Diverse Environmental Samples Research Articles

BGC Atlas: a web resource for exploring the global chemical diversity encoded in bacterial genomes.

Secondary metabolites are compounds not essential for an organism's development, but provide significant ecological and physiological benefits. These compounds have applications in medicine, biotechnology and agriculture. Their production is encoded in biosynthetic gene clusters (BGCs), groups of genes collectively directing their biosynthesis. The advent of metagenomics has allowed researchers to study BGCs directly from environmental samples, identifying numerous previously unknown BGCs encoding unprecedented chemistry. Here, we present the BGC Atlas (https://bgc-atlas.cs.uni-tuebingen.de), a web resource that facilitates the exploration and analysis of BGC diversity in metagenomes. The BGC Atlas identifies and clusters BGCs from publicly available datasets, offering a centralized database and a web interface for metadata-aware exploration of BGCs and gene cluster families (GCFs). We analyzed over 35000 datasets from MGnify, identifying nearly 1.8 million BGCs, which were clustered into GCFs. The analysis showed that ribosomally synthesized and post-translationally modified peptidesare the most abundant compound class, with most GCFs exhibiting high environmental specificity. We believe that our tool will enable researchers to easily explore and analyze the BGC diversity in environmental samples, significantly enhancing our understanding of bacterial secondary metabolites,and promote the identification of ecological and evolutionary factors shaping the biosynthetic potential of microbial communities.

Long-lasting copper carbon nanotubes for non-enzymatic electrochemical sensing of glyphosate

Accurate electrochemical detection of glyphosate (GLY) presents significant challenges due to its non-electroactive nature on conventional electrode materials. To address this challenge, copper-modified electrodes have been developed to form a complex with GLY, enhancing detection capabilities. In this study, we propose an innovative material: copper-modified carbon nanotubes on a glassy carbon electrode (Cu/CNT/GCE). The detection mechanism of GLY using Cu/CNT/GCE involves the formation of a copper-GLY complex, which inhibits the electrochemical response of copper, proportionally to the GLY concentration. This effect is enhanced by the synergistic interaction between copper and carbon nanotubes, increasing the electrochemical stability and detection capacity of the system. To evaluate the electrochemical performance of Cu/CNT/GCE, cyclic voltammetry was performed at different electrolyte concentrations and pH levels. Square wave voltammetry was employed for the electrochemical quantification of GLY, showing a linear correlation between GLY concentration and the inhibition of the copper response. The proposed sensor exhibited a low limit of detection (0.098 ppm) and a limit of quantification (0.326 ppm). Furthermore, the electrode demonstrated long-term stability, retaining 95 % of its signal after one year of storage. This stability is attributed to the carbon nanotube support, which prevents corrosion of copper particles. Recovery values ranged from 94 to 106 % for Citromax™ and Orium™ glyphosate with precision. The method showed excellent selectivity for GLY detection, even in the presence of other herbicides such as diuron and oryzalin. These properties suggest that Cu/CNT/GCE presents promising features for the electrochemical monitoring of GLY in diverse environmental samples.

Interference-resistant gold nanoparticle assay for detecting Enterococcus in fresh and marine waters

Enterococci are common indicators of fecal contamination and are used to assess the quality of fresh and marine water, sand, soil, and sediment. However, samples collected from these environments contain various cells and other factors that can interfere with the assays used to detect enterococci. We developed a novel assay for the sensitive and specific detection of enterococci that is resistant to interference from other cells and environmental factors. Our interference-resistant assay used 30-nm gold nanoparticles (AuNPs), streptavidin, and a biotinylated Enterococcus antibody. Enterococci inhibited the interaction between streptavidin and biotin and led to the disaggregation of AuNPs. The absence of enterococci led to the aggregation of AuNPs, and this difference was easily detected by spectrophotometry. This interference-resistant AuNP assay was able to detect whole cells of Enterococcus in the range of 10 to 107 CFU/mL within 3 h, had high specificity for enterococci, and was unaffected by the presence of other intestinal bacteria, such as Escherichia coli. Our examination of fresh and marine water samples demonstrated no interference from other cells or environmental factors. The interference-resistant AuNP assay described here has the potential to be used as a rapid, simple, and effective method for monitoring enterococci in diverse environmental samples.

