Shotgun Metagenomic Sequencing Research Articles

Rapid inference of antibiotic resistance and susceptibility for Klebsiella pneumoniae by clinical shotgun metagenomic sequencing

ObjectivesThe study aimed to develop a genotypic antimicrobial resistance testing method for Klebsiella pneumoniae using metagenomic sequencing data. MethodsWe utilized Lasso regression on assembled genomes to identify genetic resistance determinants for six antibiotics (Gentamicin, Tobramycin, Imipenem, Meropenem, Ceftazidime, Trimethoprim/Sulfamethoxazole). The genetic features were weighted, grouped into clusters to establish classifier models. Origin species of detected antibiotic resistant gene (ARG) was determined by novel strategy integrating “possible species,” “gene copy number calculation” and “species-specific kmers.” The performance of the method was evaluated on retrospective case studies. ResultsOur study employed machine learning on 3928 K. pneumoniae isolates, yielding stable models with AUCs > 0.9 for various antibiotics. GenseqAMR, a read-based software, exhibited high accuracy (AUC 0.926–0.956) for short-read datasets. The integration of a species-specific kmer strategy significantly improved ARG-species attribution to an average accuracy of 96.67%. In a retrospective study of 191 K. pneumoniae-positive clinical specimens (0.68–93.39% genome coverage), GenseqAMR predicted 84.23% of AST results on average. It demonstrated 88.76–96.26% accuracy for resistance prediction, offering genotypic AST results with a shorter turnaround time (mean ± SD: 18.34 ± 0.87 hours) than traditional culture-based AST (60.15 ± 21.58 hours). Furthermore, a retrospective clinical case study involving 63 cases showed that GenseqAMR could lead to changes in clinical treatment for 24 (38.10%) cases, with 95.83% (23/24) of these changes deemed beneficial. ConclusionsIn conclusion, GenseqAMR is a promising tool for quick and accurate AMR prediction in Klebsiella pneumoniae, with the potential to improve patient outcomes through timely adjustments in antibiotic treatment.

Gut microbiome composition and functionality impact the responsiveness to a dairy-based product containing galacto-oligosaccharides for improving sleep quality in adults.

Sleep quality and duration can be impacted by diet, and has been linked to gut microbiota composition and function as the result of communication via the microbiota-gut-brain axis. As one strategy to improve sleep quality could be through the modulation of the gut microbiome, we assessed the effects of a dairy-based product containing whey protein, galacto-oligosaccharides, tryptophan, vitamins and minerals after a 3 weeks intervention on gut microbiota composition and (gut-brain related) functions on basis of 67 healthy subjects with moderate sleep disturbances. Associations of the gut microbiota with sleep quality and with response/non-response to the treatment were revealed by shotgun metagenomics sequencing of faecal DNA samples, and subsequent analyses of microbiota taxonomy and generic functionality. A database of manually curated Gut-Brain Modules (GBMs) was applied to analyse specific microbial functions/pathways that have the potential to interact with the brain. A moderate discriminating effect of the DP treatment on gut microbiota composition was revealed which could be mainly attributed to a decrease in Pseudomonas resinovorans, Flintibacter sp. KGM00164, Intestinimonas butyriciproducens, and Flavonifractor plautii. As interindividual variance in microbiota composition could have given rise to a heterogenous responsiveness of the subjects in the intervention group, we zoomed in on the differences between responders and non-responders. A significant difference in baseline microbiota composition between responders and non-responders was apparent, showing lower Bifidobacterium longum and Bifidobacterium adolescentis, and higher Faecalibacterium prausnitzii relative abundances in responders. The findings provide leads with respect to the effectiveness and potential underlying mechanisms of mode of action in sleep improvement that could support future nutritional interventions to aid sleep improvement.

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.

Plasma Microbial Cell-free DNA Next-generation Sequencing Can Be a Useful Diagnostic Tool in Patients With Osteoarticular Infections.

