Search Results (298)

16S rRNA amplicon sequencing has been an effective method for profiling microbial taxonomy in microbiome research, as it offers lower per-sample costs and higher sample throughput than shotgun metagenomics. Although 16S rRNA sequencing offers clear advantages over shotgun sequencing, it depends on precise trimming of low-quality bases at the 3′ ends of reads. Given the widespread use of 16S rRNA amplicon sequencing, there is an increasing demand for analysis tools that can identify errors in the 3′ region of reads and remove erroneous bases. While various algorithms for predicting trim locations are widely employed, most are command-line standalone tools, which pose challenges for users with limited computational background or resources. Furthermore, in the absence of biological or experimental priors such as amplicon size, trim position predictions may be unreliable. Here, we introduce PixelCut, a fully automated trim-position prediction framework that requires no hyperparameters or prior biological information for accurate prediction. Unlike most available algorithms that operate on raw FASTQ data, PixelCut analyzes the per-base quality report generated by FastQC to infer trimming positions. Based on the recommended quality score threshold from the quality report, PixelCut inspects the quality scores across bases and automatically determines the recommended trim position using character recognition techniques based on computer vision. We have also developed a user-friendly web application to make the method accessible to those without programming expertise, while offering a command-line version for advanced users. Through comprehensive computer simulations, we show that PixelCut produces taxonomic profiling results that are consistent with those from popular trim-location prediction algorithms. Full article

Antimicrobial resistance (AMR) poses a critical public health threat, with rising multidrug resistance cases compromising treatment effectiveness. Knowledge about the resistome in dairy production systems remains limited, particularly regarding lactating cows. This study investigated the microbiome and resistome across the hospital, fresh, and mid-lactation pens on 18 conventional dairy farms in California and Ohio using shotgun metagenomic sequencing of pooled fecal samples. Pooled fecal pat samples were collected as part of a larger field study using a quasi-experimental design that assigned farms to the training intervention group (six per state) or the control group (three per state). For the training intervention group, farm worker(s), identified as having the task of diagnosing and treating adult cows on the farm, participated in a training program on antimicrobial stewardship practices. Pooled fecal samples (n = 7) were collected at enrollment and 3 months after the intervention was completed on each participating farm (n = 18). A total of 10,221 bacterial species and 345 AMR genes conferring resistance to 22 antimicrobial classes were identified. The hospital pen exhibited a higher AMR gene diversity compared to fresh and mid-lactation pens (p < 0.05). Several AMR genes showed bimodal distribution, suggesting complex transmission mechanisms. Network analysis revealed distinct gene correlation profiles across pens, with the hospital pen showing fewer gene interactions. Our findings suggest that farm-level antimicrobial drug use may not be the sole or primary driver of resistome composition in pooled fecal samples from dairy cattle, highlighting the need to investigate other factors influencing AMR dynamics in livestock systems. Full article

The microbiome of howler monkeys is being studied as a potential indicator of forest health. This explorative research aimed to analyze the microbiome, antibiotic resistance genes, and virulence factors of the howler monkey (Alouatta seniculus) in two Colombian Andean forests. A total of six samples were collected from three monkeys in two different forests. The samples were processed and sequenced using 16S rRNA V3-V4 metabarcoding and shotgun metagenomics. No significant differences in microbial diversity were observed between locations. A total of 43 possible resistance genes were identified, 11 of which were associated with plasmids, while 66 virulence genes were detected. The bacterial genera with the highest number of resistance genes were Escherichia and Enterococcus, whereas Escherichia and Citrobacter exhibited the highest number of virulence factors. The bacteria were predominantly resistant to fluoroquinolones, macrolides and beta-lactams, while adherence was the dominant virulence mechanism. This exploratory study suggests that the locations provide similar habitats for howler monkeys and that the presence of resistance genes is primarily due to intrinsic bacterial resistance mechanisms and natural resistance in wild populations despite the environmental presence of bacterial genera with resistance genes and virulence factors. However, acquisition through interaction with domestic animals was not evaluated. Full article

