Metagenomic Approach Research Articles

Degradation of logs installed underground for a half century and the microbial communities involved

The use of wood in civil engineering projects is attractive from the perspective of utilizing renewable materials and reducing CO2 emissions. Furthermore, civil engineering presents an appealing opportunity to expand the demand for wood. Wood degradation rates are low in anaerobic environments, such as underground conditions. In this study, the degradation of wood after 55 years in an underground environment was examined by analyzing logs that had been driven underground for a soil stabilization project. Log density was measured and wood cell walls were observed under a microscope to estimate the degree of degradation, and the bacterial and fungal communities living in the sapwood of the logs were examined using a metagenomic approach. The results showed that the wood density was hardly reduced, and that anaerobic cellulolytic bacteria were dominant in the microbial communities in the wood. The degree of wood degradation over 55 years was low, indicating that these logs retained their ground improvement function for at least half a century.

Gut Microbiome modulation in patients treated with an enriched nutraceutical composition: a multi target strategy

Introduction: The contribution of gut microbiota to health and disease is now well accepted and under continuous investigation. It is the most representative in the body (about 70% of the total) with over 400 different species of microorganisms living there. It is exclusive to each individual and therefore represents a real biological imprint, capable of distinguishing humans from one another. Gut microbiota works synergically with the host, promoting health by performing protective, metabolic and structural functions. Thus, when a modification of the personal normal gut microbiota composition occurs, a predisposition to develop different diseases may increase. Probiotics are functional food that may modulate gut microbiota stimulating protective bacterial properties and inhibiting detrimental activity. We have previously reported a beneficial effect from the administration of an enriched nutraceutical composition containing probiotics in reducing irritable bowel syndrome-related symptoms and improving metabolic profile by lowering triglycerides. However, if these effects are attributed to gut microbiota remain unknown. Objective: This analysis aims at evaluating microbiota modulation in subjects before and after Triobiotix® administration using a metagenomic approach Methods: Triobiotix® is an enriched nutraceutical composition containing maltodextrin, hyaluroic acid and mineralized seaweed extract Lithothamnion Calcareum plus a mix of five different lactic bacterial species. A total of 46 subjects with availability of sample test before and after treatment were included in the present analysis selected from 265 subjects. A fecal sample was collected before and up to 90 days after the administration of Triobiotix®. Feces were analyzed by Wellmicro® gut test that uses the next-generation sequencing of the 16S rRNA gene on Illumina MiSeq. Additionally, for 171 subjects a 90-day clinical evaluation was available. Results: Treatment with this enriched nutraceutical for up to 3 months resulted in a positive modulation of the main indices of improved gut microbiota, such as biodiversity and dysbiosis. Improvement on metabolic functions, such as glycemic and lipid metabolisms, as well as on immunological and anti-inflammatory activities have been described, thus indicating that the observed clinical benefits associated with the administration of this nutraceutical formulation might be also attributed to a gut microbiota modulation. Improvement in main IBS-related symptoms and bloating was maintained up to 90 days as well as microbiota diversity. Conclusions: Our analysis suggests that the gut microbiota of subjects taking Triobiotix® rapidly returns to a high diversity with reduced dysbiosis after treatment with associated improvement of main physiological functions linked to brain, heart and liver. Keywords: gut microbiota; microbiome, metagenomic; Nutraceuticals

Physicochemistry and comparative metagenomics of a tropical estuary persistently inundated with anthropogenic pollutants.

