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Alzheimer's disease (AD) is a complex neurodegenerative disorder significantly impairing cognitive faculties, memory, and physical abilities. To characterize the modulation of the gut microbiota in an in vivo AD model, we performed shotgun metagenomics sequencing on 3xTgAD mice at key time points (i.e., 2, 6, and 12 months) of AD progression. Fecal samples from both 3xTgAD and wild-type mice were collected, DNA extracted, and sequenced. Quantitative taxon abundance assessment using MetaPhlAn 4 ensured precise microbial community representation. The analysis focused on species-level genome bins (SGBs) including both known and unknown SGBs (kSGBs and uSGBs, respectively) and also comprised higher taxonomic categories such as family-level genome bins (FGBs), class-level genome bins (CGBs), and order-level genome bins (OGBs). Our bioinformatic results pinpointed the presence of extensive gut microbial diversity in AD mice and showed that the largest proportion of AD- and aging-associated microbiome changes in 3xTgAD mice concern SGBs that belong to the Bacteroidota and Firmicutes phyla, along with a large set of uncharacterized SGBs. Our findings emphasize the need for further advanced bioinformatic studies for accurate classification and functional analysis of these elusive microbial species in relation to their potential bridging role in the gut-brain axis and AD pathogenesis. [ABSTRACT FROM AUTHOR]

The key association between gut dysbiosis and cancer is already known. Here, we used whole-genome shotgun sequencing (WGS) and gas chromatography/mass spectrometry (GC/MS) to conduct metagenomic and metabolomic analyses to identify common and distinct taxonomic configurations among 40, 45, 71, 34, 50, 60, and 40 patients with colorectal cancer, stomach cancer, breast cancer, lung cancer, melanoma, lymphoid neoplasms and acute myeloid leukemia (AML), respectively, and compared the data with those from sex- and age-matched healthy controls (HC). α-diversity differed only between the lymphoid neoplasm and AML groups and their respective HC, while β-diversity differed between all groups and their HC. Of 203 unique species, 179 and 24 were under- and over-represented, respectively, in the case groups compared with HC. Of these, Faecalibacillus intestinalis was under-represented in each of the seven groups studied, Anaerostipes hadrus was under-represented in all but the stomach cancer group, and 22 species were under-represented in the remaining five case groups. There was a marked reduction in the gut microbiome cancer index in all case groups except the AML group. Of the short-chain fatty acids and amino acids tested, the relative concentration of formic acid was significantly higher in each of the case groups than in HC, and the abundance of seven species of Faecalibacterium correlated negatively with most amino acids and formic acid, and positively with the levels of acetic, propanoic, and butanoic acid. We found more differences than similarities between the studied malignancy groups, with large variations in diversity, taxonomic/metabolomic profiles, and functional assignments. While the results obtained may demonstrate trends rather than objective differences that correlate with different types of malignancy, the newly developed gut microbiota cancer index did distinguish most of the cancer cases from HC. We believe that these data are a promising step forward in the search for new diagnostic and predictive tests to assess intestinal dysbiosis among cancer patients. [ABSTRACT FROM AUTHOR]

Background: As a nexus of routine antibiotic use and zoonotic pathogen presence, the livestock farming environment is a potential hotspot for the emergence of zoonotic diseases and antibiotic resistant bacteria. Livestock can further facilitate disease transmission by serving as intermediary hosts for pathogens before a spillover event. In light of this, we aimed to characterize the microbiomes and resistomes of dairy workers, whose exposure to the livestock farming environment places them at risk for facilitating community transmission of antibiotic resistant genes and emerging zoonotic diseases. Results: Using shotgun sequencing, we investigated differences in the taxonomy, diversity and gene presence of 10 dairy farm workers and 6 community controls' gut metagenomes, contextualizing these samples with additional publicly available gut metagenomes. We found no significant differences in the prevalence of resistance genes, virulence factors, or taxonomic composition between the two groups. The lack of statistical significance may be attributed, in part, to the limited sample size of our study or the potential similarities in exposures between the dairy workers and community controls. We did, however, observe patterns warranting further investigation including greater abundance of tetracycline resistance genes and prevalence of cephamycin resistance genes as well as lower average gene diversity (even after accounting for differential sequencing depth) in dairy workers' metagenomes. We also found evidence of commensal organism association with tetracycline resistance genes in both groups (including Faecalibacterium prausnitzii, Ligilactobacillus animalis, and Simiaoa sunii). Conclusions: This study highlights the utility of shotgun metagenomics in examining the microbiomes and resistomes of livestock workers, focusing on a cohort of dairy workers in the United States. While our study revealed no statistically significant differences between groups in taxonomy, diversity and gene presence, we observed patterns in antibiotic resistance gene abundance and prevalence that align with findings from previous studies of livestock workers in China and Europe. Our results lay the groundwork for future research involving larger cohorts of dairy and non-dairy workers to better understand the impact of occupational exposure to livestock farming on the microbiomes and resistomes of workers. [ABSTRACT FROM AUTHOR]

Cabbage (Brassica oleracea), a crop of major economic importance worldwide, is affected by numerous diseases, which are caused by a wide range of microorganisms, including fungi, oomycetes, bacteria, and viruses, which lead to important losses in yield and quality. The increasing availability of reference genomes of plant-associated microbes together with recent advances in metagenomic approaches provide new opportunities to identify microbes linked to distinct symptomatology in Brassica leaves. In this study, shotgun metagenomics was used to investigate the microbial community in leaves of B. oleracea plants from agricultural farmlands. Compared with conventional techniques based on culture-based methods, whole-genome shotgun sequencing allows the reliable identification of the microbial population inhabiting a plant tissue at the species level. Asymptomatic and symptomatic leaves showing different disease symptoms were examined. In the asymptomatic leaves, Xanthomonas species were the most abundant taxa. The relative abundance of bacterial and fungal communities varied depending on disease symptoms on the leaf. The microbiome of the leaves showing mild to severe levels of disease was enriched in bacterial populations (Sphingomonas, Methylobacterium, Paracoccus) and to a lesser degree in some fungal taxa, such as Alternaria and Colletotrichum (e.g., in leaves with high levels of necrotic lesions). Sclerotinia species were highly abundant in severely damaged leaves (S. sclerotium, S. trifolium, S. bolearis), followed by Botrytis species. The common and specific bacterial and fungal species associated to disease symptoms were identified. Finally, the analysis of the gene functions in the metagenomic data revealed enrichment in carbohydrate-active enzymes potentially involved in pathogenicity, whose distribution also varied among disease severity groups. Understanding the B. oleracea leaf microbiome in agricultural ecosystems will pave the way for the efficient management of diseases in this crop. [ABSTRACT FROM AUTHOR]

Shotgun metagenomics sequencing experiments are finding a wide range of applications. Nonetheless, there are still limited guidelines regarding the number of sequences needed to acquire meaningful information for taxonomic profiling and antimicrobial resistance gene (ARG) identification. In this study, we explored this issue in the context of oral microbiota by sequencing with a very high number of sequences (~ 100 million), four human plaque samples, and one microbial community standard and by evaluating the performance of microbial identification and ARGs detection through a downsampling procedure. When investigating the impact of a decreasing number of sequences on quantitative taxonomic profiling in the microbial community standard datasets, we found some discrepancies in the identified microbial species and their abundances when compared to the expected ones. Such differences were consistent throughout downsampling, suggesting their link to taxonomic profiling methods limitations. Overall, results showed that the number of sequences has a great impact on metagenomic samples at the qualitative (i.e., presence/absence) level in terms of loss of information, especially in experiments having less than 40 million reads, whereas abundance estimation was minimally affected, with only slight variations observed in low-abundance species. The presence of ARGs was also assessed: a total of 133 ARGs were identified. Notably, 23% of them inconsistently resulted as present or absent across downsampling datasets of the same sample. Moreover, over half of ARGs were lost in datasets having less than 20 million reads. This study highlights the importance of carefully considering sequencing aspects and suggests some guidelines for designing shotgun metagenomics experiments with the final goal of maximizing oral microbiome analyses. Our findings suggest varying optimized sequence numbers according to different study aims: 40 million for microbiota profiling, 50 million for low-abundance species detection, and 20 million for ARG identification. Key points: • Forty million sequences are a cost-efficient solution for microbiota profiling • Fifty million sequences allow low-abundance species detection • Twenty million sequences are recommended for ARG identification [ABSTRACT FROM AUTHOR]

