Leveraging aquatic-terrestrial interfaces to capture putative habitat generalists.

Habitat type is a strong determinant of microbial composition. Habitat interfaces, such as the boundary between aquatic and terrestrial systems, present unique combinations of abiotic factors for microorganisms to contend with. Aside from the spillover of certain harmful microorganisms from agricultural soils into water (e.g. fecal coliform bacteria), we know little about the extent of soil-water habitat switching across microbial taxa. In this study, we developed a proof-of-concept system to facilitate the capture of putatively generalist microorganisms that can colonize and persist in both soil and river water. We aimed to examine the phylogenetic breadth of putative habitat switchers and how this varies across different source environments. Microbial composition was primarily driven by recipient environment type, with the strongest phylogenetic signal seen at the order level for river water colonizers. We also identified more microorganisms colonizing river water when soil was collected from a habitat interface (i.e. soil at the side of an intermittently flooded river, compared to soil collected further from water sources), suggesting that environmental interfaces could be important reservoirs of microbial habitat generalists. Continued development of experimental systems that actively capture microorganisms that thrive in divergent habitats could serve as a powerful tool for identifying and assessing the ecological distribution of microbial generalists.

Detection of Salmonella enterica and Listeria monocytogenes in alternative irrigation water by culture and qPCR-based methods in the Mid-Atlantic U.S.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results. IMPORTANCE: Detecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence of Salmonella enterica and Listeria monocytogenes in irrigation water samples examined in this study.

[Up to Date of Cyanobacterial Natural Products].

More than 2000 compounds have been reported from cyanobacteria. The most successful example is dolastatin 10, of which a related compound monomethylauristatin E is used as antibody-drug conjugate (ADC) for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Recently genome-based analyses by Piel led to the discovery of novel compounds from cyanobacteria. W. H. Gerwick found a potential as anti-SARS-CoV-2 agent in gallinamide A, which was reported as a cathepsin L inhibitor. In our group columbamides were isolated from the marine cyanobacterium Moorena bouillonii. The geometry of the double bond was determined by the coupling constant obtained using non-decoupled heteronuclear single quantum coherence (HSQC). The configuration of chloromethine in a long-chain acyl moiety was determined by the Ohrui method at room temperature using a chiral HPLC column. Columbamide D showed biosurfactant activity. One strain many compounds (OSMAC) is a method to discover new compounds by changing culture conditions. Prior to our experiments, attempts to apply OSMAC in cyanobacteria resulted in the induction or up-regulation of only known compounds. The heat shock culture of the freshwater cyanobacterium Microcystis aeruginosa up-regulated a ribosomal peptide argicyclamide C. At the same time, we discovered bis-prenylated and monoprenylated argicyclamides A and B. More recently iron-limited culture produced hydroxylated argicyclamide A. OSMAC and genome-based screening could lead the discovery of unique biologically active compounds from cyanobacteria.

What, How, When, and Where: Spatiotemporal Water Quality Hazards of Cyanotoxins in Subtropical Eutrophic Reservoirs.

Though toxins produced during harmful blooms of cyanobacteria present diverse risks to public health and the environment, surface water quality surveillance of cyanobacterial toxins is inconsistent, spatiotemporally limited, and routinely relies on ELISA kits to estimate total microcystins (MCs) in surface waters. Here, we employed liquid chromatography tandem mass spectrometry to examine common cyanotoxins, including five microcystins, three anatoxins, nodularin, cylindrospermopsin, and saxitoxin in 20 subtropical reservoirs spatially distributed across a pronounced annual rainfall gradient. Probabilistic environmental hazard analyses identified whether water quality values for cyanotoxins were exceeded and if these exceedances varied spatiotemporally. MC-LR was the most common congener detected, but it was not consistently observed with other toxins, including MC-YR, which was detected at the highest concentrations during spring with many observations above the California human recreation guideline (800 ng/L). Cylindrospermopsin was also quantitated in 40% of eutrophic reservoirs; these detections did not exceed a US Environmental Protection Agency swimming/advisory level (15,000 ng/L). Our observations have implications for routine water quality monitoring practices, which traditionally use ELISA kits to estimate MC levels and often limit collection of surface samples during summer months near reservoir impoundments, and further indicate that spatiotemporal surveillance efforts are necessary to understand cyanotoxins risks when harmful cyanobacteria blooms occur throughout the year.