Diarrhea in young ruminants is a global issue and causes significant economic losses worldwide. In addition to common pathogens like rotavirus, coronavirus, and astrovirus, new viruses can be identified through unbiased next-generation sequencing (NGS) techniques. Here, we report the initial identification of a hunnivirus from a one-month-old goat with diarrhea using shotgun metagenomic NGS. A complete hunnivirus genome was recovered. Phylogenetic tree analysis revealed that this goat hunnivirus was more closely related to cattle hunnivirus than to small ruminant hunnivirus strains, suggesting a prior cross-species transmission event. The genome was used to design primers/probes for the conserved 3D^pol RdRP gene for real-time RT-PCR to screen banked ruminant fecal samples. Screening of 144 ruminant fecal samples showed that 9 of 38 goat, 22 of 96 cattle, and 0 of 8 sheep samples were positive for hunnivirus. Sequencing of the 3D^po region was performed on selected positive samples and revealed two lineages of hunnivirus circulating in North America. Our study highlights the importance of further investigation and monitoring of fecal samples using unbiased metagenomic tools to identify potential pathogens or co-infections in ruminants. Full article

The human endometrium, previously considered a sterile environment, is now recognized as a low-biomass but biologically active microbial niche critical to reproductive health. Advances in sequencing technologies, particularly shotgun metagenomics, have provided unprecedented insights into the taxonomic and functional complexity of the endometrial microbiome. While 16S rRNA sequencing has delineated the distinction between Lactobacillus-dominant and non-dominant microbial communities, shotgun metagenomics has revealed additional diversity at the species and strain level, uncovering microbial signatures that remain undetected by amplicon-based approaches. Current evidence supports the association of Lactobacillus dominance with endometrial homeostasis and favorable reproductive outcomes. Dysbiosis, characterized by increased microbial diversity and enrichment of anaerobic taxa such as Gardnerella, Atopobium, Prevotella, and Streptococcus, is linked to chronic endometritis, implantation failure, and adverse IVF results. Beyond compositional differences, the endometrial microbiome interacts with the host through immunological, metabolic, and epigenetic mechanisms. These interactions modulate cytokine signaling, epithelial barrier integrity, and receptivity-associated gene expression, ultimately influencing embryo implantation. However, discrepancies between published studies reflect the lack of standardized protocols for sampling, DNA extraction, and bioinformatic analysis, as well as the inherent challenges of studying low-biomass environments. Factors such as geography, ethnicity, hormonal status, and antibiotic exposure further contribute to interindividual variability. Culturomics approaches complement sequencing by enabling the isolation of viable bacterial strains, offering perspectives for microbiome-based biotherapeutics. Emerging 3D endometrial models provide additional tools to dissect microbiome–host interactions under controlled conditions. Taken together, the growing body of data highlights the potential of endometrial microbiome profiling as a biomarker for reproductive success and as a target for personalized interventions. Future research should focus on integrating multi-omics approaches and functional analyses to establish causal relationships and translate findings into clinical practice. This review gives a new insight into current knowledge on the uterine microbiome and its impact on implantation success, analyzed through the lenses of microbiology, immunology, and oxidative stress. Full article