(Sensor Division Early Career Award) Portable Diagnostics for Cancer Management at Printed Strips

Point-of-care testing (PoC) is revolutionizing the healthcare sector improving patient care in daily hospital practice and allowing reaching even remote areas. In the frame of cancer management, the design and validation of PoC enabling the non-invasive, rapid detection of cancer markers is urgently required to implement liquid biopsy. However, despite substantial advances in sensing technologies, the development, preparation, and use of self-testing devices is still confined to specialist laboratories and users. Decentralized analytical devices will enormously impact daily lives, enabling people to analyze diverse clinical, environmental, and food samples, evaluate them and make predictions to improve quality of life, particularly in remote, resource-scarce areas. In recent years, paper-based analytical tools have attracted a great deal of attention; the well-known properties of paper, such as abundance, affordability, lightness, and biodegradability, combined with features of printed electrochemical sensors, have enabled the development of sustainable devices that drive (bio)sensors beyond the state of the art. Their blindness toward colored/turbid matrices (i.e., blood, soil), their portability, and the capacity of paper to autonomously filter/purge/react with target species make such devices powerful in establishing point-of-need tools for use by non-specialists. Depending on analytical requisites, different types of paper (filter, office) and configurations (1D, 2D, 3D) can be adopted. Focusing on stable blood-based markers with high-specificity, such as miRNAs, is of crucial importance. A wide overview regarding application ranging from DNA to miRNA detection will be provided, with the aim in showing the potentialities of portable electrochemical biosensors for improving society involvement in monitoring circulating nucleic acids associated to diverse conditions, i.e. cancer, Alzheimer’s disease, HIV.

Tetraphenylethylene tethered 1-(pyridine-2-yl)imidazo[1,5-a]pyridine: Synthesis, aggregation induced emission, copper(II) ion detection and imaging of latent fingerprint

The development of aggregation induced emissive probe with high sensitivity and selectivity for Cu2+ ions is a fascinating research in biomedical sciences. In this context, we have successfully synthesized an aggregation induced emissive probe for the selective detection of Cu2+ ions by incorporating a 1-(pyridine-2-yl)imidazo[1,5-a]pyridine arm in the tetraphenylethylene framework. The aggregates have been characterized using pivotal microscopic and photophysical measurements. The results exhibits that the aggregates have a spherical shape with an average size of 90 nm and a fluorescence quantum yield of ∼23 %. The aggregated fluorescent probe exhibited an excellent selectivity towards Cu2+ ions with a detection limit of 83 nM and presented a good efficacy over diverse environmental water samples. Furthermore, the quenching type is delineated as static due to complexation followed by deaggregation which was supported by dynamic light scattering and Tyndall effect experiments. In addition, the high solid state emissive property of the probe makes it a potential candidate for latent fingerprint analysis to gather level 2 and 3 information thereby playing a substantial role in forensic investigations.

Enzyme-based sensor for the real-time detection of atrazine: Evidence from electrochemical and docking studies

This study focused on strategically employing the carboxylesterase enzyme Ha006a, derived from the pesticide-resistant microorganism Helicoverpa armigera, to detect atrazine. A comprehensive analysis through biochemical, biophysical and bioinformatics approaches was conducted to determine the interaction between the Ha006a protein and the herbicide atrazine. These experimental findings elucidated the potential of leveraging the inherent pesticide sequestration mechanism of the Ha006a enzyme for sensor fabrication. Numerous optimizations were undertaken to ensure the precision, reproducibility and convenient storage of the resulting electrochemical sensor, Ha006a/MCPE. This biosensor exhibited exceptional performance in detecting atrazine, demonstrating outstanding selectivity with a lower limit of detection of 5.4 µM. The developed biosensor has emerged as a reliable and cost-effective green tool for the detection of atrazine from diverse environmental samples. The Ha006a-based biosensor fabrication has expanded the possibilities for the efficient integration of insect enzymes as analytical tools, paving the way for the design of cost-effective biosensors capable of detecting and quantifying pesticides.