Recent advances in shotgun metagenomic sequencing (sMGS) for detecting microbial cell-free DNA (mcfDNA) in peripheral blood have shown promise across various patient populations. This study evaluates the application of sMGS for diagnosing osteoarticular infections (OAIs), a condition with significant diagnostic challenges. We conducted a retrospective analysis on 73 patients suspected of OAIs at the Mayo Clinic from 2019 to 2023, incorporating mcfDNA sMGS (Karius test [KT]) into their diagnostic evaluation. We categorized the clinical impact of KT on OAI diagnoses and management into 4 distinct outcomes. (1) KT was able to confirm an established diagnosis, (2) KT supported noninfectious diseases diagnosis, (3) KT established an unsuspected diagnosis, (4) KT did not add relevant information. In our cohort, KT was performed in 73 patients. Among the infected individuals, KT yielded positive results in 22 of 43 (51.2%) cases. Of these 22 cases, 11 (50%) showed agreement with conventional diagnostic workup, whereas in 5 (22.7%) cases, the KT established an unsuspected diagnosis. Native vertebral osteomyelitis diagnosis (P < .001) or OAIs with concomitant presence of endocarditis or endovascular infection (P = .005) were statistically associated with a definite, probable, or possible diagnostic certainty of KT result. In complex OAIs, KT enhanced diagnostic accuracy by 11.6%, proving especially beneficial in diagnosing native vertebral osteomyelitis and infections with concurrent endocarditis or endovascular complications. Our findings underscore the utility of KT in the diagnostic workflow for challenging OAI cases, potentially altering clinical management for a significant subset of patients.

Gardnerella diversity and ecology in pregnancy and preterm birth.

The vaginal microbiome has been linked to negative health outcomes including preterm birth. Specific taxa, including Gardnerella spp., have been identified as risk factors for these conditions. Historically, microbiome analysis methods have treated all Gardnerella spp. as one species, but the broad diversity of Gardnerella has become more apparent. We explore the diversity of Gardnerella clades and genomic species in the vaginal microbiome of pregnant women and their associations with microbiome composition and preterm birth. Relative abundance of Gardnerella clades and genomic species and other taxa was quantified in shotgun metagenomic sequencing data from three distinct cohorts of pregnant women. We also assessed the diversity and abundance of Gardnerella variants in 16S rRNA gene amplicon sequencing data from seven previously conducted studies in differing populations. Individual microbiomes often contained multiple Gardnerella variants, and the number of clades was associated with increased microbial load, or the ratio of non-human reads to human reads. Taxon co-occurrence patterns were largely consistent across Gardnerella clades and among cohorts. Some variants previously described as rare were prevalent in other cohorts, highlighting the importance of surveying a diverse set of populations to fully capture the diversity of Gardnerella. The diversity of Gardnerella both across populations and within individual vaginal microbiomes has long been unappreciated, as has been the intra-species diversity of many other members of the vaginal microbiome. The broad genomic diversity of Gardnerella has led to its reclassification as multiple species; here we demonstrate the diversity of Gardnerella found within and between vaginal microbiomes.IMPORTANCEThe present study shows that single microbiomes can contain all currently known species of Gardnerella and that multiple similar species can exist within the same environment. Furthermore, surveys of demographically distinct populations suggest that some species appear more commonly in certain populations. Further studies in broad and diverse populations will be necessary to fully understand the ecological roles of each Gardnerella sp., how they can co-exist, and their distinct impacts on microbial communities, preterm birth, and other health outcomes.

Microbial metagenomic shifts in children with acute lymphoblastic leukaemia during induction therapy and predictive biomarkers for infection

