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882100.9937Aromatics valorization to polyhydroxyalkanoate by the ligninolytic bacteria isolated from soil sample. Polyhydroxyalkanoates (PHA) are ecofriendly alternatives to conventional plastics due to their biodegradable nature. However, the high production cost limits their applications. Exploring novel bacteria with ligninolytic potential would be crucial to advance cost-effective PHA synthesis. The current study aims to unveil soil bacteria capable of aromatics valorization to PHA. Considering this, six aromatics resistance bacteria from a soil sample were isolated through culture acclimatization strategy and their growth was analyzed in various lignin model compounds. Ralstonia sp. BPSS-1 and Arthrobacter sp. BPSS-3 presented high-cell-densities in 4-hydroxybenzoic acid (4-HBA) and benzoate, respectively. Fluorescence microscopy confirmed the strains to be PHA positive and were subsequently evaluated for PHA synthesis from 4-HBA and benzoate at a concentration of 2 g L(-1) in a nitrogen-limited M9 medium. However, applying a co-feeding strategy by the integration of 4-HBA and benzoate further increased the substrates consumption efficiency, biomass and PHA titer compared to single carbon sources. The maximum dry cell weight (DCW) and PHA yield by Ralstonia sp. BPSS-1 through the substrate co-feeding under optimized fermentation conditions was 0.69 ± 0.03, and 0.4 ± 0.02 g L(-1), respectively. The draft genome analysis confirmed the genes involved in aromatic degradation. Besides, the proposed metabolic pathway was validated by studying the expression level of key genes, analyzing key intermediates and associated enzymes activities. The FTIR, (1)H NMR and GC-MS determined the PHA functional group, chemical structure and monomers analysis, respectively. Overall, the current study highlighted the aromatic valorization potential of newly isolated PHA producing bacteria for sustainable biomanufacturing.202540032105
853610.9935New insights into bioaugmented removal of sulfamethoxazole in sediment microcosms: degradation efficiency, ecological risk and microbial mechanisms. BACKGROUND: Bioaugmentation has the potential to enhance the ability of ecological technology to treat sulfonamide-containing wastewater, but the low viability of the exogenous degraders limits their practical application. Understanding the mechanism is important to enhance and optimize performance of the bioaugmentation, which requires a multifaceted analysis of the microbial communities. Here, DNA-stable isotope probing (DNA-SIP) and metagenomic analysis were conducted to decipher the bioaugmentation mechanisms in stabilization pond sediment microcosms inoculated with sulfamethoxazole (SMX)-degrading bacteria (Pseudomonas sp. M2 or Paenarthrobacter sp. R1). RESULTS: The bioaugmentation with both strains M2 and R1, especially strain R1, significantly improved the biodegradation rate of SMX, and its biodegradation capacity was sustainable within a certain cycle (subjected to three repeated SMX additions). The removal strategy using exogenous degrading bacteria also significantly abated the accumulation and transmission risk of antibiotic resistance genes (ARGs). Strain M2 inoculation significantly lowered bacterial diversity and altered the sediment bacterial community, while strain R1 inoculation had a slight effect on the bacterial community and was closely associated with indigenous microorganisms. Paenarthrobacter was identified as the primary SMX-assimilating bacteria in both bioaugmentation systems based on DNA-SIP analysis. Combining genomic information with pure culture evidence, strain R1 enhanced SMX removal by directly participating in SMX degradation, while strain M2 did it by both participating in SMX degradation and stimulating SMX-degrading activity of indigenous microorganisms (Paenarthrobacter) in the community. CONCLUSIONS: Our findings demonstrate that bioaugmentation using SMX-degrading bacteria was a feasible strategy for SMX clean-up in terms of the degradation efficiency of SMX, the risk of ARG transmission, as well as the impact on the bacterial community, and the advantage of bioaugmentation with Paenarthrobacter sp. R1 was also highlighted. Video Abstract.202438424602