Revolutionizing medicine: Molecularly imprinted polymers as precision tools in cancer diagnosis and antibiotic detection.

Molecularly imprinted polymers (MIPs) are pivotal in medicine, mimicking biological receptors with enhanced specificity and affinity. Comprising templates, functional monomers, and cross-linkers, MIPs form stable three-dimensional polymer networks. Synthetic templates like glycan and aptamers improve efficiency, guiding the molecular imprinting process. Cross-linking determines MIPs' morphology and mechanical stability, with printable hydrogels offering biocompatibility and customizable properties, mimicking native extracellular matrix (ECM) microenvironments. Their versatility finds applications in tissue engineering, soft robotics, regenerative medicine, and wastewater treatment. In cancer research, MIPs excel in both detection and therapy. MIP-based detection systems exhibit superior sensitivity and selectivity for cancer biomarkers. They target nucleic acids, proteins, and exosomes, providing stability, sensitivity, and adaptability. In therapy, MIPs offer solutions to challenges like multidrug resistance, excelling in drug delivery, photodynamic therapy, photothermal therapy, and biological activity regulation. In microbiology, MIPs serve as adsorbents in solid-phase extraction (SPE), efficiently separating and enriching antibiotics during sample preparation. They contribute to bacterial identification, selectively capturing specific strains or species. MIPs aid in detecting antibiotic residues using fluorescent nanostructures and developing sensors for sulfadiazine detection in food samples. In summary, MIPs play a pivotal role in advancing medical technologies with enhanced sensitivity, selectivity, and versatility. Applications range from biomarker detection to innovative cancer therapies, making MIPs indispensable for the accurate determination and monitoring of diverse biological and environmental samples.

NIRis: A low-cost, versatile imaging system for near-infrared fluorescence detection of phototrophic cell colonies used in research and education.

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350€ in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device.

Molecular Insights into Bile Salt Hydrolase-Positive Lactobacilli: A PCR-Based Identification Approach

Bile salt hydrolase (BSH)-positive Lactobacilli play a crucial role in gut health and have been associated with various physiological benefits. This study presents a comprehensive molecular investigation into the identification of BSH-positive Lactobacilli using a polymerase chain reaction (PCR)-based approach. The methodology involves the targeted amplification of specific genetic markers associated with BSH activity, enabling the precise discrimination of these beneficial bacteria within complex microbial communities. The study encompasses the design and validation of PCR primers specific to BSH-encoding genes, ensuring the accurate detection of BSH-positive Lactobacilli strains. Utilizing this molecular tool, we conducted extensive screenings across diverse environmental samples, revealing the prevalence and diversity of BSH positive strains in various ecosystems.

Effects of different assembly strategies on gene annotation in activated sludge

Activated sludge comprises diverse bacteria, fungi, and other microorganisms, featuring a rich repertoire of genes involved in antibiotic resistance, pollutant degradation, and elemental cycling. In this regard, hybrid assembly technology can revolutionize metagenomics by detecting greater gene diversity in environmental samples. Nonetheless, the optimal utilization and comparability of genomic information between hybrid assembly and short- or long-read technology remain unclear. To address this gap, we compared the performance of the hybrid assembly, short- and long-read technologies, abundance and diversity of annotated genes, and taxonomic diversity by analysing 46, 161, and 45 activated sludge metagenomic datasets, respectively. The results revealed that hybrid assembly technology exhibited the best performance, generating the most contiguous and longest contigs but with a lower proportion of high-quality metagenome-assembled genomes than short-read technology. Compared with short- or long-read technologies, hybrid assembly technology can detect a greater diversity of microbiota and antibiotic resistance genes, as well as a wider range of potential hosts. However, this approach may yield lower gene abundance and pathogen detection. Our study revealed the specific advantages and disadvantages of hybrid assembly and short- and long-read applications in wastewater treatment plants, and our approach could serve as a blueprint to be extended to terrestrial environments.

