Metagenomic Sequencing Analysis Research Articles

Microbial diversity and gene abundance in denitrifying bioreactors: A comparison of the woodchip surface biofilm versus the interior wood matrix.

Excessive amounts of nitrogen (N) and phosphorus (P) can lead to eutrophication in water sources. Woodchip bioreactors have shown success in removing N from agricultural runoff, but less is known regarding P removal. Woodchip bioreactors are subsurface basins filled with woodchips installed downgradient of agricultural land to collect and treat drainage runoff. Microorganisms use the woodchips as a carbon (C) source to transform N in the runoff, with unresolved biological impacts on P. This study aims to explore microbial communities present in the bioreactor and determine whether milling woodchips to probe the microbial communities within them reveals hidden microbial diversities or potential activities. Metagenomic sequencing and bioinformatic analyses were performed on six woodchip samples (i.e., three unmilled and three milled) collected from a 10-year-old woodchip bioreactor treating agricultural tile drainage. All samples had similar DNA purity, yield, quality, and microbial diversity regardless of milling. However, when sequences were aligned against various protein libraries, our results indicated greater relative abundance of denitrification and P transformation proteins on the outside of the woodchips (unmilled), while the interior of woodchips (milled) exhibited more functional gene abundance for carbohydrate breakdown. Thus, it may be important to characterize microbial communities both within woodchips, and on woodchip surfaces, to gain a more holistic understanding of coupled biogeochemical cycles on N, P, and C in woodchip bioreactors. Based on these findings, we advise that future microbial research on woodchips (and potentially other permeable organic materials) examine both the surface biofilm and the interior organic material during initial studies. Once researchers determine where specific proteins or enzymes of interest are most prevalent, subsequent studies may then focus on either one or both aspects, as needed.

The gut microbial differences between pre-released and wild red deer: Firmicutes abundance may affect wild adaptation after release

Insufficient density of red deer has affected the stability of forest ecosystems and the recovery of large carnivores (represented by Amur tiger). Conservation translocations from captivity to the wild has become an important way to restore the red deer populations. However, the difference in gut microbes between pre-release and wild red deer may affect the feeding adaptability of red deer after release. In this study, we clarified the differences in gut microbes between pre-released and wild red deer and screened the key gut microbes of the red deer involved in feeding by using metagenomic sequencing and feeding analysis. The results showed that the microbial difference between pre-released and wild red deer was mainly related to Firmicutes represented by Eubacteriales and Clostridia, and Firmicutes abundance in pre-released red deer (68.23%) was significantly lower than that of wild red deer (74.91%, p < 0.05). The expression of microbial metabolic pathways in pre-released red deer were significantly lower than those in wild red deer (p < 0.05), including carbohydrate metabolism, amino acid metabolism, glycan biosynthesis and metabolism, etc. The combinations of Firmicutes were significantly positively correlated with the intake of plant fiber and carbohydrate (p < 0.05), and were key microbes to help red deer deal with wild plant resources. Additionally, the combinations of Firmicutes represented by Eubacteriales and Clostridia lacking in pre-released red deer contributed the most to expression of microbial metabolic pathways (importance > 1), showing a significant positive correlation (p < 0.05). This study indicates that high abundance of Firmicutes is an important guarantee for red deer to adapt to the wild feeding environment, which provides critical implications for the recovery of red deer populations and the protection of endangered ungulates.

Metagenomic insights into microbial metabolic mechanisms of a combined solid-phase denitrification and anammox process for nitrogen removal in mainstream wastewater treatment

