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AIMS: Gastrointestinal disease is a leading cause of morbidity in bottlenose dolphins (Tursiops truncatus) under managed care. Fecal microbiota transplantation (FMT) holds promise as a therapeutic tool to restore gut microbiota without antibiotic use. This prospective clinical study aimed to develop a screening protocol for FMT donors to ensure safety, determine an effective FMT administration protocol for managed dolphins, and evaluate the efficacy of FMTs in four recipient dolphins. METHODS AND RESULTS: Comprehensive health monitoring was performed on donor and recipient dolphins. Fecal samples were collected before, during, and after FMT therapy. Screening of donor and recipient fecal samples was accomplished by in-house and reference lab diagnostic tests. Shotgun metagenomics was used for sequencing. Following FMT treatment, all four recipient communities experienced engraftment of novel microbial species from donor communities. Engraftment coincided with resolution of clinical signs and a sustained increase in alpha diversity. CONCLUSION: The donor screening protocol proved to be safe in this study and no adverse effects were observed in four recipient dolphins. Treatment coincided with improvement in clinical signs.

Background: Microbiomes, essential to ecosystem processes, face strong selective forces that can drive rapid evolutionary adaptation. However, our understanding of evolutionary processes within natural systems remains limited. We investigated evolution in response to naturally occurring selenium in soils of different geological parental materials on the Western Slope of Colorado. Our study focused on examining changes in gene frequencies within microbial communities in response to selenium exposure. Results: Despite expectations of taxonomic composition shifts and increased gene content changes at high-selenium sites, we found no significant alterations in microbial diversity or community composition. Surprisingly, we observed a significant increase in differentially abundant genes within high-selenium sites. Conclusions: These findings are suggestive that selection within microbiomes primarily drives the accumulation of genes among existing microbial taxa, rather than microbial species turnover, in response to strong stressors like selenium. Our study highlights an unusual system that allows us to examine evolution in response to the same stressor annually in a non-model system, contributing to understanding microbiome evolution beyond model systems. [ABSTRACT FROM AUTHOR]

Objective: The influence of different faecal collection methods on metagenomic analyses remains under discussion, and there is no general agreement on which collection method is preferable for gut microbiome research. We compared faecal samples collected in tubes without preservatives with those containing 10 mL of 96% ethanol for gut microbiome research when the timeframe from defecation to freezing at – 80 °C was up to 24 h. We aimed to compare the collection methods on faeces from participants with inflammatory and non-inflammatory gastrointestinal disorders and healthy controls to investigate the most suitable method when considering data yield, human fraction of sequencing reads, and ease of use. We also examined the faecal sample homogeneity. Results: Faeces collected in tubes without preservatives resulted in more sequencing reads compared to faeces collected in tubes with 96% ethanol and were also easier to handle. The human fraction of total reads in faeces collected in ethanol from participants with inflammatory bowel disease was higher than all other samples. DNA extraction and sequencing from two different locations in the same faecal sample gave similar results and showed sample homogeneity. [ABSTRACT FROM AUTHOR]

Hypersaline ecosystems are distributed all over the globe. They are subjected to poly-extreme stresses and are inhabited by halophilic microorganisms possessing multiple adaptations. The halophiles have many biotechnological applications such as nutrient supplements, antioxidant synthesis, salt tolerant enzyme production, osmolyte synthesis, biofuel production, electricity generation etc. However, halophiles are still underexplored in terms of complex ecological interactions and functions as compared to other niches. The advent of metagenomics and the recent advancement of next-generation sequencing tools have made it feasible to investigate the microflora of an ecosystem, its interactions and functions. Both target gene and shotgun metagenomic approaches are commonly employed for the taxonomic, phylogenetic, and functional analyses of the hypersaline microbial communities. This review discusses different types of hypersaline niches, their residential microflora, and an overview of the metagenomic approaches used to investigate them. Various applications, hurdles and the recent advancements in metagenomic approaches have also been focused on here for their better understanding and utilization in the study of hypersaline microbiome. [ABSTRACT FROM AUTHOR]

Bacterial community of a diesel-spiked agricultural soil was monitored over a 42-day period using the metagenomic approach in order to gain insight into key phylotypes impacted by diesel contamination and be able to predict end point of bioattenuation. Soil physico-chemical parameters showed significant differences (P < 0.05) between the Polluted Soil (PS) and the Unpolluted control (US)across time points. After 21 days, the diesel content decreased by 27.39%, and at the end of 42 days, by 57.11%. Aromatics such as benzene, anthanthrene, propylbenzene, phenanthrenequinone, anthraquinone, and phenanthridine were degraded to non-detected levels within 42 days, while some medium range alkanes and polyaromatics such as acenaphthylene, naphthalene, and anthracene showed significant levels of degradation. After 21 days (LASTD21), there was a massive enrichment of the phylum Proteobacteria (72.94%), a slight decrease in the abundance of phylum Actinobacteriota (12.74%), and > 500% decrease in the abundance of the phylum Acidobacteriodota (5.26%). Day 42 (LASTD42) saw establishment of the dominance of the Proteobacteria (34.95%), Actinobacteriota, (21.71%), and Firmicutes (32.14%), and decimation of phyla such as Gemmatimonadota, Planctomycetota, and Verrucromicrobiota which play important roles in the cycling of elements and soil health. Principal component analysis showed that in PS moisture contents, phosphorus, nitrogen, organic carbon, had greater impacts on the community structure in LASTD21, while acidity, potassium, sodium, calcium and magnesium impacted the control sample. Recovery time of the soil based on the residual hydrocarbons at Day 42 was estimated to be 229.112 d. Thus, additional biostimulation may be required to achieve cleanup within one growing season. [ABSTRACT FROM AUTHOR]

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]