Using DNA metabarcoding to characterize national scale diatom-environment relationships and to develop indicators in streams and rivers of the United States

Recent advancements in DNA techniques, metabarcoding, and bioinformatics could help expand the use of benthic diatoms in monitoring and assessment programs by providing relatively quick and increasingly cost-effective ways to quantify diatom diversity in environmental samples. However, such applications of DNA-based approaches are relatively new, and in the United States, unknowns regarding their applications at large scales exist because only a few small-scale studies have been done. Here, we present results from the first nationwide survey to use DNA metabarcoding (rbcL) of benthic diatoms, which were collected from 1788 streams and rivers across nine ecoregions spanning the conterminous USA. At the national scale, we found that diatom assemblage structure (1) was strongly associated with total phosphorus and total nitrogen concentrations, conductivity, and pH and (2) had clear patterns that corresponded with differences in these variables among the nine ecoregions. These four variables were strong predictors of diatom assemblage structure in ecoregion-specific analyses, but our results also showed that diatom-environment relationships, the importance of environmental variables, and the ranges of these variables within which assemblage changes occurred differed among ecoregions. To further examine how assemblage data could be used for biomonitoring purposes, we used indicator species analysis to identify ecoregion-specific taxa that decreased or increased along each environmental gradient, and we used their relative abundances of gene reads in samples as metrics. These metrics were strongly correlated with their corresponding variable of interest (e.g., low phosphorus diatoms with total phosphorus concentrations), and generalized additive models showed how their relationships compared among ecoregions. These large-scale national patterns and nine sets of ecoregional results demonstrated that diatom DNA metabarcoding is a robust approach that could be useful to monitoring and assessment programs spanning the variety of conditions that exist throughout the conterminous United States.

A hierarchical cerium metal organic framework-graphene oxide heterostructure composite for rapid and efficient scavenging of fluoride

The rational design of functional heretrostructures with integrated advantages for the efficient adsorption and removal of fluoride is important for water pollution control. In this study, a cerium metal organic framework-graphene oxide heterostructure composite (CeMOF@GO) is successfully prepared through ultrasonic irradiation and vapor diffusion-assisted interfacial growth. CeMOF@GO with hierarchical structure boosts the mass transport of fluoride with adsorption time reduced to 25 min. By virtue of the specific ligand exchange between adsorbed H2O/OH− at metal defect sites and fluoride ions, such unique CeMOF@GO can be utilized for fluoride removal, showing satisfactory adsorption capacity (151.5 mg g−1), excellent specific affinity (with a distribution coefficient of up to 21.865 L g−1), glorious salt tolerance (8.0 mol L−1 of nitrate), wide pH applicability and eminent recycling ability. The practical application of CeMOF@GO is well demonstrated by the selective scavenging of fluoride from diverse environmental water samples. This work is expected to provide a reliable defluorination approach, and bridge the gap between academic MOF-based adsorbent research and practical environmental application.

Examining Antimicrobial Resistance in Escherichia coli: A Case Study in Central Virginia's Environment.

While environmental factors may contribute to antimicrobial resistance (AMR) in bacteria, many aspects of environmental antibiotic pollution and resistance remain unknown. Furthermore, the level of AMR in Escherichia coli is considered a reliable indicator of the selection pressure exerted by antimicrobial use in the environment. This study aimed to assess AMR variance in E. coli isolated from diverse environmental samples, such as animal feces and water from wastewater treatment plants (WWTPs) and drainage areas of different land use systems in Central Virginia. In total, 450 E. coli isolates obtained between August 2020 and February 2021 were subjected to susceptibility testing against 12 antimicrobial agents approved for clinical use by the U.S. Food and Drug Administration. Approximately 87.8% of the tested isolates were resistant to at least one antimicrobial agent, with 3.1% showing multi-drug resistance. Streptomycin resistance was the most common (73.1%), while susceptibility to chloramphenicol was the highest (97.6%). One isolate obtained from WWTPs exhibited resistance to seven antimicrobials. AMR prevalence was the highest in WWTP isolates, followed by isolates from drainage areas, wild avians, and livestock. Among livestock, horses had the highest AMR prevalence, while cattle had the lowest. No significant AMR difference was found across land use systems. This study identifies potential AMR hotspots, emphasizing the environmental risk for antimicrobial resistant E. coli. The findings will aid policymakers and researchers, highlighting knowledge gaps in AMR-environment links. This nationally relevant research offers a scalable AMR model for understanding E. coli ecology. Further large-scale research is crucial to confirm the environmental impacts on AMR prevalence in bacteria.