To overcome the significant challenges associated with nitrite supply and nitrate residues in mainstream anaerobic ammonium oxidation (anammox)-based processes, this study developed a combined solid-phase denitrification (SPD) and anammox process for low-strength nitrogen removal without the addition of nitrite. The SPD step was performed in a packed-bed reactor containing poly-3-hydroxybutyrate-co-3-hyroxyvelate (PHBV) prior to employing the anammox granular sludge reactor in the continuous-flow mode. The removal efficiency of total inorganic nitrogen reached 95.7 ± 1.2% under a nitrogen loading rate of 0.18 ± 0.01 kg N·m3·d−1, and it required 1.02 mol of nitrate to remove 1 mol of ammonium nitrogen. The PHBV particles not only served as biofilm carriers for the symbiosis of hydrolytic bacteria (HB) and denitrifying bacteria (DB), but also carbon sources that facilitated the coupling of partial denitrification and anammox in the granules. Metagenomic sequencing analysis indicated that Burkholderiales was the most abundant HB genus in SPD. The metabolic correlations between DB (Betaproteobacteria, Rhodocyclaceae, and Anaerolineae) and anammox bacteria (Candidatus Brocadiac and Kuenenia) in the granules were confirmed through microbial co-occurrence networks analysis and functional gene annotations. Additionally, the genes encoding nitrate reductase (Nap) and nitrite reductase (Nir) in DB primarily facilitated nitrate reduction, thereby supplying nitric oxide to anammox bacteria for subsequent nitrogen removal with hydrazine synthase (Hzs) and hydrazine dehydrogenase (Hdh). The findings provide insights into microbial metabolism within combined SPD and anammox processes, thus advancing the development of mainstream anammox-based processes in engineering applications.

The Conservation Implications of the Gut Microbiome for Protecting the Critically Endangered Gray Snub-Nosed Monkey (Rhinopithecus brelichi).

The gut microbiota plays a crucial role in regulating energy metabolism, facilitating nutrient absorption, and supporting immune function, thereby assisting the host in adapting to seasonal dietary changes. Here, we compare the gut microbiome composition of wild gray snub-nosed monkeys during winter (from October to December) and spring (from January to March) to understand differences in seasonal nutrient intake patterns. Snub-nosed monkeys are foregut fermenters and consume difficult-to-digest carbohydrates and lichen. To examine the digestive adaptations of gray snub-nosed monkeys, we collected 14 fresh fecal samples for DNA analysis during the winter and spring. Based on 16S rRNA sequencing, metagenomic sequencing, and functional metagenomic analyses, we identified that Firmicutes, Actinobacteria, Verrucomicrobia, and Bacteroidetes constitute a keystone bacterial group in the gut microbiota during winter and spring and are responsible for degrading cellulose. Moreover, the transition in dietary composition from winter to spring was accompanied by changes in gut microbiota composition, demonstrating adaptive responses to varying food sources and availability. In winter, the bacterial species of the genera Streptococcus were found in higher abundance. At the functional level, these bacteria are involved in fructose and mannose metabolism and galactose metabolism c-related pathways, which facilitate the breakdown of glycogen, starch, and fiber found in fruits, seeds, and mature leaves. During spring, there was an increased abundance of bacteria species from the Prevotella and Lactobacillus genera, which aid the digestion of protein-rich buds. Combined, these findings reveal how the gut microbiota adjusts to fluctuations in energy balance and nutrient intake across different seasons in this critically endangered species. Moreover, we also identified Pseudomonas in two samples; the presence of potential pathogens within the gut could pose a risk to other troop members. Our findings highlight the necessity of a conservation plan that focuses on protecting vegetation and implementing measures to prevent disease transmission for this critically endangered species.

Phylogenetic and mutational analysis of H10N3 avian influenza A virus in China: potential threats to human health.

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People's Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient's sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.

Presence and Persistence of ESKAPEE Bacteria before and after Hospital Wastewater Treatment.

The metagenomic surveillance of antimicrobial resistance in wastewater has been suggested as a methodological tool to characterize the distribution, status, and trends of antibiotic-resistant bacteria. In this study, a cross-sectional collection of samples of hospital-associated raw and treated wastewater were obtained from February to March 2020. Shotgun metagenomic sequencing and bioinformatic analysis were performed to characterize bacterial abundance and antimicrobial resistance gene analysis. The main bacterial phyla found in all the samples were as follows: Proteobacteria, Bacteroides, Firmicutes, and Actinobacteria. At the species level, ESKAPEE bacteria such as E. coli relative abundance decreased between raw and treated wastewater, but S. aureus, A. baumannii, and P. aeruginosa increased, as did the persistence of K. pneumoniae in both raw and treated wastewater. A total of 172 different ARGs were detected; blaOXA, blaVEB, blaKPC, blaGES, mphE, mef, erm, msrE, AAC(6'), ant(3″), aadS, lnu, PBP-2, dfrA, vanA-G, tet, and sul were found at the highest abundance and persistence. This study demonstrates the ability of ESKAPEE bacteria to survive tertiary treatment processes of hospital wastewater, as well as the persistence of clinically important antimicrobial resistance genes that are spreading in the environment.