BackgroundEmerging evidence has indicated a link between the gut microbiota and acute lymphoblastic leukaemia (ALL). However, the acute changes in gut microbiota during chemotherapy and the predictive value of baseline gut microbiota in infectious complication remain largely unknown.MethodsFaecal samples (n = 126) from children with ALL (n = 49) undergoing induction chemotherapy were collected at three timepoints, i.e., initiation of chemotherapy (baseline, T0), 7 days (T1) and 33 days (T2) after initiation of chemotherapy. Gut microbiome profile was performed via metagenomic shotgun sequencing. The bioBakery3 pipeline (Kneaddata, Metaphlan 3 and HUMAnN) was performed to assign taxonomy and functional annotations. Gut microbiome at T0 were used to predict infection during chemotherapy.ResultsThe microbial diversities and composition changed significantly during chemotherapy, with Escherichia coli, Klebsiella pneumoniae and Bifidobacterium longum being the most prominent species. The microbial metabolic pathways were also significantly altered during chemotherapy, including the pathway of pyruvate fermentation to acetate and lactate, and assimilatory sulfate reduction pathway. The receiver operating characteristic (ROC) models based on Bifidobacterium longum at T0 could predict infectious complications during the first month of chemotherapy with the area under the curve (AUC) of 0.720.ConclusionsOur study provides new insights into the acute changes in microbial and functional characteristics in children with ALL during chemotherapy. The baseline gut microbiota could be potential biomarkers for infections during chemotherapy.Trial registrationThe study was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University (2021-KY-171-01) and registered on http://www.chictr.org.cn (ChiCTR2200065406, Registration Date: November 4, 2022).

A universal oral microbiome-based signature for periodontitis.

We analyzed eight oral microbiota shotgun metagenomic sequencing cohorts from five countries and three continents, identifying 54 species biomarkers and 26 metabolic biomarkers consistently altered in health and disease states across three or more cohorts. Additionally, machine learning models based on taxonomic profiles achieved high accuracy in distinguishing periodontitis patients from controls (internal and external areas under the receiver operating characteristic curves of 0.86 and 0.85, respectively). These results support metagenome-based diagnosis of periodontitis and provide a foundation for further research and effective treatment strategies.

Exploration of mobile genetic elements in the ruminal microbiome of Nellore cattle

Metagenomics has made it feasible to elucidate the intricacies of the ruminal microbiome and its role in the differentiation of animal production phenotypes of significance. The search for mobile genetic elements (MGEs) has taken on great importance, as they play a critical role in the transfer of genetic material between organisms. Furthermore, these elements serve a dual purpose by controlling populations through lytic bacteriophages, thereby maintaining ecological equilibrium and driving the evolutionary progress of host microorganisms. In this study, we aimed to identify the association between ruminal bacteria and their MGEs in Nellore cattle using physical chromosomal links through the Hi-C method. Shotgun metagenomic sequencing and the proximity ligation method ProxiMeta were used to analyze DNA, getting 1,713,111,307 bp, which gave rise to 107 metagenome-assembled genomes from rumen samples of four Nellore cows maintained on pasture. Taxonomic analysis revealed that most of the bacterial genomes belonged to the families Lachnospiraceae, Bacteroidaceae, Ruminococcaceae, Saccharofermentanaceae, and Treponemataceae and mostly encoded pathways for central carbon and other carbohydrate metabolisms. A total of 31 associations between host bacteria and MGE were identified, including 17 links to viruses and 14 links to plasmids. Additionally, we found 12 antibiotic resistance genes. To our knowledge, this is the first study in Brazilian cattle that connect MGEs with their microbial hosts. It identifies MGEs present in the rumen of pasture-raised Nellore cattle, offering insights that could advance biotechnology for food digestion and improve ruminant performance in production systems.

Shotgun metagenomic sequencing of swab samples from Japanese university campuses.

We obtained shotgun metagenome sequences from swab samples obtained through 3-minute swabbing of different surfaces and the air within buildings at three university campuses in part of the Greater Tokyo Area in Japan. These data aid in understanding built environment microbial communities and elucidate various microbial profiles across different locations.

Statistical and computational methods for integrating microbiome, host genomics, and metabolomics data.

Large-scale microbiome studies are progressively utilizing multiomics designs, which include the collection of microbiome samples together with host genomics and metabolomics data. Despite the increasing number of data sources, there remains a bottleneck in understanding the relationships between different data modalities due to the limited number of statistical and computational methods for analyzing such data. Furthermore, little is known about the portability of general methods to the metagenomic setting and few specialized techniques have been developed. In this review, we summarize and implement some of the commonly used methods. We apply these methods to real data sets where shotgun metagenomic sequencing and metabolomics data are available for microbiome multiomics data integration analysis. We compare results across methods, highlight strengths and limitations of each, and discuss areas where statistical and computational innovation is needed.