Background: Despite effective antiretroviral therapy, people with HIV (PWH) experience persistent systemic inflammation and increased morbidity and mortality. Modulating the gut microbiome through fecal microbiota transplantation (FMT) represents a novel therapeutic strategy. We aimed to evaluate proteomic changes in inflammatory pathways following repeated, low-dose FMT versus placebo. Methods: This double-masked, placebo-controlled pilot study assessed the proteomic impacts of weekly FMT versus placebo treatment over 8 weeks on systemic inflammation in 29 PWH receiving stable antiretroviral therapy (ART). Three stool donors with high Faecalibacterium and butyrate profiles were selected, and their individual stools were used for FMT capsule preparation. Proteomic changes in 345 inflammatory proteins in plasma were quantified using the proximity extension assay, with samples collected at baseline and at weeks 1, 8, and 24. Concurrently, we characterized shifts in the gut microbiota composition and annotated functions through shotgun metagenomics. We fitted generalized additive models to evaluate the dynamics of protein expression. We selected the most relevant proteins to explore their correlations with microbiome composition and functionality over time using linear mixed models. Results: FMT significantly reduced the plasma levels of 45 inflammatory proteins, including established mortality predictors such as IL6 and TNF-α. We found notable reductions persisting up to 16 weeks after the final FMT procedure, including in the expression of proteins such as CCL20 and CD22. We identified changes in 46 proteins, including decreases in FT3LG, IL6, IL10RB, IL12B, and IL17A, which correlated with multiple bacterial species. We found that specific bacterial species within the Ruminococcaceae, Succinivibrionaceae, Prevotellaceae families, and the Clostridium genus, in addition to their associated genes and functions, were significantly correlated with changes in inflammatory markers. Conclusions: Targeting the gut microbiome through FMT effectively decreased inflammatory proteins in PWH, with sustained effects. These findings suggest the potential of the microbiome as a therapeutic target to mitigate inflammation-related complications in this population, encouraging further research and development of microbiome-based interventions. 3bGeWVUV_8zBqrmtjgQWmW Video Abstract [ABSTRACT FROM AUTHOR]

Medication can affect the gut microbiota composition and function. The aim of this study was to investigate connections between use of common non-antibiotic medicines and the gut microbiota composition and function in a large Swedish cohort (N = 2223). Use of 67 medications and polypharmacy (≥ 5 medications), based on self-reported and prescription registry data, were associated with the relative abundance of 881 gut metagenomic species (> 5% prevalence) and 103 gut metabolic modules (GMMs). Altogether, 97 associations of 26 medications with 40 species and of four medications with five GMMs were observed (false discovery rate < 5%). Several earlier findings were replicated like the positive associations of proton pump inhibitors (PPIs) with numerous oral species, and those of metformin with Escherichia species and with lactate consumption I and arginine degradation II. Several new associations were observed between, among others, use of antidepressants, beta-blockers, nonsteroidal anti-inflammatory drugs and calcium channel blockers, and specific species. Polypharmacy was positively associated with Enterococcus faecalis, Bacteroides uniformis, Rothia mucilaginosa, Escherichia coli and Limosilactobacillus vaginalis, and with 13 GMMs. We confirmed several previous findings and identified numerous new associations between use of medications/polypharmacy and the gut microbiota composition and functional potential. Further studies are needed to confirm the new findings. [ABSTRACT FROM AUTHOR]

Wastewater treatment facilities can filter out some plastics before they reach the open environment, yet microplastics often persist throughout these systems. As they age, microplastics in wastewater may both leach and sorb pollutants and fragment to provide an increased surface area for bacterial attachment and conjugation, possibly impacting antimicrobial resistance (AMR) traits. Despite this, little is known about the effects of persistent plastic pollution on microbial functioning. To address this knowledge gap, we deployed five different artificially weathered plastic types and a glass control into the final maturation pond of a municipal wastewater treatment plant in Ōtautahi-Christchurch, Aotearoa/New Zealand. We sampled the plastic-associated biofilms (plastisphere) at 2, 6, 26, and 52 weeks, along with the ambient pond water, at three different depths (20, 40, and 60 cm from the pond water surface). We investigated the changes in plastisphere microbial diversity and functional potential through metagenomic sequencing. Bacterial 16S ribosomal RNA genes composition did not vary among plastic types and glass controls (P = 0.997) but varied among sampling times [permutational multivariate analysis of variance (PERMANOVA), P = 0.001] and depths (PERMANOVA, P = 0.011). Overall, there was no polymer-substrate specificity evident in the total composition of genes (PERMANOVA, P = 0.67), but sampling time (PERMANOVA, P = 0.002) and depth were significant factors (PERMANOVA, P = 0.001). The plastisphere housed diverse AMR gene families, potentially influenced by biofilm-meditated conjugation. The plastisphere also harbored an increased abundance of genes associated with the biodegradation of nylon, or nylon-associated substances, including nylon oligomer-degrading enzymes and hydrolases. IMPORTANCE Plastic pollution is pervasive and ubiquitous. Occurrences of plastics causing entanglement or ingestion, the leaching of toxic additives and persistent organic pollutants from environmental plastics, and their consequences for marine macrofauna are widely reported. However, little is known about the effects of persistent plastic pollution on microbial functioning. Shotgun metagenomics sequencing provides us with the necessary tools to examine broad-scale community functioning to further investigate how plastics influence microbial communities. This study provides insight into the functional consequence of continued exposure to waste plastic by comparing the prokaryotic functional potential of biofilms on five types of plastic [linear low-density polyethylene (LLDPE), nylon-6, polyethylene terephthalate, polylactic acid, and oxygendegradable LLDPE], glass, and ambient pond water over 12 months and at different depths (20, 40, and 60 cm) within a tertiary maturation pond of a municipal wastewater treatment plant. [ABSTRACT FROM AUTHOR]

An animal charcoal contaminated cottage industry soil in Lagos, Nigeria (ACGT) was compared in an expostfacto study with a nearby unimpacted soil (ACGC). Hydrocarbon content was higher than regulatory limits in ACGT (180.2 mg/kg) but lower in ACGC (19.28 mg/kg). Heavy metals like nickel, cadmium, chromium and lead were below detection limit in ACGC. However, all these metals, except cadmium, were detected in ACGT, but at concentrations below regulatory limits. Furthermore, copper (253.205 mg/kg) and zinc (422.630 mg/kg) were above regulatory limits in ACGT. Next generation sequencing revealed that the procaryotic community was dominated by bacteria in ACGC (62%) while in ACGT archaea dominated (76%). Dominant phyla in ACGC were Euryarchaeota (37%), Pseudomonadota (16%) and Actinomycetota (12%). In ACGT it was Euryarchaeota (76%), Bacillota (9%), Pseudomonadota (7%) and Candidatus Nanohaloarchaeota (5%). Dominant Halobacteria genera in ACGT were Halobacterium (16%), Halorientalis (16%), unranked halophilic archaeon (13%) Salarchaeum (6%) and Candidatus Nanohalobium (5%), whereas ACGC showed greater diversity dominated by bacterial genera Salimicrobium (7%) and Halomonas (3%). Heavy metals homeostasis genes, especially for copper, were fairly represented in both soils but with bacterial taxonomic affiliations. Sites like ACGT, hitherto poorly studied and understood, could be sources of novel bioresources. [ABSTRACT FROM AUTHOR]