855420.9934Nanomaterial-Enhanced Hybrid Disinfection: A Solution to Combat Multidrug-Resistant Bacteria and Antibiotic Resistance Genes in Wastewater. This review explores the potential of nanomaterial-enhanced hybrid disinfection methods as effective strategies for addressing the growing challenge of multidrug-resistant (MDR) bacteria and antibiotic resistance genes (ARGs) in wastewater treatment. By integrating hybrid nanocomposites and nanomaterials, natural biocides such as terpenes, and ultrasonication, this approach significantly enhances disinfection efficiency compared to conventional methods. The review highlights the mechanisms through which hybrid nanocomposites and nanomaterials generate reactive oxygen species (ROS) under blue LED irradiation, effectively disrupting MDR bacteria while improving the efficacy of natural biocides through synergistic interactions. Additionally, the review examines critical operational parameters-such as light intensity, catalyst dosage, and ultrasonication power-that optimize treatment outcomes and ensure the reusability of hybrid nanocomposites and other nanomaterials without significant loss of photocatalytic activity. Furthermore, this hybrid method shows promise in degrading ARGs, thereby addressing both microbial and genetic pollution. Overall, this review underscores the need for innovative wastewater treatment solutions that are efficient, sustainable, and scalable, contributing to the global fight against antimicrobial resistance.202439591087
974030.9934Nitrogen-Doped Carbon Dots Facilitate CRISPR/Cas for Reducing Antibiotic Resistance Genes in the Environment. The continued acquisition and propagation of antibiotic resistance genes (ARGs) in the environment confound efforts to manage the global rise in antibiotic resistance. Here, CRISPR-Cas9/sgRNAs carried by nitrogen-doped carbon dots (NCDs) were developed to precisely target multi-"high-risk" ARGs (tet, cat, and aph(3')-Ia) commonly detected in the environment. NCDs facilitated the delivery of Cas9/sgRNAs to Escherichia coli (E. coli) without cytotoxicity, achieving sustained elimination of target ARGs. The elimination was optimized using different weight ratios of NCDs and Cas9 protein (1:1, 1:20, and 1:40), and Cas9/multi sgRNAs were designed to achieve multi-cleavage of ARGs in either a single strain or mixed populations. Importantly, NCDs successfully facilitated Cas9/multi sgRNAs for resensitization of antibiotic-resistant bacteria in soil (approaching 50%), whereas Cas9/multi sgRNAs alone were inactivated in the complex environment. This work highlights the potential of a fast and precise strategy to minimize the reservoir of antibiotic resistance in agricultural system.202438335532
855240.9933Sustainable material platforms for multi-log removal of antibiotic-resistant bacteria and genes from wastewater: A review. Antibiotic-resistant bacteria (ARB) and the associated resistance genes (ARGs) are now recognized as emerging contaminants that can disseminate via wastewater streams, posing significant risks to both human and ecosystem health. Conventional physicochemical treatment approaches (e.g., chlorination, ozonation, advanced oxidation processes) typically suppress these contaminants but may also result in the formation of hazardous by-products. This critical review comprehensibly evaluates bio-based and other sustainable materials designed for the removal of ARB and ARGs from aqueous environments. The materials are systematically categorized into (i) biopolymers and their composites (chitosan, alginate, cellulose), (ii) carbon-rich adsorbents and (photo-)catalysts (biochar, activated carbon, graphene), (iii) metal- and semiconductor-based nanomaterials, and (iv) nature-based treatment solutions (constructed wetlands, soil-aquifer treatment, clay sorbents). Observed log-reduction value range from 2 to 7 for ARB with platforms such as zinc oxide/activated-carbon alginate beads, Fe/N-doped biochars, and graphene-supramolecular-porphyrin hybrids demonstrating high multifunctional efficacy. Mechanistic studies reveal that removal involves synergistic adsorption, photodynamic or Fenton-like oxidation, cell-membrane disruption, and inhibition of horizontal gene transfer. This review emphasizes the advancing potential of sustainable material solutions for mitigating antibiotic resistance and highlights the urgent need to develop scalable, environmentally sustainable treatment methods for protecting water resources and public health.202540763861