The effect of daily usage of Listerine Cool Mint mouthwash on the oropharyngeal microbiome: a substudy of the PReGo trial.

Introduction. ListerineÒ is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear.Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome.Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome.Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a double-blind single-centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP). Fifty-nine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non-host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA-like differential expression analysis.Results. Streptococcus was the most abundant genus in most samples (n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6-44.8), followed by Prevotella [13.5% (IQR 4.8-22.6)] and Veillonella [10.0% (IQR 4.0-16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine (P = 0.006, pseudo-F = 2.29) or placebo (P = 0.003, pseudo-F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline.Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

Time restricted eating (TRE) and immunotherapy outcomes in head and neck squamous cell carcinoma (HNSCC) via modulation of microbial butyrate and tryptophan metabolic pathways.

e18028 Background: Metastatic HNSCC continues to have very poor prognosis. Only a fraction of patients respond to immune checkpoint blocker therapy (ICB), with no effective treatment options for non-responders. Gut microbial composition and diversity have been associated with response to ICB in many cancers including HNSCC. We have previously demonstrated that TRE significantly improved progression free survival via modulation of gut microbiota. We herein report our mechanistic data demonstrating critical role of microbially mediated butyric and tryptophan metabolic pathways in augmenting antitumor immune response to ICB. Methods: Paired blood and stool samples, at baseline and at first response assessment, from 43 HNSCC patients enrolled in the TRE study (NCT05083416) was collected and analyzed using untargeted and targeted metabolomics, focusing on tryptophan and short chain fatty acid pathway. Further shot gun metagenomic sequencing was conducted on stool samples. Finally, we performed an in vitro luciferase cytotoxicity assay using SCC-4 human cancer cells to demonstrate TRE mediated antitumor immune response. We co-cultured MHC-I and NY-ESO-1, and NY-ESO-1-specific TCR-transduced T cells. tumor cells and the T cells under different conditions of butyrate concentration and T cell-to-tumor cell ratio (1:5, 1:10) and evaluated T cell mediated tumors killing. Results: We noted significantly higher abundance of clostridium butyricum (OR-1004, p-0.0002) with corresponding higher expression of butyrate kinase enzyme in stool of patients who observed TRE (OR-1.9, p-0.0003) vs those who did not, leading to higher concentration of fecal butyric acid and hydroxy indole acetic acid (HIAA) (OR-4.2, p-0.003), both of which were independently associated with longer progression free survival (HR-0.9, p&lt;0.01). Higher abundance of butyric acid in gut is known to enhance the gut barrier function with consequent decrease in plasma butyrate and HIAA, lower systemic levels of which, was found to be associated with longer progression free survival in our cohort (OR-0.9, p-0.05). Both butyrate and 5HIAA are known activators of immunosuppressive aryl hydrocarbon receptor (AhR) expressed on cytotoxic CD8T cells, inhibiting CD8 mediated antitumor response. In line with our hypothesis, we observed that butyrate and 5HIAA impaired CD8 T mediated T cell killing of tumor cells in a dose-dependent manner. Conclusions: These results suggest that TRE favorably modulates ICB response via enrichment of gut microbiota with butyrate producing microbial organism, leading to increased abundance of gut butyrate and microbial tryptophan metabolites and their consequent reduction in systemic circulation, ultimately enhancing antitumor immune response.

A Shotgun Metagenomics Investigation into Labeling Inaccuracies in Widely Sold Probiotic Supplements in the USA.