Gut microbiota enable wood-feeding insects to digest recalcitrant diets. Two DNA-based analyses were performed. Amplicon sequencing of gut microbiota samples from Cryptocercus punctulatus showed inter-individual heterogeneity with visually distinct ordination patterns; however, no statistically significant differences were detected. Shotgun metagenomics was used to compare the taxonomic and functional profiles of C. punctulatus gut microbiota with those of other xylophagous Dictyoptera. Despite taxonomic differences, C. punctulatus microbiota revealed functional convergence with termites (Mastotermes darwiniensis and Nasutitermes sp.). Carbohydrate metabolism was performed by different bacterial phyla across all insects. All insect species possessed metabolic potential for cellulose, hemicellulose, pectin, and starch digestion, but lignin degradation capabilities were not detected. Termites showed higher abundance of chitin and xylan degradation pathways and nitrogen fixation genes, though nitrogen fixation was also present in Cryptocercus cockroaches. Genes for oxidative stress tolerance were present across all species but were most abundant in cockroaches, particularly, Cryptocercus. All insects harbored antibiotic resistance genes, with highest levels found in cockroaches. These findings indicate that metabolic requirements for wood digestion shape gut microbial community assembly across xylophagous insects, with distinct microbial taxa contributing to cellulose and hemicellulose breakdown. Moreover, the widespread presence of antibiotic resistance genes raises concerns about the potential transmission of antibiotic resistance within insect-associated microbiomes. Full article

Serotonin, a tryptophan metabolite, exerts a significant influence on both brain and gut functionality. While previous research has elucidated the intricate dynamics of the gut–brain axis, the interplay between serotonin pathway metabolites and gut microbiota in individuals undergoing hemodialysis remains largely unexplored. Therefore, this study aimed to investigate gut microbiota composition corresponding to serotonin pathway metabolite levels among patients with hemodialysis. A total of 85 patients undergoing hemodialysis were selected. Their gut microbiota was analyzed using shotgun metagenomic sequencing profiling. The serotonin pathway metabolites, including 5-hydroxytryptophan (5-HTP), serotonin, 5-methoxytryptophan (5-MTP), 5-methoxytryptamine, melatonin, and 6-hydroxymelatonin, were analyzed with the liquid chromatograph–tandem mass spectrometer. The robust linear discriminant analysis Effect Size (LEfSe) was employed to reveal the gut microbiota signature according to levels of serotonin pathway metabolites. A significant β-diversity difference in 5-Methoxytryptamine (p = 0.037) was found, while no variance in α-diversity was detected. Using LefSe analysis, we identified an enriched Tannerellaceae family in the high-hydroxytryptophan (5-HTP) group, the Odoribacteraceae family in the high-serotonin group, the Eubacteriales order in the high-5-methoxytryptophan (5-MTP) group, the Prevotella copri species in the high-5-Methoxytryptamine group, and the Clostridium genus in the high-melatonin group. In contrast, an enriched Clostridiaceae family in the low-5-HTP group, the Clostridiaceae family in the low-serotonin group, and the Bacteroides ovatus species in the low-5-MTP group were found. Distinct gut microbiota signatures linked to serotonin pathway metabolites were identified in patients undergoing hemodialysis. These findings provide insights for future gut–brain axis research and may guide methods to modulate gut microbiota to influence serotonin metabolites. Full article

In piglets, the gut microbiota matures in a segment-specific manner during the nursing period, while fecal-based studies provide limited functional resolution across intestinal sites. We profiled the ileum using 16s rRNA gene sequencing and assessed segmental functions by shotgun metagenomics at selected ages. Ileal species richness and diversity were relatively stable across days. Lactobacillus were prominent from day 7, with stage-associated taxa including Lactobacillus johnsonii, Lactobacillus delbrueckii, Ligilactobacillus salivarius, and Limosilactobacillus pontis. Through metagenomic functional analysis, at 21 days, genes were enriched in butanoate metabolism, and Limosilactobacillus pontis as a potential probiotic played an important role in it. At day 28, metagenomic analysis indicated higher relative abundance in the ileum of pathways linked to cysteine and methionine metabolism and lysine biosynthesis, largely carried by Limosilactobacillus mucosae, Limosilactobacillus oris, and Limosilactobacillus pontis. These data describe the composition and function of the ileum in the intestines of piglets and indicate a differentiation period around day 21 to day 28. Full article