854850.9933Persulfate salts to combat bacterial resistance in the environment through antibiotic degradation and biofilm disruption. Antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) have become a critical topic among researchers because of the excessive use of antibiotics in human and animal health care. Globally, it poses a serious threat to human health and the environment. Antibiotics are often poorly metabolized, with 30-90 % excreted into the environment, contaminating aquatic and ground ecosystems, and fostering resistance. Advanced oxidation processes (AOPs), particularly sulfate radical-based AOPs (SR-AOPs), offer promising solutions for degrading antibiotics and resistant biofilms. Persulfate (PS) and Peroxymonosulfate (PMS) are key oxidants in these processes, generating sulfate and hydroxyl radicals when activated by heat, UV light, or transition metals. PS with a redox potential of E°=2.01 V is an affordable and effective oxidant. However, PS requires activation for the degradation of contaminants. PMS is stable across a broad pH range and produces both sulfate and hydroxyl radicals, allowing it to function independently without activation. Thus, PMS serving as a versatile agent for environmental treatment. This review broadly describes the degradation mechanisms of different classes of antibiotics and biofilms. Despite these promising developments, SR-AOPs still face challenges in managing complex wastewater systems, which often contain multiple pollutants. Moreover, gaps remain in understanding of the toxicity of reaction intermediates and in optimizing the large-scale application of these processes. Future research should focus on the in-situ generation of sulfate radicals, combining different activation methods to enhance degradation efficiency, and developing sustainable and cost-effective approaches for large-scale wastewater treatment.202540532556
787360.9933Wheat straw pyrochar more efficiently decreased enantioselective uptake of dinotefuran by lettuce and dissemination of antibiotic resistance genes than hydrochar in an agricultural soil. Remediation of soils pollution caused by dinotefuran, a chiral pesticide, is indispensable for ensuring human food security. In comparison with pyrochar, the effect of hydrochar on enantioselective fate of dinotefuran, and antibiotic resistance genes (ARGs) profiles in the contaminated soils remain poorly understood. Therefore, wheat straw hydrochar (SHC) and pyrochar (SPC) were prepared at 220 and 500 °C, respectively, to investigate their effects and underlying mechanisms on enantioselective fate of dinotefuran enantiomers and metabolites, and soil ARG abundance in soil-plant ecosystems using a 30-day pot experiment planted with lettuce. SPC showed a greater reduction effect on the accumulation of R- and S-dinotefuran and metabolites in lettuce shoots than SHC. This was mainly resulted from the lowered soil bioavailability of R- and S-dinotefuran due to adsorption/immobilization by chars, together with the char-enhanced pesticide-degrading bacteria resulted from increased soil pH and organic matter content. Both SPC and SHC efficiently reduced ARG levels in soils, owing to lowered abundance of ARG-carrying bacteria and declined horizontal gene transfer induced by decreased dinotefuran bioavailability. The above results provide new insights for optimizing char-based sustainable technologies to mitigate pollution of dinotefuran and spread of ARGs in agroecosystems.202336996986
855570.9932Combating Antibiotic Resistance in Persulfate-Based Advanced Oxidation Processes: Activation Methods and Energy Consumption. Antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) have become increasing concerning issues, threatening human health. Persulfate-based advanced oxidation processes (PS-AOPs), due to their remarkable potential in combating antibiotic resistance, have garnered significant attention in the field of disinfection in recent years. In this review, we systematically evaluated the efficacy and underlying mechanism of PS integration with various activation methods for the elimination of ARB/ARGs. These approaches encompass physical methods, catalyst activation, and hybrid techniques with photocatalysis, ozonation, and electrochemistry. Additionally, we employed Chick's model and electrical energy per log order (EE/O) to assess the performance and energy efficiency, respectively. This review aims at providing a guide for future investigation on PS-AOPs for antibiotic resistance control.202539864723
767480.9931Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing. The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.202438374282