Safety and preliminary efficacy of different doses of fecal microbiota capsule transplantation combined with anti-PD-1 inhibitor for patients with advanced malignant solid tumors.

e14622 Background: Chemotherapy combined with anti-PD-1 inhibitors has now become the standard first-line(1L) treatment for most malignant solid tumors. However, most Phase III studies have found that overall progression-free survival (PFS) with 1L combined subsequent immuno-maintenance therapy (IMT) is still less than 1 year. Fecal microbiota transplantation (FMT) has been shown to synergize with PD-1 antibodies to enhance efficacy. In this study, we administered oral microbiota capsule transplantation to patients(pts) during 1L IMT to evaluate its safety, preliminary efficacy, optimal dosage, and explore the changes in gut microbiota composition and serum metabolism (ChiCTR2100054928). Methods: This is a dose-escalation, exploratory clinical trial. The donor capsules were derived from the feces of a healthy 10-year-old child from a rural area. Twelve late-stage pan-cancer pts who had received standard 1L chemotherapy + PD-1 antibodies and achieved stable or responsive disease were enrolled. The first 3 patients were in the i group (5 capsules po tid d1-3). If no dose-limiting toxicities (DLTs) were observed, another 3 patients were escalated to the i+1 group (10 capsules po bid d1-3). If no DLTs were observed again, another 3 patients were escalated to the i+2 group (10capsules po tid d1-3), and so forth, until the i+3 dose group (20 capsules po bid d1-3). Simultaneously, patients continued receiving the original PD-1 IMT. Fecal samples were collected at baseline (S0), after 10 days (S1), 25days (S2), and 50days (S3) of FMT. Results: From March 2023 to December 2023, 12 pts were successfully enrolled. As of the data cutoff date (December 31, 2023), the median follow-up time was 9.05 months (6.5-9.5 months), and the median PFS has not been reached, with 3 patients experiencing disease progression. Four patients (33.3%) experienced Grade 1-2 FMT-related toxicities, including diarrhea, bloating, and abdominal pain, with no Grade 3 or higher adverse events observed. Fecal metagenomic sequencing analysis showed that the relative abundance levels of Blautia and Faecalibacterium significantly increased after FMT, peaking at day 25 and remaining higher than pre-transplant levels at day 50. We also found that patients in the i+1 group exhibited the greatest changes in microbial composition before and after transplantation, with high absolute and relative transplantation indices. Further analysis of host serum metabolism revealed an increase in molecules related to fatty acid metabolism, such as L-carnitine, stearic acid, and arachidic acid, especially a more pronounced increase in the i+1 group. Conclusions: This preliminary study demonstrates that the combination of oral microbiota capsule and immunotherapy can open up a more economical, safe, and effective avenue for 1L maintenance immunotherapy in tumors. Clinical trial information: ChiCTR2100054928.

Safety and preliminary efficacy of different doses of fecal microbiota capsule transplantation combined with anti-PD-1 inhibitor for patients with advanced malignant solid tumors.