Roseateles amylovorans sp. nov., isolated from freshwater.

A Gram-stain-negative, rod-shaped, amylolytic bacterial strain, designated as bsSlp3-1T, was isolated from the Slepian water system, a freshwater reservoir. Strain bsSlp3-1T was found to be aerobic, oxidase-positive and catalase-negative, grew at 5-37 °C (optimum, 28 °C), pH 5.0-9.5 (optimum, pH 7.0) and low NaCl concentration (up to 1.0 %). Comparative analysis of 16S rRNA gene sequence similarity revealed that strain bsSlp3-1T clustered with Roseateles species and is closely related to Roseateles depolymerans KCTC 42856T (98.7 %) and Roseateles terrae CCUG 52222T (98.6 %). Whole-genome comparisons using average nucleotide identity and digital DNA-DNA hybridization values suggested that strain bsSlp3-1T represents a novel species within the genus Roseateles and is most closely related to Roseateles aquatilis CCUG 48205T (81.2 and 25.6 %, respectively). The genome of strain bsSlp3-1T consisted of a single circular chromosome with size 6 289 366 bp and DNA G+C content of 66.8 mol%. The predominant cellular fatty acids of bsSlp3-1T were cis-9-hexadecanoic and hexadecenoic acids. According to the data obtained in this work, strain bsSlp3-1T represents a novel Roseateles species for which the name Roseateles amylovorans sp. nov. is proposed. The type strain is bsSlp3-1T (=BIM B-1768T=NBIMCC 9098T=VKM B-3671T).

Flavobacterium psychrotrophum sp. nov. and Flavobacterium panacagri sp. nov., Isolated from Freshwater and Soil.

Two novel bacterial strains CJ74T and CJ75T belonging to the genus Flavobacterium were isolated from freshwater of Han River and ginseng soil, South Korea, respectively. Strain CJ74T was Gram-stain-negative, aerobic, rod-shaped, non-motile, and non-flagellated, and did not produce flexirubin-type pigments. Strain CJ75T was Gram-stain-negative, aerobic, rod-shaped, motile by gliding, and non-flagellated, and produced flexirubin-type pigments. Both strains were shown to grow optimally at 30 °C in the absence of NaCl on R2A medium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains CJ74T and CJ75T belonged to the genus Flavobacterium and were most closely related to Flavobacterium niveum TAPW14T and Flavobacterium foetidum CJ42T with 96.17% and 97.29% 16S rRNA sequence similarities, respectively. Genomic analyses including the reconstruction of phylogenomic tree, average nucleotide identity, and digital DNA-DNA hybridization suggested that they were novel species of the genus Flavobacterium. Both strains contained menaquinone 6 (MK-6) as the primary respiratory quinone and phosphatidylethanolamine as a major polar lipid. The predominant fatty acids of both strains were iso-C15:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). Based on the polyphasic taxonomic study, strains CJ74T and CJ75T represent novel species of the genus Flavobacterium, for which names Flavobacterium psychrotrophum sp. nov. and Flavobacterium panacagri sp. nov. are proposed, respectively. The type strains are CJ74T (=KACC 19819T =JCM 32889T) and CJ75T (=KACC 23149T =JCM 36132T).

Most treatments to control freshwater algal blooms are not effective: Meta-analysis of field experiments.

Harmful algal blooms negatively impact freshwater, estuarine, and marine systems worldwide, including those used for drinking water, recreation, and aquaculture, through the production of toxic and nontoxic secondary metabolites as well as hypoxic events that occur when algal blooms degrade. Consequently, water resource managers often utilize chemical, bacterial, physical, and/or plant-based treatments to control algal blooms and improve water quality. However, awareness of available treatments may be limited, and there is ambiguity among the effects of algal bloom treatments across studies. Such variation within the literature and lack of knowledge of other tested treatments leave uncertainty for water resource managers when deciding what treatments are best to control algal blooms and improve water quality. Our primary objective was to synthesize data from 39 published and unpublished studies that used one of 28 chemical, bacterial, physical, and/or plant-based treatments in field experiments on various water quality measurements, including phytoplankton pigments and cell density, cyanobacterial toxins (microcystin), and common off-flavors (i.e., taste and odor compounds; geosmin and 2-methylisoborneol). We hypothesized that treatments would improve water quality. Across all studies and treatment types (227 effect sizes), water quality improvements were observed when measured at the time of greatest decline following treatment or at the end of the experiment. However, these findings were primarily mediated by only four chemicals, namely copper sulfate, hydrogen peroxide, peracetic acid, and simazine. None of the bacterial, physical, or plant-based treatments were shown to significantly improve water quality by themselves. Results from this synthesis quantitatively showed that most treatments fail to improve water quality in the field and highlight the need for more research on existing and alternative treatments.