Background: Alterations of the gut microbiota have been increasingly implicated in the pathogenesis of dementia through mechanisms involving systemic inflammation, immune dysregulation, and gut–brain axis disruption. Clinical evidence, however, remains fragmented. Objectives: This systematic review aimed to characterize gut microbiota profiles in individuals with mild cognitive impairment (MCI) or Alzheimer’s dementia (AD), explore mechanistic associations with neurodegeneration, and evaluate the impact of microbiota-targeted interventions on cognitive outcomes. Methods: Following PRISMA 2020 guidelines and a registered protocol (PROSPERO CRD420251074832), PubMed/Medline was searched through May 2025. Eligible studies included randomized controlled trials (RCTs) and cohort and case–control studies assessing microbiota composition or interventions in participants with MCI or AD. Results: Twenty-one studies were included (1 RCT, 20 observational; sample size 22–302). Most used 16S rRNA sequencing; one used shotgun metagenomics. Across cohorts, MCI and AD patients consistently showed reduced short-chain fatty acid-producing bacteria (Faecalibacterium, Ruminococcaceae, Lachnospiraceae) and increased pro-inflammatory taxa (Escherichia/Shigella, Enterobacteriaceae, Bacteroides). Several studies reported reduced microbial diversity. Specific taxa, including Akkermansia muciniphila and Faecalibacterium, were associated with amyloid burden, hippocampal atrophy, and cognitive decline. Environmental and dietary factors influenced microbial composition and cognition. The RCT reported that probiotic supplementation improved inflammatory markers and BDNF levels, although changes in microbiota composition were inconsistent. Conclusions: Gut dysbiosis is strongly associated with cognitive impairment and markers of neurodegeneration. Modulation of the microbiota through diet and probiotics emerges as a promising avenue for dementia prevention and management, though robust longitudinal and interventional studies are needed to confirm causality and therapeutic efficacy. Full article

Prickly pear by-products contain dietary fibre and bioactive components like polyphenols and flavonols, which can reduce total gas and methane emissions. To this end, an in vitro trial was carried out in duplicate utilizing three diets containing hay, concentrate, and two prickly pear by-products obtained after grinding the fruit peel and pastazzo (pulp + peel + seeds), which were ensiled with the addition of 12% wheat bran (raw weight). Based on the ingredient intake recorded in the in vivo study for 12 lactating ewes fed the three diets, an in vitro rumen fermentation study with the innovative Gas Endeavour system (GES) was performed, and the Gage R&R statistical method was used to evaluate the accuracy of the total gas and methane production detected by the GES device. Fermented liquor samples for each diet were used to calculate the disappearance of organic matter and neutral detergent fibre. Shotgun metagenome sequencing analysis was used to evaluate the effect of diet on the rumen fluid microbiota, and it was found that the parameters of repeatability and reproducibility of the total gas and the methane produced after 24 h were satisfactory. Prickly pear by-products display high fermentability for the peel and low fermentability for pastazzo silage, which generates lower total gas and methane emissions. This diminished methane gas production is not correlated with the relative abundance of methanogens. The different chemical and nutritional composition of the three diets altered the rumen bacteria, albeit only slightly, with particular reference to the Succinivibrio and Selenomonas genera. In conclusion, prickly pear peel silage displayed acceptable fermentation traits, which could support its utilization in sheep diets. Full article

The gut microbiome of pigs is important for energy supply and impacts the animals’ health. Additionally, the microbiota can act as a reservoir for antimicrobial resistance genes (ARG). Different husbandry systems in pig farming can influence the microbiome and the overall composition of the resistome, i.e., the complete collection of ARG. In this study, pooled fecal samples were collected repeatedly in one pig farm over a period of two years. One group of animals was kept in organic husbandry conditions with access to an outdoor run, while the other group was kept according to conventional standards. Shotgun metagenomic sequencing was performed on the samples. Additionally, E. coli isolates were subjected to whole-genome sequencing and antimicrobial susceptibility testing. Significant differences were observed in alpha and beta diversity in the microbiome between the two husbandry systems. Families enriched in the organic group included Prevotellaceae, Lachnospiraceae, and Cellulosilyticaceae, while Methanobacteriaceae showed a higher abundance in the conventional group. In the resistome, the differences were smaller, and the dominant genes were the same in both groups. However, there was a significant difference in beta diversity. In addition, the overall frequency of ARG, normalized by 16S rRNA gene content, was on average higher in the organic group. Overall, the husbandry system shaped the microbiome and—albeit to a lesser extent—the resistome in pigs from the same farm. Full article