Sfela is a white brined Greek cheese of protected designation of origin (PDO) produced in the Peloponnese region from ovine, caprine milk, or a mixture of the two. Despite the PDO status of Sfela, very few studies have addressed its properties, including its microbiology. For this reason, we decided to investigate the microbiome of two PDO industrial Sfela cheese samples along with two non-PDO variants, namely Sfela touloumotiri and Xerosfeli. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS), 16S rDNA amplicon sequencing and shotgun metagenomics analysis were used to identify the microbiome of these traditional cheeses. Cultured-based analysis showed that the most frequent species that could be isolated from Sfela cheese were Enterococcus faecium, Lactiplantibacillus plantarum, Levilactobacillus brevis, Pediococcus pentosaceus and Streptococcus thermophilus. Shotgun analysis suggested that in industrial Sfela 1, Str. thermophilus dominated, while industrial Sfela 2 contained high levels of Lactococcus lactis. The two artisanal samples, Sfela touloumotiri and Xerosfeli, were dominated by Tetragenococcus halophilus and Str. thermophilus, respectively. Debaryomyces hansenii was the only yeast species with abundance > 1% present exclusively in the Sfela touloumotiri sample. Identifying additional yeast species in the shotgun data was challenging, possibly due to their low abundance. Sfela cheese appears to contain a rather complex microbial ecosystem and thus needs to be further studied and understood. This might be crucial for improving and standardizing both its production and safety measures. [ABSTRACT FROM AUTHOR]

Background: Recent endeavours in metagenomics, exemplified by projects such as the human microbiome project and TARA Oceans, have illuminated the complexities of microbial biomes. A robust bioinformatic pipeline and meticulous evaluation of their methodology have contributed to the success of these projects. The soil environment, however, with its unique challenges, requires a specialized methodological exploration to maximize microbial insights. A notable limitation in soil microbiome studies is the dearth of soil-specific reference databases available to classifiers that emulate the complexity of soil communities. There is also a lack of in-vitro mock communities derived from soil strains that can be assessed for taxonomic classification accuracy. Results: In this study, we generated a custom in-silico mock community containing microbial genomes commonly observed in the soil microbiome. Using this mock community, we simulated shotgun sequencing data to evaluate the performance of three leading metagenomic classifiers: Kraken2 (supplemented with Bracken, using a custom database derived from GTDB-TK genomes along with its own default database), Kaiju, and MetaPhlAn, utilizing their respective default databases for a robust analysis. Our results highlight the importance of optimizing taxonomic classification parameters, database selection, as well as analysing trimmed reads and contigs. Our study showed that classifiers tailored to the specific taxa present in our samples led to fewer errors compared to broader databases including microbial eukaryotes, protozoa, or human genomes, highlighting the effectiveness of targeted taxonomic classification. Notably, an optimal classifier performance was achieved when applying a relative abundance threshold of 0.001% or 0.005%. The Kraken2 supplemented with bracken, with a custom database demonstrated superior precision, sensitivity, F1 score, and overall sequence classification. Using a custom database, this classifier classified 99% of in-silico reads and 58% of real-world soil shotgun reads, with the latter identifying previously overlooked phyla using a custom database. Conclusion: This study underscores the potential advantages of in-silico methodological optimization in metagenomic analyses, especially when deciphering the complexities of soil microbiomes. We demonstrate that the choice of classifier and database significantly impacts microbial taxonomic profiling. Our findings suggest that employing Kraken2 with Bracken, coupled with a custom database of GTDB-TK genomes and fungal genomes at a relative abundance threshold of 0.001% provides optimal accuracy in soil shotgun metagenome analysis. [ABSTRACT FROM AUTHOR]

The taxonomic characterization of ancient microbiomes is a key step in the rapidly growing field of paleomicrobiology. While PCR amplification of the 16S ribosomal RNA (rRNA) gene is a widely used technique in modern microbiota studies, this method has systematic biases when applied to ancient microbial DNA. Shotgun metagenomic sequencing has proven to be the most effective method in reconstructing taxonomic profiles of ancient dental calculus samples. Nevertheless, shotgun sequencing approaches come with inherent limitations that could be addressed through hybridization enrichment capture. When employed together, shotgun sequencing and hybridization capture have the potential to enhance the characterization of ancient microbial communities. Here, we develop, test, and apply a hybridization enrichment capture technique to selectively target 16S rRNA gene fragments from the libraries of ancient dental calculus samples generated with shotgun techniques. We simulated data sets generated from hybridization enrichment capture, indicating that taxonomic identification of fragmented and damaged 16S rRNA gene sequences was feasible. Applying this enrichment approach to 15 previously published ancient calculus samples, we observed a 334-fold increase of ancient 16S rRNA gene fragments in the enriched samples when compared to unenriched libraries. Our results suggest that 16S hybridization capture is less prone to the effects of background contamination than 16S rRNA amplification, yielding a higher percentage of on-target recovery. While our enrichment technique detected low abundant and rare taxa within a given sample, these assignments may not achieve the same level of specificity as those achieved by unenriched methods. [ABSTRACT FROM AUTHOR]

Complementary feeding induces dramatic ecological shifts in the infant gut microbiota toward more diverse compositions and functional metabolic capacities, with potential implications for immune and metabolic health. The aim of this study was to examine whether the age at which solid foods are introduced differentially affects the microbiota in predominantly breastfed infants compared with predominantly formula-fed infants. We performed whole-genome shotgun metagenomic sequencing of infant stool samples from a cohort of six-month-old Australian infants enrolled in a nested study within the ORIGINS Project longitudinal birth cohort. Infants born preterm or those who had been administered antibiotics since birth were excluded. The taxonomic composition was highly variable among individuals at this age. Predominantly formula-fed infants exhibited a higher microbiome diversity than predominantly breastfed infants. Among the predominantly breastfed infants, the introduction of solid foods prior to five months of age was associated with higher alpha diversity than solid food introduction after six months of age, primarily due to the loss of Bifidobacterium infantis. In contrast, the age at which solid food was introduced was not associated with the overall change in diversity among predominantly formula-fed infants but was associated with compositional changes in Escherichia abundance. Examining the functional capacity of the microbiota in relation to these changes, we found that the introduction of solid foods after six months of age was associated with elevated one-carbon compound metabolic pathways in both breastfed and formula-fed infants, although the specific metabolic sub-pathways differed, likely reflecting different taxonomic compositions. Our findings suggest that the age of commencement of solid foods influences the gut microbiota composition differently in predominantly breastfed infants than in predominantly formula-fed infants. [ABSTRACT FROM AUTHOR]

Background Shotgun and targeted metagenomic sequencing have been shown in separate studies to be potentially useful for culture-free pathogen identification in blood and/or plasma of patients with infective endocarditis (IE). However, the 2 approaches have not been directly compared. The aim of this study was to compare shotgun metagenomic sequencing with targeted metagenomic sequencing (tMGS) for organism identification in blood or plasma of patients with IE. Methods Patients with possible or definite IE were prospectively enrolled from October 2020 to July 2021. Shotgun metagenomic sequencing was performed with the Karius test, which uses microbial cell-free DNA (mcfDNA) sequencing to detect, identify, and quantitate DNA-based pathogens in plasma. tMGS was performed using a 16S ribosomal RNA (rRNA) polymerase chain reaction assay targeting the V1 to V3 regions of the 16S rRNA gene. Results were compared using the McNemar test of paired proportions. Results Samples from 34 patients were investigated. The Karius test was positive in 24/34 (71%), including 3/6 (50%) with blood culture–negative endocarditis (BCNE), which was not significantly different from the positivity rate of tMGS (P =.41). Results of the Karius test were concordant with tMGS in 75% of cases. The Karius test detected 2 cases of methicillin-resistant Staphylococcus aureus among the 7 S. aureus detections, in accordance with results of phenotypic susceptibility testing. The combination of blood cultures, the Karius test, and tMGS found a potential causative pathogen in 33/34 (97%), including 5/6 with BCNE. Conclusions The Karius test and tMGS yielded comparable detection rates; however, beyond organism identification, the Karius test generated potentially useful antibiotic resistance data. [ABSTRACT FROM AUTHOR]