908190.9931Identification and reconstruction of novel antibiotic resistance genes from metagenomes. BACKGROUND: Environmental and commensal bacteria maintain a diverse and largely unknown collection of antibiotic resistance genes (ARGs) that, over time, may be mobilized and transferred to pathogens. Metagenomics enables cultivation-independent characterization of bacterial communities but the resulting data is noisy and highly fragmented, severely hampering the identification of previously undescribed ARGs. We have therefore developed fARGene, a method for identification and reconstruction of ARGs directly from shotgun metagenomic data. RESULTS: fARGene uses optimized gene models and can therefore with high accuracy identify previously uncharacterized resistance genes, even if their sequence similarity to known ARGs is low. By performing the analysis directly on the metagenomic fragments, fARGene also circumvents the need for a high-quality assembly. To demonstrate the applicability of fARGene, we reconstructed β-lactamases from five billion metagenomic reads, resulting in 221 ARGs, of which 58 were previously not reported. Based on 38 ARGs reconstructed by fARGene, experimental verification showed that 81% provided a resistance phenotype in Escherichia coli. Compared to other methods for detecting ARGs in metagenomic data, fARGene has superior sensitivity and the ability to reconstruct previously unknown genes directly from the sequence reads. CONCLUSIONS: We conclude that fARGene provides an efficient and reliable way to explore the unknown resistome in bacterial communities. The method is applicable to any type of ARGs and is freely available via GitHub under the MIT license.201930935407
7833100.9931Defect-Rich Cu(2)O Nanospheres as a Fenton-Like Catalyst for Cu(III) Generation: Enhanced Inactivation of Antibiotic-Resistant Bacteria and Genes. Cupryl species (Cu(III)) are promising oxidants for degrading recalcitrant organic contaminants and harmful microorganisms in water. In this study, defect-rich cuprous oxide (D-Cu(2)O) nanospheres (NSs) are introduced as a Fenton-like catalyst to generate Cu(III) for the inactivation of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). D-Cu(2)O, in the presence of H(2)O(2), achieved inactivation efficiencies 3.2, 3.0, and 2.4 times higher than those of control Cu(2)O for ARB, extracellular ARGs (e-ARGs), and intracellular ARGs (i-ARGs), respectively. Experimental evidence from oxidant scavenging tests, Cu(III)-periodate complexation assays, electron paramagnetic resonance (EPR), and in situ Raman spectroscopy confirmed that D-Cu(2)O significantly enhanced Cu(III) generation when reacting with H(2)O(2) compared to control Cu(2)O. Density functional theory (DFT) calculations further revealed that unsaturated copper atoms in D-Cu(2)O enhance H(2)O(2) adsorption by improving the structural accessibility of adjacent oxygen atoms. This facilitates electron transfer processes and promotes subsequent Cu(III) generation. The D-Cu(2)O/H(2)O(2) system demonstrated excellent reusability, maintaining a 4-log reduction of ARB over five cycles, and proved effective across various water matrices and microbial species. These findings highlight the potential of the D-Cu(2)O/H(2)O(2) system, driven by defect engineering, as a robust platform for enhancing water safety and advancing sustainable disinfection technologies.202540795282
9092110.9931Antimicrobial and Antiviral Nanofibers Halt Co-Infection Spread via Nuclease-Mimicry and Photocatalysis. The escalating spread of drug-resistant bacteria and viruses is a grave concern for global health. Nucleic acids dominate the drug-resistance and transmission of pathogenic microbes. Here, imidazolium-type poly(ionic liquid)/porphyrin (PIL-P) based electrospun nanofibrous membrane and its cerium (IV) ion complex (PIL-P-Ce) are developed. The obtained PIL-P-Ce membrane exhibits high and stable efficiency in eradicating various microorganisms (bacteria, fungi, and viruses) and decomposing microbial antibiotic resistance genes and viral nucleic acids under light. The nuclease-mimetic and photocatalytic mechanisms of the PIL-P-Ce are elucidated. Co-infection wound models in mice with methicillin-resistant S. aureus and hepatitis B virus demonstrate that PIL-P-Ce integrate the triple effects of cationic polymer, photocatalysis, and nuclease-mimetic activities. As revealed by proteomic analysis, PIL-P-Ce shows minimal phototoxicity to normal tissues. Hence, PIL-P-Ce has potential as a "green" wound dressing to curb the spread of drug-resistant bacteria and viruses in clinical settings.202438647392
