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Some of the most commonly identified freshwater toxins are anatoxin-a (ATX-a), cylindrospermopsin (CYN), and microcystin-LR (MC-LR). The aim of this paper was to compare different methods of extracting and concentrating these cyanotoxins and check the impact of selected physical factors on the accumulation of biomass of Dolichospermum flos-aquae, Microcystis aeruginosa, and Raphidiopsis raciborskii. The effect of different cyanobacteria cultivation conditions on the amount of cyanotoxins synthesized showed no significant changes over time in the average concentration of all tested toxins in the medium compared to the control. Mixing cultures increases the intracellular content of ATX-a. Aerating also positively affects the concentration of MC-LR intracellularly. In order to optimize the solid phase extraction (SPE) process of toxins, the C18 phase or activated carbon was used. In general, higher toxin recoveries were achieved when using the C18 phase. The best result was achieved for ATX-a, 94% recovery with elution using methanol with 0.1% trifluoroacetic acid (TFA). For MC-LR, the best recovery was 59%, and for CYN 22%. The study evaluated the various methods to release cyanotoxins from cyanobacteria showed that: the highest ATX-a concentration (0.60 μg/mg d.w) was obtained using MilliQ water and microwave treatment for 10-15 s. For MC-LR, the highest extracted amount (6.73 μg/mg d.w) resulted from methanol treatment and boiling at 100 °C for 15 min. CYN extraction was the most effective by using MilliQ water and alternative freezing/thawing (1.54 μg/mg d.w). In conclusion, changing the optimal parameters of cyanobacterial cultivation, only slightly affects the increase in biomass accumulation and synthesis of cyanobacterial toxins. In the case of ATX, the key is the use of the TFA additive in the SPE process. No single method has been identified as the ideal approach for isolating various intracellular cyanotoxins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cyanobacteria play an essential role in nutrient cycling in aquatic ecosystems. However, certain species adversely affect the environment and human health by causing harmful cyanobacterial algal blooms (cyanoHABs) and producing cyanotoxins. To address this issue, continuous cyanoHAB monitoring has been considered; however, a gold standard has not yet been established. In this study, we aimed to develop a dual DNA-targeting capacitive-type biosensor for rapid field-ready monitoring of Raphidiopsis raciborskii , a causative species of cyanoHAB. To enhance the sensing signal, a plate-like Au-BiOCl nanocomposite was synthesized using a spontaneous carbonation process without additional additives. The alternating-current electrothermal flow (ACEF) technique was applied to enable rapid DNA and probe binding within 10 min. The limits of detection (LODs) for R. raciborskii RubisCO large subunit ( rbcL ) and RNA polymerase beta subunit ( rpoB ) genes diluted in deionized (DI) water were 4.89 × 10 -17 and 3.89 × 10 -17 M, respectively. Furthermore, the LODs of R. raciborskii rbcl and rpoB diluted in freshwater containing HAB were 2.55 × 10 -16 and 3.84 × 10 -16 M, respectively, demonstrating the field-ready applicability of the device. The fabricated cyanobacterial DNA-sensing platform enabled powerful species-specific detection using a small sample volume and low target concentration without a nucleic acid amplification step, dramatically reducing the detection time. This study has considerable implications for detecting HABs, early warning systems, and species-specific environmental monitoring technology.

Abandoned shipwrecks are sitting at the bottom of oceans, lakes, and rivers around the world. Over time, microbial-comprised biofilms can help protect wrecks against chemical corrosion or contribute to their deterioration through microbiologically influenced corrosion (MIC) by organisms including iron-oxidizing bacteria (FeOB) and sulfate-reducing bacteria (SRB). Assessing the community assembly of these biofilms will give us a better understanding of the role these microbes play in MIC and the factors that influence it. Here, we determine if microbial community composition differs across a shallow freshwater ferrous-hulled shipwreck environment. Results suggest that there was a statistically significant difference among the sample types indicating the wreck environments around Accomac influenced the community composition. This is consistent with previous observations within an estuarine, shallow-water wreck environment. Bacteroidota , Chloroflexota , and Cyanobacteriota were the primary taxa responsible for differences among these wreck environments. Interestingly, port-side biofilm communities were significantly different than those on the starboard side suggesting physical factors of the environment drove niche partitioning on each side of the wreck. Similarly, FeOB enrichments and known FeOB taxa were found across the entire wreck but were primarily found in samples associated with the port side of the wreck. Amplicon sequencing identified both known FeOB and SRB taxa with a higher proportion of FeOB than SRB. Overall, these results indicate that there is niche partitioning of the microbial communities as well as with corrosion-causing taxa within a shallow freshwater wreck site which may lead to variation in how microbes may contribute to the protection or deterioration of these ferrous-hulled wrecks., Importance: The overall structure, abundance, and diversity of microbial communities on shipwrecks have recently been studied in marine aquatic environments. While previous studies have looked at the microbial communities associated with shallow-water ferrous-hulled wrecks in marine environments, studies focusing on freshwater wreck systems are limited. The purpose of this study was to determine microbial community diversity and composition trends across the Accomac shipwreck environment. Furthermore, shipwrecks are colonized by corrosion-causing taxa, such as iron-oxidizing bacteria and sulfate-reducing bacteria which have been shown to influence the biocorrosion of ferrous-hulled structures. Identification of various microbes in biofilms, as well as corrosion-causing microbes, can help researchers identify the role they play in aquatic ecosystem development and persistence as well as artificial reef integrity. Understanding how microbes assemble on wrecks will provide insight into preservation strategies to prevent deterioration of these wrecks over time, as well as limiting biocorrosion of similar structures., Competing Interests: The authors declare no conflict of interest.

Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show that Rhodoluna lacicola and Aurantimicrobium photophilum , which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities., Importance: To advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments., Competing Interests: The authors declare no conflict of interest.

The gut microbiota of vertebrates is malleable and may be shaped by both intrinsic and extrinsic factors. Here, the effect that geography has on the cloacal microbiota of two species of Australian freshwater chelonians, eastern longneck turtle (Chelodina longicollis) and Macquarie River turtle (Emydura macquarii), captured from waterbodies with different levels of anthropogenic pressure was investigated. We analysed the microbiota composition, structure and diversity through 16S rRNA gene amplicon sequencing. It was hypothesised that animals from less disturbed environments would harbour a more diverse cloacal microbial population. The cloacal microbiotas from 93 turtles (C. longicollis n = 78; E. macquarii n = 15), from five locations, were analysed. For both species, the most predominant phylum was Proteobacteria. Cloacal microbiota alpha diversity varied significantly between the C. longicollis from all locations, but no differences were found for E. macquarii. In C. longicollis, turtles from wetlands within the centre of Melbourne had the lowest alpha diversity metrics, while the highest alpha diversity values were seen in turtles captured from an undisturbed rural waterbody. Beta diversity, obtained by weighted UniFrac distance, showed significant differences between locations of capture for both species of turtles in this investigation. For C. longicollis, 87 biomarkers were identified responsible for explaining differences between locations, and in E. macquarii, 42 biomarkers were found. This is the first study to explore the cloacal microbiota composition of the eastern longneck turtle and gives greater insight into microbial community structures in Macquarie River turtles. Our study demonstrated that cloacal microbiota composition of freshwater turtles was significantly influenced by locality and that disrupted environments may reduce microbial diversity in C. longicollis. Interestingly, we discovered that the effects of location contrasted significantly between species for alpha diversity with differences discovered for C. longicollis but not E. macquarii. However, for both species, beta diversity was notably influenced by habitat type. These results highlight the need to interpret chelonian microbiota data in the context of geography and human disturbance of the environment., Competing Interests: Declarations Ethics Approval This study was approved by the University of Melbourne Office of Research Ethics and Integrity (Ethics ID: 2022–24808-32226–4) and all experiments were performed in accordance with relevant guidelines and regulations. Turtles were trapped and sampled under permit 10010480 from the Department of Environment, Land, Water and Planning, and permit number RP1497 from the Victorian Fisheries Authority. All turtles were released alive at their point of capture immediately after sampling had been completed. Competing Interests The authors declare no competing interests., (© 2024. The Author(s).)