The physicochemistry, metabolic properties, and microbial community structure of a tropical estuary persistently inundated with anthropogenic pollutants were elucidated using diverse analytical tools and a shotgun metagenomics approach. The physicochemistry of the Awoye estuary surface water (AEW) and sediment (AES) revealed higher values in the sediment for most of the parameters analyzed, while aside from copper and zinc, the concentrations of the detected heavy metals (Cd, Cr, Pb, Fe, As, Ni, Hg, Mn, Se) in the water and sediment were higher than the acceptable thresholds. Hydrocarbon content analysis revealed increasingly high concentrations of high molecular weight polycyclic aromatic hydrocarbons (HMW PAHs) in the sediment. Structurally, the predominant taxa in the AEW metagenome are Proteobacteria (50.35%), Alphaproteobacteria (43.31%), Brevundimonas (49.96%), and Leptolyngbya boryana (14.93%), while in the sediment (AES) metagenome, Proteobacteria (53.03%), Gammaproteobacteria (28.66%), Azospirillum (6.51%), and Acidihalobacter prosperus (7.56%) were preponderant. Statistical analysis of the two microbiomes (AEW, AES) revealed significant statistical differences (P < 0.05) at all the hierarchical levels. Functional characterization of the two metagenomes revealed extensive adaptations of the sediment microbiome to various environmental stressors as evident in the high numbers of putative genes involved in the degradation of diverse classes of aromatic hydrocarbons, efflux, detoxification, and transport of heavy metals, and metabolism of organic/inorganic nutrients. Findings from this study revealed that the estuary sediment is the sink for most of the anthropogenic pollutants and harbors the more adapted microbiome that could serve as a potential bioresource for the bioremediation of the perturbed estuary.

Microbiome shifts elicited by ornamental lighting of granite facades identified by MinION sequencing

Night-time outdoor illumination in combination with natural sunlight can influence the visible phototrophic colonizers (mainly algae) growing on stone facades; however, the effects on the microbiome (invisible to the naked eye) are not clear. The presence of stone-dwelling microbes, such as bacteria, diatoms, fungi, viruses and archaea, drives further biological colonization, which may exacerbate the biodeterioration of substrates. Considering the microbiome is therefore important for conservation of the built heritage. The impact of the following types of lighting on the relative abundance and diversity of the microbiome on granite ashlars was evaluated in a year-long outdoor pilot study: no lighting; lighting with a metal halide lamp (a traditional lighting system currently used to illuminate monuments); and lighting with a novel LED lamp (an environmentally sound prototype lamp with a biostatic effect, halting biological colonization by phototrophs, currently under trial). Culturable fractions of microbiome and whole-genome sequencing by metabarcoding with Oxford Nanopore Sequencing (MinION) was conducted for bacteria and fungi in order to complement both community characterization strategies. In addition, the possible biodeteriorative profiles of the isolated strains, relative to calcium carbonate precipitation/solubilisation and iron oxidation/reduction, were investigated by plate assays. Alpha and beta diversity indexes were also determined, along with the abundance of biocide and antibiotic resistance genes. Culture-dependent microbiological analysis failed to properly show changes in community composition, for which metagenomic approaches like MinION are better suited. Thus, MinION analysis identified shifts in the granite microbiome elicited by ornamental lighting. The novel LED lamp with the biostatic effect on phototrophs caused an increase in the diversity of bacteria and fungi. In this case, the microbiome was more similar to that in the unlit samples. In the samples illuminated by the metal halide lamp, dominance of bacteria was favoured and the presence of fungi was negligible.

Physicochemical properties of acid sulphate soil profoundly influence the composition of rhizobacterial community of rice (Oryza sativa L.)