7855120.9931Combat against antibiotic resistance genes during photo-treatment of magnetic Zr-MOFs@Layered double hydroxide heterojunction: Conjugative transfer risk mitigating and bacterial inactivation. The dissemination of antimicrobial resistance (AMR) in wastewater treatment poses a severe threat to the global ecological environment. This study explored the effectiveness of photocatalysis in inactivating antibiotic resistant bacteria (ARB) and quantitatively clarified the inhibiting rate of the transfer of antibiotics resistance genes (ARGs). Herein, the magnetic heterojunction as UiO-66-NH(2)@CuFe LDH-Fe(3)O(4) (UN-66@LDH-Fe) effectively facilitated the electron-hole separation and accelerated the photogenerated charge transfer, thereby guaranteeing the stable practical application in aeration tanks. Notably, the internal electric field of heterogeneous photocatalyst resulted in significant increase of ARGs inactivation, achieving 5.63 log of ARB, 3.66 log of tetA and 3.57 log of Ampr genes were photodegraded under optimal reaction conditions within 6 h. Based on the complex microbial and molecular mechanism of multiple-ARB communities inactivation in photo-treatment, the photogenerated reactive oxygen species (ROSs, ·OH and ·O(2)(-)) effectively destroyed bacterial membrane protein, thereby the intracellular ROSs and redox cycles further induced oxidative stress, attributing to the abundance reduction of ARGs and their host bacteria. Moreover, long-term (7 days) continuous operation preliminarily verified the practical potential in reducing AMR spread and developing wastewater treatment efficacy. Overall, this study presented an advantageous synergistic strategy for mitigating the AMR-associated environmental risk in wastewater treatment.202540188541
7813130.9931A framework predicting removal efficacy of antibiotic resistance genes during disinfection processes with machine learning. Disinfection has been applied widely for the removal of antibiotic resistance genes (ARGs) to curb the spread of antibiotic resistance. Quantitative polymerase chain reaction (qPCR) is the most used method to quantify the damage of DNA thus calculating the ARG degradation during disinfection but suffers the deviation due to the limitation of amplicon length. In contrast, transformation assay more accurately measures ARG deactivation based on expression of disinfected ARG in the receiving bacteria but is typically laborious and material-intensive. This work applied machine learning (ML) to develop a framework by using qPCR results as a proxy to estimate the transformation assay measurements during disinfection with chlorine (FAC), ultraviolet (UV(254)), ozone (O(3)), and hydrogen peroxide/ultraviolet (UV/H(2)O(2)) for multiple kinds of ARGs. ARG degradation rates and deactivation rates were well predicted with the optimal correlation coefficient (R(2)) of all test sets > 0.926 and > 0.871, respectively. Besides, by concatenating the ARG degradation and deactivation predictive models, ARG removal efficiency under given disinfection conditions was directly predicted as the loss of transformation activity with R(2) > 0.828. Furthermore, an online platform was built to provide users with access to the developed ML models for rapid and accurate evaluation of ARG removal efficiency.202540179779
8556140.9931Bubbles Expand the Dissemination of Antibiotic Resistance in the Aquatic Environment. Antibiotic resistance is a global health challenge, and the COVID-19 pandemic has amplified the urgency to understand its airborne transmission. The bursting of bubbles is a fundamental phenomenon in natural and industrial processes, with the potential to encapsulate or adsorb antibiotic-resistant bacteria (ARB). However, there is no evidence to date for bubble-mediated antibiotic resistance dissemination. Here, we show that bubbles can eject abundant bacteria to the air, form stable biofilms over the air-water interface, and provide opportunities for cell-cell contact that facilitates horizontal gene transfer at and over the air-liquid interface. The extracellular matrix (ECM) on bacteria can increase bubble attachment on biofilms, increase bubble lifetime, and, thus, produce abundant small droplets. We show through single-bubble probe atomic force microscopy and molecular dynamics simulations that hydrophobic interactions with polysaccharides control how the bubble interacts with the ECM. These results highlight the importance of bubbles and its physicochemical interaction with ECM in facilitating antibiotic resistance dissemination and fulfill the framework on antibiotic resistance dissemination.202337379503