Antimicrobial resistance in southeast Asian water environments: A systematic review of current evidence and future research directions.

Antimicrobial resistance has been a serious and complex issue for over a decade. Although research on antimicrobial resistance (AMR) has mainly focused on clinical and animal samples as essential for treatment, the AMR situation in aquatic environments may vary and have complicated patterns according to geographical area. Therefore, this study aimed to examine recent literature on the current situation and identify gaps in the AMR research on freshwater, seawater, and wastewater in Southeast Asia. The PubMed, Scopus, and ScienceDirect databases were searched for relevant publications published from January 2013 to June 2023 that focused on antimicrobial resistance bacteria (ARB) and antimicrobial resistance genes (ARGs) among water sources. Based on the inclusion criteria, the final screening included 41 studies, with acceptable agreement assessed using Cohen's inter-examiner kappa equal to 0.866. This review found that 23 out of 41 included studies investigated ARGs and ARB reservoirs in freshwater rather than in seawater and wastewater, and it frequently found that Escherichia coli was a predominant indicator in AMR detection conducted by both phenotypic and genotypic methods. Different ARGs, such as blaTEM, sul1, and tetA genes, were found to be at a high prevalence in wastewater, freshwater, and seawater. Existing evidence highlights the importance of wastewater management and constant water monitoring in preventing AMR dissemination and strengthening effective mitigation strategies. This review may be beneficial for updating current evidence and providing a framework for spreading ARB and ARGs, particularly region-specific water sources. Future AMR research should include samples from various water systems, such as drinking water or seawater, to generate contextually appropriate results. Robust evidence regarding standard detection methods is required for prospective-era work to raise practical policies and alerts for developing microbial source tracking and identifying sources of contamination-specific indicators in aquatic environment markers.

Cyanotoxins, biosynthetic gene clusters, and factors modulating cyanotoxin biosynthesis.

Cyanobacterial harmful algal blooms (CHABs) are a global environmental concern that encompasses public health issues, water availability, and water quality owing to the production of various secondary metabolites (SMs), including cyanotoxins in freshwater, brackish water, and marine ecosystems. The frequency, extent, magnitude, and duration of CHABs are increasing globally. Cyanobacterial species traits and changing environmental conditions, including anthropogenic pressure, eutrophication, and global climate change, together allow cyanobacteria to thrive. The cyanotoxins include a diverse range of low molecular weight compounds with varying biochemical properties and modes of action. With the application of modern molecular biology techniques, many important aspects of cyanobacteria are being elucidated, including aspects of their diversity, gene-environment interactions, and genes that express cyanotoxins. The toxicological, environmental, and economic impacts of CHABs strongly advocate the need for continuing, extensive efforts to monitor cyanobacterial growth and to understand the mechanisms regulating species composition and cyanotoxin biosynthesis. In this review, we critically examined the genomic organization of some cyanobacterial species that lead to the production of cyanotoxins and their characteristic properties discovered to date.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

The effect of Paenibacillus on IDEXX Enterolert results from freshwater stream environments.

Enterolert, a fluorogenic substrate test, is used as a quantitative method for determining freshwater concentrations of Enterococcus for water quality indicators. However, there is some evidence from recent studies suggesting that Enterolert may not suppress false positives due to pollution sources in waterbodies. In this study, we evaluated this method by analyzing field water and sediment samples from four freshwater streams. We also performed a laboratory microcosm study from two of the stream sediments. The Enterolert method was investigated by phenotypic and genomic analyses for accuracy of isolating and quantifying Enterococcus and/or Streptococcus. Additionally, we tested isolates from Enterolert panels for antibiotic resistance. Results from the field and microcosm studies from initial to final time points indicated that false positives were predominantly Paenibacillus spp. and other non-fecal indicator bacteria. Furthermore, the microcosm study indicated shifts from lactic acid to non-lactic acid bacteria between initial to final time points, but Enterococcus concentrations from Enterolert panels remained stable for the duration of the study for both stream sediments. Antibiotic resistance indicated no distinct pattern of resistance or susceptibility to a suite of antibiotics. However, all isolates tested were resistant to bacitracin and nalidixic acid. In conclusion, we found that Enterolert was not exclusively selective for Enterococcus from freshwater environments and that sediment and polluted waterbodies have the potential to skew the presumed concentrations. More research is needed to evaluate the effectiveness and selectivity of the medium used for the fluorogenic substrate test for Enterococcus enumeration.