Water from the Lake Okeechobee watershed historically flowed south through the Everglades. Hydrologic alterations created the Lake Okeechobee Waterway, where lake water is periodically shunted east to the St. Lucie Estuary (C-44 canal) and west to the Caloosahatchee River and Estuary (C-43 canal). Within the last two decades, Microcystis blooms have developed in Lake Okeechobee and been discharged to the downstream urbanized estuaries, resulting in negative environmental and human health impacts. To better understand drivers of cyanobacterial blooms across this modified waterway, two cruises were conducted from the St. Lucie Estuary through Lake Okeechobee to the Caloosahatchee River Estuary during 2019 and 2020. Opportunistic sampling was also conducted during Microcystsis blooms. Cruise stations were sampled for environmental parameters, dissolved nutrients, chlorophyll a, cyanobacterial cell concentrations, and microcystins, as well as particulate organic matter (POM) nutrient properties. Higher ammonium (NH 4 + ), nitrate + nitrite (NO 3 - ), dissolved inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), total dissolved phosphorus (TDP), and POM stable N isotope (δ 15 N) values were observed in the estuaries and Kissimmee River than in Lake Okeechobee. The nitrogen to phosphorus ratio (N:P), microcystins, and Microcystis cell concentrations were higher in Lake Okeechobee than documented over past decades. During Microcystis blooms, high NH 4 + , SRP, total dissolved nitrogen (TDN), TDP, and sucralose were observed with elevated algal δ 15 N. These results demonstrate the importance of local basin contributions, including those within the lake, to estuarine Microcystis blooms. This suggests that decreasing nutrient loading within the St. Lucie and Caloosahatchee estuaries would help to mitigate these urban blooms. High POM δ 15 N values, NO 3 - concentrations, and N:P ratios in the Kissimmee River suggest that expanding urbanization north of the lake represents an increasing human N source contributing to cyanobacterial blooms in Lake Okeechobee., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Three Gram-stain-negative, aerobic, short rod-shaped and motile strains (FXH3W T , SHGZ20W and SMYT11W T ) were isolated from freshwater environments in China. Comparisons based on the 16S rRNA gene sequences indicated that strains FXH3W T and SHGZ20W showed the highest 16S rRNA gene sequence similarity of about 99.6% to ' Luteimonas cellulosilyticus ' MIC 1.5, and strain SMYT11W T showed the highest 16S rRNA gene sequence similarity of 99.8% to Luteimonas fraxinea D4P002 T , respectively. Observing the phylogenetic trees reconstructed based on the 16S rRNA gene sequences, the species of genera Luteimonas and Lysobacter were not monophyletic and often mixed together. The further reconstructed phylogenomic tree and Genome Taxonomy Database also showed that the species of both genera were polyphyletic, implying that the current taxonomic status of the species of both genera was questionable. The calculated OrthoANIu, digital DNA-DNA hybridization and average amino acid sequence identity (AAI) values supported that strains FXH3W T and SHGZ20W should belong to the same novel species and strain SMYT11W T should also represent an independent novel species. Combining the AAI values and phylogenomic analyses, the species of genera Luteimonas and Lysobacter should be reassigned to 12 genera ( Luteimonas , Lysobacter , Cognatiluteimonas , Noviluteimonas , Pseudoluteimonas , Solilutibacter , Agrilutibacter , Cognatilysobacter , Marilutibacter , Novilysobacter , Montanilutibacter and Aerolutibacter ). The AAI values 69.5-76.0% were also proposed as the Lysobacteraceae -specific thresholds for genus delineation. Strain SMYT11W T should represent a novel species of the genus Luteimonas , for which the name Luteimonas flava sp. nov. (type strains SMYT11W T =GDMCC 1.4275 T =KCTC 8304 T ) is proposed. Strains FXH3W T and SHGZ20W should represent a novel species of a new genus Aquilutibacter rugosus gen. nov., sp. nov. The type strain of the type species is FXH3W T (=GDMCC 1.4096 T =KCTC 8154 T ).

To ensure sustainable aquaculture, it is essential to understand the path 'from feed to fish', whereby the gut microbiome plays an important role in digestion and metabolism, ultimately influencing host health and growth. Previous work has reported the taxonomic composition of the Atlantic salmon (Salmo salar) gut microbiome; however, functional insights are lacking. Here we present the Salmon Microbial Genome Atlas consisting of 211 high-quality bacterial genomes, recovered by cultivation (n = 131) and gut metagenomics (n = 80) from wild and farmed fish both in freshwater and seawater. Bacterial genomes were taxonomically assigned to 14 different orders, including 35 distinctive genera and 29 previously undescribed species. Using metatranscriptomics, we functionally characterized key bacterial populations, across five phyla, in the salmon gut. This included the ability to degrade diet-derived fibres and release vitamins and other exometabolites with known beneficial effects, which was supported by genome-scale metabolic modelling and in vitro cultivation of selected bacterial species coupled with untargeted metabolomic studies. Together, the Salmon Microbial Genome Atlas provides a genomic and functional resource to enable future studies on salmon nutrition and health., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Treated sewage contains a large diversity of pathogens that can be transmitted to the environment and, directly or indirectly, infect humans through water use (i.e., consumption, bathing, or irrigation). In urban environments, wastewater normally flows into wastewater treatment plants (WWTPs), where it is subjected to different processes in order to eliminate the greatest amount of waste. However, there are inequalities among European countries concerning wastewater management. In this context, we evaluate the potential of freshwater mussels to improve water quality (i.e., reduce bacterial abundance) in rivers receiving primary, secondary, or tertiary sewage-treated effluents. Additionally, because freshwater mussels are declining at a global scale and empty niches are progressively occupied by non-native counterparts, we evaluate if depauperate communities and the Asian clams, Corbicula genus, can provide equivalent ecosystem services (i.e., water quality improvement by biofiltration) formerly provided by diverse native communities. For this, an analysis of the bacterial biodiversity of the samples filtered by the different bivalve communities was carried out. The experimental approach was performed by metabarcoding the 16S rRNA gene using Illumina technologies. According to the results obtained, secondary treatment processes were effective in reducing the bacterial diversity. Furthermore, the waters filtered by the bivalves presented a lower bacterial abundance for certain genera. Biofiltration differs, however, among species, with Corbicula reducing a large number of taxa much more efficiently than native freshwater mussels in both diverse and depauperated communities. These results are likely related to Corbicula being a generalist species in front of native mussels, which may be more selective. Considering it is not possible to eradicate Corbicula from European rivers, its filtering capacity should be considered when managing freshwater ecosystems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Noé Ferreira-Rodriguez reports financial support was provided by Government of Galicia, Department of Culture Education and Universities. Margarita Poza reports financial support was provided by Carlos III Health Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Lakes and wetlands play pivotal roles in global organic matter storage, receiving significant inputs of organic material. However, the co-metabolic processes governing the decomposition of these organic materials and their impact on greenhouse gas emissions remain inadequately understood. This study aims to assess the effects of mixed decomposition involving macrophytes and cyanobacteria on carbon emissions. A series of microcosms was established to investigate the decomposition of macrophyte residues and algae over a period of 216 days. A two-component kinetic model was utilized to estimate methane (CH 4 ) production rates. Gas isotope technology was employed to discern the contributions of CH 4 produced by macrophyte residues or algae. Quantitative PCR and analysis of 16S rRNA gene amplicons were employed to assess changes in functional genes and microbial communities. There were significant differences in the cumulative carbon release from the decomposition of different plant types due to the addition of carbon sources. After adding algae, the cumulative emission of CH 4 increased significantly. The δ 13 C-CH 4 partitioning indicated that CH 4 originated exclusively from the fresh organic carbon of macrophyte residues, while it shifted to algae source after adding algae. The synergistic effect of the mixed decomposition on the CH 4 emissions was greater than the sum of the individual decompositions. The microbial community richness was higher in the single plant residue treatment compared to the mixed treatment with algae addition, while microbial evenness in the sediment increased steadily in each treatment. Our findings emphasize the pronounced co-metabolic effect observed during the mixed decomposition of macrophytes and cyanobacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Biofilms are mucilaginous-organic layers produced by microbial activity including viruses. Growing biofilms form microbial mats which enhance sediment stability by binding particles with extracellular polymeric substances and promoting growth through nutrient cycling and organic matter accumulation. They preferentially develop at the sediment-water interface of both marine and non-marine environments, and upon the growing surfaces of modern tufa and travertine. In this context, however, little is known about the factors, environmental or anthropogenic, which affect viral communities in freshwater spring settings. To explore this issue, geochemical and metagenomic data were subjected to multidimensional analyses (Principal Component Analysis, Classical Multidimensional Scaling, Partial Least Squares analysis and cluster analysis based on beta-diversity), and these show that viral composition is specific and dependent on environment. Indeed, waters precipitating tufa and travertine do vary in their geochemistry with their viruses showing distinct variability between sites. These differences between virus groups allow the formulation of a viral proxy, based on the Caudoviricetes/Megaviricetes ratio established on the most abundant groups of viruses. This ratio may be potentially used in analysing ancient DNA preserved in carbonate formations as an additional source of information on the microbiological community during sedimentation., (© 2024. The Author(s).)