The soils of kari lands of Kuttanad, the ‘Rice bowl’ of Kerala, India are characterized as acid sulphate as they comprise of pyrite deposits. Productivity in these soils is at stake due to several constraints like high acidity and salinity, metal toxicity, nutrient unavailability, redox fluctuations, besides seasonal flooding. Sustainable management of acid sulphate soil is a critical priority to improve the rice output from these areas. Such soils would harbor unique innate microbial communities with definite abilities which could be exploited further for their sustainable amelioration. Development of inoculant technology with soil and crop specific beneficial microbial agents is expected to boost the production potential of kari soils. However, the soil parameters would impose a great influence on the rhizobacterial community development in these geologically distinct soils. We studied the rhizobacterial communities (at the family level), associated with rice grown in five acid sulphate (Purakkad, Vaikom, Ambalappuzha, Thakazhi and Kallara) as well as one non-acid sulphate (Muhamma) soil series of geographically unique Kuttanad region. We also examined the effects of soil physicochemical attributes on shaping the rhizosphere bacterial community assemblage. The soil physicochemical attributes were analyzed using standard procedures and correlations existing amongst them were also determined. A metagenomic approach was adopted to study the rhizobacterial communities (family level) and were correlated with soil parameters using canonical correspondence analysis (CCA). Compared to other acid sulphate regions, Thakazhi and Kallara soils indicated higher electrical conductivity, available nitrogen, potassium, organic carbon, aluminium as well as iron and lowest pH and available phosphorus. Intense significant relationships were exhibited amongst the acid sulphate properties and soil nutrient contents. The taxa summary after the Illumina MiSeq sequencing revealed the abundant rhizobacterial families in the soil samples as Anaerolineaceae, Ktedonobacteriaceae, Acidothermaceae, Acidimicrobiaceae, Clostridiaceae, Nocardioidaceae, Xanthobacteraceae, Methanobacteriaceae, Sphingomonadaceae and Peptostreptococcaceae. Acidothermaceae (14%) and Acidimicrobiaceae (12%) were found abundant exclusively in highly acid sulphate soil samples. Moreover, only a few shared taxa were observed between the soil samples, which denoted the uniqueness of each sample in terms of rhizobacterial communities. The shared taxa between highly acidic sampling areas include members of Acidothermaceae, Ktedonobacteraceae, Acidimicrobiaceae, Micrococcaceae, Stellaceae and Anaerolineaceae. CCA showed that pH, EC and Al content were the soil properties governing the bacterial assembly in the rhizosphere of actively tillering rice grown in acid sulphate soil followed by P and K. The data generated in the present study are considered to be of use in developing consortia of kari soil specific rice rhizosphere microbial agents which could be used as inoculants in future.

Metagenomic analysis reveals houseflies as indicators for monitoring environmental antibiotic resistance genes.

Given the threat to public health posed by antibiotic resistance transmission, environmental monitoring is essential for tracking antibiotic resistance genes (ARGs). Houseflies, being ubiquitous organisms capable of carrying and disseminating ARGs, serve as suitable indicators for environmental monitoring. In this study, we employ metagenomic approaches to investigate housefly body surface samples from five typical sites associated with human activities. The investigation reveals microbiome diversity among the samples, along with variations in the occurrence and mobility potential of ARGs. Metagenomic analysis indicates that the composition of ARGs on housefly body surfaces is influenced by environmental ARGs, which may be enriched on the housefly body surface. The resistance genes related to multidrug, β-lactam, bacitracin, and tetracycline were the predominant ARGs detected, with multidrug-related ARGs consistently exhibiting dominance. Furthermore, the abundance of ARGs in the different housefly body surface samples was found to correlate with the population density and mobility of the sampling site. Natural environments exhibited the lowest ARG abundance, while areas with higher population density and limited population mobility displayed higher ARG abundance. This study emphasizes the effectiveness of houseflies as monitors for environmental ARGs and underscores their potential for assessing and controlling antibiotic resistance risks in urban environments.

Multiomics comparative analysis of feces AMRGs of Duroc pigs and Tibetan and the effect of fecal microbiota transplantation on AMRGs upon antibiotic exposure.

To the best of our knowledge, this study represents the first comprehensive analysis of antimicrobial resistance gene (AMRGs) expression in the fecal microbiota of Tibetan and Duroc pigs, employing an integrated metagenomic and metatranscriptomic approach. Our findings indicate a higher risk of AMRGs transmission in the feces of Duroc pigs compared to Tibetan pigs. Given the escalating antimicrobial resistance crisis, novel therapeutic interventions are imperative to mitigate gut colonization by pathogens and AMRGs. In this regard, we investigated the impact of fecal microbiota from Tibetan and Duroc pig sources on AMRGs excretion in Duroc Landrace Yorkshire (DLY) piglets' feces following acute antibiotic exposure. Remarkably, only fecal microbiota sourced from Tibetan pigs exhibited a reduction in AMRGs excretion in DLY piglets' feces. This underscores the significance of evaluating the presence of AMRGs within donor fecal microbiota for effective AMRGs decolonization strategies.