e14622 Background: Chemotherapy combined with anti-PD-1 inhibitors has now become the standard first-line(1L) treatment for most malignant solid tumors. However, most Phase III studies have found that overall progression-free survival (PFS) with 1L combined subsequent immuno-maintenance therapy (IMT) is still less than 1 year. Fecal microbiota transplantation (FMT) has been shown to synergize with PD-1 antibodies to enhance efficacy. In this study, we administered oral microbiota capsule transplantation to patients(pts) during 1L IMT to evaluate its safety, preliminary efficacy, optimal dosage, and explore the changes in gut microbiota composition and serum metabolism (ChiCTR2100054928). Methods: This is a dose-escalation, exploratory clinical trial. The donor capsules were derived from the feces of a healthy 10-year-old child from a rural area. Twelve late-stage pan-cancer pts who had received standard 1L chemotherapy + PD-1 antibodies and achieved stable or responsive disease were enrolled. The first 3 patients were in the i group (5 capsules po tid d1-3). If no dose-limiting toxicities (DLTs) were observed, another 3 patients were escalated to the i+1 group (10 capsules po bid d1-3). If no DLTs were observed again, another 3 patients were escalated to the i+2 group (10capsules po tid d1-3), and so forth, until the i+3 dose group (20 capsules po bid d1-3). Simultaneously, patients continued receiving the original PD-1 IMT. Fecal samples were collected at baseline (S0), after 10 days (S1), 25days (S2), and 50days (S3) of FMT. Results: From March 2023 to December 2023, 12 pts were successfully enrolled. As of the data cutoff date (December 31, 2023), the median follow-up time was 9.05 months (6.5-9.5 months), and the median PFS has not been reached, with 3 patients experiencing disease progression. Four patients (33.3%) experienced Grade 1-2 FMT-related toxicities, including diarrhea, bloating, and abdominal pain, with no Grade 3 or higher adverse events observed. Fecal metagenomic sequencing analysis showed that the relative abundance levels of Blautia and Faecalibacterium significantly increased after FMT, peaking at day 25 and remaining higher than pre-transplant levels at day 50. We also found that patients in the i+1 group exhibited the greatest changes in microbial composition before and after transplantation, with high absolute and relative transplantation indices. Further analysis of host serum metabolism revealed an increase in molecules related to fatty acid metabolism, such as L-carnitine, stearic acid, and arachidic acid, especially a more pronounced increase in the i+1 group. Conclusions: This preliminary study demonstrates that the combination of oral microbiota capsule and immunotherapy can open up a more economical, safe, and effective avenue for 1L maintenance immunotherapy in tumors. Clinical trial information: ChiCTR2100054928.

Characterization of the gut butyrate-producing bacteria and lipid metabolism in African green monkey as a natural host of SIV infection.

Natural hosts of simian immunodeficiency virus (SIV), such as the African green monkey (AGM), possess the ability to avoid acquired immune deficiency syndrome (AIDS) despite lifelong infection. The underlying mechanisms are not completely understood. This study aimed to characterize the gut microbiome and metabolite profiles of different nonhuman primates (NHPs) to provide potential insight into AIDS resistance. Fresh feces from Cynomolgus macaques (CMs), and Rhesus macaques (RMs), SIV- AGMs (AGM_N), and SIV+ AGMs (AGM_P) were collected and used for metagenomic sequencing and metabonomic analysis. Compared with CMs and RMs, significant decreases in the abundances of Streptococcus, Alistipes, Treponema, Bacteroides, and Methanobrevibacter (P < 0.01), and significant increases in the abundances of Clostridium, Eubacterium, Blautia, Roseburia, Faecalibacterium, and Dialister (P < 0.01) were detected in AGM_N. Compared with AGM_N, a trend toward increased abundances of Streptococcus and Roseburia were found in AGM_P. The levels of metabolites involved in lipid metabolism and butanoate metabolism significantly differed among AGM_P, AGM_N and CM (P < 0.05). Our data, for the first time, demonstrated distinguishing features in the abundances of butyrate-producing bacteria and lipid metabolism capacities between different NHP hosts of SIV infection. These findings may correlate with the different characteristics observed among these hosts in the maintenance of intestinal epithelial barrier integrity, regulation of inflammation, and provide insights into AIDS resistance in AGMs.

Performance of Halo-Alkali-Tolerant Endophytic Bacteria on Hybrid Pennisetum and Bacterial Community under Varying Soil Conditions.