The occurrence and spread of antibiotic resistance genes (ARGs) in environmental microorganisms, particularly in poly-extremophilic bacteria, remain underexplored and have received limited attention. This study aims to investigate the prevalence of ARGs and metal resistance genes (MRGs) in shotgun metagenome sequences obtained from water and salt crust samples collected from Lake Afdera and the Assale salt plain in the Danakil Depression, northern Ethiopia. Potential ARGs were characterized by the comprehensive antibiotic research database (CARD), while MRGs were identified by using BacMetScan V.1.0. A total of 81 ARGs and 39 MRGs were identified at the sampling sites. We found a copA resistance gene for copper and the β-lactam encoding resistance genes were the most abundant the MRG and ARG in the study area. The abundance of MRGs is positively correlated with mercury (Hg) concentration, highlighting the importance of Hg in the selection of MRGs. Significant correlations also exist between heavy metals, Zn and Cd, and ARGs, which suggests that MRGs and ARGs can be co-selected in the environment contaminated by heavy metals. A network analysis revealed that MRGs formed a complex network with ARGs, primarily associated with β-lactams, aminoglycosides, and tetracyclines. This suggests potential co-selection mechanisms, posing concerns for both public health and ecological balance. [ABSTRACT FROM AUTHOR]

Background: Multi-species biofilms pose a problem in various environments, especially food-processing environments. The diversity of microorganisms in these biofilms plays a critical role in their integrity and protection against external biotic and abiotic factors. Compared to single-species biofilms, mixed-species biofilms are more resistant to various stresses, including antimicrobials like sanitizers. Therefore, understanding the microbiome composition and diversity in biofilms and their metabolic potential is a priority when developing intervention techniques to combat foodborne pathogens in food processing environments. Methods: This study aimed to describe and compare the microbiome profile of 75 drain biofilm samples obtained from five different locations (Hotscale, Hotbox, Cooler, Processing, & Grind room) of three beef-processing plants (Plant A, B & C) taken over two timepoints 2017-18 (T1) and 2021 (T2) by shotgun sequencing. Results: Core microbiome analysis found Pseudomonas, Psychrobacter, and Acinetobacter to be the top three prevalent genera among the plants and locations. Alpha diversity analysis demonstrated a high diversity of microbiome present in all the plants and locations across the time points. Functional analysis showed the high metabolic potential of the microbial community with abundance of genes in metabolism, cell-adhesion, motility, and quorum sensing. Moreover, Quaternary Ammonium Compound (QAC) resistance genes were also observed, this is significant as QAC sanitizers are commonly used in many food processing facilities. Multi-functional genes such as transposases, polymerases, permeases, flagellar proteins, and Mobile Genetic Elements (MGEs) were found suggesting these are dynamic microbial communities that work together to protect themselves against environmental stresses through multiple defense mechanisms. Conclusion: This study provides a framework for understanding the collective microbial network spanning a beef processing system. The results can be used to develop intervention strategies to best control these highly communicative microbial networks. [ABSTRACT FROM AUTHOR]

The impacts of hexavalent chromium (Cr) contamination on the microbiome, soil physicochemistry, and heavy metal resistome of a tropical agricultural soil were evaluated for 6 weeks in field-moist microcosms consisting of a Cr-inundated agricultural soil (SL9) and an untreated control (SL7). The physicochemistry of the two microcosms revealed a diminution in the total organic matter content and a significant dip in macronutrients phosphorus, potassium, and nitrogen concentration in the SL9 microcosm. Heavy metals analysis revealed the detection of seven heavy metals (Zn, Cu, Fe, Cd, Se, Pb, Cr) in the agricultural soil (SL7), whose concentrations drastically reduced in the SL9 microcosm. Illumina shotgun sequencing of the DNA extracted from the two microcosms showed the preponderance of the phyla, classes, genera, and species of Actinobacteria (33.11%), Actinobacteria_class (38.20%), Candidatus Saccharimonas (11.67%), and Candidatus Saccharimonas aalborgensis (19.70%) in SL7, and Proteobacteria (47.52%), Betaproteobacteria (22.88%), Staphylococcus (16.18%), Staphylococcus aureus (9.76%) in SL9, respectively. Functional annotation of the two metagenomes for heavy metal resistance genes revealed diverse heavy metal resistomes involved in the uptake, transport, efflux, and detoxification of various heavy metals. It also revealed the exclusive detection in SL9 metagenome of resistance genes for chromium (chrB, chrF, chrR, nfsA, yieF), cadmium (czcB/czrB, czcD), and iron (fbpB, yqjH, rcnA, fetB, bfrA, fecE) not annotated in SL7 metagenome. The findings from this study revealed that Cr contamination induces significant shifts in the soil microbiome and heavy metal resistome, alters the soil physicochemistry, and facilitates the loss of prominent members of the microbiome not adapted to Cr stress. [ABSTRACT FROM AUTHOR]

Fibre-based dietary interventions are at the forefront of gut microbiome modulation research, with a wealth of 16S rRNA information to demonstrate the prebiotic effects of isolated fibres. However, there is a distinct lack of data relating to the effect of a combination of soluble and insoluble fibres in a convenient-to-consume fruit juice food matrix on gut microbiota structure, diversity, and function. Here, we aimed to determine the impact of the MOJU Prebiotic Shot, an apple, lemon, ginger, and raspberry fruit juice drink blend containing chicory inulin, baobab, golden kiwi, and green banana powders, on gut microbiota structure and function. Healthy adults (n = 20) were included in a randomised, double-blind, placebo-controlled, cross-over study, receiving 60 mL MOJU Prebiotic Shot or placebo (without the fibre mix) for 3 weeks with a 3-week washout period between interventions. Shotgun metagenomics revealed significant between-group differences in alpha and beta diversity. In addition, the relative abundance of the phyla Actinobacteria and Desulfobacteria was significantly increased as a result of the prebiotic intervention. Nine species were observed to be differentially abundant (uncorrected p-value of <0.05) as a result of the prebiotic treatment. Of these, Bifidobacterium adolescentis and CAG-81 sp900066785 (Lachnospiraceae) were present at increased abundance relative to baseline. Additionally, KEGG analysis showed an increased abundance in pathways associated with arginine biosynthesis and phenylacetate degradation during the prebiotic treatment. Our results show the effects of the daily consumption of 60 mL MOJU Prebiotic Shot for 3 weeks and provide insight into the functional potential of B. adolescentis. [ABSTRACT FROM AUTHOR]

The microbiomes living in the rhizosphere soil of the tomato plant contribute immensely to the state of health of the tomato plant alongside improving sustainable agriculture. With the aid of shotgun metagenomics sequencing, we characterized the putative functional genes (plant-growth-promoting and disease-resistant genes) produced by the microbial communities dwelling in the rhizosphere soil of healthy and powdery mildew-diseased tomato plants. The results identified twenty-one (21) plant growth promotion (PGP) genes in the microbiomes inhabiting the healthy rhizosphere (HR) which are more predomiant as compared to diseased rhizosphere (DR) that has nine (9) genes and four (4) genes in bulk soil (BR). Likewise, we identified some disease-resistant genes which include nucleotide binding genes and antimicrobial genes. Our study revealed fifteen (15) genes in HR which made it greater in comparison to DR that has three (3) genes and three (3) genes in bulk soil. Further studies should be conducted by isolating these microorganisms and introduce them to field experiments for cultivation of tomatoes. [ABSTRACT FROM AUTHOR]