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 (" E. coli ") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day -1 in the marine waters and -0.89 to -3.2 day -1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators., Competing Interests: The authors declare no conflict of interest.

Background: The biofloc system (BFS) provides a sustainable aquaculture system through its efficient in situ water quality maintenance by the microbial biomass, besides continuous availability of these protein-rich microbes as feed to enhance growth and immunity of the reared organism. This study explores the gill architecture, growth performance, digestive enzyme activity, intestinal microbial composition, and histology of three freshwater fish species, Puntius gonionotus, Pangasianodon hypophthalmus, and Heteropneustus fossilis reared in biofloc based polyculture system., Results: The three species in T2 showed significantly higher WG and SGR, followed by T1 and T3. The wet mount of gill architecture showed smaller inter-filament gaps in gill arches of silver barb followed by stinging catfish and stripped catfish, but showed no correlation with the weight gain. However, silver barb being an omnivore and filter-feeder, accumulated a more diverse microbial community, both in T1 and BFS (T2 and T3), while the bottom feeder H. fossilis exhibited unique gut bacterial adaptability. The presence of floc in T2 and T3 enhanced bacterial abundance in water and fish gut, but their microbial diversities significantly reduced compared to T1 receiving only feed. Next-generation sequencing revealed that the Pseudomonas dominated in gut of P. gonionotus and P. hypophthalmus in T1, Enterobacterales and Fusobacterium prevailed in those of T2 and T3, respectively. In contrast, gut of H. fossilis had the highest proportion of Clostridium in T1, while Rhizobiaceae dominated in T3. Similarly in floc samples, Enterococcus dominated in T1 while Micrococcales and Rhizobiaceae dominated in T2 and T3, respectively. A positive correlation of enterobacteria, with the digestive enzyme activities and growth patterns was observed in all treatments., Conclusion: The present study revealed feeding behaviour to play crucial role in distinguishing the gut microbial composition patterns in fishes reared in Biofloc System. Further it revealed the requirement of supplementary feed along with floc in these three species for higher growth in the biofloc system., (© 2024. The Author(s).)

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (ΦT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology.IMPORTANCEFunctional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes., Competing Interests: The authors declare no conflict of interest.

Clostridioides difficile is an intestinal pathogen of humans and animals. In community-associated infections, the environment is suggested to play a significant role in overall transmission routes. Although the prevalence of C. difficile in freshwater and soil has been widely studied, little is known about its presence in sediments. In this study, we tested 15 sediment samples collected from various freshwater sources. C. difficile was isolated from all sampled sites, yielding a total of 171 strains grouped into 26 ribotypes, with 001/072 and 014/020 being the most prevalent. Genome sequencing of 37 isolates from 17 PCR ribotypes confirmed the presence of highly related strains in the geographically distant and unlinked water samples. Eight divergent PCR ribotypes from clades C-II and C-III were found in six samples. In each sample, the unbound fraction (supernatant after sediment wash) and bound fraction (sonicated sediment sample) were subjected to enrichment. Sonication was only slightly better than washing in terms of sample positivity (14 positive samples with sonication and 11 with washing). However, sonication substantially increased the diversity of the PCR ribotypes obtained (23 in sonicated samples vs nine in washed samples). In conclusion, sediments are a rich source for investigating the diversity of environmental C. difficile, including isolates from divergent lineages. Selection of the isolation method can significantly impact the diversity of captured PCR ribotypes.IMPORTANCE Clostridioides difficile , a pathogenic bacterium that can cause intestinal infections in humans and animals, thrives in the gut but also disperses widely through spores found in the environment. Clinical and environmental strains often overlap with common PCR ribotypes, which are consistently isolated worldwide. Environmental studies have mostly focused on water and soil, but sediments have been very poorly studied. In this study, we investigated the presence of C. difficile in various freshwater sediments and evaluated the effectiveness of two different isolation approaches on positivity rates and strain diversity. C. difficile was found to be highly prevalent in sediments, with an isolation rate of 100%. Sonication proved to be more effective than simple washing for capturing a greater diversity of PCR ribotypes. Overall, this study underscores the widespread presence of C. difficile in freshwater sediments and emphasizes the importance of continued surveillance and monitoring to understand its ecology and transmission dynamics., Competing Interests: The authors declare no conflict of interest.

Antibiotic resistance genes (ARGs) are a growing problem that is widespread in river-lake ecosystems, where they pose a threat to the aquatic environment's health and public safety. These systems serve as critical nodes in water management, as they facilitate the equitable allocation of water resources through long-term and frequent water diversions. However, hydrological disturbances associated with water-regulation practices can influence the dynamics of their potential host microorganisms and associated resistance genes. Consequently, identifying the key ARGs and their resistance mechanisms in heavily regulated waters is vital for safeguarding human health and that of river-lake ecosystems. In this study, we examined the impact of water-regulation factors on ARGs and their hosts within a river-lake continuum using 16S rRNA and metagenomic sequencing. We found that a significant increase in ARG abundance during regulation periods (p < 0.05), especially in the aquatic environment. Key resistance genes were macB, tetA, evgS, novA, and msbA, with increased efflux pinpointed as their principal resistance mechanism. Network analysis identified Flavobacteriales, Acinetobacter, Pseudomonas, Burkholderiaceae, and Erythrobacter as key potential host microorganisms, which showed increased abundance within the water column during regulation periods (p < 0.05). Flow velocity and water depth both drove the host microorganisms and critical ARGs. Our findings underscore the importance of monitoring and mitigating the antibiotic resistance risk during water transfers in river-lake systems, thereby supporting informed management and conservation strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Viruses, the majority of which are uncultivated, are among the most abundant biological entities on Earth. From altering microbial physiology to driving community dynamics, viruses are fundamental members of microbiomes. While the number of studies leveraging viral metagenomics (viromics) for studying uncultivated viruses is growing, standards for viromics research are lacking. Viromics can utilize computational discovery of viruses from total metagenomes of all community members (hereafter metagenomes) or use physical separation of virus-specific fractions (hereafter viromes). However, differences in the recovery and interpretation of viruses from metagenomes and viromes obtained from the same samples remain understudied., Results: Here, we compare viral communities from paired viromes and metagenomes obtained from 60 diverse samples across human gut, soil, freshwater, and marine ecosystems. Overall, viral communities obtained from viromes had greater species richness and total viral genome abundances than those obtained from metagenomes, although there were some exceptions. Despite this, metagenomes still contained many viral genomes not detected in viromes. We also found notable differences in the predicted lytic state of viruses detected in viromes vs metagenomes at the time of sequencing. Other forms of variation observed include genome presence/absence, genome quality, and encoded protein content between viromes and metagenomes, but the magnitude of these differences varied by environment., Conclusions: Overall, our results show that the choice of method can lead to differing interpretations of viral community ecology. We suggest that the choice of whether to target a metagenome or virome to study viral communities should be dependent on the environmental context and ecological questions being asked. However, our overall recommendation to researchers investigating viral ecology and evolution is to pair both approaches to maximize their respective benefits. Video Abstract., (© 2024. The Author(s).)