Compound Similarity Network as a Novel Data Mining Strategy for High-Throughput Investigation of Degradation Pathways of Organic Pollutants in Industrial Wastewater Treatment.

Identification of degradation products and pathways is crucial for investigating emerging pollutants and evaluation of wastewater treatment methods. Nontargeted analysis is a powerful tool to comprehensively investigate the degradation pathways of organic pollutants in real-world wastewater samples but often generates large data sets, making it difficult to effectively locate the exact information on interests. Herein, to efficiently establish the linkages among compounds in the same degradation pathways, we introduce a compound similarity network (CSN) as a novel data mining strategy for LC-MS-based nontargeted analysis of complex wastewater samples. Different from molecular networks that cluster compounds based on MS/MS spectra similarity, our CSN strategy harnesses molecular fingerprints to establish linkages among compounds and thus is spectra-independent. The effectiveness of CSN was demonstrated by nontargeted identification of degradation pathways and products of organic pollutants in leather industrial wastewater that underwent laboratory-scale activated carbon adsorption (ACD) and ozonation treatments. Utilizing CSN in interpreting nontargeted data, we tentatively annotated 4324 compounds in the untreated leather industrial wastewater, 3246 after ACD, and 3777 after ACD/ozonation. We located 145 potential degradation pathways of organic pollutants in the ACD/ozonation process using CSN and validated 7 pathways with 15 chemical standards. CSN also revealed 5 clusters of emerging pollutants, from which 3 compounds were selected for in vitro cytotoxicity study to evaluate their potential biohazards as new pollutants. As CSN offers an efficient way to connect massive compounds and to find multiple degradation pathways in a high-throughput manner, we anticipate that it will find wide applications in nontargeted analysis of diverse environmental samples.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Glyphosate detection via a nanomaterial-enhanced electrochemical molecularly imprinted polymer sensor

Glyphosate (GLY) is a widely used herbicide with an important role in agriculture. It effectively controls weeds, enhancing agricultural yield and product quality. However, its use raises significant concerns such as potential risks to non-target ecosystems and human health. In response to these concerns, we develop an electrochemical sensor with a molecularly imprinted polymer (MIP) and gold nanoparticles for GLY detection. The sensor includes a screen-printed carbon electrode (SPCE) functionalized with gold nanoparticles and a self-assembled polyvinyl carboxylic acid chloride (PVC-COOH) layer. GLY compounds interact with carboxylic groups and are encapsulated by a polymer of methacrylic acid (MAA) cross-linked with ethylene glycol dimethacrylate (EGDMA). Electrochemical performance was assessed using differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Morphological characterization was performed using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM). The sensor exhibits impressive selectivity, detecting GLY within a range of 273–1200 pg/mL with minimal interference from other pesticides. It boasts a low detection limit of 0.8 pg/mL (signal-to-noise ratio S/N = 3) by DPV and 0.001 pg/mL by EIS. The sensor’s versatility extends to various sample types, including surface water, agricultural wastewater, soil, and cucumber, demonstrating high recovery rates (> 96.05%) and low relative standard deviation (RSD) (< 5.7%). The developed MIP sensor is proven to be a valuable tool for rapid and highly sensitive detection of GLY in diverse environmental and agri-food samples.

Occurrence and distribution of microplastics in the adjacent environment of Yellow River Delta, China

In the precent study, the microplastics (MPs) pollution level was evaluated in diverse environmental samples from the Yellow River Delta. The results indicated that the abundance of MPs in water, sediment and soil samples ranged from 0.50 to 7.83 items·L−1, 200 to 4200 items·kg−1, and 100 to 1400 items·kg−1, respectively. Film form of MPs was dominant in water, while fiber MPs were dominant in both sediment and soil samples. In all samples, most MPs were < 1 mm in size. White was the main color in water, black was the main color in sediment and soil samples. The most common MPs type was polyethylene (33 %) in water, while rayon accounted for the majority of MPs in sediment (42 %) and soil (70 %) samples. The redundancy analysis results showed that MPs in water and sediment were more affected by water quality, while soil MPs were easily affected by landscape pattern.