Halo-alkali soil threatens agriculture, reducing growth and crop yield worldwide. In this study, physicochemical and molecular techniques were employed to explore the potential of halo-alkali-tolerant endophytic bacteria strains Sphingomonas sp. pp01, Bacillus sp. pp02, Pantoea sp. pp04, and Enterobacter sp. pp06 to enhance the growth of hybrid Pennisetum under varying saline conditions. The strains exhibited tolerance to high salt concentrations, alkaline pH, and high temperatures. Under controlled conditions, all four strains showed significant growth-promoting effects on hybrid Pennisetum inoculated individually or in combination. However, the effects were significantly reduced in coastal saline soil. The best growth-promoting effect was achieved under greenhouse conditions, increasing shoot fresh and dry weights of hybrid Pennisetum by up to 457.7% and 374.7%, respectively, using irrigating trials. Metagenomic sequencing analysis revealed that the diversity and composition of rhizosphere microbiota underwent significant changes after inoculation with endophytic bacteria. Specifically, pp02 and co-inoculation significantly increased the Dyella and Pseudomonas population. Firmicutes, Mycobacteria, and Proteobacteria phyla were enriched in Bacillus PP02 samples. These may explain the best growth-promoting effects of pp02 and co-inoculation on hybrid Pennisetum under greenhouse conditions. Our findings reveal the performance of endophytic bacterial inoculants in enhancing beneficial microbiota, salt stress tolerance, and hybrid Pennisetum growth.

Metagenomics analysis reveals effects of salinity fluctuation on diversity and ecological functions of high and low nucleic acid content bacteria

Salinity is a critical environmental factor in marine ecosystems and has complex and wide-ranging biological effects. However, the effects of changing salinity on diversity and ecological functions of high nucleic acid (HNA) and low nucleic acid (LNA) bacteria are not well understood. In this study, we used 16S rRNA sequencing and metagenomic sequencing analysis to reveal the response of HNA and LNA bacterial communities and their ecological functions to salinity, which was decreased from 26 ‰ to 16 ‰. The results showed that salinity changes had significant effects on the community composition of HNA and LNA bacteria. Among LNA bacteria, 14 classes showed a significant correlation between relative abundance and salinity. Salinity changes can lead to the transfer of some bacteria from HNA bacteria to LNA bacteria. In the network topology relationship, the complexity of the network between HNA and LNA bacterial communities gradually decreased with decreased salinity. The abundance of some carbon and nitrogen cycling genes in HNA and LNA bacteria varied with salinity. Overall, this study demonstrates the effects of salinity on diversity and ecological functions and suggests the importance of salinity in regulating HNA and LNA bacterial communities and functions.

Investigating Distinct Skin Microbial Communities and Skin Metabolome Profiles in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder influenced by genetic predisposition, environmental factors, immune dysregulation, and skin barrier dysfunction. The skin microbiome and metabolome play crucial roles in modulating the skin's immune environment and integrity. However, their specific contributions to AD remain unclear. We aimed to investigate the distinct skin microbial communities and skin metabolic compounds in AD patients compared to healthy controls (HCs). Seven patients with AD patients and seven HCs were enrolled, from whom skin samples were obtained for examination. The study involved 16S rRNA metagenomic sequencing and bioinformatics analysis as well as the use of gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) to detect metabolites associated with AD in the skin. We observed significant differences in microbial diversity between lesional and non-lesional skin of AD patients and HCs. Staphylococcus overgrowth was prominent in AD lesions, while Cutibacterium levels were decreased. Metabolomic analysis revealed elevated levels of several metabolites, including hypoxanthine and glycerol-3-phosphate in AD lesions, indicating perturbations in purine metabolism and energy production pathways. Moreover, we found a positive correlation between hypoxanthine and glycerol-3-phosphate and clinical severity of AD and Staphylococcus overgrowth. These findings suggest potential biomarkers for monitoring AD severity. Further research is needed to elucidate the causal relationships between microbial dysbiosis, metabolic alterations, and AD progression, paving the way for targeted therapeutic interventions.

Crosstalk between human immunodeficiency virus infection and salivary bacterial function in men who have sex with men.