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 10 7 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We aimed to evaluate the ability of naturally occurring colonies of Microcystis, embedded in a thick mucilage, to persist in estuarine waters. In two batch experiments, we examined the dynamics of microbial communities, including cyanobacteria and associated heterotrophic bacteria, sampled from the field during both a cyanobacterial bloom (non-limiting nutrient condition) and the post-bloom period (limiting nutrient condition), and subjected them to a salinity gradient representative of the freshwater-marine continuum. We demonstrated that both Microcystis aeruginosa and M. wesenbergii survived high salinities due to osmolyte accumulation. Specifically, prolonged exposure to high salinity led to betaine accumulation in the cyanobacterial biomass. The relative abundance of the mcyB gene remained around 30%, suggesting no selection for toxic genotypes with salinity or nutrient changes. Microcystins were predominantly intracellular, except at high salinity levels (>15), where more than 50% of the total microcystin concentration was extracellular. In both nutrient conditions, over 70% of the heterotrophic bacterial community belonged to the Gammaproteobacteria family, followed by the Bacteroidota. Bacterial community composition differed in both size fractions, as well as along the salinity gradient over time. Finally, genus-specific core microbiomes were identified and conserved even under highly stressful conditions, suggesting interactions that support community stability and resilience., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

A sulphur-oxidizing bacterium, designated strain SCUT-2 T , was isolated from freshwater sediment collected from the Pearl River in Guangzhou, PR China. This strain was an obligate chemolithoautotroph, utilizing reduced sulphur compounds (elemental sulphur, thiosulphate, tetrathionate and sulphite) as the electron donor. Growth of strain SCUT-2 T was observed at 20-40 ℃ (optimum at 30 °C), pH 5.0-9.0 (optimum at 6.0), and NaCl concentration range of 0-9 g L -1 (optimum at 1 g L -1 ). The major cellular fatty acids were C 16:0 ω7c and cyclo-C 17:0 . The DNA G + C content of the complete genome sequence was 66.8 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain SCUT-2 T formed a lineage within the genus Thiobacillus, showing gene sequence identity of 98.0% with its closest relative Thiobacillus thioparus THI 115. The genome of strain SCUT-2 T contains multiple genes encoding sulphur-oxidizing enzymes that catalyse the oxidation of reduced sulphur compounds, partial genes that are necessary for denitrification, and the genes encoding cbb 3 -type cytochrome c oxidase, aa 3 -type cytochrome c oxidase and bd-type quinol oxidase. Facultative anaerobic growth occurs when using nitrate as the electron acceptor and thiosulphate as the electron donor. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic analysis, strain SCUT-2 T (= GDMCC 1.4108 T  = JCM 39443 T ) is deemed to represent a novel Thiobacillus species, for which we propose the name Thiobacillus sedimenti sp. nov., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Proteus faecis is a gram-negative facultative anaerobic rod-shaped bacterium capable of swarming motility. It has been isolated from numerous sources such as humans, animals, and refuse and is considered potentially pathogenic towards humans. In this study, bacteria were isolated from the blowhole of a Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) living in captivity in China. One bacterium, P. faecis porpoise, was isolated and whole genome sequencing done. Biofilm formation, motility and antimicrobial resistance were also investigated. To find putative virulence factors, the genome of P. faecis strain porpoise was compared to the genomic sequences of eight other P. faecis isolates using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) ( https://www.bv-brc.org/ ). The goal of this study was to initially characterize the pathogenicity of this bacterium isolated from a cetacean species using both pathogenomics and conventional approaches., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Domestic wastewater is a significant reservoir of antibiotic resistance genes, which pose environmental and public health risks. We aimed to define an antibiotic resistome signature, represented by core genes, i.e., shared by ≥ 90% of the metagenomes of each of three conceptual environmental compartments - wastewater (influent, sludge, effluent), freshwater, and agricultural soil. The definition of resistome signatures would support the proposal of a framework for monitoring treatment efficacy and assessing the impact of treated wastewater discharge into the environment, such as freshwater and agricultural soil. Metagenomic data from 163 samples originating from wastewater (n = 81), freshwater (n = 58), and agricultural soils (n = 24) across different regions (29 countries, 5 continents), were analysed regarding antibiotic resistance diversity, based on annotation against a database that merged CARD and ResFinder databases. The relative abundance of the total antibiotic resistance genes (corresponding to the ratio between the antibiotic resistance genes and total reads number) was not statistically different between raw and treated wastewater, being significantly higher than in freshwater or agricultural soils. The latter had the significantly lowest relative abundance of antibiotic resistance genes. Genes conferring resistance to aminoglycosides, beta-lactams, and tetracyclines were among the most abundant in wastewater environments, while multidrug resistance was equally distributed across all environments. The wastewater resistome signature included 27 antibiotic resistance genes that were detected in at least 90% of the wastewater resistomes, and that were not frequent in freshwater or agricultural soil resistomes. Among these were genes responsible for resistance to tetracyclines (n = 8), macrolide-lincosamide-streptogramin B (n = 7), aminoglycosides (n = 4), beta-lactams (n = 3), multidrug (n = 2), sulphonamides (n = 2), and polypeptides (n = 1). This comprehensive assessment provides valuable insights into the dynamics of antibiotic resistance in urban wastewater systems and their potential ecological implications in diverse environmental settings. Furthermore, provides guidance for the implementation of One Health monitoring approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In recirculating aquaculture systems (RAS), maintaining water quality in aquaculture tanks is a paramount factor for effective fish production. A down-flow hanging sponge (DHS) reactor, a trickling filter system used for water treatment of RAS that employs sponges to retain biomass, has high nitrification activity. However, nitrification in seawater RAS requires a long start-up time owing to the high salinity stress. Therefore, this study aimed to evaluate the nitrification characteristics and changes in the microbial community during the conversion of freshwater to seawater in a DHSreactor fed with ammonia-based artificial seawater. The total ammonia nitrogen concentration reached 1.0 mg-N·L -1 (initial concentration 10 mg-N·L -1 ) within 11 days of operation, and nitrate production was observed. The 16 S rRNA gene sequence of the DHS-retained sludge indicated that the detection rate of the ammonia-oxidizing archaeon Candidatus Nitrosocosmicus decreased from 23.9 % to 14.0 % and 25.8-17.6 % in the upper and lower parts of the DHS reactor, respectively, after the introduction of seawater. In contrast, the nitrite-oxidizing bacteria Nitrospira spp. increased from 0.1 % to 9.5 % and from 0.5 % to 10.5 %, respectively. The ammonia oxidation rates of 0.12 ± 0.064 and 0.051 ± 0.0043 mg-N·g-MLVSS -1 ·h -1 on the 37th day in the upper and bottom layers, respectively. Thus, nitrification in the DHS reactor performed well, even under high-salinity conditions with short operational days. This finding makes the transition from freshwater to saltwater fish in the RAS system simple and economical, and has the potential for early start-up of the RAS., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Takahiro Watari reports financial support was provided by Japan Society for the Promotion of Science. Takahiro Watari reports financial support was provided by Japan Science and Technology Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microorganisms can colonize to the surface of microplastics (MPs) to form biofilms, termed "plastisphere", which could significantly change their physiochemical properties and ecological roles. However, the biofilm characteristics and the deep mechanisms (interaction, assembly, and biogeochemical cycles) underlying plastisphere in wetlands currently lack a comprehensive perspective. In this study, in situ biofilm formation experiments were performed in a park with different types of wetlands to examine the plastisphere by extrinsic addition of PVC MPs in summer and winter, respectively. Results from the spectroscopic and microscopic analyses revealed that biofilms attached to the MPs in constructed forest wetlands contained the most abundant biomass and extracellular polymeric substances. Meanwhile, data from the high-throughput sequencing showed lower diversity in plastisphere compared with soil bacterial communities. Network analysis suggested a simple and unstable co-occurrence pattern in plastisphere, and the null model indicated increased deterministic process of heterogeneous selection for its community assembly. Based on the quantification of biogeochemical cycling genes by high-throughput qPCR, the relative abundances of genes involving in carbon degradation, carbon fixation, and denitrification were significantly higher in plastisphere than those of soil communities. This study greatly enhanced our understanding of biofilm formation and ecological effects of MPs in freshwater wetlands., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protists are less studied for their role and diversity in ecosystems. Notably, protists have played and still play an important role in microbialites. Microbialites, or lithified microbial mats, represent the oldest evidence of fossil biofilms (~3.5 Gyr). Modern microbialites may offer a unique proxy to study the potential role of protists within a geological context. We examined protist diversity in freshwater (Kelly and Pavilion Lake in British Columbia, Canada) and marine (Highborne Cay, Bahamas) to hypersaline (Shark Bay, Australia) microbialites to decipher their geomicrobiological role. The freshwater microbialite communities were clearly distinct from their marine and hypersaline counterparts. Chlorophytes had higher numerical abundance in freshwater microbialites; whereas pennate diatoms dominated numerically in marine microbialites. Despite the differences, protists across ecosystems may have adopted similar roles and functions. We suggest a consistent biogeochemical role of protists across microbialites globally; but that salinity may shape protist composition and evolution in these ecosystems., (© 2024. The Author(s).)