Persistence of human- and cattle-associated Bacteroidales and mitochondrial DNA markers in freshwater mesocosms.

Accurate identification of the origins of non-point source pollution is essential for the effective control of fecal pollution. Host-associated Bacteroidales and mitochondrial DNA (mtDNA) markers have been developed to identify the sources of human and cattle fecal pollution. However, the differences in persistence between these two types of markers under different environmental conditions are still poorly understood. Here, we conducted mesocosm experiments to investigate the influence of indigenous microbiota and nutrients on the decay of Bacteroidales and mtDNA markers associated with humans and cattle. Raw sewage or cattle feces were inoculated into mesocosms containing natural eutrophic water, sterile eutrophic water or artificial freshwater. The Bacteroidales markers HF183 (human) and CowM3 (cattle) and mtDNA markers HcytB (human) and QMIBo (cattle) were quantified using the quantitative polymerase chain reaction (qPCR) assays. All markers but HF183 decreased the fastest in the presence of indigenous microbiota. Nutrients caused a decrease in the persistence of HF183; however, no significant nutrient effects were observed for HcytB, CowM3, and QMIBo. The time to reach one log reduction (T90) for HF183 and HcytB was similar; CowM3 reached T90 earlier than QMIBo in all the treatments but eutrophic water. E. coli persisted longer than both Bacteroidales and mtDNA markers in the mesocosms regardless of inoculum type. Additionally, 16S rRNA gene amplicon sequencing was used to determine the changes in bacterial communities accompanying the marker decay. Analysis using the SourceTracker software showed that bacterial communities in the mesocosms became more dissimilar to those in the corresponding inoculants over time. Our results indicate that environmental factors are important determinants of genetic markers' persistence, but their impact can vary depending on the genetic markers. The cattle Bacteroidales markers may be more suitable for determining recent fecal contamination than cattle mtDNA.

First redescription and molecular phylogeny of Trimyema claviforme Kahl, 1933 with the description of a Chinese population of Plagiopyla nasuta Stein, 1860 (Ciliophora, Plagiopylea).

Ciliates belonging to the class Plagiopylea are obligate anaerobes that are often neglected due to their cryptic lifestyles, difficulty of observation, and overall under-sampling. Here, we investigate two species, namely Trimyema claviforme Kahl, 1933 and Plagiopyla nasuta Stein, 1860, collected in China from marine and freshwater anaerobic sediments, respectively. A complete morphological dataset, together with SSU rRNA gene sequence data were obtained and used to diagnose the species. No molecular sequencing had ever been performed on Trimyema claviforme, with its ciliature also previously unknown. Based on these novel data presented here, the ciliate is characterized by a claviform cell shape, with a size of 35-45 × 10-20 µm in vivo, 28-39 longitudinal somatic ciliary rows forming five ciliary girdles (four complete girdles and a shorter one), two dikinetids left to anterior end of oral kinety 1, and an epaulet. A Chinese population of the well-known ciliate P. nasuta was investigated, and morphological comparisons revealed phenotypic stability of the species. The phylogenetic analyses supported previous findings about the monophyly of the families Trimyemidae and Plagiopylidae, with Trimyema claviforme branching off early in the genus Trimyema. The Chinese population of P. nasuta clusters together with two other populations with full support corroborating their conspecificity.

Nutrient availability and grazing influence the strength of priority effects during freshwater bacterial community coalescence.