While probiotics are generally considered safe, concerns persist regarding the accuracy of labels on these supplements and their potential contribution to the spread of antibiotic resistance genes. Given that probiotics are predominantly ingested with a view towards obtaining particular health benefits. The objective of this study is to assess the composition of 50 widely available probiotic supplements in the USA using shotgun metagenome sequencing. The study also determines the potential resistome profile, and the functional characteristics of these products. This study finds that 67% of products does not contain any labeling inaccuracies. Antimicrobial Resistance Genes (ARGs) are identified in several products, particularly Bacillus-based products carrying between 10 and 56 genes. The risk posed by the presence of these ARGs requires further study. Functional analysis reveals differences in metabolic profiles among probiotic supplements, indicating the importance of strain-level selection for personalized probiotics. This study provides updated and comprehensive analysis to evaluate a snapshot of the USA market. The study demonstrates that label inaccuracies occur on approximately one third of popular dietary supplement products sold in the USA, supporting the need for improved approaches to marketing and quality control. Further, the risk of antibiotic resistance, especially in Bacillus-based formulations, should be assessed.

Tumor microbiome differences in early-onset versus average-onset pancreatic adenocarcinoma.

4016 Background: The rise in early-onset gastrointestinal cancers includes an increased risk of pancreatic adenocarcinoma (PDAC), but the underlying pathogenesis is incompletely understood. The tumor microbiome is an increasing focus of research given its impact on tumor pathogenesis and outcomes. We evaluated microbiome profiles in resected specimens from early-onset PDAC (eoPDAC) and average-onset PDAC (aoPDAC). Methods: PDAC specimens were selected consecutively from Cleveland Clinic and Roswell Park Cancer Institute biorepositories and categorized on age at diagnosis ( &lt; 50 years defined as eoPDAC, and &gt; 50 years as aoPDAC). The tissue microbiome was characterized using shotgun metagenomic sequencing. Alpha diversity, denoting the richness and evenness of microbial species within a specific sample, was compared using the Wilcoxon rank-sum test. The differential test of Beta-diversity distances (Bray Curtis distance), representing microbial taxonomic differences between the specimens was performed using Permutational MANOVA. All p-values were adjusted for multiple testing. Results: The study comprised 44 individuals with resected PDAC (24 eoPDAC and 20 aoPDAC). After quality checks, 63 tissue specimens were selected for analysis (13 tumor and 10 adjacent normal specimens for eoPDAC, 20 each of tumor and adjacent normal specimens for aoPDAC). eoPDAC tumor demonstrated significantly higher alpha diversity compared to adjacent normal tissue (P = 0.02) and aoPDAC (P = 0.0062) while aoPDAC did not (tumor vs normal alpha diversity P = 0.31). Beta diversity analysis demonstrated significantly different diversity of genera between all the groups (P &lt; 0.05). Consistent with this finding, the majority of the species - 259/302 (85.8%) in eo vs aoPDAC comparison and 262/366 (71.6%) for eoPDAC vs normal comparison did not overlap. Differential abundance analysis revealed a significant variation of tumor microbiome in eoPDAC vs aoPDAC (P &lt; 0.05). Prevalent bacterial genera (tumor specimens) are listed in the Table. eoPDAC tumor tissues were enriched in Enterobacter, Neisseria,and Escherichia genera, while aoPDAC tumor tissues were enriched in Klebsiella and Bacillus genera. Conclusions: eoPDAC has a distinct microbiome profile compared to aoPDAC. The correlation with several microbial genera associated with pancreatic diseases is a significant finding and warrants further exploration. This is relevant considering the rising incidence of eoPDAC of unknown etiology, and ongoing efforts to develop microbiome-based biomarkers for screening and treatment. [Table: see text]

From parent to progeny

How infants acquire their gut microbial communities and the various factors influencing these dynamics remain unclear. In this issue of Cell Host & Microbe, Selma-Royo etal. and Dubois etal. use shotgun metagenomic sequencing to understand the transmission of microbes from parents to infants and delve into factors modifying this process.

Evaluating gut microbiome as a biomarker of pathological complete response in patients with early locally advanced triple negative breast cancer (LA TNBC): A pilot study.