Skin microbiomes provide vital functions, yet knowledge about the drivers and processes structuring their species assemblages is limited-especially for non-model organisms. In this study, fish skin microbiome was assessed by high throughput sequencing of amplicon sequence variants from metabarcoding of V3-V4 regions in the 16S rRNA gene on fish hosts subjected to the following experimental manipulations: (i) translocation between fresh and brackish water habitats to investigate the role of environment; (ii) treatment with an antibacterial disinfectant to reboot the microbiome and investigate community assembly and priority effects; and (iii) maintained alone or in pairs to study the role of social environment and inter-host dispersal of microbes. The results revealed that fish skin microbiomes harbour a highly dynamic microbial composition that was distinct from bacterioplankton communities in the ambient water. Microbiome composition first diverged as an effect of translocation to either the brackish or freshwater habitat. When the freshwater individuals were translocated back to brackish water, their microbiome composition converged towards the fish microbiomes in the brackish habitat. In summary, external environmental conditions and individual-specific factors jointly determined the community composition dynamics, whereas inter-host dispersal had negligible effects. The dynamics of the microbiome composition was seemingly non-affected by reboot treatment, pointing towards high resilience to disturbance. The results emphasised the role of inter-individual variability for the unexplained variation found in many host-microbiome systems, although the mechanistic underpinnings remain to be identified., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Small water bodies, although supporting high biodiversity, are often understudied in the Alpine region. In this work, we characterized the planktic and benthic bacterial communities, as well as the water chemistry, of a wide physiographic range of 19 freshwater bodies within an Alpine Biosphere Reserve, including ponds, pasture ponds, peat bogs, shallow lakes, and lakes. We collected both water and surface sediment samples, followed by metabarcoding analysis based on the V3-V4 regions of the 16S rRNA gene. We investigated the changes in biodiversity and the distribution of unique and shared amplicon sequence variants (ASVs) between water (11,829 ASVs) and surface sediment (19,145 ASVs) habitats, as well as across different freshwater typologies. The majority of ASVs (78 %) were unique to a single sample, highlighting the variability and uniqueness of bacterial communities in such freshwater bodies. Most freshwater environments showed higher α-diversity in sediment samples (median, 1469 ASVs) compared to water (468 ASVs). We found that water and sediment habitats harboured unique bacterial communities with significant differences in their taxonomic compositions. Benthic bacteria were associated with several biogeochemical and degradative processes occurring in the sediments, with no notable differences among freshwater typologies and with phylogenetically and ecologically similar species. Conversely, planktic communities showed greater heterogeneity: small water bodies and peat bogs were characterized by higher relative abundances of Patescibacteria (up to 33 %), while lakes and shallow lakes were dominated by Actinobacteriota (up to 36 %). Cyanobacteria (426 ASVs) were generally distributed at low abundances in both water and sediment habitats. Overall, our results provided essential insights into the bacterial ecology of understudied environments such as ponds and pasture ponds and highlighted the importance of further exploring their rich pelagic and benthic bacterial biodiversity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Filamentous multicellular cable bacteria perform centimeter-scale electron transport in a process that couples oxidation of an electron donor (sulfide) in deeper sediment to the reduction of an electron acceptor (oxygen or nitrate) near the surface. While this electric metabolism is prevalent in both marine and freshwater sediments, detailed electronic measurements of the conductivity previously focused on the marine cable bacteria ( Candidatus Electrothrix), rather than freshwater cable bacteria, which form a separate genus ( Candidatus Electronema) and contribute essential geochemical roles in freshwater sediments. Here, we characterize the electron transport characteristics of Ca . Electronema cable bacteria from Southern California freshwater sediments. Current-voltage measurements of intact cable filaments bridging interdigitated electrodes confirmed their persistent conductivity under a controlled atmosphere and the variable sensitivity of this conduction to air exposure. Electrostatic and conductive atomic force microscopies mapped out the characteristics of the cell envelope's nanofiber network, implicating it as the conductive pathway in a manner consistent with previous findings in marine cable bacteria. Four-probe measurements of microelectrodes addressing intact cables demonstrated nanoampere currents up to 200 μm lengths at modest driving voltages, allowing us to quantify the nanofiber conductivity at 0.1 S/cm for freshwater cable bacteria filaments under our measurement conditions. Such a high conductivity can support the remarkable sulfide-to-oxygen electrical currents mediated by cable bacteria in sediments. These measurements expand the knowledgebase of long-distance electron transport to the freshwater niche while shedding light on the underlying conductive network of cable bacteria., Competing Interests: TY, MC, CN, SX, ME No competing interests declared, (© 2023, Yang, Chavez et al.)

Microcystis, a type of cyanobacteria known for producing microcystins (MCs), is experiencing a global increase in blooms. They have been recently recognized as potential contributors to the widespread of antibiotic resistance genes (ARGs). By reviewing approximately 150 pieces of recent studies, a hypothesis has been formulated suggesting that significant fluctuations in MCs concentrations and microbial community structure during Microcystis blooms could influence the dynamics of waterborne ARGs. Among all MCs, microcystin-LR (MC-LR) is the most widely distributed worldwide, notably abundant in reservoirs during summer. MCs inhibit protein phosphatases or increase reactive oxygen species (ROS), inducing oxidative stresses, enhancing membrane permeability, and causing DNA damage. This further enhances selective pressures and horizontal gene transfer (HGT) chances of ARGs. The mechanisms by which Microcystis regulates ARG dissemination have been systematically organized for the first time, focusing on the secretion of MCs and the alterations of bacterial community structure. However, several knowledge gaps remain, particularly concerning how MCs interfere with the electron transport chain and how Microcystis facilitates HGT of ARGs. Concurrently, the predominance of Microcystis forming the algal microbial aggregates is considered a hotspot for preserving and transferring ARGs. Yet, Microcystis can deplete the nutrients from other taxa within these aggregates, thereby reducing the density of ARG-carrying bacteria. Therefore, further studies are needed to explore the 'symbiotic - competitive' relationships between Microcystis and ARG-hosting bacteria under varied nutrient conditions. Addressing these knowledge gaps is crucial to understand the impacts of the algal aggregates on dynamics of waterborne antibiotic resistome, and underscores the need for effective control of Microcystis to curb the spread of antibiotic resistance. Constructed wetlands and photocatalysis represent advantageous strategies for halting the spread of ARGs from the perspective of Microcystis blooms, as they can effectively control Microcystis and MCs while maintaining the stability of aquatic ecosystem., Competing Interests: Declaration of competing interest Authors read and approved the final manuscript. There are no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx- dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria . Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties., Importance: Bacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced by Sphingomonas sp. LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering., Competing Interests: The authors declare no conflict of interest.

Background: Picocyanobacteria from the genera Prochlorococcus, Synechococcus, and Cyanobium are the most widespread photosynthetic organisms in aquatic ecosystems. However, their freshwater populations remain poorly explored, due to uneven and insufficient sampling across diverse inland waterbodies., Results: In this study, we present 170 high-quality genomes of freshwater picocyanobacteria from non-axenic cultures collected across Central Europe. In addition, we recovered 33 genomes of their potential symbiotic partners affiliated with four genera, Pseudomonas, Mesorhizobium, Acidovorax, and Hydrogenophaga. The genomic basis of symbiotic interactions involved heterotrophs benefiting from picocyanobacteria-derived nutrients while providing detoxification of ROS. The global abundance patterns of picocyanobacteria revealed ecologically significant ecotypes, associated with trophic status, temperature, and pH as key environmental factors. The adaptation of picocyanobacteria in (hyper-)eutrophic waterbodies could be attributed to their colonial lifestyles and CRISPR-Cas systems. The prevailing CRISPR-Cas subtypes in picocyanobacteria were I-G and I-E, which appear to have been acquired through horizontal gene transfer from other bacterial phyla., Conclusions: Our findings provide novel insights into the population diversity, ecology, and evolutionary strategies of the most widespread photoautotrophs within freshwater ecosystems. Video Abstract., (© 2024. The Author(s).)