Life on the edge: Microbial diversity, resistome, and virulome in soils from the union glacier cold desert

The high-latitude regions of Antarctica remain among the most remote, extreme, and least explored areas on Earth. Still, microbial life has been reported in these environments, with limited information on their genetic properties and functional capabilities. Although diverse autochthonous multidrug-resistant bacteria were found in Antarctic Peninsula soils, posing whether these soils could act as a source of resistance determinants that could emerge among pathogens, we still lack information regarding the resistome of areas closer to the South Pole. Moreover, no previous studies have evaluated the pathogenic potential of microbes inhabiting Antarctic soils. In this work, we combined metagenomic and culture-dependent approaches to investigate the microbial diversity, resistome, virulome, and mobile genetic elements (MGEs) in soils from Union Glacier, a cold desert in West Antarctica. Despite the extreme conditions, several bacterial phyla were found, predominating Actinomycetota and Pseudomonadota, with limited archaeal and fungal taxa. Contrastive with Ecology Glacier soils from King George Island, the Union Glacier soil bacterial community is significantly less diverse, mainly attributed to scarce moisture. We recovered >80 species-level representative genomes (SRGs) of predominant bacteria and an ammonia-oxidating nitrogen- and carbon-fixing archaeon from a novel species of Nitrosocosmicus. Several resistance and virulence genes were found in Union Glacier soils, similar to those in other Antarctic cold desert areas but significantly distinct from those observed in maritime Antarctica and other non-cryosphere biomes. Furthermore, we characterized bacterial isolates resistant to up to 24 clinical antibiotics, mainly Pseudomonas, Arthrobacter, Plantibacter, and Flavobacterium. Moreover, some isolates produced putative virulence factors, including siderophores, pyocyanins, and exoenzymes with hemolytic, lecithinase, protease, and DNAse activity. This evidence uncovers a largely unexplored resistome and virulome hosted by deep Antarctica's soil microbial communities and the presence of bacteria with pathogenic potential, highlighting the relevance of One Health approaches for environmental surveillance in this continent.

Effect of inoculated sludge concentration on start-up of anammox reactor: Nitrogen removal performance and metabolic pathways

The anammox process is efficient for nitrogen removal but faces challenges due to slow bacterial growth and limited inoculated sludge supply. This study examined the effects of different inoculated sludge concentrations (3.5, 7, and 14 g/L) on start-up and nitrogen metabolism in anammox reactors. Three identical reactors were operated under controlled conditions, with comprehensive analysis of nitrogen removal efficiency, sludge characteristics, and microbial community dynamics through metagenomic and transcriptomic approaches. Results demonstrated that higher inoculated sludge concentrations accelerated reactor start-up, with the 14 g/L reactor achieving stable operation in 13 days compared to 44 days for the 3.5 g/L reactor. However, the improvement in nitrogen removal rate showed a boundary effect, not proportional to the increase in sludge concentration. Notably, reactors with higher inoculated sludge concentrations exhibited lower sludge loads but higher sludge yield coefficients. Metagenomic analysis revealed Candidatus Kuenenia as the dominant anammox bacteria, with decreasing hydrazine dehydrogenase (hdh) gene expression levels observed at higher sludge concentrations, suggesting hydrazine synthesis as a potential rate-limiting step. This study provides novel insights into the optimal range of inoculated sludge concentration for anammox reactor start-up and elucidates the underlying metabolic mechanisms, offering valuable guidance for practical engineering applications.

Metagenomic insights into microbial community, functional annotation, and antibiotic resistance genes in Himalayan Brahmaputra River sediment, India.