Development of a Novel Internally Controlled HrpB1 Gene-Based Real-Time qPCR Assay for Detection of Burkholderia pseudomallei.

Melioidosis, caused by category B bioterrorism agent Burkholderia pseudomallei, is a seasonal disease of tropical and subtropical regions with a high mortality rate. An early and culture-independent detection ofB. pseudomalleiis required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences ofB. pseudomalleiand development of quantitative polymerase chain reaction (qPCR) assay. A novel B. pseudomallei-specific target sequence was identified by in silico analysis for the qPCR assay development. The specificity of the developed assay was assessed using purified DNA of 65 different bacterial cultures, and the sensitivity was estimated using a cloned target gene. Further, a type III secretion protein HrpB1 (HrpB1) gene-based duplex qPCR assay incorporating suitable extraction and amplification control was developed, and its viability was assessed in different clinical and environmental matrices for the detection of B. pseudomallei. In this study, an 80-nucleotide-longB. pseudomallei-specific region within the geneHrpB1was identified by computational analysis. The developedHrpB1-based qPCR assay was highly specific forB. pseudomalleidetection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with theHrpB1-recombinant plasmid was found to be five copies per qPCR reaction. The assay's detection limit was found to be 5 × 102 CFU/mL for human blood and urine, 5 × 101 CFU/mL in river water, and 2 × 103 CFU/gm in paddy field soil. The results of the study showed the applicability of a novelHrpB1-based qPCR assay for sensitive and specific detection ofB. pseudomalleiin diverse clinical and environmental samples.

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

BackgroundWhile genome-resolved metagenomics has revolutionized our understanding of microbial and genetic diversity in environmental samples, assemblies of short-reads often result in incomplete and/or highly fragmented metagenome-assembled genomes (MAGs), hampering in-depth genomics. Although Nanopore sequencing has increasingly been used in microbial metagenomics as long reads greatly improve the assembly quality of MAGs, the recommended DNA quantity usually exceeds the recoverable amount of DNA of environmental samples. Here, we evaluated lower-than-recommended DNA quantities for Nanopore library preparation by determining sequencing quality, community composition, assembly quality and recovery of MAGs.ResultsWe generated 27 Nanopore metagenomes using the commercially available ZYMO mock community and varied the amount of input DNA from 1000 ng (the recommended minimum) down to 1 ng in eight steps. The quality of the generated reads remained stable across all input levels. The read mapping accuracy, which reflects how well the reads match a known reference genome, was consistently high across all libraries. The relative abundance of the species in the metagenomes was stable down to input levels of 50 ng. High-quality MAGs (> 95% completeness, ≤ 5% contamination) could be recovered from metagenomes down to 35 ng of input material. When combined with publicly available Illumina reads for the mock community, Nanopore reads from input quantities as low as 1 ng improved the quality of hybrid assemblies.ConclusionOur results show that the recommended DNA amount for Nanopore library preparation can be substantially reduced without any adverse effects to genome recovery and still bolster hybrid assemblies when combined with short-read data. We posit that the results presented herein will enable studies to improve genome recovery from low-biomass environments, enhancing microbiome understanding.

A sensitive method to determine 210Po and 210Pb in environmental samples by alpha spectrometry using CuS micro-precipitation

A new sensitive method to determine polonium-210 (210Po) and lead-210 (210Pb) in a diversity of environmental samples was developed. For fresh and marine waters, Po was pre-concentrated using a titanium (III) hydroxide (Ti(OH)3) co-precipitation. Solid environmental samples were digested with nitric acid (HNO3) and hydrogen peroxide (H2O2). The alpha thin layer source was prepared using CuS micro-precipitation and 210Po was measured by alpha spectrometry. Lead-210 was left to decay for up to a year and indirectly measured via its progeny, 210Po. The chemical recoveries for 210Po and 210Pb were high, 90% and 97%, respectively, for a large variety of samples and a very low minimum detectable activity (MDA) was obtained. The method was validated using standardized solutions and certified reference materials.