discrepancies between the methods suggest the need for integrated approaches to improve the pathogen detection accuracy. This study demonstrates the potential of metagenomics in advancing food safety research and supports its adoption as a complementary tool alongside culture-based methods for comprehensive foodborne pathogen surveillance and ARG profiling in Malaysian food systems.

P1343 Impact of Biologic Therapy on Gut Microbiome in Crohn's Disease: An Indian Patient Cohort Study

Abstract Background The advent of biologic therapy has revolutionized the treatment paradigm in IBD. In developing countries including India, where affordability and accessibility drive the treatment choice, it is important to understand the mechanisms behind biologic therapy and pave way to the future to predict response rates. This prospective study examines the impact of biologic therapy on the gut microbiome in moderate-to severe CD patients in India. Methods Fecal samples were collected from healthy controls(n=36) and 39 CD patients at baseline and after completing induction doze [Infliximab (n=16), Vedolizumab (n=6), and Ustekinumab (n=17)]. Individuals on recent antibiotics(<8weeks)/probiotics/NSAIDs were excluded. Clinical response was assessed at 6-weeks, with follow-up at 6 months to classify responders (CDAI<150) and non-responders(CDAI>150). DNA was isolated from 114 fecal samples and subjected to whole-genome shotgun metagenomics to generate a minimum of 6GB data per sample. After removal of human DNA read contamination, taxonomy and functional annotation was assigned using Kraken2, Bracken, and Human3 tools and statistical significance by using Wilcoxon Test (R-Studio). Results The gut microbiome in CD at baseline exhibited reduced diversity, lower SCFA-producing bacteria and increased pro-inflammatory bacteria like Collinsella and Escherichia compared to healthy cohort. The human DNA content in fecal samples was significantly higher in CD compared to healthy cohort with mean of 19.2% and 0.06% respectively. Post biologic therapy, there was significant restoration of the gut microbiome with increase in bacterial diversity, increased abundance of SCFA-producing Firmicutes and Phascolaractobacterium , probiotic Bifidobacterium species and reduction in pathogenic bacteria Enterococcus gallinarium. The average human DNA content also decreased to 9.6%. On sub-analysis, between responders (n=31) and non-responders; pathogenic Klebsiella pneumonia, Fusobacterium and Corynebacterium were found to be higher in non-responders compared to responders (Fig2A). Further, 19 metabolic pathways significantly differed between responders and non-responders, which included those pathways responsible for degradation of carbohydrates and generation of precursor metabolites and energy higher in the former (Fig2B). Conclusion There is increased gut dysbiosis in CD relative to healthy cohort which appears to get restored upon biologic therapy. The findings suggest the biologic therapy may play a role in restoring the compromised intestinal barrier in CD. A microbiome-based signature to predict response rates on biologic therapy can be seen in future.

DOP022 Shared and Distinct Features of the Gut Microbiome in Immune-mediated Inflammatory Diseases: Initial Analysis from the INTEGRATE Cohort Study

Abstract Background Inflammatory bowel disease (IBD) and ankylosing spondylitis (AS) share overlapping pathophysiological mechanisms as immune-mediated inflammatory diseases. While gut microbial dysbiosis has been implicated in both conditions, the shared and distinct features of the oral-gut microbiome axis remain poorly understood. This study aims to characterize the oral and gut microbial signatures and their relationship with environmental factors in IBD and AS to identify common pathogenic pathways and disease-specific patterns. Methods The INTEGRATE study is a nationwide prospective observational study in Korea targeting recruitment of 1,100 IBD patients, 700 AS patients, and 2,244 healthy controls over five years (Table 1). Systematic collection includes clinical data and biological samples (saliva, stool, blood, intestinal tissues) for multi-omics analyses. Microbial characterization employs full-length 16S rRNA gene sequencing and shotgun metagenomics for saliva and stool samples, complemented by metabolomic analyses of saliva, stool, and plasma. This initial analysis includes gut microbiome data from 254 IBD patients, 156 AS patients, and 194 healthy controls. Results Alpha diversity analysis revealed a gradient pattern, with IBD patients showing lowest gut microbiome diversity, AS patients intermediate, and healthy controls highest (p<1.0e-12 for all indices, Fig. 1). In IBD patients, alpha diversity significantly correlated with disease activity indices (Harvey-Bradshaw Index score in Crohn's disease, total Mayo score in ulcerative colitis) and inflammatory markers (CRP, r=-0.13, p=0.042). Blood urea nitrogen levels showed positive correlation with alpha diversity in both IBD (r=0.21, p=0.00062) and AS cohorts (r=0.20, p=0.015). Beta diversity analysis demonstrated distinct clustering patterns (UniFrac distance), with AS patients positioned intermediately between IBD patients and healthy controls (Healthy vs IBD: R²=0.0502, Healthy vs AS: R²=0.0148, IBD vs AS: R²=0.0198; all p=0.001). Species-level analysis through full-length 16S rRNA sequencing identified specific taxa (Clostridium bolteae, Blautia hansenii) significantly enriched in both IBD and AS patients compared to controls. Conclusion Our initial analysis demonstrates distinct gut microbiome signatures across IMIDs, with a clear diversity gradient and shared taxonomic features between IBD and AS. These findings, coupled with specific clinical correlations, provide a foundation for understanding common pathogenic pathways. Ongoing multi-omics analyses and quantitative microbiome profiling will further elucidate the role of the oral-gut microbiome axis in these IMIDs, potentially identifying novel microbial biomarkers for precision medicine.