Four methane-oxidizing bacteria, designated as strains WSC-6 T , WSC-7 T , SURF-1 T , and SURF-2 T , were isolated from Saddle Mountain Creek in southwestern Oklahoma, USA, and the Sanford Underground Research Facility (SURF) in Lead, South Dakota, USA. The strains were Gram-negative, motile, short rods that possessed intracytoplasmic membranes characteristic of type I methanotrophs. All four strains were oxidase-negative and weakly catalase-positive. Colonies ranged from pale pink to orange in colour. Methane and methanol were the only compounds that could serve as carbon and energy sources for growth. Strains WSC-6 T and WSC-7 T grew optimally at lower temperatures (25 and 20 °C, respectively) compared to strains SURF-1 T and SURF-2 T (40 °C). Strains WSC-6 T and SURF-2 T were neutrophilic (optimal pH of 7.5 and 7.3, respectively), while strains WSC-7 T and SURF-1 T were slightly alkaliphilic, with an optimal pH of 8.8. The strains grew best in media amended with ≤0.5% NaCl. The major cellular fatty acids were C 14 : 0 , C 16 : 1   ω 8 c , C 16 : 1   ω 7 c , and C 16 : 1   ω 5 c . The DNA G+C content ranged from 51.5 to 56.0 mol%. Phylogenetic analyses indicated that the strains belonged to the genus Methylomonas , with each exhibiting 98.6-99.6% 16S rRNA gene sequence similarity to closely related strains. Genome-wide estimates of relatedness (84.5-88.4% average nucleotide identity, 85.8-92.4% average amino acid identity and 27.4-35.0% digital DNA-DNA hybridization) fell below established thresholds for species delineation. Based on these combined results, we propose to classify these strains as representing novel species of the genus Methylomonas , for which the names Methylomonas rivi (type strain WSC-6 T =ATCC TSD-251 T =DSM 112293 T ), Methylomonas rosea (type strain WSC-7 T =ATCC TSD-252 T =DSM 112281 T ), Methylomonas aurea (type strain SURF-1 T =ATCC TSD-253 T =DSM 112282 T ), and Methylomonas subterranea (type strain SURF-2 T =ATCC TSD-254 T =DSM 112283 T ) are proposed.

Freshwater samples (n = 199) were obtained from 41 sites with contrasting land-uses (avian, low impact, dairy, urban, sheep and beef, and mixed sheep, beef and dairy) and the E. coli phylotype of 3980 isolates (20 per water sample enrichment) was determined. Eight phylotypes were identified with B1 (48.04%), B2 (14.87%) and A (14.79%) the most abundant. Escherichia marmotae (n = 22), and Escherichia ruysiae (n = 1), were rare (0.68%) suggesting that these environmental strains are unlikely to confound water quality assessments. Phylotypes A and B1 were overrepresented in dairy and urban sites (p < 0.0001), whilst B2 were overrepresented in low impact sites (p < 0.0001). Pathogens ((Salmonella, Campylobacter, Cryptosporidium or Giardia) and the presence of diarrhoeagenic E. coli-associated genes (stx and eae) were detected in 89.9% (179/199) samples, including 80.5% (33/41) of samples with putative non-recent faecal inputs. Quantitative PCR to detect microbial source tracking targets from human, ruminant and avian contamination were concordant with land-use type and E. coli phylotype abundance. This study demonstrated that a potential recreational health risk remains where pathogens occurred in water samples with low E. coli concentration, potential non-recent faecal sources, low impact sites and where human, ruminant and avian faecal sources were absent., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Three closely related, aerobic, Gram-stain-negative, motile, rod-shaped bacterial strains (PS-2 T , PS-17, and PS-19) were isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Colonies are pinkish-colored. The optimum growth occurred at 28-30 °C, and the pH was 6.5-7. The major cellular fatty acids were C16:1 ω7c, iso-C15.0, C17:1 ω8c, C18:1 ω7c, and C16:0. The predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and amino lipids. The genome size of strain PS-2 T is 4.8 Mbp, and the G + C content was 46.0%. The major fraction of genes were associated with biological processes (45.64%), followed by molecular function (29.86%) and cellular components (24.49%). The unique genes identified in strain PS-2 T secreted cyanophycinase, UDP-N-acetylglucosamine 2-epimerase, methyltransferase, kynureninase, ADA regulatory protein, biphenyl degradation, thermostable carboxypeptidase 1, tetrathionate respiration, etc. In addition, alanine and glutamate racemases were present. The 16S rRNA gene sequences shared 98.83-99.24% similarity with the closely related type strains of Shewanella. The ANI and AAI of strain PS-2 T with reference type strains of the genus Shewanella were below 95-96%, and the corresponding dDDH values were below 70%. A phylogenetic tree based on 16S rRNA gene sequences and genome-wide core genes revealed that strain PS-2 T clustered with Shewanella oneidensis LMG 19005 T in both phylogenetic trees. Based on the polyphasic analysis, the new isolates (PS-2 T , PS-17, and PS-19) represent a novel species of Shewanella, for which Shewanella cutis sp. nov. is proposed. The type strain is PS-2 T (= TBRC 15838 T  = NBRC 115342 T )., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

A Gram-strain-negative, aerobic, yellow-colored, non-motile, and rod-shaped bacterial strain, designated IMCC34852 T , was isolated from a freshwater stream in the Republic of Korea. Cellular growth occurred at 10-37 °C, pH 6.0-9.0, and with 0-0.5% (w/v) NaCl. The 16S rRNA gene sequence analysis showed that strain IMCC34852 T belonged to the genus Flavobacterium and that the strain was most closely related to F. cheonhonense ARSA-15  T (97.6%), F. buctense T7 T (96.7%), F. silvisoli RD-2-33  T (96.1%), and F. paronense KNUS1 T (96.1%). The whole-genome sequence of strain IMCC34852 T was 3.2 Mbp in size, with a DNA G + C content 37.3%. The average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) values between strain IMCC34852 T and its related species were all below 79.8% and 22.7%, respectively, which are significantly lower than the thresholds of 95% for ANI and 70% for DDH for species delineation. The major respiratory quinone of strain IMCC34852 T was menaquinone-6 (MK-6) and the predominant cellular fatty acids were iso-C 15:0 (32.6%), iso-C 16:0 (11.7%), iso-C 15:1 G (10.3%), and iso-C 14:0 (6.7%). The major polar lipids of the strain were phosphatidylethanolamine, two unidentified aminolipids and six unidentified lipids. Based on these results, it was concluded that strain IMCC34852 T represents a novel species in the genus Flavobacterium, for which the name Flavobacterium rivulicola sp. nov is proposed. The type strain of the proposed novel species is IMCC34852 T (= KACC 23133  T  = KCTC 82066  T  = NBRC 114419  T )., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Environmental concentrations of antimicrobials can inhibit Cyanobacteria, but little is known about their effects on Cyanobacteria-blooming freshwater ecosystem. Here, a 21 days' outdoor freshwater mesocosm experiment was established to study effects of single and combined tetracycline, triclocarban and zinc at environmental concentrations on microbial community, microbial function and antimicrobial resistance using amplicon- and metagenomic-based methods. Results showed that three chemicals reshaped the microbial community with magnified effects by chemical combinations. Relative abundance of Cyanobacteria was decreased in all chemical groups, especially from 74.5 to 0.9% in combination of three chemicals. Microbial community networks were more simplified after exposure. Proteobacteria and Bacteroidetes predominated in Cyanobacteria-degraded ecosystems, and their relative abundances were significantly correlated with antibiotic resistome, suggesting that they might host antibiotic resistance genes. Notably, relative abundance (copy per 16 S rRNA gene) of total antibiotic resistome reached five to nine folds higher than the initial abundance in chemical-combined groups. The affected antibiotic resistance genes referred to a wide range of antibiotic classes. However, weak effects were detected on biocide/metal resistance and microbial virulence. Three chemicals posed complicated effects on microbial function, some of which had consistent variations across the groups, while some varied greatly in chemical groups. The findings highlight sensitivity of Cyanobacteria-blooming ecosystem to antimicrobials., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nitrification by aquarium biofilters transforms ammonia waste (NH 3 /NH 4 + ) to less toxic nitrate (NO 3 - ) via nitrite (NO 2 - ). Prior to the discovery of complete ammonia-oxidizing ("comammox" or CMX) Nitrospira , previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquaria. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples (average CMX amoA genes: 2.2 × 10 3 ± 1.5 × 10 3 copies/ng) and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples (average AOA amoA genes: 1.1 × 10 3 ± 2.7 × 10 3 copies/ng) and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters (average of 3.2 × 10 1 ± 1.1 × 10 2 copies of AOB amoA genes/ng of DNA), except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results demonstrate that comammox Nitrospira plays a previously overlooked, but important role in home aquarium biofilter nitrification., Importance: Nitrification is a crucial process that converts toxic ammonia waste into less harmful nitrate that occurs in aquarium biofilters. Prior research found that ammonia-oxidizing archaea (AOA) were dominant over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Our study profiled microbial communities of aquarium biofilters and quantified the abundance of all currently known groups of aerobic ammonia oxidizers. The findings reveal that complete ammonia-oxidizing (comammox) Nitrospira were present in all freshwater aquarium biofilter samples in high abundance, challenging our previous understanding of aquarium nitrification. We also highlight niche adaptation of ammonia oxidizers based on salinity. The network analysis of freshwater biofilter microbial communities revealed significant positive correlations among nitrifiers and other community members, suggesting intricate interactions within biofilter communities. Overall, this study expands our understanding of nitrification in aquarium biofilters, emphasizes the role of comammox Nitrospira , and highlights the value of aquaria as microcosms for studying nitrifier ecology., Competing Interests: Subsequent to when this research was conducted, J.D.N. received a research grant from the Natural Sciences and Engineering Research Council of Canada (NSERC), with industry partner Hagen Industries, to explore the possibility of using AOA and comammox Nitrospira to improve existing aquarium biological supplements.