The Brahmaputra, a major transboundary river of the Himalayas flowing predominantly through Northeast India, particularly Assam, is increasingly endangered by contamination due to rapid urbanization and anthropogenic pressures. These environmental changes pose significant risks at the microbial level, affecting nutrient cycling and productivity, and thereby impacting river ecosystem health. The next-generation sequencing technology using a metagenomics approach has revolutionized our understanding of the microbiome and its critical role in various aquatic environments. The present study aimed to investigate the structure of the bacterial community and its functional potentials within the sediments of the Brahmaputra River, India, using high-throughput shotgun metagenomics. Additionally, this study sought to explore the presence of antimicrobial resistance genes in the river's sediment. Shotgun metagenomics revealed a diverse bacterial community comprising 31 phyla, 52 classes, 291 families, 1,016 genera, and 3,630 species. Dominant phyla included Pseudomonadota (62.47-83.48%), Actinobacteria (11.10-24.89%), Bacteroidetes (0.97-3.82%), Firmicutes (0.54-3.94%), Cyanobacteria (0.14-1.70%), and Planctomycetes (0.30-0.78%). Functional profiling highlighted significant involvement in energy metabolism, amino acid and central carbon metabolism, stress response, and degradation pathways, emphasizing the microbial community's role in ecosystem functioning and resilience. Notably, 50 types of antibiotic resistance genes (ARGs) were detected, with resistance profiles spanning multidrug, aminoglycoside, β-lactam, fluoroquinolone, rifampicin, sulfonamide, and tetracycline classes. Network analysis underscored the intricate relationships among ARG subtypes, suggesting potential mechanisms of resistance propagation. Furthermore, plasmid-related genes and 185 virulence factor genes (VFGs) were identified, indicating additional layers of microbial adaptation and potential pathogenicity within the river sediments. This comprehensive microbial and functional profiling of the Brahmaputra's sediment metagenome provides crucial insights into microbial diversity, resistance potential, and ecological functions, offering a foundation for informed management and mitigation strategies to preserve river health and mitigate pollution impacts.

Bacterial Diversity, Metabolic Profiling, and Application Potential of Antarctic Soil Metagenomes.

Antarctica has attracted increasing interest in understanding its microbial communities, metabolic potential, and as a source of microbial hydrolytic enzymes with industrial applications, for which advances in next-generation sequencing technologies have greatly facilitated the study of unculturable microorganisms. In this work, soils from seven sub-Antarctic islands and Union Glacier were studied using a whole-genome shotgun metagenomic approach. The main findings were that the microbial community at all sites was predominantly composed of the bacterial phyla Actinobacteria and Cyanobacteria, and the families Streptomycetaceae and Pseudonocardiaceae. Regarding the xenobiotic biodegradation and metabolism pathway, genes associated with benzoate, chloroalkane, chloroalkene, and styrene degradation were predominant. In addition, putative genes encoding industrial enzymes with predicted structural properties associated with improved activity at low temperatures were found, with catalases and malto-oligosyltrehalose trehalohydrolase being the most abundant. Overall, our results show similarities between soils from different Antarctic sites with respect to more abundant bacteria and metabolic pathways, especially at higher classification levels, regardless of their geographic location. Furthermore, our results strengthen the potential of Antarctic soils as a source of industrially relevant enzymes.

Metagenomics approaches in the discovery and development of new bioactive compound of 8-demethoxy-10-deoxysteffimycin from mangrove sediments.

A metagenomic library consisting of 15,000 clones was constructed from the mangrove sediment. An antimicrobially active clone from the metagenomic library PS49 was identified by function- based screening. This paper presents the results of the biochemical characterization and metagenomic library screening of the marine-derived antibiotic, 8-demethoxy-10-deoxysteffimycin. Plasmid libraries were constructed, and clones were produced using a metagenomic approach. Out of 15,000 clones, 81 clones were screened for antimicrobial activity, and five potential clones were selected. The activity of one clone was characterized and named as PS49. The bioactive compounds from the selected clone were checked for antimicrobial, antioxidant, and anticancer activities. The clone PS49 was tested against various pathogens including bacteria and fungi and it showed inhibitory effects against all the tested pathogens. The antimicrobially active fractions were then crystallized and subjected to spectroscopic analysis such as FTIR, NMR and LC-MS analysis. The substance from clone PS49 has finally been recognized, and the compound from clone PS49 has been identified as 8-demethoxy-10-deoxysteffimycin. The substances isolated from the PS49 clone exhibited strong anticancer activity against skin cancer-cell lines SK-MEL2. The compounds showed a reduction in cell viability with an increase in the compound concentration. The compounds obtained from clone PS49 showed an IC50 value of 85µg/ml.

Rhodobacteraceae are Prevalent and Ecologically Crucial Bacterial Members in Marine Biofloc Aquaculture.