593 Background: It has previously been shown that microbial metabolic pathways are differentially activated in immunotherapy resistance, hence can serve as potential biomarker as well as target for therapeutic intervention. With the advent of Immune Checkpoint Blockers (ICB) in the neoadjuvant (NA) setting of locally advanced (LA) TNBC, we hypothesized that similar microbial metabolite mediated resistance may be at play. We are presenting data demonstrating differences in microbiome and metabolites in these patients. Methods: 28 patients who received NA pembrolizumab and chemotherapyfor LA TNBC were identified at Moffitt Cancer Center and Tampa General Hospital between February 2022 to October 2023. Blood and stool samples were collected at therapy initiation, at completion of neoadjuvant treatment and/or when the patients developed grade 3 or higher toxicity to therapy. We evaluated stool metagenomics and untargeted stool/plasma metabolomics as correlatives. Patient characteristics were as follows: 21 Caucasian, 3 African-American, 2 Hispanic, 2 unknown, median age 51 years, median body mass index (BMI) 28.5 kg/m2. 17 patients had stage II, while 11 patients had stage III TNBC. Results: Response was assessed in 27 patients, of which 11 had pCR (ypT0/is ypN0) while 16 had residual disease (RD). 1 patient progressed, while on NA therapy, while 8 patients developed grade 3-4 immune related adverse events leading to treatment discontinuation and death in one patient. We conducted shallow shotgun metagenomic sequencing and untargeted metabolomic profiling on pre and post treatment fecal and plasma samples respectively to identify gut microbial diversity, composition and differentially abundant metabolites associated with pCR.There wasno difference in alpha and beta diversity; however significant enrichment of microbiota with genus Akkermansia HR 0.04, p 0.03 was noted in those with pCR. We observed higher abundance of tryptophan metabolizing clostridium species HR 50541, p &lt;0.001, along with differential activation of microbial tryptophan pathway in those with residual disease, specifically higher abundance of hydroxyindole acetic acid HR 1.54 P&lt;0.005, indole propionic acid HR 1.75 p 0.05 and indole butyric acid HR 1.86 p 0.04. Conclusions: To our knowledge this is the first analysis of differences in the microbiome and microbial metabolism in patients with LA TNBC treated with NA ICB. Despite being a small study, it shows a more aggressive disease pattern with higher RD than noted in KEYNOTE 522 and shows significant concern for serious immune mediated adverse effects. This highlights the importance of biomarkers to stratify patients most likely to benefit from ICB. Enrichment of microbiota and immunosuppressive tryptophan metabolites were associated with pCR and should be validated in large studies as biomarker of response to NA ICB in TNBC.

Shotgun metagenomic analysis of microbiota dynamics during long-term backslopping fermentation of traditional fermented milk in a controlled laboratory environment

Traditional fermented milks are produced through an inoculation process that involves the deliberate introduction of microorganisms that have been adapted and perpetuated across successive generations. However, the changes in the microbiota of traditional fermented milk during long-term inoculation fermentation in a laboratory environment remain unclear. In this study, we collected 5 samples of traditional fermented milk samples from 5 different counties in Tibet (3 kurut products) and Xinjiang (2 tarag products) of China, which served as starter cultures for a 9-mo continuous inoculation fermentation experiment. We analyzed the inter- and intra-population variations in the microbial communities of the collected samples, representing their macrodiversity and microdiversity, using shotgun metagenomic sequencing. Across all samples, we obtained a total of 186 high-quality metagenomic-assembled genomes, including 7 genera and 13 species with a relative abundance of more than 1%. The majority of these genomes were annotated as Lactobacillus helveticus (60.46%), Enterococcus durans (9.52%), and Limosilactobacillus fermentum (6.23%). We observed significant differences in species composition and abundance among the 5 initial inoculants. During the long-term inoculation fermentation, we found an overall increasing trend in species diversity, composition, and abundances of carbohydrate metabolism module-encoding genes in the fermented milk bacterial metagenome, while the fermented milk virome exhibited a relatively narrow range of variation. Lactobacillus helveticus, a dominant species in traditional fermented milk, displayed high stability during the long-term inoculation fermentation. Our study provides valuable insights for the industrial production of traditional fermented milk.

Hackflex library preparation enables low-cost metagenomic profiling.