8811150.9930Mechanisms controlling the transformation of and resistance to mercury(II) for a plant-associated Pseudomonas sp. strain, AN-B15. Bioremediation using mercury (Hg)-volatilizing and immobilizing bacteria is an eco-friendly and cost-effective strategy for Hg-polluted farmland. However, the mechanisms controlling the transformation of and resistance to Hg(II) by these bacteria remain unknown. In this study, a plant-associated Pseudomonas sp. strain, AN-B15 was isolated and determined to effectively remove Hg(II) under both nutrient-poor and nutrient-rich conditions via volatilization by transforming Hg(II) to Hg(0) and immobilization by transforming Hg(II) to mercury sulfide and Hg-sulfhydryl. Genome and transcriptome analyses revealed that the molecular mechanisms involved in Hg(II) resistance in AN-B15 were a collaborative process involving multiple metabolic systems at the transcriptional level. Under Hg(II) stress, AN-B15 upregulated genes involved in the mer operon and producing the reducing power to rapidly volatilize Hg(II), thereby decreasing its toxicity. Hydroponic culture experiments also revealed that inoculation with strain AN-B15 alleviated Hg-induced toxicity and reduced the uptake of Hg(II) in the roots of wheat seedlings, as explained by the volatilization and immobilization of Hg(II) and plant growth-promoting traits of AN-B15. Overall, the results from the in vitro assays provided vital information that are essential for understanding the mechanism of Hg(II) resistance in plant-associated bacteria, which can also be applied for the bioremediation of Hg-contamination in future.202234915295
7856160.9930Boosting Low-Dose Ferrate(VI) Activation by Layered FeOCl for the Efficient Removal of Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes via Enhancing Fe(IV)/Fe(V) Generation. Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments pose threats to ecosystem safety and human health, which could not be efficiently removed by conventional disinfection techniques. Herein, layered FeOCl with coordinatively unsaturated Fe sites were fabricated and used to activate Fe(VI) for the efficient ARB/ARG removal in the present study. We found that highly reactive Fe(IV)/Fe(V) intermediates were generated in the FeOCl/Fe(VI) system, rapidly disinfecting 1 × 10(7) CFU mL(-1) ARB to below the limit of detection within only 6 min. Via the combination of in situ characterization and theoretical calculations, we revealed that Fe(VI) was preferentially adsorbed onto Fe sites on the (010) plane of FeOCl and subsequently activated to produce reactive Fe(IV)/Fe(V) through direct electron transfer. Meanwhile, O(2)(•-) generated from O(2) activation on the FeOCl surface enhanced Fe(VI) conversion to Fe(IV)/Fe(V). During the disinfection process, intracellular/extracellular ARGs and DNA bases were simultaneously degraded, inhibiting the potential horizontal gene transfer process. The FeOCl/Fe(VI) system could effectively disinfect ARB under complex water matrices and in real water samples including tap water, lake water, and groundwater. When integrated into a continuous-flow reactor, the FeOCl/Fe(VI) system with excellent stability successively disinfected ARB. Overall, the FeOCl/Fe(VI) system showed great promise for eliminating ARB/ARGs from water.202540739812
7697170.9930Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing. The effects of sample multiplexing on the detection sensitivity of antimicrobial resistance genes (ARGs) and pathogenic bacteria in metagenomic sequencing remain underexplored in newer sequencing technologies such as Oxford Nanopore Technologies (ONT), despite its critical importance for surveillance applications. Here, we evaluate how different multiplexing levels (four and eight samples per flowcell) on two ONT platforms, GridION and PromethION, influence the detection of ARGs, bacterial taxa and pathogens. While overall resistome and bacterial community profiles remained comparable across multiplexing levels, ARG detection was more comprehensive in the four-plex setting with low-abundance genes. Similarly, pathogen detection was more sensitive in the four-plex, identifying a broader range of low abundant bacterial taxa compared to the eight-plex. However, triplicate sequencing of the same microbiomes revealed that these differences were primarily due to sequencing variability rather than multiplexing itself, as similar inconsistencies were observed across replicates. Given that eight-plex sequencing is more cost-effective while still capturing the overall resistome and bacterial community composition, it may be the preferred option for general surveillance. Lower multiplexing levels may be advantageous for applications requiring enhanced sensitivity, such as detailed pathogen research. These findings highlight the trade-off between multiplexing efficiency, sequencing depth, and cost in metagenomic studies.202540611965