Bioflocs are microbial aggregates primarily composed of heterotrophic bacteria that play essential ecological roles in maintaining animal health, gut microbiota, and water quality in biofloc aquaculture systems. Despite the global adoption of biofloc aquaculture for shrimp and fish cultivation, our understanding of biofloc microbiota-particularly the dominant bacterial members and their ecological functions-remains limited. In this study, we employed integrated metataxonomic and metagenomic approaches to demonstrate that the family Rhodobacteraceae of Alphaproteobacteria consistently dominates the biofloc microbiota and plays essential ecological roles. We first analyzed a comprehensive metataxonomic dataset consisting of 200 16S rRNA gene amplicons collected across three Asian countries: South Korea, China, and Vietnam. Taxonomic investigation identified Rhodobacteraceae as the dominant and consistent bacterial members across the datasets. The predominance of this taxon was further validated through metagenomics approaches, including read taxonomy and read recruitment analyses. To explore the ecological roles of Rhodobacteraceae, we applied genome-centric metagenomics, reconstructing 45 metagenome-assembled genomes. Functional annotation of these genomes revealed that dominant Rhodobacteraceae genera, such as Marivita, Ruegeria, Dinoroseobacter, and Aliiroseovarius, are involved in vital ecological processes, including complex carbohydrate degradation, aerobic denitrification, assimilatory nitrate reduction, ammonium assimilation, and sulfur oxidation. Overall, our study reveals that the common practice of carbohydrate addition in biofloc aquaculture systems fosters the growth of specific heterotrophic bacterial communities, particularly Rhodobacteraceae. These bacteria contribute to maintaining water quality by removing toxic nitrogen and sulfur compounds and enhance animal health by colonizing gut microbiota and exerting probiotic effects.

Metagenomic inference of microbial community composition and function in the weathering crust aquifer of a temperate glacier

Bacterial, fungal, and algal communities that colonize aquatic systems on glacial ice surfaces mediate biogeochemical reactions that alter meltwater composition and affect meltwater production and storage. In this study, we sought to improve understanding of microbial communities inhabiting the shallow aquifer that forms seasonally within the ice surface of a glacier’s ablation zone (i.e., the weathering crust aquifer). Using a metagenomic approach, we compared gene contents of microbial assemblages in the weathering crust aquifer (WCA) of the Matanuska Glacier (Alaska, USA) to those recovered from supraglacial features and englacial ice. High abundances of Pseudomonadota, Cyanobacteriota, Actinomycetota, and Bacteroidota were observed across all samples, while taxa in class Gammaproteobacteria were found at significantly higher abundances in the weathering crust aquifer. The weathering crust aquifer samples also contained higher abundances of Dothideomycetes and Microbotryomyetes; fungal classes commonly observed in snow and other icy ecosystems. Phylogenetic analysis of 18S rRNA and rbcL gene sequences indicated high abundances of algae in the WCA that are closely related (&amp;gt; 98% and &amp;gt; 93% identity, respectively) to taxa of Ancylonema (Streptophyta) and Ochromonas (Ochrophyta) reported from glacial ice surfaces in Svalbard and Antarctic sea ice. Many functional gene categories (e.g., homeostasis, cellular regulation, and stress responses) were enriched in samples from the weathering crust aquifer compared to those from proximal englacial and supraglacial habitats, providing evidence for ecological specialization in the communities. The identification of phagotrophic phytoflagellate taxa and genes involved in mixotrophy implies that combined phototrophic and heterotrophic production may assist with persistence in the low light, low energy, and ephemeral conditions of the weathering crust environment. The compositional and functional differences we have documented indicate distinct microbial distributions and functional processes occur in the weathering crust aquifer environment, and we discuss how deciphering these nuances is essential for developing a more complete understanding of ecosystem biogeochemistry in supraglacial hydrological systems.

Metagenomic insights into enhancing protein content and digestibility in Jack Bean (Canavalia ensiformis) tempeh: Unraveling microbial dynamics during fermentation

Jack beans (Canavalia ensiformis) are a locally abundant Indonesian bean variety boasting a significant protein content that remains underutilized. The production of tempeh from Jack beans presents a promising avenue for tapping into this valuable vegetable protein source. The tempeh-making process involves several key stages, including soaking the ingredients, inoculating with a mold starter, and fermenting the mixture. Throughout each stage, protein content preservation and effective utilization are paramount. This study uses high-throughput sequencing technologies to characterize protein hydrolysis in Jack beans during fermentation. The research aims to investigate how microorganisms participating in the fermentation of Jack bean tempeh influence protein content and protein digestibility. The findings reveal that prolonging the fermentation time of Jack beans, utilizing a 1.5 % tempeh mold starter, and fermenting for 36 h resulted in a notable increase in protein content by 66.63 % and enhanced protein digestibility in vitro by 48.82 %. This improvement may be attributed to the predominant activities of Bacillus and Rhizopus during the fermentation process by the metagenomics approach.