Objectives The aim of this study is to evaluate the effectiveness of a CRISPR-based human and bacterial ribosomal RNA (rRNA) depletion kit (JUMPCODE Genomics) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shotgun metagenomic sequencing in weakly positive respiratory samples. Methods Shotgun metagenomics was performed on 40 respiratory specimens collected from solid organ transplant patients and deceased intensive care unit patients at UCLA Medical Center in late 2020 to early 2021. Human and bacterial rRNA depletion was performed on remnant library pools prior to sequencing by Illumina MiSeq. Data quality was analyzed using Geneious Prime, whereas the identification of SARS-CoV-2 variants and lineages was determined by Pangolin. Results The average genome coverage of the rRNA-depleted respiratory specimens increased from 72.55% to 93.71% in overall samples and from 29.3% to 83.3% in 15 samples that failed to achieve sufficient genome coverage using the standard method. Moreover, rRNA depletion enhanced genome coverage to over 85% in 11 (73.3%) of 15 low viral load samples with cycle threshold values up to 35, resulting in the identification of genotypes. Conclusion The CRISPR-based human and bacterial rRNA depletion enhanced the sensitivity of SARS-CoV-2 shotgun metagenomic sequencing, especially in low viral load samples. [ABSTRACT FROM AUTHOR]

Traditional fermented foods, which are well-known microbial resources, are also bright national cultural inheritances. Recently, traditional fermented foods have received great attention due to their potential probiotic properties. Based on shotgun metagenomic sequencing data, we analyzed the microbial diversity, taxonomic composition, metabolic pathways, and the potential benefits and risks of fermented foods through a meta-analysis including 179 selected samples, as well as our own sequencing data collected from Hainan Province, China. As expected, raw materials, regions (differentiated by climatic zones), and substrates were the main driving forces for the microbial diversity and taxonomic composition of traditional fermented foods. Interestingly, a higher content of beneficial bacteria but a low biomass of opportunistic pathogens and antibiotic resistance genes were observed in the fermented dairy products, indicating that fermented dairy products are the most beneficial and reliable fermented foods. In contrast, despite the high microbial diversity found in the fermented soy products, their consumption risk was still high due to the enrichment of opportunistic pathogens and transferable antibiotic resistance genes. Overall, we provided the most comprehensive assessment of the microbiome of fermented food to date and generated a new view of its potential benefits and risks related to human health. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to investigate the specific alterations in gut microbiome and serum metabolome and their interactions in patients with polycystic ovary syndrome (PCOS). Methods: The stool samples from 32 PCOS patients and 18 healthy controls underwent the intestinal microbiome analysis using shotgun metagenomics sequencing approach. Serum metabolome was analyzed by ultrahigh performance liquid chromatography quadrupole time-of-flight mass spectrometry. An integrative network by combining metagenomics and metabolomics datasets was constructed to explore the possible interactions between gut microbiota and circulating metabolites in PCOS, which was further assessed by fecal microbiota transplantation (FMT) in a rat trial. Results: Fecal metagenomics identified 64 microbial strains significantly differing between PCOS and healthy subjects, half of which were enriched in patients. These changed species showed an ability to perturb host metabolic homeostasis (including insulin resistance and fatty acid metabolism) and inflammatory levels (such as PI3K/Akt/mTOR signaling pathways) by expressing sterol regulatory element-binding transcription factor-1, serine/threonine-protein kinase mTOR, and 3-oxoacyl-[acyl-cattier-protein] synthase III, possibly suggesting the potential mechanisms of gut microbiota underlying PCOS. By integrating multi-omics datasets, the panel comprising seven strains (Achromobacter xylosoxidans, Pseudomonas sp. M1, Aquitalea pelogenes, Porphyrobacter sp. HL-46, Vibrio fortis, Leisingera sp. ANG-Vp, and Sinorhizobium meliloti) and three metabolites [ganglioside GM3 (d18:0/16:0), ceramide (d16:2/22:0), and 3Z,6Z,9Z-pentacosatriene] showed the highest predictivity of PCOS (AUC: 1.0) with sensitivity of 0.97 and specificity of 1.0. Moreover, the intestinal microbiome modifications by FMT were demonstrated to regulate PCOS phenotypes including metabolic variables and reproductive hormones. Conclusion: Our findings revealed key microbial and metabolite features and their interactions underlying PCOS by integrating multi-omics approaches, which may provide novel insights into discovering clinical diagnostic biomarkers and developing efficient therapeutic strategies for PCOS. [ABSTRACT FROM AUTHOR]

Background and aims: Birth mode and other early life factors affect a newborn's microbial colonization with potential long-term health effects. Individual variations in early life gut microbiota development, especially their effects on the functional repertoire of microbiota, are still poorly characterized. This study aims to provide new insights into the gut microbiome developmental trajectories during the first year of life. Methods: Our study comprised 78 term infants sampled at 3 weeks, 3 months, 6 months, and 12 months (n = 280 total samples), and their mothers were sampled in late pregnancy (n = 50). Fecal DNA was subjected to shotgun metagenomic sequencing. Infant samples were studied for taxonomic and functional maturation, and maternal microbiota was used as a reference. Hierarchical clustering on taxonomic profiles was used to identify the main microbiota developmental trajectories in the infants, and their associations with perinatal and postnatal factors were assessed. Results: In line with previous studies, infant microbiota composition showed increased alpha diversity and decreased beta diversity by age, converging toward an adult-like profile. However, we did not observe an increase in functional alpha diversity, which was stable and comparable with the mother samples throughout all the sampling points. Using a de novo clustering approach, two main infant microbiota clusters driven by Bacteroidaceae and Clostridiaceae emerged at each time point. The clusters were associated with birth mode and their functions differed mainly in terms of biosynthetic and carbohydrate degradation pathways, some of which consistently differed between the clusters for all the time points. The longitudinal analysis indicated three main microbiota developmental trajectories, with the majority of the infants retaining their characteristic cluster until 1 year. As many as 40% of vaginally delivered infants were grouped with infants delivered by C-section due to their clear and persistent depletion in Bacteroides. Intrapartum antibiotics, any perinatal or postnatal factors, maternal microbiota composition, or other maternal factors did not explain the depletion in Bacteroides in the subset of vaginally born infants. Conclusion: Our study provides an enhanced understanding of the compositional and functional early life gut microbiota trajectories, opening avenues for investigating elusive causes that influence non-typical microbiota development. [ABSTRACT FROM AUTHOR]

Here, we present a longitudinal shotgun sequencing metagenomics study of 16 healthy, Danish women in the reproductive age. The aim of the study was to investigate whether lactobacilli, orally consumed, had any impact on the vaginal microbiome and its functional potential. The 16 women aged 19–45 years were recruited from Copenhagen, Denmark. One baseline vaginal sample (Day 0) and two study samples (Days 25–30 and Days 55–60, respectively), were sampled. The vaginal samples were analyzed by shotgun metagenomics. We detected 26 species in the vaginal microbiota of the 16 women, of which six belonged to the Lactobacillus genus. We observed three vaginal microbiome clusters mainly dominated by Gardnerella vaginalis, Lactobacillus iners, or Lactobacillus crispatus. The oral probiotic had no detectable effect on either the composition or the functional potential of the vaginal microbiota. Most of the study subjects (11 out of 16 women) exhibited only minor changes in the vaginal microbiome during the treatment with probiotics. Any compositional changes could not be associated to the probiotic treatment. Future studies may benefit from an increased number of participants, and administration of the probiotics during conditions with bacterial imbalance (e.g., during/after antibiotic treatment) or the use of different Lactobacillus spp. known to colonize the vagina. [ABSTRACT FROM AUTHOR]