When bacterial communities mix, immigration history can fundamentally affect the community composition as a result of priority effects. Priority effects arise when an early immigrant exhausts resources and/or alters habitat conditions, thereby influencing the establishment success of the late arriver. The strength of priority effects is context-dependent and expected to be stronger if environmental conditions favour the growth of the first arriver. In this study, we conducted a two-factorial experiment testing the importance of nutrient availability and grazing on the strength of priority effects in complex aquatic bacterial communities. We did so by mixing two dissimilar communities, simultaneously, and with a 38 h time-delay. Priority effects were measured as the invasion resistance of the first community to the invading second community. We found stronger priority effects in treatments with high nutrient availability and absence of grazing, but in general, the arrival timing was less important than the selection by nutrients and grazing. At the population level, the results were complex, but priority effects may have been driven by bacteria belonging to for example, the genera Rhodoferax and Herbaspirillum. Our study highlights the importance of arrival timing in complex bacterial communities, especially if environmental conditions favour rapid community growth.

Cable bacteria regulate sedimentary phosphorus release in freshwater sediments.

Previous studies have demonstrated that e-SOx can regulate the sedimentary release of phosphorus (P) in brackish and marine sediments. When e-SOx is active, an iron (Fe) and manganese (Mn) oxide rich layer is formed near the sediment surface, which prevents P release. When e-SOx becomes inactive, the metal oxide layer is reduced via sulfide-mediated dissolution, and P is subsequently released to the water column. Cable bacteria have been shown to also occur in freshwater sediments. In these sediments, sulfide production is limited, and the metal oxide layer would thus dissolve less efficiently, leaving the P trapped at the sediment surface. This lack of an efficient dissolution mechanism implies that e-SOx could play an important role in the regulation of P availability in eutrophied freshwater streams. To test this hypothesis, we incubated sediments from a eutrophic freshwater river to investigate the impact of cable bacteria on sedimentary cycling of Fe, Mn and P. High-resolution depth profiling of pH, O2 and ΣH2S complemented with FISH analysis and high-throughput gene sequencing showed that the development of e-SOx activity was closely linked to the enrichment of cable bacteria in incubated sediments. Cable bacteria activity caused a strong acidification in the suboxic zone, leading to the dissolution of Fe and Mn minerals and consequently a strong release of dissolved Fe2+ and Mn2+ to the porewater. Oxidation of these mobilized ions at the sediment surface led to the formation of a metal oxide layer that trapped dissolved P, as shown by the enrichment of P-bearing metal oxides in the top layer of the sediment and low phosphate in the pore and overlying water. After e-SOx activity declined, the metal oxide layer did not dissolve and P remained trapped at the surface. Overall, our results suggested cable bacteria can play an important role to counteract eutrophication in freshwater systems.

Large-scale phylogenomics of aquatic bacteria reveal molecular mechanisms for adaptation to salinity.

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transitions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive challenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome transitions, resulting in species-level separation between aquatic biomes.

A novel microplate-based spectrophotometric method for the quantitative assessment of freshwater bacterial coaggregation kinetics.

Coaggregation, the specific recognition and adhesion of genetically distinct bacteria, is proposed to contribute to the development of freshwater biofilms. This work aimed to develop a microplate-based system to measure and model the kinetics of freshwater bacterial coaggregation. Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 were evaluated for coaggregation ability using 24-well microplates containing novel dome shaped wells (DSWs) and standard flat-bottom wells. Results were compared to a tube-based visual aggregation assay. The DSWs facilitated the reproducible detection of coaggregation via spectrophotometry and the estimation of coaggregation kinetics using a linked mathematical model. Quantitative analysis using DSWs was more sensitive than the visual tube aggregation assay and subject to substantially less variation than flat-bottom wells. Collectively these results demonstrate the utility of the DSW-based method and improve upon the current toolkit for studying freshwater bacterial coaggregation.

Microplastic biodegradability dependent responses of plastisphere antibiotic resistance to simulated freshwater-seawater shift in onshore marine aquaculture zones.