Simple, reference-independent assessment to empirically guide correction and polishing of hybrid microbial community metagenomic assembly.

Hybrid metagenomic assembly of microbial communities, leveraging both long- and short-read sequencing technologies, is becoming an increasingly accessible approach, yet its widespread application faces several challenges. High-quality references may not be available for assembly accuracy comparisons common for benchmarking, and certain aspects of hybrid assembly may benefit from dataset-dependent, empiric guidance rather than the application of a uniform approach. In this study, several simple, reference-free characteristics-particularly coding gene content and read recruitment profiles-were hypothesized to be reliable indicators of assembly quality improvement during iterative error-fixing processes. These characteristics were compared to reference-dependent genome- and gene-centric analyses common for microbial community metagenomic studies. Two laboratory-scale bioreactors were sequenced with short- and long-read platforms, and assembled with commonly used software packages. Following long read assembly, long read correction and short read polishing were iterated up to ten times to resolve errors. These iterative processes were shown to have a substantial effect on gene- and genome-centric community compositions. Simple, reference-free assembly characteristics, specifically changes in gene fragmentation and short read recruitment, were robustly correlated with advanced analyses common in published comparative studies, and therefore are suitable proxies for hybrid metagenome assembly quality to simplify the identification of the optimal number of correction and polishing iterations. As hybrid metagenomic sequencing approaches will likely remain relevant due to the low added cost of short-read sequencing for differential coverage binning or the ability to access lower abundance community members, it is imperative that users are equipped to estimate assembly quality prior to downstream analyses.

An INDEL genomic approach to explore population diversity of phytoplankton

BackgroundAlthough metabarcoding and metagenomic approaches have generated large datasets on worldwide phytoplankton species diversity, the intraspecific genetic diversity underlying the genetic adaptation of marine phytoplankton to specific environmental niches remains largely unexplored. This is mainly due to the lack of biological resources and tools for monitoring the dynamics of this diversity in space and time.ResultsTo gain insight into population diversity, a novel method based on INDEL markers was developed on Bathycoccus prasinos (Mamiellophyceae), an abundant and cosmopolitan species with strong seasonal patterns. Long read sequencing was first used to characterize structural variants among the genomes of six B. prasinos strains sampled from geographically distinct regions in the world ocean. Markers derived from identified insertions/deletions were validated by PCR then used to genotype 55 B. prasinos strains isolated during the winter bloom 2018–2019 in the bay of Banyuls-sur-Mer (Mediterranean Sea, France). This led to their classification into eight multi-loci genotypes and the sequencing of strains representative of local diversity, further improving the available genetic diversity of B. prasinos. Finally, selected markers were directly tracked on environmental DNA sampled during 3 successive blooms from 2018 to 2021, showcasing a fast and cost-effective approach to follow local population dynamics.ConclusionsThis method, which involves (i) pre-identifying the genetic diversity of B. prasinos in environmental samples by PCR, (ii) isolating cells from selected environmental samples and (iii) identifying genotypes representative of B. prasinos diversity for sequencing, can be used to comprehensively describe the diversity and population dynamics not only in B. prasinos but also potentially in other generalist phytoplankton species.