Engaging in anal sexual intercourse markedly increases the risk of developing HIV among men who have sex with men (MSM); oral sexual activities tend to uniquely introduce gut-derived microbes to salivary microbiota, which, combined with an individual's positive HIV status, may greatly perturb oral microecology. However, till date, only a few published studies have addressed this aspect. Based on 16S rRNA sequencing data of bacterial taxa, MicroPITA picks representative samples for metagenomic analysis, effectively revealing how the development and progression of the HIV disease influences oral microbiota in MSM. Therefore, we collected samples from 11 HIV-negative and 44 HIV-positive MSM subjects (stage 0 was defined by HIV RNA positivity, but negative or indeterminate antibody status; stages 1, 2, and 3 were defined by CD4+ T lymphocyte counts ≥ 500, 200-499, and ≤ 200 or opportunistic infection) and selected 25 representative saliva samples (5 cases/stage) using MicroPITA. Metagenomic sequencing analysis were performed to explore whether positive HIV status changes salivary bacterial KEGG function and metabolic pathway in MSM. The core functions of oral microbiota were maintained across each of the five groups, including metabolism, genetic and environmental information processing. All HIV-positive groups displayed KEGG functions of abnormal proliferation, most prominently at stage 0, and others related to metabolism. Clustering relationship analysis tentatively identified functional relationships between groups, with bacterial function being more similar between stage 0-control groups and stage 1-2 groups, whereas the stage 3 group exhibited large functional changes. Although we identified most metabolic pathways as being common to all five groups, several unique pathways formed clusters for certain groups; the stage 0 group had several, while the stage 2 and 3 groups had few, such clusters. The abundance of K03046 was positively correlated with CD4 counts. As HIV progresses, salivary bacterial function and metabolic pathways in MSM progressively changes, which may be related to HIV promoting abnormal energy metabolism and exacerbate pathogen virulence. Further, infection and drug resistance of acute stage and immune cell destruction of AIDS stage were abnormally increased, predicting an increased risk for MSM individuals to develop systemic and oral diseases.

Metagenomic nanopore sequencing for exploring the nature of antimicrobial metabolites of Bacillus haynesii

Multidrug-resistant (MDR) pathogens are a rising global health worry that imposes an urgent need for the discovery of novel antibiotics particularly those of natural origin. In this context, we aimed to use the metagenomic nanopore sequence analysis of soil microbiota coupled with the conventional phenotypic screening and genomic analysis for identifying the antimicrobial metabolites produced by promising soil isolate(s). In this study, whole metagenome analysis of the soil sample(s) was performed using MinION™ (Oxford Nanopore Technologies). Aligning and analysis of sequences for probable secondary metabolite gene clusters were extracted and analyzed using the antiSMASH version 2 and DeepBGC. Results of the metagenomic analysis showed the most abundant taxa were Bifidobacterium, Burkholderia, and Nocardiaceae (99.21%, followed by Sphingomonadaceae (82.03%) and B. haynesii (34%). Phenotypic screening of the respective soil samples has resulted in a promising Bacillus isolate that exhibited broad-spectrum antibacterial activities against various MDR pathogens. It was identified using microscopical, cultural, and molecular methods as Bacillus (B.) haynesii isolate MZ922052. The secondary metabolite gene analysis revealed the conservation of seven biosynthetic gene clusters of antibacterial metabolites namely, siderophore lichenicidin VK21-A1/A2 (95% identity), lichenysin (100%), fengycin (53%), terpenes (100%), bacteriocin (100%), Lasso peptide (95%) and bacillibactin (53%). In conclusion, metagenomic nanopore sequence analysis of soil samples coupled with conventional screening helped identify B. haynesii isolate MZ922052 harboring seven biosynthetic gene clusters of promising antimicrobial metabolites. This is the first report for identifying the bacteriocin, lichenysin, and fengycin biosynthetic gene clusters in B. haynesii MZ922052.

Improving Bacterial Metagenomic Research through Long-Read Sequencing.

Metagenomic sequencing analysis is central to investigating microbial communities in clinical and environmental studies. Short-read sequencing remains the primary approach for metagenomic research; however, long-read sequencing may offer advantages of improved metagenomic assembly and resolved taxonomic identification. To compare the relative performance for metagenomic studies, we simulated short- and long-read datasets using increasingly complex metagenomes comprising 10, 20, and 50 microbial taxa. Additionally, we used an empirical dataset of paired short- and long-read data generated from mouse fecal pellets to assess real-world performance. We compared metagenomic assembly quality, taxonomic classification, and metagenome-assembled genome (MAG) recovery rates. We show that long-read sequencing data significantly improve taxonomic classification and assembly quality. Metagenomic assemblies using simulated long reads were more complete and more contiguous with higher rates of MAG recovery. This resulted in more precise taxonomic classifications. Principal component analysis of empirical data demonstrated that sequencing technology affects compositional results as samples clustered by sequence type, not sample type. Overall, we highlight strengths of long-read metagenomic sequencing for microbiome studies, including improving the accuracy of classification and relative abundance estimates. These results will aid researchers when considering which sequencing approaches to use for metagenomic projects.