8703180.9930New Dimensions in Microbial Ecology-Functional Genes in Studies to Unravel the Biodiversity and Role of Functional Microbial Groups in the Environment. During the past decades, tremendous advances have been made in the possibilities to study the diversity of microbial communities in the environment. The development of methods to study these communities on the basis of 16S rRNA gene sequences analysis was a first step into the molecular analysis of environmental communities and the study of biodiversity in natural habitats. A new dimension in this field was reached with the introduction of functional genes of ecological importance and the establishment of genetic tools to study the diversity of functional microbial groups and their responses to environmental factors. Functional gene approaches are excellent tools to study the diversity of a particular function and to demonstrate changes in the composition of prokaryote communities contributing to this function. The phylogeny of many functional genes largely correlates with that of the 16S rRNA gene, and microbial species may be identified on the basis of functional gene sequences. Functional genes are perfectly suited to link culture-based microbiological work with environmental molecular genetic studies. In this review, the development of functional gene studies in environmental microbiology is highlighted with examples of genes relevant for important ecophysiological functions. Examples are presented for bacterial photosynthesis and two types of anoxygenic phototrophic bacteria, with genes of the Fenna-Matthews-Olson-protein (fmoA) as target for the green sulfur bacteria and of two reaction center proteins (pufLM) for the phototrophic purple bacteria, with genes of adenosine-5'phosphosulfate (APS) reductase (aprA), sulfate thioesterase (soxB) and dissimilatory sulfite reductase (dsrAB) for sulfur oxidizing and sulfate reducing bacteria, with genes of ammonia monooxygenase (amoA) for nitrifying/ammonia-oxidizing bacteria, with genes of particulate nitrate reductase and nitrite reductases (narH/G, nirS, nirK) for denitrifying bacteria and with genes of methane monooxygenase (pmoA) for methane oxidizing bacteria.201627681913
8490190.9929Unveiling the kinetics and mechanism of carbonate radicals in antibiotic resistance dissemination. The escalating contamination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic systems has driven extensive investigations into their interactions with reactive intermediates. However, the reaction kinetics and mechanisms underlying the degradation of ARB and ARGs by CO(3)(•-) remain unelucidated. This study quantifies the reaction rate constants between CO(3)(•-) and ARB, ARGs, and critical biomolecules including extracellular polymeric substances, phospholipids, amino acids, and deoxynucleotides. Results demonstrate negligible CO(3)(•-) reactivity with phospholipids and amino acids (< 10(5) M(-1)·s(-1)), yet remarkably high reaction rates (∼10(7) M(-1)·s(-1)) with ARB, ARGs and deoxynucleotides. Mechanistic studies demonstrate that CO(3)(•-) enhances membrane fluidity by attenuating inter-lipid interactions and reducing lipid ordering (Δη = -60.44 %), thereby driving transmembrane co-transport with HCO(3)⁻. Notably, CO(3)(•-) exhibits an extended reaction radius (1.30-2.31 μm, far exceeding typical membrane-targeting radicals) and exploits its low membrane affinity to achieve deep cellular penetration. Within cells, it selectively oxidizes biomolecules, primarily via deoxyguanosine modification, inducing ARGs damage and systemic biomolecular dysregulation. Integrated transcriptomic and metabolomic analyses confirm these genotoxic impacts, revealing perturbations at transcriptional regulation and metabolic pathway levels. These results establish CO(3)(•-) as a potential key agent in suppressing antibiotic resistance dissemination, resolving a critical knowledge gap in environmental radical-ARB/ARGs interactions.202541101271