Colistin is considered the last line therapy for treating multidrug-resistant (MDR) bacterial infections in humans. Therefore, the spread of colistin resistance poses a serious threat to human, and environmental health. Though Bangladesh is known as a hotspot of AMR, limited studies have been carried out regarding the status of colistin resistance. Information on the emerging bacterial resistance is inevitable for protecting public health. Nowadays, wastewater analysis has been prioritized for metagenomics-enabled AMR surveillance. Our study on the metagenomic analysis of untreated hospital effluents first detected the colistin resistance-conferring mcr-5.1 gene in the hospital environment of Bangladesh. Phylogenetic tree and in silico AMR analysis confirmed the detection of this mcr-5 variant, which is located in a plasmid contig. The plasmid was untypeable and belonged to the bacteria from the Enterobacteriaceae family. The mcr-5.1 operon was embedded in a Tn3 transposon, suggesting the mobility of the gene. Tnshfr1 transposon, chromate resistance protein ChrB, DNA invertase hin, and two MFS-type proteins were present in the genetic environment of mcr-5.1. Our findings provide evidence of the occurrence of mcr-5.1 in a hospital environment in Bangladesh, which calls for immediate attention and effective measures to contain the dissemination of colistin resistance in the environment.

Abstract Background Microbiomes, essential to ecosystem processes, face strong selective forces that can drive rapid evolutionary adaptation. However, our understanding of evolutionary processes within natural systems remains limited. We investigated evolution in response to naturally occurring selenium in soils of different geological parental materials on the Western Slope of Colorado. Our study focused on examining changes in gene frequencies within microbial communities in response to selenium exposure. Results Despite expectations of taxonomic composition shifts and increased gene content changes at high-selenium sites, we found no significant alterations in microbial diversity or community composition. Surprisingly, we observed a significant increase in differentially abundant genes within high-selenium sites. Conclusions These findings are suggestive that selection within microbiomes primarily drives the accumulation of genes among existing microbial taxa, rather than microbial species turnover, in response to strong stressors like selenium. Our study highlights an unusual system that allows us to examine evolution in response to the same stressor annually in a non-model system, contributing to understanding microbiome evolution beyond model systems.

Abstract Objective The influence of different faecal collection methods on metagenomic analyses remains under discussion, and there is no general agreement on which collection method is preferable for gut microbiome research. We compared faecal samples collected in tubes without preservatives with those containing 10 mL of 96% ethanol for gut microbiome research when the timeframe from defecation to freezing at – 80 °C was up to 24 h. We aimed to compare the collection methods on faeces from participants with inflammatory and non-inflammatory gastrointestinal disorders and healthy controls to investigate the most suitable method when considering data yield, human fraction of sequencing reads, and ease of use. We also examined the faecal sample homogeneity. Results Faeces collected in tubes without preservatives resulted in more sequencing reads compared to faeces collected in tubes with 96% ethanol and were also easier to handle. The human fraction of total reads in faeces collected in ethanol from participants with inflammatory bowel disease was higher than all other samples. DNA extraction and sequencing from two different locations in the same faecal sample gave similar results and showed sample homogeneity.

Abstract Background Systemic lupus erythematosus (SLE) is linked to host gut dysbiosis. Here we performed faecal gut microbiome sequencing to investigate SLE-pathogenic gut microbes and their potential mechanisms. Methods There were 134 healthy controls (HCs) and 114 SLE cases for 16 S ribosomal RNA (rRNA) sequencing and 97 HCs and 124 SLE cases for shotgun metagenomics. Faecal microbial changes and associations with clinical phenotypes were evaluated, and SLE-associated microbial genera were identified in amplicon analysis. Next, metagenomic sequencing was applied for accurate identification of microbial species and discovery of their metabolic pathways and immunogenic peptides both relevant to SLE. Finally, contribution of specific taxa to disease development was confirmed by oral gavage into lupus-prone MRL/lpr mice. Results SLE patients had gut microbiota richness reduction and composition alteration, particularly lupus nephritis and active patients. Proteobacteria/Bacteroidetes (P/B) ratio was remarkably up-regulated, and Escherichia was identified as the dominantly expanded genus in SLE, followed by metagenomics accurately located Escherichia coli and Escherichia unclassified species. Significant associations primarily appeared among Escherichia coli, metabolic pathways of purine nucleotide salvage or peptidoglycan maturation and SLE disease activity index (SLEDAI), and between multiple epitopes from Escherichia coli and disease activity or renal involvement phenotype. Finally, gavage with faecal Escherichia revealed that it upregulated lupus-associated serum traits and aggravated glomerular lesions in MRL/lpr mice. Conclusion We characterize a novel SLE exacerbating Escherichia outgrowth and suggest its contribution to SLE procession may be partially associated with metabolite changes and cross-reactivity of gut microbiota-associated epitopes and host autoantigens. The findings could provide a deeper insight into gut Escherichia in the procession of SLE.