Metagenomic insight into the enrichment of antibiotic resistance genes in activated sludge upon exposure to nanoplastics

Activated sludge is an important reservoir for the co-occurring emerging contaminants including nanoplastics (NPs) and antibiotic resistance genes (ARGs). However, the impacts and potential mechanisms of NPs on the fate of ARGs in activated sludge are not fully understood. Herein, we used metagenomic approach to investigate the responses of ARGs, host bacteria, mobile genetic elements (MGEs), and functional genes to polystyrene (PS) NPs at environmentally relevant (0.5 mg/L) and high stress concentrations (50 mg/L) in activated sludge. The results showed that 0.5 and 50 mg/L PS NPs increased the relative abundance of ARGs in the activated sludge by 58.68% and 46.52%, respectively (p < 0.05). Host tracking analysis elucidated that the hosts of ARGs were significantly enriched by PS NPs (p < 0.05), with Proteobacteria being the predominant host bacteria. Additionally, the occurrence of new ARGs hosts and the enrichment of MGEs and functional genes (i.e., genes related to SOS response, cell membrane permeability, and secretion system, etc.) indicated that PS NPs promoted horizontal gene transfer (HGT) of ARGs. Finally, path modeling analysis revealed that the proliferation of ARGs caused by PS NPs was primarily attributed to the enhancement of HGT and the enrichment of host bacteria. Our findings contribute to a comprehensive understanding of the spread risk of ARGs in activated sludge under NPs pollution.

High utility of bronchoalveolar lavage fluid metagenomic next-generation sequencing approach for etiological diagnosis of pneumonia

BackgroundFor patients with pneumonia, the rapid detection of pathogens is still a major global problem in clinical practice because traditional diagnostic techniques for infection are time-consuming and insensitive. Metagenomic next-generation sequencing (mNGS) is a novel technique that has the potential to improve pathogen diagnosis. This study aimed to investigate the microbiological diagnostic ability of mNGS compared with conventional culture and to determine the optimal time to test patients for pneumonia.MethodsA prospective study using data from June 2020 to June 2021 was performed at a tertiary teaching hospital in China. We included 56 patients from all adult patients with a clinical diagnosis of pneumonia. Blood and bronchoalveolar lavage fluid (BALF) samples were taken for simultaneous mNGS and conventional culture testing.ResultsAll 56 patients underwent both conventional culture and mNGS. Of these patients, 37 were diagnosed with severe pneumonia and 17 were diagnosed with non-severe pneumonia. The top three pathogenic bacteria detected by mNGS were Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Enterococcus faecium was detected more frequently in the non-severe pneumonia group (4 vs. 0, p < 0.05). The findings revealed that the detection rate of mNGS (84%) was superior to that of conventional culture methods (48%). Notably, the percentage of mNGS-positive BALF samples (46/56, 82.14%) was significantly greater than that of blood samples (27/56, 48.21%). The etiological comparison demonstrated that mNGS-positive samples, which received clinical approval, tended to be associated with a more normalized temperature, lower PCO2 levels, and a higher SOFA score than mNGS-negative samples (p = 0.022, p = 0.0.028, and p = 0.038, respectively).ConclusionsIn this study, we discovered that the etiology of lung infections frequently involves multiple pathogens. The use of mNGS in BALF is instrumental for detecting nonviral pathogens associated with lung infections. Although the rate of positive blood NGS results is significantly influenced by various clinical factors, for patients suspected of having viral, Legionella, or tsutsugamushi infections, plasma mNGS could serve as a complementary diagnostic tool.

SALINITY-Induced Changes in Diversity, Stability, and Functional Profiles of Microbial Communities in Different Saline Lakes in Arid Areas

Saline lakes, characterized by high salinity and limited nutrient availability, provide an ideal environment for studying extreme halophiles and their biogeochemical processes. The present study examined prokaryotic microbial communities and their ecological functions in lentic sediments (with the salinity gradient and time series) using 16S rRNA amplicon sequencing and a metagenomic approach. Our findings revealed a negative correlation between microbial diversity and salinity. The notable predominance of Archaea in high-salinity lakes signified a considerable alteration in the composition of the microbial community. The results indicate that elevated salinity promotes homogeneous selection pressures, causing substantial alterations in microbial diversity and community structure, and simultaneously hindering interactions among microorganisms. This results in a notable decrease in the complexity of microbial ecological networks, ultimately influencing the overall ecological functional responses of microbial communities such as carbon fixation, sulfur, and nitrogen metabolism. Overall, our findings reveal salinity drives a notable predominance of Archaea, selects for species adapted to extreme conditions, and decreases microbial community complexity within saline lake ecosystems.