A novel Gram-negative, white-pigmented, and auxin-producing strain, 20NA77.5 T , was isolated from fresh water during cyanobacterial bloom period. Pairwise comparison of the 16S rRNA gene sequences showed that strain 20NA77.5 T belonged to the genus Undibacterium and exhibited the highest sequence similarity to the type strains of Undibacterium danionis (98.00%), Undibacterium baiyunense (97.93%), Undibacterium macrobrachii (97.92%), and Undibacterium fentianense (97.71%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain 20NA77.5 T and its related type strains were below 79.93 and 23.80%, respectively. The predominant fatty acids (> 10% of the total fatty acids) were C 16:0 and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c). The genomic DNA G + C content of strain 20NA77.5 T was found to be 48.61%. Based on the phylogenetic distinctness, chemotaxonomic features, and phenotypic features, strain 20NA77.5 T is considered to represent a novel species of the genus Undibacterium, for which the name Undibacterium cyanobacteriorum sp. nov is proposed. The type strain is 20NA77.5 T (= KCTC 8005 T  = LMG 33136 T )., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The fish gut microbiome is well known for its role in degrading nutrients to improve the host's digestion and absorption efficiency. In this study, we focused on the core physiological adaptability during the various reproductive stages of the black Amur bream ( Megalobrama terminalis ) to explore the interaction mechanisms among the fish host gut mucosal structure, gut enzyme activity, and gut microbial metabolism in the course of the host's reproductive cycle. Our findings showed that M. terminalis exhibited locomotion metabolic type (aids in sporting) in the reproductive stage, and a change to visceral metabolic type (aids in digestion) during non-reproductive and post-reproductive stage phases. The impact of metabolic type selection and energy demand during various reproductive stages on fish nutrition strategy and digestive function was substantial. Our resulted showed that mitochondria in intestinal epithelial cells of reproductive M. terminalis appeared autophagy phenomenon, and the digestive enzyme activities in the intestines of reproductive M. terminalis were lower than those in the non-reproductive and post-reproductive individuals. Moreover, these differences in nutrition strategy have a prominent impact on the gut microbiome of reproductive M. terminalis , compared to non-reproductive and post-reproductive samples. Our findings showed that reproductive females had lower levels of alpha diversity compared to non-reproductive and post-reproductive females. Our results also showed a greater functional variety and an increase in functional genes related to carbohydrate, lipid, amino acid, cofactors, and vitamin metabolic pathways in the NRS and PRS group. It is noteworthy that an enrichment of genes encoding putative enzymes implicated in the metabolism of taurine and hypotaurine was observed in the RS samples. Our findings illustrated that the stability and resilience of the gut bacterial community could be shaped in the wild fish host-microbiome interactions during reproductive life history.

Urban ecosystems are subjected to multiple anthropogenic stresses, which impact aquatic communities. Artificial light at night (ALAN) for instance can significantly alter the composition of algal communities as well as the photosynthetic cycles of autotrophic organisms, possibly leading to cellular oxidative stress. The combined effects of ALAN and chemical contamination could increase oxidative impacts in aquatic primary producers, although such combined effects remain insufficiently explored. To address this knowledge gap, a one-month experimental approach was implemented under controlled conditions to elucidate effects of ALAN and dodecylbenzyldimethylammonium chloride (DDBAC) on aquatic biofilms. DDBAC is a biocide commonly used in virucidal products, and is found in urban aquatic ecosystems. The bioaccumulation of DDBAC in biofilms exposed or not to ALAN was analyzed. The responses of taxonomic composition, photosynthetic activity, and fatty acid composition of biofilms were examined. The results indicate that ALAN negatively affects photosynthetic yield and chlorophyll production of biofilms. Additionally, exposure to DDBAC at environmental concentrations induces lipid peroxidation, with an increase of oxylipins. This experimental study provides first insights on the consequences of ALAN and DDBAC for aquatic ecosystems. It also opens avenues for the identification of new biomarkers that could be used to monitor urban pollution impacts in natural environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: The purpose of this work was to study extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021., Methods and Results: Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n = 642) and AmpC (n = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 gene groups by PCR. Genes belonging to blaCTX-M-1 and blaCTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No blaCTX-M-2 group's gene was found. A subset of strains (n = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. β-lactamase genes identified were blaCTX-M (n = 21), with blaCTX-M-15 mostly represented (n = 15), as well as blaTEM (n = 11), blaOXA (n = 7), blaSHV (n = 9), and carbapenemase (CP) genes were observed in several strains-blaKPC-3 (n = 19), blaNDM-1 (n = 1), blaVIM-1 (n = 2), and blaOXA-244 (n = 2)-even from freshwaters., Conclusions: ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Distance-decay (DD) equations can discern the biogeographical pattern of organisms and genes in a better way with advanced statistical methods. Here, we developed a data Compilation, Arrangement, and Statistics framework to advance quantile regression (QR) into the generation of DD equations for antibiotic resistance genes (ARGs) across various spatial scales using freshwater reservoirs as an illustration. We found that QR is superior at explaining dissemination potential of ARGs to the traditionally used least squares regression (LSR). This is because our model is based on the 'law of limiting factors', which reduces influence of unmeasured factors that reduce the efficacy of the LSR method. DD equations generated from the 99th QR model for ARGs were 'S all = 90.03e -0.01 Dall ' in water and 'S all = 92.31e -0.011 Dall ' in sediment. The 99th QR model was less impacted by uneven sample sizes, resulting in a better quantification of ARGs dissemination. Within an individual reservoir, the 99th QR model demonstrated that there is no dispersal limitation of ARGs at this smaller spatial scale. The QR method not only allows for construction of robust DD equations that better display dissemination of organisms and genes across ecosystems, but also provides new insights into the biogeography exhibited by key parameters, as well as the interactions between organisms and environment., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The freshwater microbiome harbours numerous copiotrophic bacteria that rapidly respond to elevated substrate concentrations. We hypothesized that their high centimetre-scale beta diversity in lake water translates into pronounced metabolic variability, and that a large fraction of microbial 'metabolic potential' originates from point sources such as fragile organic aggregates. Three experiments were conducted in pre-alpine Lake Zurich over the course of a harmful cyanobacterial bloom: Spatially explicit 9 ml 'syringe' samples were collected in situ at centimetre distances along with equally sized 'mixed' samples drawn from pre-homogenized lake water and incubated in BIOLOG EcoPlate substrate arrays. Fewer compounds promoted bacterial growth in the syringe than in the mixed samples, in particular during the pre- and late bloom periods. Community analysis of enrichments on three frequently utilized substrates revealed both pronounced heterogeneity and functional redundancy. Bacterial consortia had higher richness in mixed than in syringe samples and differed in composition. Members of the Enterobacter cloacae complex dominated the EcoPlate assemblages during the mid-bloom period irrespective of treatment or substrate. We conclude that small-scale functional dispersal limitation among free-living copiotrophs in lake water reduces local biotransformation potential, and that lacustrine blooms of harmful cyanobacteria can be environmental reservoirs for metabolically versatile potential pathogens., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