Abstract 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 Background Despite effective antiretroviral therapy, people with HIV (PWH) experience persistent systemic inflammation and increased morbidity and mortality. Modulating the gut microbiome through fecal microbiota transplantation (FMT) represents a novel therapeutic strategy. We aimed to evaluate proteomic changes in inflammatory pathways following repeated, low-dose FMT versus placebo. Methods This double-masked, placebo-controlled pilot study assessed the proteomic impacts of weekly FMT versus placebo treatment over 8 weeks on systemic inflammation in 29 PWH receiving stable antiretroviral therapy (ART). Three stool donors with high Faecalibacterium and butyrate profiles were selected, and their individual stools were used for FMT capsule preparation. Proteomic changes in 345 inflammatory proteins in plasma were quantified using the proximity extension assay, with samples collected at baseline and at weeks 1, 8, and 24. Concurrently, we characterized shifts in the gut microbiota composition and annotated functions through shotgun metagenomics. We fitted generalized additive models to evaluate the dynamics of protein expression. We selected the most relevant proteins to explore their correlations with microbiome composition and functionality over time using linear mixed models. Results FMT significantly reduced the plasma levels of 45 inflammatory proteins, including established mortality predictors such as IL6 and TNF-α. We found notable reductions persisting up to 16 weeks after the final FMT procedure, including in the expression of proteins such as CCL20 and CD22. We identified changes in 46 proteins, including decreases in FT3LG, IL6, IL10RB, IL12B, and IL17A, which correlated with multiple bacterial species. We found that specific bacterial species within the Ruminococcaceae, Succinivibrionaceae, Prevotellaceae families, and the Clostridium genus, in addition to their associated genes and functions, were significantly correlated with changes in inflammatory markers. Conclusions Targeting the gut microbiome through FMT effectively decreased inflammatory proteins in PWH, with sustained effects. These findings suggest the potential of the microbiome as a therapeutic target to mitigate inflammation-related complications in this population, encouraging further research and development of microbiome-based interventions. Video Abstract

Abstract Medication can affect the gut microbiota composition and function. The aim of this study was to investigate connections between use of common non-antibiotic medicines and the gut microbiota composition and function in a large Swedish cohort (N = 2223). Use of 67 medications and polypharmacy (≥ 5 medications), based on self-reported and prescription registry data, were associated with the relative abundance of 881 gut metagenomic species (> 5% prevalence) and 103 gut metabolic modules (GMMs). Altogether, 97 associations of 26 medications with 40 species and of four medications with five GMMs were observed (false discovery rate

We used Hi-C proximity ligation with shotgun sequencing to retrieve metagenome-assembled genomes (MAGs) from the fecal microbiomes of two domestic cats (Felis catus). The genomes were assessed for completeness and contamination, classified taxonomically, and annotated for putative antimicrobial resistance (AMR) genes.

Abstract 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 Soybean cultivation in tropical regions relies on symbioses with nitrogen-fixing Bradyrhizobium and plant growth-promoting bacteria (PGPBs), reducing environmental impacts of N fertilizers and pesticides. We evaluate the effects of soybean inoculation with different bacterial consortia combined with PGPBs or microbial secondary metabolites (MSMs) on rhizosoil chemistry, plant physiology, plant nutrition, grain yield, and rhizosphere microbial functions under field conditions over three growing seasons with four treatments: standard inoculation of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens consortium (SI); SI plus foliar spraying with Bacillus subtilis (SI + Bs); SI plus foliar spraying with Azospirillum brasilense (SI + Az); and SI plus seed application of MSMs enriched in lipo-chitooligosaccharides extracted from B. diazoefficiens and Rhizobium tropici (SI + MSM). Rhizosphere microbial composition, diversity, and function was assessed by metagenomics. The relationships between rhizosoil chemistry, plant nutrition, grain yield, and the abundance of microbial taxa and functions were determined by generalized joint attribute modeling. The bacterial consortia had the most significant impact on rhizosphere soil fertility, which in turn affected the bacterial community, plant physiology, nutrient availability, and production. Cluster analysis identified microbial groups and functions correlated with shifts in rhizosoil chemistry and plant nutrition. Bacterial consortia positively modulated specific genera and functional pathways involved in biosynthesis of plant secondary metabolites, amino acids, lipopolysaccharides, photosynthesis, bacterial secretion systems, and sulfur metabolism. The effects of the bacterial consortia on the soybean holobiont, particularly the rhizomicrobiome and rhizosoil fertility, highlight the importance of selecting appropriate consortia for desired outcomes. These findings have implications for microbial-based agricultural practices that enhance crop productivity, quality, and sustainability. Graphical Abstract

Abstract Antibiotic resistance is a worldwide problem that imposes a devastating effect on developing countries and requires immediate interventions. Initially, most of the antibiotic drugs were identified by culturing soil microbes. However, this method is prone to discovering the same antibiotics repeatedly. The present study employed a shotgun metagenomics approach to investigate the taxonomic diversity, functional potential, and biosynthetic capacity of microbiomes from two natural agricultural farmlands located in Bekeka and Welmera Choke Kebelle in Ethiopia for the first time. Analysis of the small subunit rRNA revealed bacterial domain accounting for 83.33% and 87.24% in the two selected natural farmlands. Additionally, the analysis showed the dominance of Proteobacteria representing 27.27% and 28.79% followed by Actinobacteria making up 12.73% and 13.64% of the phyla composition. Furthermore, the analysis revealed the presence of unassigned bacteria in the studied samples. The metagenome functional analysis showed 176,961 and 104, 636 number of protein-coding sequences (pCDS) from the two samples found a match with 172,655 and 102, 275 numbers of InterPro entries, respectively. The Genome ontology annotation suggests the presence of 5517 and 3293 pCDS assigned to the “biosynthesis process”. Numerous Kyoto Encyclopedia of Genes and Genomes modules (KEGG modules) involved in the biosynthesis of terpenoids and polyketides were identified. Furthermore, both known and novel Biosynthetic gene clusters, responsible for the production of secondary metabolites, such as polyketide synthases, non-ribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptides (Ripp), and Terpene, were discovered. Generally, from the results it can be concluded that the microbiomes in the selected sampling sites have a hidden functional potential for the biosynthesis of secondary metabolites. Overall, this study can serve as a strong preliminary step in the long journey of bringing new antibiotics to the market.