The excessive proliferation of cyanobacteria in surface waters is a widespread problem worldwide, leading to the contamination of drinking water sources. Short- and long-term solutions for managing cyanobacterial blooms are needed for drinking water supplies. The goal of this research was to investigate the cyanobacteria community composition using shotgun metagenomics in a short term, in situ mesocosm experiment of two lakes following their coagulation with ferric sulfate (Fe2(SO4)3) as an option for source water treatment. Among the nutrient paramenters, dissolved nitrogen was related to Microcystis in both Missisquoi Bay and Petit Lac St. François, while the presence of Synechococcus was related to total nitrogen, dissolved nitrogen, dissolved organic carbon, and dissolved phosphorus. Results from the shotgun metagenomic sequencing showed that Dolichospermum and Microcystis were the dominant genera in all of the mesocosms in the beginning of the sampling period in Missisquoi Bay and Petit Lac St. François, respectively. Potentially toxigenic genera such as Microcystis were correlated with intracellular microcystin concentrations. A principal component analysis showed that there was a change of the cyanobacterial composition at the genus level in the mesocosms after two days, which varied across the studied sites and sampling time. The cyanobacterial community richness and diversity did not change significantly after its coagulation by Fe2(SO4)3 in all of the mesocosms at either site. The use of Fe2(SO4)3 for an onsite source water treatment should consider its impact on cyanobacterial community structure and the reduction of toxin concentrations. [ABSTRACT FROM AUTHOR]

The fiber, vitamin, and antioxidant contents of fruits contribute to a balanced human diet. In countries such as Argentina, several tropical fruits are witnessing a high yield in the harvest season, with a resulting surplus. Fruit fermentation using autochthonous starter cultures can provide a solution for food waste. However, limited knowledge exists about the microbiota present on the surfaces of fruits and the preceding flowers. In the present exploratory study, the microbiomes associated with the surfaces of tropical fruits from Northern Argentina, such as white guava, passion fruit and papaya were investigated using a shotgun metagenomic sequencing approach. Hereto, one sample composed of 14 white guava fruits, two samples of passion fruits with each two to three fruits representing the almost ripe and ripe stage of maturity, four samples of papaya with each two to three fruits representing the unripe, almost ripe, and ripe stage of maturity were processed, as well as a sample of closed and a sample of open Japanese medlar flowers. A considerable heterogeneity was found in the composition of the fruits' surface microbiota at the genus and species level. While bacteria dominated the microbiota of the fruits and flowers, a small number of the metagenomic sequence reads corresponded with yeasts and filamentous fungi. A minimal abundance of bacterial species critical in lactic acid and acetic acid fermentations was found. A considerable fraction of the metagenomic sequence reads from the fruits' surface microbiomes remained unidentified, which suggested that intrinsic species are to be sequenced or discovered. [ABSTRACT FROM AUTHOR]

Background: Shotgun metagenomics yields ever richer and larger data volumes on the complex communities living in diverse environments. Extracting deep insights from the raw reads heavily depends on the availability of fast, accurate and user-friendly biodiversity analysis tools. Results: Because environmental samples may contain strains and species that are not covered in reference databases and because protein sequences are more conserved than the genes encoding them, we explore the alternative route of taxonomic profiling based on protein coding regions translated from the shotgun metagenomics reads, instead of directly processing the DNA reads. We therefore developed the Unipept MetaGenomics Analysis Pipeline (UMGAP), a highly versatile suite of open source tools that are implemented in Rust and support parallelization to achieve optimal performance. Six preconfigured pipelines with different performance trade-offs were carefully selected, and benchmarked against a selection of state-of-the-art shotgun metagenomics taxonomic profiling tools. Conclusions: UMGAP's protein space detour for taxonomic profiling makes it competitive with state-of-the-art shotgun metagenomics tools. Despite our design choices of an extra protein translation step, a broad spectrum index that can identify both archaea, bacteria, eukaryotes and viruses, and a highly configurable non-monolithic design, UMGAP achieves low runtime, manageable memory footprint and high accuracy. Its interactive visualizations allow for easy exploration and comparison of complex communities. [ABSTRACT FROM AUTHOR]

Metagenomics – shotgun sequencing of all DNA fragments from a community DNA extract – is routinely used to describe the composition, structure, and function of microorganism communities. Advances in DNA sequencing and the availability of genome databases increasingly allow the use of shotgun metagenomics on eukaryotic communities. Metagenomics offers major advances in the recovery of biomass relationships in a sample, in comparison to taxonomic marker gene‐based approaches (metabarcoding). However, little is known about the factors which influence metagenomics data from eukaryotic communities, such as differences among organism groups, the properties of reference genomes, and genome assemblies.We evaluated how shotgun metagenomics records composition and biomass in artificial soil invertebrate communities at different sequencing efforts. We generated mock communities of controlled biomass ratios from 28 species from all major soil mesofauna groups: mites, springtails, nematodes, tardigrades, and potworms. We shotgun sequenced these communities and taxonomically assigned them with a database of over 270 soil invertebrate genomes.We recovered over 95% of the species, and observed relatively high false‐positive detection rates. We found strong differences in reads assigned to different taxa, with some groups (e.g., springtails) consistently attracting more hits than others (e.g., enchytraeids). Original biomass could be predicted from read counts after considering these taxon‐specific differences. Species with larger genomes, and with more complete assemblies, consistently attracted more reads than species with smaller genomes. The GC content of the genome assemblies had no effect on the biomass–read relationships. Results were similar among different sequencing efforts.The results show considerable differences in taxon recovery and taxon specificity of biomass recovery from metagenomic sequence data. The properties of reference genomes and genome assemblies also influence biomass recovery, and they should be considered in metagenomic studies of eukaryotes. We show that low‐ and high‐sequencing efforts yield similar results, suggesting high cost‐efficiency of metagenomics for eukaryotic communities. We provide a brief roadmap for investigating factors which influence metagenomics‐based eukaryotic community reconstructions. Understanding these factors is timely as accessibility of DNA sequencing and momentum for reference genomes projects show a future where the taxonomic assignment of DNA from any community sample becomes a reality. [ABSTRACT FROM AUTHOR]

Precipitation is one of the major constraints influencing the diversity, structure, and activity of soil microbial communities in desert ecosystems. However, the effect of changes in precipitation on soil microbial communities in arid soil microbiomes remains unresolved. In this study, using 16S rRNA gene high-throughput sequencing and shotgun metagenome sequencing, we explored changes in taxonomic composition and functional potential across two zones in the Namib Desert with contrasting precipitation regime. We found that precipitation regime had no effect on taxonomic and functional alpha-diversity, but that microbial community composition and functional potential (beta-diversity) changed with increased precipitation. For instance, Acidobacteriota and 'resistance to antibiotics and toxic compounds' related genes were relatively more abundant in the high-rainfall zone. These changes were largely due to a small set of microbial taxa, some of which were present in low abundance (i.e. members of the rare biosphere). Overall, these results indicate that key climatic factors (i.e. precipitation) shape the taxonomic and functional attributes of the arid soil microbiome. This research provides insight into how changes in precipitation patterns associated with global climate change may impact microbial community structure and function in desert soils. [ABSTRACT FROM AUTHOR]

Background: Diosmectite, a natural colloidal clay, has been used worldwide for a number of approved indications, including the treatment of chronic functional diarrhea. Here, we used high-resolution whole metagenome shotgun sequencing to assess the impact of a 5 weeks administration of diosmectite (3 g/sachet, 3 sachets/day) on the fecal microbiota of 35 adults with functional chronic diarrhea. Results: Gut microbiota was not impacted by diosmectite administration. In particular, richness remained stable and no microbial species displayed a significant evolution. Segregating patients either by diosmectite response (non responder, early responder, late responder) or by nationality (Great-Britain or Netherlands) yielded the same results. Conclusion: We concluded that no microbiota-related physiological alterations are expected upon long-term treatment with diosmectite. Trial registration: Clinicaltrials.gov NCT03045926 [ABSTRACT FROM AUTHOR]