Background: Autism Spectrum Disorder (ASD) is increasingly associated with alterations in gut microbiota, intestinal permeability, and immune dysregulation. However, integrative studies exploring these mechanisms in Latin American populations are lacking. Objective: To characterize gut microbiota profiles in Colombian children with ASD and evaluate the effects of two microbiota-targeted interventions, an anti-inflammatory diet and a probiotic formulation, on microbial diversity and taxonomic composition. Methods: In a two-phase study, shotgun metagenomic sequencing was performed on fecal samples from 23 children with ASD and 7 typically developing (TD) controls. In the second phase, 17 children with ASD were randomized to receive a 12-week intervention (anti-inflammatory diet, probiotics, or no intervention). Alpha diversity indices (Shannon, Pielou, and Chao1) and differential abundance analyses were conducted. Results: Compared to TD children, those with ASD showed a higher Firmicutes/Bacteroidetes ratio and a significantly increased abundance of genera such as Clostridioides, Thomasclavelia, Alistipes, and Coprococcus. The presence of functional gastrointestinal disorders (FGIDs) in ASD patients is associated with reduced microbial richness. POST-intervention, the anti-inflammatory diet group showed that no statistically significant changes in alpha diversity were observed, although a slight upward trend was noted and significant enrichment of six bacterial genera, including Moraxella and Eubacterium. The probiotic group exhibited a significant increase in Romboutsia and a decrease in Lachnospira. Cytokine–microbiota networks in ASD were fragmented and dominated by IFN-γ and MCP-1 hubs, indicating systemic immune activation. Interventions induced functional remodeling: The anti-inflammatory diet increased the number of beneficial genera (Eubacterium, Adlercreutzia) and shifted networks toward positive correlations involving IL-8 and MIP-1β. Probiotics increased Romboutsia, reduced Lachnospira, and restructured networks with regulatory cytokines (SDF-1α, Eotaxin) and SCFA-producing taxa (Blautia, Roseburia). Conclusions: Children with ASD in Colombia displayed distinct microbial profiles characterized by pro-inflammatory taxa and altered richness. Both the anti-inflammatory diet and probiotics produced compositional shifts in the gut microbiota, although global changes in diversity were limited. These findings support the potential of microbiota-targeted nutritional strategies for ASD and underscore the need for precision interventions tailored to specific clinical and microbial phenotypes. Full article

Hashimoto’s thyroiditis (HT) and alopecia areata (AA) are organ-specific autoimmune diseases that frequently co-occur, suggesting shared immunological and microbial pathways. The gut microbiome has emerged as a key modulator of immune function, yet disease-specific microbial signatures remain poorly defined. Fecal samples from 51 participants (HT: n = 16, AA: n = 17, healthy controls: n = 18) aged 18–65 years were analyzed using shotgun metagenomic sequencing followed by multivariate statistical analyses. While alpha and beta diversity did not differ significantly across groups, taxonomic profiling revealed disease-specific microbial patterns. Bacteroides fragilis was significantly enriched in HT, suggesting a potential role in immune modulation; although mechanisms such as polysaccharide A production and molecular mimicry have been proposed in previous studies, their involvement in HT remains to be confirmed. Microbacterium sp. T32 was elevated in both HT and AA, indicating its potential as a shared autoimmune marker. Functional analysis showed increased fermentation and amino acid biosynthesis in AA, contrasting with reduced metabolic activity and elevated carbohydrate biosynthesis in HT. HT and AA exhibit distinct gut microbial and metabolic signatures. Bacteroides fragilis and Microbacterium sp. T32 may serve as potential microbial correlates for autoimmune activity, offering new insights into disease pathogenesis and targets for microbiome-based interventions. Full article