Aims Biofilms are complex microbial cell aggregates that attach to different surfaces in nature, industrial environments, or hospital settings. In photovoltaic panels (PVs), biofilms are related to significant energy conversion losses. In this study, our aim was to characterize the communities of microorganisms and the genes involved in biofilm formation. Methods and results In this study, biofilm samples collected from a PV system installed in southeastern Brazil were analyzed through shotgun metagenomics, and the microbial communities and genes involved in biofilm formation were investigated. A total of 2030 different genera were identified in the samples, many of which were classified as extremophiles or producers of exopolysaccharides. Bacteria prevailed in the samples (89%), mainly the genera Mucilaginibacter, Microbacterium, Pedobacter, Massilia , and Hymenobacter. The functional annotation revealed >12 000 genes related to biofilm formation and stress response. Genes involved in the iron transport and synthesis of c-di-GMP and c-AMP second messengers were abundant in the samples. The pathways related to these components play a crucial role in biofilm formation and could be promising targets for preventing biofilm formation in the PV. In addition, Raman spectroscopy analysis indicated the presence of hematite, goethite, and ferrite, consistent with the mineralogical composition of the regional soil and metal-resistant bacteria. Conclusions Taken together, our findings reveal that PV biofilms are a promising source of microorganisms of industrial interest and genes of central importance in regulating biofilm formation and persistence. [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]

Soybean cultivation in tropical regions relies on symbioses with nitrogen-fixing Bradyrhizobium and plant growth-promoting bacteria (PGPBs), reducing environmental impacts of N fertilizers and pesticides. We evaluate the effects of soybean inoculation with different bacterial consortia combined with PGPBs or microbial secondary metabolites (MSMs) on rhizosoil chemistry, plant physiology, plant nutrition, grain yield, and rhizosphere microbial functions under field conditions over three growing seasons with four treatments: standard inoculation of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens consortium (SI); SI plus foliar spraying with Bacillus subtilis (SI + Bs); SI plus foliar spraying with Azospirillum brasilense (SI + Az); and SI plus seed application of MSMs enriched in lipo-chitooligosaccharides extracted from B. diazoefficiens and Rhizobium tropici (SI + MSM). Rhizosphere microbial composition, diversity, and function was assessed by metagenomics. The relationships between rhizosoil chemistry, plant nutrition, grain yield, and the abundance of microbial taxa and functions were determined by generalized joint attribute modeling. The bacterial consortia had the most significant impact on rhizosphere soil fertility, which in turn affected the bacterial community, plant physiology, nutrient availability, and production. Cluster analysis identified microbial groups and functions correlated with shifts in rhizosoil chemistry and plant nutrition. Bacterial consortia positively modulated specific genera and functional pathways involved in biosynthesis of plant secondary metabolites, amino acids, lipopolysaccharides, photosynthesis, bacterial secretion systems, and sulfur metabolism. The effects of the bacterial consortia on the soybean holobiont, particularly the rhizomicrobiome and rhizosoil fertility, highlight the importance of selecting appropriate consortia for desired outcomes. These findings have implications for microbial-based agricultural practices that enhance crop productivity, quality, and sustainability. [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]

Antibiotic resistance is a worldwide problem that imposes a devastating effect on developing countries and requires immediate interventions. Initially, most of the antibiotic drugs were identified by culturing soil microbes. However, this method is prone to discovering the same antibiotics repeatedly. The present study employed a shotgun metagenomics approach to investigate the taxonomic diversity, functional potential, and biosynthetic capacity of microbiomes from two natural agricultural farmlands located in Bekeka and Welmera Choke Kebelle in Ethiopia for the first time. Analysis of the small subunit rRNA revealed bacterial domain accounting for 83.33% and 87.24% in the two selected natural farmlands. Additionally, the analysis showed the dominance of Proteobacteria representing 27.27% and 28.79% followed by Actinobacteria making up 12.73% and 13.64% of the phyla composition. Furthermore, the analysis revealed the presence of unassigned bacteria in the studied samples. The metagenome functional analysis showed 176,961 and 104, 636 number of protein-coding sequences (pCDS) from the two samples found a match with 172,655 and 102, 275 numbers of InterPro entries, respectively. The Genome ontology annotation suggests the presence of 5517 and 3293 pCDS assigned to the "biosynthesis process". Numerous Kyoto Encyclopedia of Genes and Genomes modules (KEGG modules) involved in the biosynthesis of terpenoids and polyketides were identified. Furthermore, both known and novel Biosynthetic gene clusters, responsible for the production of secondary metabolites, such as polyketide synthases, non-ribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptides (Ripp), and Terpene, were discovered. Generally, from the results it can be concluded that the microbiomes in the selected sampling sites have a hidden functional potential for the biosynthesis of secondary metabolites. Overall, this study can serve as a strong preliminary step in the long journey of bringing new antibiotics to the market. [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]

ABSTRACTThe gut-to-lung axis is critical during respiratory infections, including influenza A virus (IAV) infection. In the present study, we used high-resolution shotgun metagenomics and targeted metabolomic analysis to characterize influenza-associated changes in the composition and metabolism of the mouse gut microbiota. We observed several taxonomic-level changes on day (D)7 post-infection, including a marked reduction in the abundance of members of the Lactobacillaceae and Bifidobacteriaceae families, and an increase in the abundance of Akkermansia muciniphila. On D14, perturbation persisted in some species. Functional scale analysis of metagenomic data revealed transient changes in several metabolic pathways, particularly those leading to the production of short-chain fatty acids (SCFAs), polyamines, and tryptophan metabolites. Quantitative targeted metabolomics analysis of the serum revealed changes in specific classes of gut microbiota metabolites, including SCFAs, trimethylamine, polyamines, and indole-containing tryptophan metabolites. A marked decrease in indole-3-propionic acid (IPA) blood level was observed on D7. Changes in microbiota-associated metabolites correlated with changes in taxon abundance and disease marker levels. In particular, IPA was positively correlated with some Lactobacillaceae and Bifidobacteriaceae species (Limosilactobacillus reuteri, Lactobacillus animalis) and negatively correlated with Bacteroidales bacterium M7, viral load, and inflammation markers. IPA supplementation in diseased animals reduced viral load and lowered local (lung) and systemic inflammation. Treatment of mice with antibiotics targeting IPA-producing bacteria before infection enhanced viral load and lung inflammation, an effect inhibited by IPA supplementation. The results of this integrated metagenomic-metabolomic analysis highlighted IPA as an important contributor to influenza outcomes and a potential biomarker of disease severity.