Shotgun metagenomics studies have improved our understanding of microbial population dynamics and have revealed significant contributions of microbes to gut homeostasis. They also allow in silico inference of the metagenome. While they link the microbiome with metabolic abnormalities associated with disease phenotypes, they do not capture microbial gene expression patterns that occur in response to the multitude of stimuli that constantly ambush the gut environment. Metatranscriptomics closes that gap, but its implementation is more expensive and tedious. We assessed the metabolic perturbations associated with gut inflammation using shotgun metagenomics and metatranscriptomics. Shotgun metagenomics detected changes in abundance of bacterial taxa known to be SCFA producers, which favors gut homeostasis. Bacteria in the phylum Firmicutes were found at decreased abundance, while those in phyla Bacteroidetes and Proteobacteria were found at increased abundance. Surprisingly, inferring the coding capacity of the microbiome from shotgun metagenomics data did not result in any statistically significant difference, suggesting functional redundancy in the microbiome or poor resolution of shotgun metagenomics data to profile bacterial pathways, especially when sequencing is not very deep. Obviously, the ability of metatranscriptomics libraries to detect transcripts expressed at basal (or simply low) levels is also dependent on sequencing depth. Nevertheless, metatranscriptomics informed about contrasting roles of bacteria during inflammation. Functions involved in nutrient transport, immune suppression and regulation of tissue damage were dramatically upregulated, perhaps contributed by homeostasis-promoting bacteria. Functions ostensibly increasing bacteria pathogenesis were also found upregulated, perhaps as a consequence of increased abundance of Proteobacteria. Bacterial protein synthesis appeared downregulated. In summary, shotgun metagenomics was useful to profile bacterial population composition and taxa relative abundance, but did not inform about differential gene content associated with inflammation. Metatranscriptomics was more robust for capturing bacterial metabolism in real time. Although both approaches are complementary, it is often not possible to apply them in parallel. We hope our data will help researchers to decide which approach is more appropriate for the study of different aspects of the microbiome. [ABSTRACT FROM AUTHOR]

Summary: The extent to which ectomycorrhizal (ECM) fungi decay soil organic matter (SOM) has implications for accurately predicting forest ecosystem response to climate change. Investigating the distribution of gene traits associated with SOM decay among ectomycorrhizal fungal communities could improve understanding of SOM dynamics and plant nutrition. We hypothesized that soil inorganic nitrogen (N) availability structures the distribution of ECM fungal genes associated with SOM decay and, specifically, that ECM fungal communities occurring in inorganic N‐poor soils have greater SOM decay potential.To test this hypothesis, we paired amplicon and shotgun metagenomic sequencing of 60 ECM fungal communities associating with Quercus rubra along a natural soil inorganic N gradient.Ectomycorrhizal fungal communities occurring in low inorganic N soils were enriched in gene families involved in the decay of lignin, cellulose, and chitin. Ectomycorrhizal fungal community composition was the strongest driver of shifts in metagenomic estimates of fungal decay potential. Our study simultaneously illuminates the identity of key ECM fungal taxa and gene families potentially involved in the decay of SOM, and we link rhizomorphic and medium‐distance hyphal morphologies with enhanced SOM decay potential.Coupled shifts in ECM fungal community composition and community‐level decay gene frequencies are consistent with outcomes of trait‐mediated community assembly processes. [ABSTRACT FROM AUTHOR]

Background: Next-Generation-Sequencing (NGS) enables detection of microorganisms present in biological and other matrices of various origin and nature, allowing not only the identification of known phyla and strains but also the discovery of novel ones. The large amount of metagenomic shotgun data produced by NGS require comprehensive and user-friendly pipelines for data analysis, that speed up the bioinformatics steps, relieving the users from the need to manually perform complex and time-consuming tasks. Results: We describe here HOME-BIO (sHOtgun MEtagenomic analysis of BIOlogical entities), an exhaustive pipeline for metagenomics data analysis, comprising three independent analytical modules designed for an inclusive analysis of large NGS datasets. Conclusions: HOME-BIO is a powerful and easy-to-use tool that can be run also by users with limited computational expertise. It allows in-depth analyses by removing low-complexity/ problematic reads, integrating the analytical steps that lead to a comprehensive taxonomy profile of each sample by querying different source databases, and it is customizable according to specific users' needs. [ABSTRACT FROM AUTHOR]

In this perspective article, we review the past use of ribosomal sequences to address scientific and diagnostic questions. We highlight a variety of sequencing approaches including metagenomics and DNA barcoding and their different demands and requirements. Meta-ribosomalomics is introduced as an unbiased approach to exploit high-throughput sequencing datasets for eukaryotic and prokaryotic ribosomal sequences. Prerequisites, benefits, drawbacks, and future perspectives are elaborated and compared to other sequencing approaches. [ABSTRACT FROM AUTHOR]

It is thought that modern wheat genotypes have lost their capacity to associate with soil microbes that would help them acquire nutrients from the soil. To test this hypothesis, ten ancestral and modern wheat genotypes were seeded in a field experiment under low fertilization conditions. The rhizosphere soil was collected, its DNA extracted and submitted to shotgun metagenomic sequencing. In contrast to our hypothesis, there was no significant difference in the global rhizosphere metagenomes of the different genotypes, and this held true when focusing the analyses on specific taxonomic or functional categories of genes. Some genes were significantly more abundant in the rhizosphere of one genotype or another, but they comprised only a small portion of the total genes identified and did not affect the global rhizosphere metagenomes. Our study shows for the first time that the rhizosphere metagenome of wheat is stable across a wide variety of genotypes when growing under nutrient poor conditions. [ABSTRACT FROM AUTHOR]

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection. [ABSTRACT FROM AUTHOR]

Lambic beer production processes are characterized by a temporal succession of well-adapted microbial species. Temporal metagenomic analysis of a Belgian, traditional, lambic beer production process, which was examined microbiologically and metabolomically before, confirmed that the microbial diversity is limited. Moreover, it allowed to link the consumption and production of certain compounds to specific microbial groups or species. Fermentation characteristics, such as the conversion of malic acid into lactic acid and acetoin production, were retrieved and could be attributed to specific microorganisms, namely Pediococcus damnosus and Acetobacter species, respectively. Traits previously ascribed to brewery-specific Dekkera bruxellensis strains were confirmed during the lambic beer production process examined multiphasically; in particular, the higher production of 4-ethylguaiacol compared to 4-ethylphenol was further shown by mass spectrometric analysis. Moreover, the absence of phenolic acid decarboxylase in Brettanomyces custersianus was shown culture-independently and could explain its late occurrence during the maturation phase. Furthermore, the potential of maltooligosaccharide degradation could be ascribed metagenomically to not only Brettanomyces species but also Saccharomyces kudriavzevii , possibly explaining their degradation early in the lambic beer production process. Also, acetic acid bacteria (AAB) seemed to be able to consume maltooligosaccharides via their conversion into trehalose. Furthermore, these AAB possessed esterase genes, potentially capable of forming ethyl acetate, which may contribute to the flavor of lambic beer. Improved knowledge on the reasons behind certain community dynamics and the role of the different microorganisms in terms of potential functionality could improve brewery practices to assure to produce more quality-stable end-products. [ABSTRACT FROM AUTHOR]

The effects of cadmium (Cd) contamination on the microbial community structure, soil physicochemistry and heavy metal resistome of a tropical agricultural soil were evaluated in field-moist soil microcosms. A Cd-contaminated agricultural soil (SL5) and an untreated control (SL4) were compared over a period of 5 weeks. Analysis of the physicochemical properties and heavy metals content of the two microcosms revealed a statistically significant decrease in value of the soil physicochemical parameters (P < 0.05) and concentration of heavy metals (Cd, Pb, Cr, Zn, Fe, Cu, Se) content of the agricultural soil in SL5 microcosm. Illumina shotgun sequencing of the DNA extracted from the two microcosms showed the predominance of the phyla, classes, genera and species of Proteobacteria (37.38%), Actinobacteria (35.02%), Prevotella (6.93%), and Conexibacter woesei (8.93%) in SL4, and Proteobacteria (50.50%), Alphaproteobacteria (22.28%), Methylobacterium (9.14%), and Methylobacterium radiotolerans (12,80%) in SL5, respectively. Statistically significant (P < 0.05) difference between the metagenomes was observed at genus and species delineations. Functional annotation of the two metagenomes revealed diverse heavy metal resistome for the uptake, transport, efflux and detoxification of various heavy metals. It also revealed the exclusive detection in SL5 metagenome of members of RND (resistance nodulation division) protein czcCBA efflux system (czcA, czrA, czrB), CDF (cation diffusion facilitator) transporters (czcD), and genes for enzymes that protect the microbial cells against cadmium stress (sodA, sodB, ahpC). The results obtained in this study showed that Cd contamination significantly affects the soil microbial community structure and function, modifies the heavy metal resistome, alters the soil physicochemistry and results in massive loss of some autochthonous members of the community not adapted to the Cd stress. [ABSTRACT FROM AUTHOR]