Tall fescue toxicosis, caused by ingestion of endophyte-infected tall fescue (Lolium arundinaceum), impairs growth and reproduction in beef cattle and results in over USD 3 billion annual loss to the U.S. livestock industry. While the effects on host metabolism and rumen function have been described, the impact on the rectal microbiome remains poorly understood. In this study, we performed whole-genome shotgun metagenomic sequencing on fecal samples collected before and after a 30-day toxic fescue seed supplementation from eight pregnant Angus × Simmental cows and heifers. We generated 157 Gbp of sequencing data in 16 metagenomes, and assembled 13.1 Gbp de novo microbial contigs, identifying 22 million non-redundant microbial genes from the cattle rectum microbiome. Fescue toxicosis significantly reduced alpha diversity (p < 0.01) and altered beta diversity (PERMANOVA p < 0.01), indicating microbial dysbiosis. We discovered significant enrichment of 31 bacterial species post-treatment, including multiple core rumen taxa. Ruminococcaceae bacterium P7 showed an average of 16-fold increase in fecal abundance (p < 0.01), making it the top-featured species in linear discriminant analysis. Functional pathway analysis revealed a shift from energy metabolism to antimicrobial resistance and DNA replication following toxic seed consumption. Comparative analysis showed increased representation of core rumen taxa in rectal microbiota post-treatment, suggesting disrupted rumen function. These findings demonstrate that fescue toxicosis alters both the composition and functional landscape of the hindgut microbiota. Ruminococcaceae bacterium P7 emerges as a promising biomarker for monitoring fescue toxicosis through non-invasive fecal sampling, with potential applications in herd-level diagnostics and mitigation strategies. Full article

Metagenomic investigation of gut microbiome is a comprehensive and rapid technique for the analysis and diagnosis of numerous diseases. The gut microbiome is an intricate ecosystem, coordinated by the interaction of various microbes and the metabolites produced by them, which helps in developing and sustaining immunity and homeostasis. A healthy gut microbiome is driven by different factors, such as nutrition, lifestyle, etc. The current study examines the association of diet to gut microbiome dysbiosis and its role in various disease conditions. Gut microbiome data was collected from 73 patients and tested at BioAro Inc. lab, using shotgun metagenomics through next generation sequencing. It was then analyzed and compared with data from 20 healthy subjects from HMP database. An in-house bioinformatics pipeline (PanOmiQ) and Pathogen Fast Identifier were utilized for secondary analysis, while tertiary analysis was accomplished using R software. Results showed a higher number of opportunistic pathogen microorganisms in the gut microbiome of subjects consuming a meat diet, as compared to those consuming a plant diet. These opportunistic pathogens included Ruminococcus torques (>3.34%), Ruminococcus gnavus (>2.22%), and Clostridium symbiosum (>1.87%). The study also found a higher relative abundance of these pathogens in cancer patients, as compared to healthy subjects. We also observed a highly significant (p < 0.0001) correlation of a meat diet with obesity in comparison to the subjects on a plant diet and the healthy subjects. Our findings suggest that patients following a plant diet have a lower relative abundance of pathogens that are associated with cancer and obesity. These findings provide critical insight into how we can use shotgun metagenomics to study the composition and diversity of the gut microbiome and the effects of a diet on the gut microbiome and its role in metabolic diseases. This is the first report investigating gut microbiota using shotgun metagenomics, correlating with different diseases and diet followed, which might impact the presence of opportunistic pathogens or keystones species. Additionally, it can provide valuable insights to physicians and dietetic practitioners for providing personalized treatment or customizing a diet plan. Full article