This study examined the impact of varying salt concentrations on microbiota, physicochemical properties, and metabolites in a secondary fortified fermentation process using multi-omics techniques. It aimed to determine the influence of salt stress on microbiota shifts and metabolic activities. The findings demonstrated that moderate salt reduction (MS) was found to enhance moromi's flavor and quality, while mitigating the negative effects of excessive low salt (LS). MS samples had 1.22, 1.13, and 2.92 times more amino acid nitrogen (AAN), non-volatiles, and volatiles, respectively, than high salt (HS) samples. In contrast, lactic acid and biogenic amines in LS samples were 1.56 g/100 g and 4115.11 mg/kg, respectively, decreasing to 0.15 g/100 g and 176.76 mg/kg in MS samples. Additionally, the contents of ethanol and small peptides increased in MS due to the growth of specific functional microorganisms such as Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii, while food-borne pathogens were inhibited. Network analysis revealed that the core microbial interactions were enhanced in MS samples, promoting a balanced fermentation environment. Redundancy analysis (RDA) and correlation analyses underscored that the physicochemical properties significantly impacted bacterial community structure and the correlations between key microbes and flavor compounds. These findings provided a theoretical foundation for developing innovative reduced-salt fermentation techniques, contributing to the sustainable production of high-quality soy sauce.

Citrus (Citrus nobilis L.) is one of the main fruit crops in Dak Lak Province of Vietnam; however, a dataset on the endophytic microbiome of this plant has yet to be discovered. This article presented the endophytic microbial dataset from roots of healthy Citrus nobilis L. collected in Dak Lak for the first time. We found that 4 kingdoms, 30 phyla, 58 classes, 125 orders, 242 families, 722 genera, and 1637 species of endophytic microorganisms were identified from the sample. Actinomycetota was shown to be the main phylum (64.36 %) and biosynthesis to be the most abundant function (55.64 %) of the endophytic microbial community. Data provided insights into the composition and functional diversity of the Citrus nobilis L. endophytic microbiome, especially novel microbial resources. They could be used for the next works towards applying the endophytic microbiome for sustainable citrus production.

Abstract Background People living with HIV (PLWH) are at increased risk of acquisition of multidrug resistant organisms due to higher rates of predisposing factors. The gut microbiome is the main reservoir of the collection of antimicrobial resistance determinants known as the gut resistome. In PLWH, changes in gut microbiome have been linked to immune activation and HIV-1 associated complications. Specifically, gut dysbiosis defined by low microbial gene richness has been linked to low Nadir CD4 + T-cell counts. Additionally, sexual preference has been shown to strongly influence gut microbiome composition in PLWH resulting in different Prevotella or Bacteroides enriched enterotypes, in MSM (men-who-have–sex-with-men) or no-MSM, respectively. To date, little is known about gut resistome composition in PLWH due to the scarcity of studies using shotgun metagenomics. The present study aimed to detect associations between different microbiome features linked to HIV-1 infection and gut resistome composition. Results Using shotgun metagenomics we characterized the gut resistome composition of 129 HIV-1 infected subjects showing different HIV clinical profiles and 27 HIV-1 negative controls from a cross-sectional observational study conducted in Barcelona, Spain. Most no-MSM showed a Bacteroides-enriched enterotype and low microbial gene richness microbiomes. We did not identify differences in resistome diversity and composition according to HIV-1 infection or immune status. However, gut resistome was more diverse in MSM group, Prevotella-enriched enterotype and gut micorbiomes with high microbial gene richness compared to no-MSM group, Bacteroides-enriched enterotype and gut microbiomes with low microbial gene richness. Additionally, gut resistome beta-diversity was different according to the defined groups and we identified a set of differentially abundant antimicrobial resistance determinants based on the established categories. Conclusions Our findings reveal a significant correlation between gut resistome composition and various host variables commonly associated with gut microbiome, including microbiome enterotype, microbial gene richness, and sexual preference. These host variables have been previously linked to immune activation and lower Nadir CD4 + T-Cell counts, which are prognostic factors of HIV-related comorbidities. This study provides new insights into the relationship between antibiotic resistance and clinical characteristics of PLWH.

ObjectivesThe 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.MethodsShotgun 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.ResultsThe 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.ConclusionThe CRISPR-based human and bacterial rRNA depletion enhanced the sensitivity of SARS-CoV-2 shotgun metagenomic sequencing, especially in low viral load samples.

Inter-individual differences in the gut microbiome are linked to alterations in inflammation and blood-brain barrier permeability, which may increase the risk of depression in people with HIV (PWH). The microbiome profile of blood, which is considered by many to be typically sterile, remains largely unexplored. We aimed to characterize the blood plasma microbiome composition and assess its association with major depressive disorder (MDD) in PWH and people without HIV (PWoH). In this cross-sectional, observational cohort, we used shallow-shotgun metagenomic sequencing to characterize the plasma microbiome of 151 participants (84 PWH and 67 PWoH), all of whom underwent a comprehensive neuropsychiatric assessment. The microbial composition did not differ between PWH and PWoH or between participants with MDD and those without it. Using the songbird model, we computed the log ratio of the highest and lowest 30% of the ranked classes associated with HIV and MDD. We found that HIV infection and lifetime MDD were enriched in a set of differentially abundant inflammatory classes, such as Flavobacteria and Nitrospira. Our results suggest that the circulating plasma microbiome may increase the risk of MDD related to dysbiosis-induced inflammation in PWH. If confirmed, these findings may indicate new biological mechanisms that could be targeted to improve treatment of MDD in PWH.

Introduction: Agricultural drainage ditches are essential for maintaining flow efficiency and are often managed by brushing (i.e., vegetation clearing along ditch banks) and dredging. These maintenance practices not only impact flow but also the ditches' capacity to process and assimilate agricultural chemicals. However, the influence of ditch management on microbial activities, essential in processing and transforming these contaminants, remains underexplored. Methods: We conducted a four-year surface water sampling campaign in two long-established (over 40 years) agricultural drainage ditches situated in eastern Ontario, Canada. One drainage ditch underwent intensive bank brushing and channel dredging, while the other remained unmanaged. We assessed the impact of these contrasting ditch management practices on the diversity and compositional structure of surface water microbiome communities, with a particular focus on the distribution of genes associated with nitrogen (N) cycling, using shotgun metagenomics sequencing and an assembly-based metagenome approach. Results and discussion: From 117 surface water samples, we reconstructed 157 metagenome-assembled genomes (MAGs), predominantly from the Patescibacteria superphylum. Brushing alone, conducted about 10 months prior to dredging, had an insignificant impact on the aquatic microbial community. However, dredging led to a significant reduction in community diversity and abundance of genes affiliated with denitrification, dissimilatory nitrate reduction to ammonia, and complete nitrification through comammox. Ditch management resulted in noticeable shifts in the microbial community, evidenced by the enrichment of taxa in Polaromonas, Emticicia, Flectobacillus, and Patescibacteria in the water of the managed ditch sites. The metagenomes of these taxa harbor genes involved in various nitrogen biotransformation pathways. Interestingly, following dredging, the levels of ammonia and ammonium, nitrite, and total Kjeldahl N in the managed ditch did not increase, but significantly decreased; likely due to ditch substrate removal and potentially increased flow efficiency and dilution. Being one of the few studies conducted to date, this study provided unique insights into the consequences of drainage ditch management on freshwater microbiomes involved in N cycling. [ABSTRACT FROM AUTHOR]