The rising global demand for agricultural products is leading to the widespread application of pesticides, such as spinetoram, resulting in environmental pollution and ecotoxicity to nontarget organisms in aquatic ecosystems. This research focused on assessing the toxicity of spinetoram at various concentrations (0, 0.01, 0.1, 0.5, 1.0, and 3.0 mg L -1 ) on two common freshwater microalgae, Chlorella vulgaris and Microcystis aeruginosa, to shed light on the ecotoxicological effects of insecticides. Our findings demonstrate that M. aeruginosa is more sensitive to spinetoram than is C. vulgaris, with a concentration-dependent reduction in the growth rate observed for M. aeruginosa, whereas only the highest concentration of spinetoram adversely affected C. vulgaris. At a concentration of 0.01 mg L -1 , the growth rate of M. aeruginosa unexpectedly increased beginning on day 7, indicating a potential hormetic effect. Although initial exposure to spinetoram improved the photosynthetic efficiency of both microalgae strains at all concentrations, detrimental effects became apparent at higher concentrations and with prolonged exposure. The photosynthetic efficiency of C. vulgaris recovered, in contrast to that of M. aeruginosa, which exhibited limited recovery. Spinetoram more significantly inhibited the effective quantum yield of PSII (EQY) in M. aeruginosa than in C. vulgaris. Although spinetoram is not designed to target phytoplankton, its toxicity can disrupt primary productivity and modify phytoplankton-consumer interactions via bottom-up control mechanisms. This study enhances our understanding of spinetoram's ecotoxicity and potential effects on aquatic ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Faecal contamination of surface waters has the potential to spread not only pathogenic organisms but also antimicrobial resistant organisms. During the bathing season of 2021, weekly water samples, from six selected coastal bathing locations (n = 93) and their freshwater tributaries (n = 93), in Northern Ireland (UK), were examined for concentrations of faecal indicator bacteria Escherichia coli and intestinal enterococci. Microbial source tracking involved detection of genetic markers from the genus Bacteroides using PCR assays for the general AllBac marker, the human HF8 marker and the ruminant BacR marker for the detection of human, and ruminant sources of faecal contamination. The presence of beta-lactamase genes blaOXA-48, blaKPC, and blaNDM-1 was determined using PCR assays for the investigation of antimicrobial resistance genes that are responsible for lack of efficacy in major broad-spectrum antibiotics. The beta-lactamase gene blaOXA-48 was found in freshwater tributary samples at all six locations. blaOXA-48 was detected in 83% of samples that tested positive for the human marker and 69% of samples that tested positive for the ruminant marker over all six locations. This study suggests a risk of human exposure to antimicrobial resistant bacteria where bathing waters receive at least episodically substantial transfers from such tributaries., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as β-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05-1.36 g L -1 after 8 days. The species secreted extracellular β-galactosidase (up to 40.84 ± 0.23 U L -1 ) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae-bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53-80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting β-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive β-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams., (© 2024. The Author(s).)

The central carbon (C) metabolic network is responsible for most of the production of energy and biosynthesis in microorganisms and is therefore key to a mechanistic understanding of microbial life in soil communities. Many upland soil communities have shown a relatively high C flux through the pentose phosphate (PP) or the Entner-Doudoroff (ED) pathway, thought to be related to oxidative damage control. We tested the hypothesis that the metabolic organization of the central C metabolic network differed between two ecosystems, an anoxic marsh soil and oxic upland soil, and would be affected by altering oxygen concentrations. We expected there to be high PP/ED pathway activity under high oxygen concentrations and in oxic soils and low PP/ED activity in reduced oxygen concentrations and in marsh soil. Although we found high PP/ED activity in the upland soil and low activity in the marsh soil, lowering the oxygen concentration for the upland soil did not reduce the relative PP/ED pathway activity as hypothesized, nor did increasing the oxygen concentration in the marsh soil increase the PP/ED pathway activity. We speculate that the high PP/ED activity in the upland soil, even when exposed to low oxygen concentrations, was related to a high demand for NADPH for biosynthesis, thus reflecting higher microbial growth rates in C-rich soils than in C-poor sediments. Further studies are needed to explain the observed metabolic diversity among soil ecosystems and determine whether it is related to microbial growth rates.IMPORTANCEWe observed that the organization of the central carbon (C) metabolic processes differed between oxic and anoxic soil. However, we also found that the pentose phosphate pathway/Entner-Doudoroff (PP/ED) pathway activity remained high after reducing the oxygen concentration for the upland soil and did not increase in response to an increase in oxygen concentration in the marsh soil. These observations contradicted the hypothesis that oxidative stress is a main driver for high PP/ED activity in soil communities. We suggest that the high PP/ED activity and NADPH production reflect higher anabolic activities and growth rates in the upland soil compared to the anaerobic marsh soil. A greater understanding of the molecular and biochemical processes in soil communities is needed to develop a mechanistic perspective on microbial activities and their relationship to soil C and nutrient cycling. Such an increased mechanistic perspective is ecologically relevant, given that the central carbon metabolic network is intimately tied to the energy metabolism of microbes, the efficiency of new microbial biomass production, and soil organic matter formation., Competing Interests: The authors declare no conflict of interest.

The microbial degradation of polyethylene (PE) and polypropylene (PP) resins in rivers and lakes has emerged as a crucial issue in the management of microplastics. This study revealed that as the flow rate decreased longitudinally, ammonia nitrogen (NH 4 + -N), heavy fraction of organic carbon (HFOC), and small-size microplastics (< 1 mm) gradually accumulated in the deep and downstream estuarine sediments. Based on their surface morphology and carbonyl index, these sediments were identified as the potential hot zone for PE/PP degradation. Within the identified hot zone, concentrations of PE/PP-degrading genes, enzymes, and bacteria were significantly elevated compared to other zones, exhibiting strong intercorrelations. Analysis of niche differences revealed that the accumulation of NH 4 + -N and HFOC in the hot zone facilitated the synergistic coexistence of key bacteria responsible for PE/PP degradation within biofilms. The findings of this study offer a novel insight and comprehensive understanding of the distribution characteristics and synergistic degradation potential of PE/PP in natural freshwater environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The unexpected foodborne outbreak in Singapore in 2015 has accentuated Group B Streptococcus (GBS, Streptococcus agalactiae ) sequence type 283 as an emerging foodborne pathogen transmitted via the consumption of contaminated raw freshwater fish. Isolation-based workflows utilizing conventional microbiological and whole-genome sequencing methods are commonly used to support biosurveillance efforts critical for the control management of this emerging foodborne pathogen. However, these isolation-based workflows tend to have relatively long turnaround times that hamper a timely response for implementing risk mitigation. To address this gap, we have developed a metagenomics-based workflow for the simultaneous detection and genomic characterization of GBS in raw freshwater fish. Notably, our validation results showed that this metagenomics-based workflow could achieve comparable accuracy and potentially better detection limits while halving the turnaround time (from 2 weeks to 5 days) relative to an isolation-based workflow. The metagenomics-based workflow was also successfully adapted for use on a portable long-read nanopore sequencer, demonstrating its potential applicability for real-time point-of-need testing. Using GBS in freshwater fish as an example, this work represents a proof-of-concept study that supports the feasibility and validity of metagenomics as a rapid and accurate test methodology for the detection and genomic characterization of foodborne pathogens in complex food matrices., Importance: The need for a rapid and accurate food microbiological testing method is apparent for a timely and effective foodborne outbreak response. This is particularly relevant for emerging foodborne pathogens such as Group B Streptococcus (GBS) whose associated food safety risk might be undercharacterized. By using GBS in raw freshwater fish as a case example, this study describes the development of a metagenomics-based workflow for rapid food microbiological safety testing and surveillance. This study can inform as a working model for various foodborne pathogens in other complex food matrices, paving the way for future methodological development of metagenomics for food microbiological safety testing., Competing Interests: The authors declare no conflict of interest.

A neutrophilic iron-oxidizing and -reducing bacterium, strain MIZ03 T , was previously isolated from a wetland in Ibaraki, Japan. Here, we report the detailed characteristics of this strain. It was motile with a single polar flagellum, and Gram-stain-negative. It could grow not only chemolithoautotrophically but also chemoorganotrophically by aerobic respiration and fermentation. Major cellular fatty acids were C 16 : 1  ω 7 c /C 16 : 1  ω 6 c , and C 16 : 0 . Phylogenetic analyses indicated that strain MIZ03 T belonged to the genus Rhodoferax . This strain was closely related to Rhodoferax ferrireducens with 98.5 % of 16S rRNA gene sequence similarity. Based on its phenotypic and genomic based characteristics, we conclude that strain MIZ03 T represents a new species in the genus Rhodoferax . We propose the name Rhodoferax lithotrophicus sp. nov. to accommodate this strain. The type strain is MIZ03 T (=JCM 34246 T =DSM 113266 T ). We also propose the name Rhodoferax koreensis sp. nov., of which the type strain is DCY110 T (=KCTC 52288 T =JCM 31441 T ), for the effectively, but not yet validly, published name ' Rhodoferax koreense '.