Shotgun metagenomic sequencing provides valuable insights into microbial communities, but the high cost of library preparation with standard kits and protocols is a barrier for many. New methods such as Hackflex use diluted commercially available reagents to greatly reduce library preparation costs. However, these methods have not been systematically validated for metagenomic sequencing. Here, we evaluate Hackflex performance by sequencing metagenomic libraries from known mock communities as well as mouse fecal samples prepared by Hackflex, Illumina DNA Prep, and Illumina TruSeq methods. Hackflex successfully recovered all members of the Zymo mock community, performing best for samples with DNA concentrations <1ng/μL. Furthermore, Hackflex was able to delineate microbiota of individual inbred mice from the same breeding stock at the same mouse facility, and statistical modeling indicated that mouse ID explained a greater fraction of the variance in metagenomic composition than did library preparation method. These results show that Hackflex is suitable for generating inventories of bacterial communities through metagenomic sequencing.

Mitochondrial DNA variants and microbiota: An experimental strategy to identify novel therapeutic potential in chronic inflammatory diseases

We previously demonstrated that mice carrying natural mtDNA variants of the FVB/NJ strain (m.7778 G>T in the mt-Atp8 gene in mitochondrial complex V), namely C57BL/6 J-mtFVB/NJ (B6-mtFVB), exhibited (i) partial protection from experimental skin inflammatory diseases in an anti-murine type VII collagen antibody-induced skin inflammation model and psoriasiform dermatitis model; (ii) significantly altered metabolites, including short-chain fatty acids, according to targeted metabolomics of liver, skin and lymph node samples; and (iii) a differential composition of the gut microbiota according to bacterial 16 S rRNA gene sequencing of stool samples compared to wild-type C57BL/6 J (B6) mice. To further dissect these disease-contributing factors, we induced an experimental antibody-induced skin inflammatory disease in gnotobiotic mice. We performed shotgun metagenomic sequencing of caecum contents and untargeted metabolomics of liver, CD4+ T cell, and caecum content samples from conventional B6-mtFVB and B6 mice. We identified D-glucosamine as a candidate mediator that ameliorated disease severity in experimental antibody-induced skin inflammation by modulating immune cell function in T cells, neutrophils and macrophages. Because mice carrying mtDNA variants of the FVB/NJ strain show differential disease susceptibility to a wide range of experimental diseases, including diet-induced atherosclerosis in low-density lipoprotein receptor knockout mice and collagen antibody-induced arthritis in DBA/1 J mice, this experimental approach is valuable for identifying novel therapeutic options for skin inflammatory conditions and other chronic inflammatory diseases to which mice carrying specific mtDNA variants show differential susceptibility.

Bacteroidales reduces growth rate through serum metabolites and cytokines in Chinese Ningdu yellow chickens

Increasing evidence has indicated that the gut microbiome plays an important role in chicken growth traits. However, the cecal microbial taxa associated with the growth rates of the Chinese Ningdu yellow chickens are unknown. In this study, shotgun metagenomic sequencing was used to identify cecal bacterial species associated with the growth rate of the Chinese Ningdu yellow chickens. We found that nine cecal bacterial species differed significantly between high and low growth rate chickens, including three species (Succinatimonas hippei, Phocaeicola massiliensis, and Parabacteroides sp. ZJ-118) that were significantly enriched in high growth rate chickens. We identified six Bacteroidales that were significantly enriched in low growth rate chickens, including Barnesiella sp. An22, Barnesiella sp. ET7, and Bacteroidales bacterium which were key biomarkers in differentiating high and low growth rate chickens and were associated with alterations in the functional taxa of the cecal microbiome. Untargeted serum metabolome analysis revealed that 8 metabolites showing distinct enrichment patterns between high and low growth rate chickens, including triacetate lactone and N-acetyl-a-neuraminic acid, which were at higher concentrations in low growth rate chickens and were positively and significantly correlated with Barnesiella sp. An22, Barnesiella sp. ET7, and Bacteroidales bacterium. Furthermore, the results suggest that serum cytokines, such as IL-5, may reduce growth rate and are related to changes in serum metabolites and gut microbes (e.g., Barnesiella sp. An22 and Barnesiella sp. ET7). These results provide important insights into the effects of the cecal microbiome, serum metabolism and cytokines in Ningdu yellow chickens.