Background: Respiratory viruses significantly impact global morbidity and mortality, causing more disease in humans than any other infectious agent. Beyond pathogens, various viruses and bacteria colonize the respiratory tract without causing disease, potentially influencing respiratory diseases' pathogenesis. Nevertheless, our understanding of respiratory microbiota is limited by technical constraints, predominantly focusing on bacteria and neglecting crucial populations like viruses. Despite recent efforts to improve our understanding of viral diversity in the human body, our knowledge of viral diversity associated with the human respiratory tract remains limited. Methods: Following a comprehensive search in bibliographic and sequencing data repositories using keyword terms, we retrieved shotgun metagenomic data from public repositories (n = 85). After manual curation, sequencing data files from 43 studies were analyzed using EVEREST (pipEline for Viral assEmbly and chaRactEriSaTion). Complete and high-quality contigs were further assessed for genomic and taxonomic characterization. Results: Viral contigs were obtained from 194 out of the 868 FASTQ files processed through EVEREST. Of the 1842 contigs that were quality assessed, 8% (n = 146) were classified as complete/high-quality genomes. Most of the identified viral contigs were taxonomically classified as bacteriophages, with taxonomic resolution ranging from the superkingdom level down to the species level. Captured contigs were spread across 25 putative families and varied between RNA and DNA viruses, including previously uncharacterized viral genomes. Of note, airway samples also contained virus(es) characteristic of the human gastrointestinal tract, which have not been previously described as part of the lung virome. Additionally, by performing a meta-analysis of the integrated datasets, ecological trends within viral populations linked to human disease states and their biogeographical distribution along the respiratory tract were observed. Conclusion: By leveraging publicly available repositories of shotgun metagenomic data, the present study provides new insights into viral genomes associated with specimens from the human respiratory tract across different disease spectra. Further studies are required to validate our findings and evaluate the potential impact of these viral communities on respiratory tract physiology. [ABSTRACT FROM AUTHOR]

The exploitation of exciting features of plastics for diverse applications has resulted in significant plastic waste generation, which negatively impacts environmental compartments, metabolic processes, and the well-being of aquatic ecosystems biota. A shotgun metagenomic approach was deployed to investigate the microbial consortia, degradation pathways, and enzyme systems involved in the degradation of plastics in a tropical lentic pond sediment (APS). Functional annotation of the APS proteome (ORFs) using the PlasticDB database revealed annotation of 1015 proteins of enzymes such as depolymerase, esterase, lipase, hydrolase, nitrobenzylesterase, chitinase, carboxylesterase, polyesterase, oxidoreductase, polyamidase, PETase, MHETase, laccase, alkane monooxygenase, among others involved in the depolymerization of the plastic polymers. It also revealed that polyethylene glycol (PEG), polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), polylactic acid (PLA), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), and nylon have the highest number of annotated enzymes. Further annotation using the KEGG GhostKOALA revealed that except for terephthalate, all the other degradation products of the plastic polymers depolymerization such as glyoxylate, adipate, succinate, 1,4-butanediol, ethylene glycol, lactate, and acetaldehyde were further metabolized to intermediates of the tricarboxylic acid cycle. Taxonomic characterization of the annotated proteins using the AAI Profiler and BLASTP revealed that Pseudomonadota members dominate most plastic types, followed by Actinomycetota and Acidobacteriota. The study reveals novel plastic degraders from diverse phyla hitherto not reported to be involved in plastic degradation. This suggests that plastic pollution in aquatic environments is prevalent with well-adapted degrading communities and could be the silver lining in mitigating the impacts of plastic pollution in aquatic environments. [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]

Cyanobacteria are known to produce diverse secondary metabolites that are toxic to aquatic ecosystems and human health. However, data about the cyanotoxins occurrence and cyanobacterial diversity in Pakistan's drinking water reservoirs is scarce. In this study, we first investigated the presence of microcystin, saxitoxin, and anatoxin in 12 water bodies using an enzyme-linked immunosorbent assay (ELISA). The observed cyanotoxin values for the risk quotient (RQ) determined by ELISA indicated a potential risk for aquatic life and human health. Based on this result, we made a more in-depth investigation with a subset of water bodies (served as major public water sources) to analyze the cyanotoxins dynamics and identify potential producers. We therefore quantified the distribution of 17 cyanotoxins, including 12 microcystin congeners using a high-performance liquid chromatography-high-resolution tandem mass spectrometry/mass spectrometry (HPLC-HRMS/MS). Our results revealed for the first time the co-occurrence of multiple cyanotoxins and the presence of cylindrospermopsin in an artificial reservoir (Rawal Lake) and a semi-saline lake (Kallar Kahar). We also quantified several microcystin congeners in a river (Panjnad) with MC-LR and MC-RR being the most prevalent and abundant. To identify potential cyanotoxin producers, the composition of the cyanobacterial community was characterized by shotgun metagenomics sequencing. Despite the noticeable presence of cyanotoxins, Cyanobacteria were not abundant. Synechococcus was the most abundant cyanobacterial genus found followed by a small amount of Anabaena, Cyanobium, Microcystis, and Dolichospermum. Moreover, when we looked at the cyanotoxins genes coverage, we never found a complete microcystin mcy operon. To our knowledge, this is the first snapshot sampling of water bodies in Pakistan. Our results would not only help to understand the geographical spread of cyanotoxin in Pakistan but would also help to improve cyanotoxin risk assessment strategies by screening a variety of cyanobacterial toxins and confirming that cyanotoxin quantification is not necessarily related to producer abundance. [ABSTRACT FROM AUTHOR]

After Amazonia, the Congo Basin represents the second-largest tropical rainforest area in the world. This basin harbours remarkable biodiversity, yet much of its microbiological diversity within its waters, soils, and populations remains largely unexplored and undiscovered. While many initiatives to characterize global biodiversity are being undertaken, few are conducted in Africa and none of them concern the Congo Basin specifically in urban areas. In this context, we assessed the microbial diversity present in gutter water in the city of Pointe-Noire, Congo. This town has interesting characteristics as the population density is high and it is located between the Atlantic Ocean and the forest of Mayombe in Central Africa. The findings illuminate the microbial composition of surface water in Pointe-Noire. The dataset allows the identification of putative new bacteria through the assembly of 81 meta-genome-assembled genomes. It also serves as a valuable primary resource for assessing the presence of antibiotic-resistant genes, offering a useful tool for monitoring risks by public health authorities.