Background: Isopods have colonized all environments, partly thanks to their ability to decompose the organic matter. Their enzymatic repertoire, as well as the one of their associated microbiota, has contributed to their colonization success. Together, these holobionts have evolved several interesting life history traits to degrade the plant cell walls, mainly composed of lignocellulose. It has been shown that terrestrial isopods achieve lignocellulose degradation thanks to numerous and diverse CAZymes provided by both the host and its microbiota. Nevertheless, the strategies for lignocellulose degradation seem more diversified in isopods, in particular in aquatic species which are the least studied. Isopods could be an interesting source of valuable enzymes for biotechnological industries of biomass conversion. Results: To provide new features on the lignocellulose degradation in isopod holobionts, shotgun sequencing of 36 metagenomes of digestive and non-digestive tissues was performed from several populations of four aquatic and terrestrial isopod species. Combined to the 15 metagenomes of an additional species from our previous study, as well as the host transcriptomes, this large dataset allowed us to identify the CAZymes in both the host and the associated microbial communities. Analyses revealed the dominance of Bacteroidetes and Proteobacteria in the five species, covering 36% and 56% of the total bacterial community, respectively. The identification of CAZymes and new enzymatic systems for lignocellulose degradation, such as PULs, cellulosomes and LPMOs, highlights the richness of the strategies used by the isopods and their associated microbiota. Conclusions: Altogether, our results show that the isopod holobionts are promising models to study lignocellulose degradation. These models can provide new enzymes and relevant lignocellulose-degrading bacteria strains for the biotechnological industries of biomass conversion. [ABSTRACT FROM AUTHOR]

Background: Currently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction). Methods: Shotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen. Results: The three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration. Conclusion: This Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades. [ABSTRACT FROM AUTHOR]

The aims of this study were i) to evaluate the possibility to detect and possibly quantify microorganisms belonging to different domains experimentally spiked in smoked salmon at known concentrations using shotgun metagenomics; ii) to compare the sequencing results using four bioinformatic tools. The salmon was spiked with six species of bacteria, including potential foodborne pathogens, as well as Cryptosporidium parvum, Saccharomyces cerevisiae and Bovine alphaherpesvirus 1. After spiking, the salmon was kept refrigerated before DNA extraction, library preparation and sequencing at 7 Gbp in paired ends at 150 bp. The bioinformatic tools named MG-RAST, OneCodex, CosmosID and MgMapper were used for the sequence analysis and the data provided were compared using STAMP. All bacteria spiked in the salmon were identified using all bioinformatic tools. Such tools were also able to assign the higher abundances to the species Propionibacterium freudenreichii spiked at the highest concentration in comparison to the other bacteria. Nevertheless, different abundances were quantified for bacteria spiked in the salmon at the same cell concentration. Cryptosporidium parvum was detected by all bioinformatics tools, while Saccharomyces cerevisiae by MG-RAST only. Finally, the DNA virus was detected by CosmosID and OneCodex only. Overall, the results of this study showed that shotgun metagenomics can be applied to detect microorganisms belonging to different domains in the same food sample. Nevertheless, a direct correlation between cell concentration of each spiked microorganism and number of corresponding reads cannot be established yet. [ABSTRACT FROM AUTHOR]

• Shotgun metagenomics (SM) allowed detection of both the microbiome and the resistome from urine. • SM enabled detection of resistance to clinically relevant antibiotics. • SM was fully concordant for microbiome detection in cultures positive with one bacterium. • SM could be a rapid and powerful tool in the future for patients with urosepsis. In sepsis, early and appropriate antibiotic therapy is key but is frequently challenging due to the increasing incidence of multidrug-resistant bacteria. The feasibility of shotgun metagenomics (SM) has been scarcely assessed in urinary tract infections (UTIs). In this study, the feasibility of SM to detect both the microbiome and the resistome in patients with confirmed UTI-related sepsis was evaluated. Urine samples were obtained from 40 adult patients with UTI-related sepsis. Conventional culture was used as a reference. Following total DNA extraction and depletion of human DNA, SM was performed using Ion ProtonTM technology. Bioinformatics analysis was conducted using GeneiousⓇ software as well as online tools from the Center for Genomic Epidemiology. For the microbiome, SM was consistently concordant when urine culture was positive with only one bacterium (mainly Escherichia coli). For the resistome, results were in agreement with antimicrobial susceptibility testing with no major discrepancies. SM consistently identified bla CTX-M genes responsible for resistance to third-generation cephalosporins. Resistance to aminoglycosides and fluoroquinolones was identified in all patients. This pilot study confirms that SM can provide clinically relevant information both on the microbiome and the resistome from urine samples of patients with UTI-related sepsis. [ABSTRACT FROM AUTHOR]

High-throughput shotgun metagenomics sequencing has enabled the profiling of myriad natural communities. These data are commonly used to identify gene families and pathways that were potentially gained or lost in an environment and which may be involved in microbial adaptation. Despite the widespread interest in these events, there are no established best practices for identifying gene gain and loss in metagenomics data. Horizontal gene transfer (HGT) represents several mechanisms of gene gain that are especially of interest in clinical microbiology due to the rapid spread of antibiotic resistance genes in natural communities. Several additional mechanisms of gene gain and loss, including gene duplication, gene loss-of-function events, and de novo gene birth are also important to consider in the context of metagenomes but have been less studied. This review is largely focused on detecting HGT in prokaryotic metagenomes, but methods for detecting these other mechanisms are first discussed. For this article to be self-contained, we provide a general background on HGT and the different possible signatures of this process. Lastly, we discuss how improved assembly of genomes from metagenomes would be the most straight-forward approach for improving the inference of gene gain and loss events. Several recent technological advances could help improve metagenome assemblies: long-read sequencing, determining the physical proximity of contigs, optical mapping of short sequences along chromosomes, and single-cell metagenomics. The benefits and limitations of these advances are discussed and open questions in this area are highlighted. [ABSTRACT FROM AUTHOR]

Gut microbiota of freshwater carp (Labeo rohita) was investigated by shotgun metagenomics to understand its taxonomic composition and functional capabilities. With the presence of 36 phyla, 326 families and 985 genera, the fish gut microbiota was found to be quite diverse in nature. However, at the phylum level, more than three-fourths of gut microbes belonged to Proteobacteria. Very low prevalence of commonly used probiotic bacteria (Bacillus, Lactobacillus, Streptococcus, and Lactococcus) in fish gut suggested the need to search for alternative probiotics for aquaculture use. Biosynthesis pathways were found to be the most dominant (51%) followed by degradation (39%), energy metabolism (4%) and fermentation (2%). In conformity with herbivorous feeding habit of L. rohita, gut microbiome also had pathways for the degradation of cellulose, hemicellulose, chitin, pectin, starch, and other complex carbohydrates. High prevalence of Actinobacteria and antibiotic biosynthesis pathways in the fish gut microbiome indicated its potential for bioprospecting of potentially novel natural antibiotics. Fifty-one different types of antibiotic resistance genes (ARGs) belonging to 15 antimicrobial resistance (AMR) gene families and conferring resistance against 24 antibiotic types were detected in fish gut. Some of the ARGs for multi-drug resistance were also found to be located on sequences of plasmid origin. The presence of pathogenic bacteria and ARGs on plasmid sequences suggested the potential risk due to horizontal gene transfer in the confined gut environment. The role of ARGs in fish gut microbiome needs further investigations. [ABSTRACT FROM AUTHOR]