Variability and bias in microbiome metagenomic sequencing: an interlaboratory study comparing experimental protocols.

Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

Impact of temperature stress on Pyropia yezoensis and its inhabitant microbiota to promote aquaculture

The diverse inhabitant microbiota of Pyropia yezoensis can establish both beneficial and harmful interactions that effect community structure. It is complex to understand the seaweed microbial diversity and their ecological and functional regulations which are affected by numerous environmental factors. In this study, temperature stress was focused to understand the metagenomics of P. yezoensis microbiota, its functional annotation and the metabolites regulation of P. yezoensis. The samples of P. yezoensis from yellow sea of China were divided into two groups i.e low temperature 5°C (LT) and High temperature 25°C (HT). The genes and functional level analysis were followed by using metagenomic sequencing analysis and sequencing of 16sRNA gene. The Pyropia yezoensis samples were also analyzed through LCMS for metabolites regulation due to effects of microbiota and temperature stress. According to taxonomic classification analysis, Proteobacteria, Bacteroidetes, and Firmicutes were predominant in both samples with over-representation of these species in high temperature stress samples. A diverse relationship between the bacterial communities residing in the LT and HT samples was explored through discriminant analysis. The functional prediction of bacterial communities from KEGG analysis revealed a notable increase in HT predicted genes associated to amino acid metabolism, carbohydrate metabolism, energy metabolism, co-factors and vitamins metabolism, membrane transport, and nucleotide metabolism. The provoked chemical signaling and transportation response from the bacterial community, leading to activation of metabolic genes to utilize the available substrates of seaweed. The LCMS illustrated that the primary metabolites of Pyropia yezoensis were down regulated due to effect of high temperature which indicates a negative effect on the growth and health of Pyropia, while the secondary metabolites were up-regulated showing the defensive mechanism against unbearable stress. These up and down regulated metabolites also activated their respective pathways, suggesting the adoptive mechanism of P. yezoensis in response of high temperature.

Oral-gut microbial transmission promotes diabetic coronary heart disease

BackgroundDiabetes is a predominant driver of coronary artery disease worldwide. This study aims to unravel the distinct characteristics of oral and gut microbiota in diabetic coronary heart disease (DCHD). Simultaneously, we aim to establish a causal link between the diabetes-driven oral-gut microbiota axis and increased susceptibility to diabetic myocardial ischemia–reperfusion injury (MIRI).MethodsWe comprehensively investigated the microbial landscape in the oral and gut microbiota in DCHD using a discovery cohort (n = 183) and a validation chohort (n = 68). Systematically obtained oral (tongue-coating) and fecal specimens were subjected to metagenomic sequencing and qPCR analysis, respectively, to holistically characterize the microbial consortia. Next, we induced diabetic MIRI by administering streptozotocin to C57BL/6 mice and subsequently investigated the potential mechanisms of the oral-gut microbiota axis through antibiotic pre-treatment followed by gavage with specific bacterial strains (Fusobacterium nucleatum or fecal microbiota from DCHD patients) to C57BL/6 mice.ResultsSpecific microbial signatures such as oral Fusobacterium nucleatum and gut Lactobacillus, Eubacterium, and Roseburia faecis, were identified as potential microbial biomarkers in DCHD. We further validated that oral Fusobacterium nucleatum and gut Lactobacillus are increased in DCHD patients, with a positive correlation between the two. Experimental evidence revealed that in hyperglycemic mice, augmented Fusobacterium nucleatum levels in the oral cavity were accompanied by an imbalance in the oral-gut axis, characterized by an increased coexistence of Fusobacterium nucleatum and Lactobacillus, along with elevated cardiac miRNA-21 and a greater extent of myocardial damage indicated by TTC, HE, TUNEL staining, all of which contributed to exacerbated MIRI.ConclusionOur findings not only uncover dysregulation of the oral-gut microbiota axis in diabetes patients but also highlight the pivotal intermediary role of the increased abundance of oral F. nucleatum and gut Lactobacillus in exacerbating MIRI. Targeting the oral-gut microbiota axis emerges as a potent strategy for preventing and treating DCHD. Oral-gut microbial transmission constitutes an intermediate mechanism by which diabetes influences myocardial injury, offering new insights into preventing acute events in diabetic patients with coronary heart disease.Graphic