Abstract Hybridization capture is an emerging method making use of short oligonucleotide baits to enrich DNA libraries for genomic fragments of specific organisms thus enabling detection of their presence in environmental samples. Although it offers a primer‐independent alternative to metabarcoding, little empirical work has been dedicated to characterizing the underlying biases and coupled implications for biological interpretation. Moreover, few published bioinformatic pipelines are available for designing polynucleotide capture baits from a reference sequence collection. We designed RNA‐baits specifically targeting two chloroplast barcoding genes matK and rbcL to reveal the plant taxonomic diversity present in a given environmental sample. Our approach leverages the sensitivity of hybridization capture and the capacity of high‐throughput DNA sequencing instruments. It builds on a new and universal method based on ancestral sequence reconstruction, ultimately limiting the number of bait‐probes required and reducing experimental costs, while accessing high levels of taxonomic diversity. Our bait‐set selectively targets four main plant orders (Fagales, Pinales, Asterales, and Poales), representing ~18% of all described vascular plants. This is achieved through the use of only 4084 baits, each 80 nucleotides in length (80‐mer), capturing ~1.0–1.6 k nucleotide sequences from each taxon. Tests on mock communities revealed important factors influencing capture efficiency and relative abundance estimates, including GC‐content, the overall target length per taxa, and the bait density and mean number of mismatches to the bait sequence. Our results show that hybridization capture, like metabarcoding, requires caution when interpreting results quantitatively within (paleo)‐ecological studies. Biases detected in this work have the potential to be mitigated with bait designs that avoid extreme base compositional biases and balancing bait targets across taxa. However, we strongly recommend the use of mock communities and read simulations to quantify the accuracy of taxonomic representation when using new bait designs.

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.

Abstract 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 The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome–phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies.

Abstract Background Infant gut microbiota is highly malleable, but the long-term longitudinal impact of antibiotic exposure in early life, together with the mode of delivery on infant gut microbiota and resistome, is not extensively studied. Methods Two hundred and eight samples from 45 infants collected from birth until 2 years of age over five time points (week 1, 4, 8, 24, year 2) were analysed. Based on shotgun metagenomics, the gut microbial composition and resistome profile were compared in the early life of infants divided into three groups: vaginal delivery/no-antibiotic in the first 4 days of life, C-section/no-antibiotic in the first 4 days of life, and C-section/antibiotic exposed in first 4 days of life. Gentamycin and benzylpenicillin were the most commonly administered antibiotics during this cohort’s first week of life. Results Newborn gut microbial composition differed in all three groups, with higher diversity and stable composition seen at 2 years of age, compared to week 1. An increase in microbial diversity from week 1 to week 4 only in the C-section/antibiotic-exposed group reflects the effect of antibiotic use in the first 4 days of life, with a gradual increase thereafter. Overall, a relative abundance of Actinobacteria and Bacteroides was significantly higher in vaginal delivery/no-antibiotic while Proteobacteria was higher in C-section/antibiotic-exposed infants. Strains from species belonging to Bifidobacterium and Bacteroidetes were generally persistent colonisers, with Bifidobacterium breve and Bifidobacterium bifidum species being the major persistent colonisers in all three groups. Bacteroides persistence was dominant in the vaginal delivery/no-antibiotic group, with species Bacteroides ovatus and Phocaeicola vulgatus found to be persistent colonisers in the no-antibiotic groups. Most strains carrying antibiotic-resistance genes belonged to phyla Proteobacteria and Firmicutes, with the C-section/antibiotic-exposed group presenting a higher frequency of antibiotic-resistance genes (ARGs). Conclusion These data show that antibiotic exposure has an immediate and persistent effect on the gut microbiome in early life. As such, the two antibiotics used in the study selected for strains (mainly Proteobacteria) which were multiple drug-resistant (MDR), presumably a reflection of their evolutionary lineage of historical exposures—leading to what can be an extensive and diverse resistome. Video Abstract

Characterization of ubiquitous microorganisms has encountered many challenges, such as determining unknown microorganisms, their interactions, and unresolved functions in natural environments. Evolution in metagenomics and tools, however, has revolutionized assessment methodologies. Metagenomics has enabled unearthing the secret microbial treasure in a culture-independent manner and has proven more successful than conventional methodologies. It has provided an unparalleled platform for functional and taxonomic characterization of microbiota dwellings in altered lakes. Of late, many research articles have used metagenomics to understand microorganisms’ role in environmental clean-up. Consequently, these studies have been consolidated in the form of review articles. However, most of them are related to microbial characterization, procedure of metagenomics, and wastewater treatment, whereas only a few were directly related to lake bioremediation. Therefore, this review highlights the applications of metagenomics for unraveling microbial potential for lake rejuvenation. A paradigm shift from conventional to recent breakthroughs in metagenomics is also emphasized. The review also discusses merits, demerits of previous molecular techniques, and potential of metagenomics for understanding the microbial world in contaminated environments. Furthermore, the review discusses recent metagenomic studies for lake rejuvenation. Finally, future research directions are proposed for designing possible strategies for lake rejuvenation. HIGHLIGHTS Metagenomics is evaluated as a novel tool for studying microbial communities’ taxonomic and functional capabilities.; The paradigm shift from conventional to recent metagenomics advancements is reviewed.; Potential of metagenomics for designing an accelerated remediation process for lakes is emphasized.;