Comparing the functional gene composition of soils at opposite extremes of environmental gradients may allow testing of hypotheses about community and ecosystem function. Here, we were interested in comparing how tropical microbial ecosystems differ from those of polar climates. We sampled several sites in the equatorial rainforest of Malaysia and Brunei, and the high Arctic of Svalbard, Canada, and Greenland, comparing the composition and the functional attributes of soil biota between the two extremes of latitude, using shotgun metagenomic Illumina HiSeq2000 sequencing. Based upon "classical" views of how tropical and higher latitude ecosystems differ, we made a series of predictions as to how various gene function categories would differ in relative abundance between tropical and polar environments. Results showed that in some respects our predictions were correct: the polar samples had higher relative abundance of dormancy related genes, and lower relative abundance of genes associated with respiration, and with metabolism of aromatic compounds. The network complexity of the Arctic was also lower than the tropics. However, in various other respects, the pattern was not as predicted; there were no differences in relative abundance of stress response genes or in genes associated with secondary metabolism. Conversely, CRISPR genes, phage-related genes, and virulence disease and defense genes, were unexpectedly more abundant in the Arctic, suggesting more intense biotic interaction. Also, eukaryote diversity and bacterial diversity were higher in the Arctic of Svalbard compared to tropical Brunei, which is consistent with what may expected from amplicon studies in terms of the higher pH of the Svalbard soil. Our results in some respects confirm expectations of how tropical versus polar nature may differ, and in other respects challenge them. [ABSTRACT FROM AUTHOR]

We report a case of tick-borne relapsing fever caused by Borrelia persica in a traveler returning to Switzerland from central Asia. After the disease was diagnosed by blood smear microscopy, the causative Borrelia species was confirmed by shotgun metagenomics sequencing. PCR and sequencing techniques provide highly sensitive diagnostic tools superior to microscopy. [ABSTRACT FROM AUTHOR]

Changes in the microbial gene content and abundance can be analyzed to detect shifts in the microbiota composition due to the use of a starter culture in the food fermentation process, with the consequent shift of key metabolic pathways directly connected with product acceptance. Meat fermentation is a complex process involving microbes that metabolize the main components in meat. The breakdown of carbohydrates, proteins, and lipids can lead to the formation of volatile organic compounds (VOCs) that can drastically affect the organoleptic characteristics of the final products. The present meta-analysis, performed with the shotgun DNA metagenomic approach, focuses on studying the microbiota and its gene content in an Italian fermented sausage produced by using a commercial starter culture (a mix of Lactobacillus sakei and Staphylococcus xylosus), with the aim to discover the connections between the microbiota, microbiome, and the release of volatile metabolites during ripening. The inoculated fermentation with the starter culture limited the development of Enterobacteriaceae and reduced the microbial diversity compared to that from spontaneous fermentation. KEGG database genes associated with the reduction of acetaldehyde to ethanol (EC 1.1.1.1), acetyl phosphate to acetate (EC 2.7.2.1), and 2,3-butanediol to acetoin (EC 1.1.1.4) were most abundant in inoculated samples (I) compared to those in spontaneous fermentation samples (S). The volatilome profiles were highly consistent with the abundance of the genes; elevated acetic acid (1,173.85 µg/kg), ethyl acetate (251.58 µg/kg), and acetoin (1,100.19 µg/kg) were observed in the presence of the starters at the end of fermentation. Significant differences were found in the liking of samples based on flavor and odor, suggesting a higher preference by consumers for the spontaneous fermentation samples. Inoculated samples exhibited the lowest scores for the liking data, which were clearly associated with the highest concentration of acetic acid. [ABSTRACT FROM AUTHOR]

Picoeukaryotes are key components in marine ecosystems that play crucial roles in food webs and biogeochemical cycles. Despite their significance, many aspects of their community ecology and diversity remain understudied. Here, we investigated the taxonomic and functional diversity of picoeukaryotic communities in response to monsoonal patterns and weather disturbances brought about by storms, characterizing tropical coastal regions. To do this, water samples were collected almost weekly or bi-weekly at a single location in a tropical coastal environment covering the late northeast (NE) and southwest (SW) monsoons. We then performed high-throughput amplicon sequencing of the V4 region of the 18S rRNA gene to generate taxonomic profiles of the communities across time. Clustering based on environmental parameters grouped our samples into months associated with NE monsoon, SW monsoon, and stormy SW monsoon, demonstrating seasonality influenced by monsoons and storms, typically observed in tropical coastal waters. In comparison, clustering based on abundance only grouped the samples into NE and SW monsoon, with most communities during storm period joining the NE monsoon samples. These samples exhibited greater diversity, with smaller taxa such as Syndiniales, Prymnesiophyceae, Picozoa, Cercozoa, Stramenopiles, and Chlorophytes being the most abundant groups present. In contrast, SW monsoon samples have lower diversity but have become generally dominated by large-celled taxa, mostly diatoms. Multivariate and correlation analyses both revealed nitrate as the strongest environmental driver of the picoeukaryotic community structuring. Meanwhile, network analysis grouped the taxa into three modules, more consistent with the clustering based on environmental parameters, implying that although storms may not significantly change the community composition, they may however influence the dominating taxa. Each module was composed of a unique set of co-occurring taxa, highlighting high turnover of picoeukaryotic communities between each season. In addition, our results showed that SW monsoon-associated module had higher interconnectivity than other modules, suggesting that the interactions during this period may be less species-specific, thus, more adaptable than during NE monsoon. However, we observed that extreme fluctuations caused by storms could have possibly allowed for selection of dominant taxa. Shotgun metagenomic sequencing of representative samples from each monsoon period also revealed that differently abundant functional genes, particularly genes associated to nitrogen metabolism, might have also helped in adaptation to the changing nutrient conditions. Our observations provide new insights on the potential trajectory of microbial communities under environmental stresses, which are important in understanding the implications of emerging threats such as coastal eutrophication and climate change. [ABSTRACT FROM AUTHOR]

Periphyton communities are complex phototrophic, multispecies biofilms that develop on surfaces in aquatic environments. These communities harbor a large diversity of organisms comprising viruses, bacteria, algae, fungi, protozoans, and metazoans. However, thus far the total biodiversity of periphyton has not been described. In this study, we use metagenomics to characterize periphyton communities from the marine environment of the Swedish west coast. Although we found approximately ten times more eukaryotic rRNA marker gene sequences compared to prokaryotic, the whole metagenome-based similarity searches showed that bacteria constitute the most abundant phyla in these biofilms. We show that marine periphyton encompass a range of heterotrophic and phototrophic organisms. Heterotrophic bacteria, including the majority of proteobacterial clades and Bacteroidetes, and eukaryotic macroinvertebrates were found to dominate periphyton. The phototrophic groups comprise Cyanobacteria and the alpha-proteobacterial genus Roseobacter, followed by different micro- and macro-algae. We also assess the metabolic pathways that predispose these communities to an attached lifestyle. Functional indicators of the biofilm form of life in periphyton involve genes coding for enzymes that catalyze the production and degradation of extracellular polymeric substances, mainly in the form of complex sugars such as starch and glycogen-like meshes together with chitin. Genes for 278 different transporter proteins were detected in the metagenome, constituting the most abundant protein complexes. Finally, genes encoding enzymes that participate in anaerobic pathways, such as denitrification and methanogenesis, were detected suggesting the presence of anaerobic or low-oxygen micro-zones within the biofilms. [ABSTRACT FROM AUTHOR]

Soil microorganisms are sensitive to environment disturbances, and such alterations have consequences on microbial diversity and functions. Our hypothesis is that alpha diversity of microbial communities and functional diversity decrease from undisturbed to disturbed soils, with consequences for functional redundancy in the soil ecosystem. To test this hypothesis, we used soil DNA shotgun metagenomics approach to assess the soil microbiome in a chronosequence of land-use from a native tropical forest, followed by deforestation and cultivation of soybean croplands and pasture in different seasons. Agriculture and pasture soils were among the most diverse and presented higher functional redundancy, which is important to maintain the ecosystem functioning after the forest conversion. On the other hand, the ecosystem equilibrium in forest is maintained based on a lower alpha diversity but higher abundance of microorganisms. Our results indicate that land-use change alters the structure and composition of microbial communities; however, ecosystem functionality is overcome by different strategies based on the abundance and diversity of the communities. [ABSTRACT FROM AUTHOR]