Effects of dietary selenized glucose on intestinal microbiota and tryptophan metabolism in rats: Assessing skatole reduction potential

3-Methylindole (Skatole), a degradation product of tryptophan produced by intestinal microbial activity, significantly contributes to odor nuisance. Its adverse effects on animal welfare, human health, and environmental pollution have been noted. However, it is still unclear whether the intestinal microbiota mediates the impact of selenium (Se) on skatole production and what the underlying mechanisms remain elusive. A selenized glucose (SeGlu) derivative is a novel organic selenium compound. In this study, a diverse range of dietary SeGlu-treated levels, including SeGlu-deficient (CK), SeGlu-adequate (0.15 mg Se per L), and SeGlu-supranutritional (0.4 mg Se per L) conditions, were used to investigate the complex interaction of SeGlu on intestinal microbiome and serum metabolome changes in male Sprague-Dawley (SD) rats. The study showed that SeGlu supplementation enhanced the antioxidant ability in rats, significantly manifested in the increases of the activity of catalase (CAT) and glutathione peroxidase (GSH-Px), while no change in the level of malonaldehyde (MDA). Metagenomic sequencing analysis verified that the SeGlu treatment group significantly increased the abundance of beneficial microorganisms such as Clostridium, Ruminococcus, Faecalibacterium, Lactobacillus, and Alloprevotella while reducing the abundance of opportunistic pathogens such as Bacteroides and Alistipes significantly. Further metabolomic analysis revealed phenylalanine, tyrosine, and tryptophan biosynthesis changes in the SeGlu treatment group. Notably, the biosynthesis of indole, a critical pathway, was affected by SeGlu treatment, with several crucial enzymes implicated. Correlation analysis demonstrated strong associations between specific bacterial species - Treponema, Bacteroides, and Ruminococcus, and changes in indole and derivative concentrations. Moreover, the efficacy of SeGlu-treated fecal microbiota was confirmed through fecal microbiota transplantation, leading to a decrease in the concentration of skatole in rats. Collectively, the analysis of microbiota and metabolome response to diverse SeGlu levels suggests that SeGlu is a promising dietary additive in modulating intestinal microbiota and reducing odor nuisance in the livestock and poultry industry.

Reverse-engineered exclusive enteral nutrition in pediatric Crohn's disease: A pilot trial.

In pediatric Crohn's disease (CD), commercial formulas used as exclusive enteral nutrition (EEN) are effective at inducing remission. This study aims to assess the impact of a whole-food blended smoothie as EEN on CD activity and the intestinal microbiome. A 4-week prospective trial assessed the impact of EEN with a whole-food smoothie on newly diagnosed mild-to-moderate active pediatric CD. The smoothie with a multivitamin were developed to meet age-appropriate nutritional requirements. Assessment over 4 weeks included Pediatric Crohn's Disease Activity Index (PCDAI), serum laboratories, fecal calprotectin (FCP), and stool collection for metagenomic shotgun sequencing and microbiota composition analysis. Clinical remission was defined as PCDAI ≤ 10 at week 4. Ten participants were enrolled with median age 14.5 years, and 8 completed the trial. Baseline mean PCDAI was 26.3 ± 9.1 and mean FCP 1149 ± 718 µg/g. At week 4, 80% of participants achieved clinical remission. FCP decreased by over half in 60% of participants, with FCP below 250 µg/g in 60% and below 100 µg/g in 40%. Microbiome analysis showed a significant increase in species richness over 4 weeks (p = 0.01). Compared to baseline, the relative abundance at week 2 and at week 4 was significantly increased for Bifidobacterium and Streptococcus and decreased for Blautia (p < 0.05 for all). A whole-food blended smoothie was effective for inducing clinical remission and decreasing FCP in pediatric CD similar to commercial EEN formulas. Further research may give insight into data-driven whole-food dietary approaches for CD management. gov NCT03508193.