MPs carrying ARGs can travel between freshwater and seawater due to intensive land-sea interaction in onshore marine aquaculture zones (OMAZ). However, the response of ARGs in plastisphere with different biodegradability to freshwater-seawater shift is still unknown. In this study, ARG dynamics and associated microbiota on biodegradable poly (butyleneadipate-co-terephthalate) (PBAT) and non-biodegradable polyethylene terephthalate (PET) MPs were investigated through a simulated freshwater-seawater shift. The results exhibited that freshwater-seawater shift significantly influenced ARG abundance in plastisphere. The relative abundance of most studied ARGs decreased rapidly in plastisphere after they entered seawater from freshwater but increased on PBAT after MPs entered freshwater from seawater. Besides, the high relative abundance of multi-drug resistance (MDR) genes occurred in plastisphere, and the co-change between most ARGs and mobile genetic elements indicated the role of horizontal gene transfer on ARG regulation. Proteobacteria was dominant phylum in plastisphere and the dominant genera, such as Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Afipia, Gemmobacter and Enhydrobacter, were significantly associated with qnrS, tet and MDR genes in plastisphere. Moreover, after MPs entered new water environment, the ARGs and microbiota genera in plastisphere changed significantly and tended to converge with those in receiving water. These results indicated that MP biodegradability and freshwater-seawater interaction influenced potential hosts and distributions of ARGs, of which biodegradable PBAT posed a high risk in ARG dissemination. This study would be helpful for understanding the impact of biodegradable MP pollution on spread of antibiotic resistance in OMAZ.

Ten Novel Species Belonging to the Genus Flavobacterium, Isolated from Freshwater Environments: F. praedii sp. nov., F. marginilacus sp. nov., F. aestivum sp. nov., F. flavigenum sp. nov., F. luteolum sp. nov., F. gelatinilyticum sp. nov., F. aquiphilum sp. nov., F. limnophilum sp. nov., F. lacustre sp. nov., and F. eburneipallidum sp. nov.

Eleven bacterial strains were isolated from freshwater environments and identified as Flavobacterium based on 16S rRNA gene sequence analyses. Complete genome sequences of the 11 strains ranged from 3.45 to 5.83 Mb with G + C contents of 33.41-37.31%. The average nucleotide identity (ANI) values showed that strains IMCC34515T and IMCC34518 belonged to the same species, while the other nine strains represented each separate species. The ANI values between the strains and their closest Flavobacterium species exhibited ≤ 91.76%, indicating they represent each novel species. All strains had similar characteristics such as being Gram-stain-negative, rod-shaped, and contained iso-C15:0 as the predominant fatty acid, menaquinone-6 as the respiratory quinone, and phosphatidylethanolamine and aminolipids as major polar lipids. Genomic, phylogenetic, and phenotypic characterization confirmed that the 11 strains were distinct from previously recognized Flavobacterium species. Therefore, Flavobacterium praedii sp. nov. (IMCC34515T = KACC 22282 T = NBRC 114937 T), Flavobacterium marginilacus sp. nov. (IMCC34673T = KACC 22284 T = NBRC 114940 T), Flavobacterium aestivum sp. nov. (IMCC34774T = KACC 22285 T = NBRC 114941 T), Flavobacterium flavigenum sp. nov. (IMCC34775T = KACC 22286 T = NBRC 114942 T), Flavobacterium luteolum sp. nov. (IMCC34776T = KACC 22287 T = NBRC 114943 T), Flavobacterium gelatinilyticum sp. nov. (IMCC34777T = KACC 22288 T = NBRC 114944 T), Flavobacterium aquiphilum sp. nov. (IMCC34779T = KACC 22289 T = NBRC 114945 T), Flavobacterium limnophilum sp. nov. (IMCC36791T = KACC 22290 T = NBRC 114947 T), Flavobacterium lacustre sp. nov. (IMCC36792T = KACC 22291 T = NBRC 114948 T), and Flavobacterium eburneipallidum sp. nov. (IMCC36793T = KACC 22292 T = NBRC 114949 T) are proposed as novel species.

The Variations of Microbial Diversity and Community Structure Along Different Stream Orders in Wuyi Mountains.

The surface water is an important habitat for freshwater microorganisms, but there is a lack of understanding of the pattern of microbial diversity and structure in stream continuums of small subtropical forest watersheds. Therefore, this study aimed to understand the variations in microbial diversity and community structure along stream orders (1-5) in the small subtropical forest catchments of the Wuyi Mountains. Using GIS software, 20 streams were chosen and classified into 5 orders. Illumina sequencing was used to analyze the dynamics of microbial communities, along with stream orders and hydro-chemical properties of stream water were also determined. Our results indicated that the bacterial and fungal richness (ACE index) was higher in low-order (1 and 2 orders) streams than in high-order (3, 4, and 5 orders) streams, with the highest value in the order 2 streams (P < 0.05). The water temperature and dissolved oxygen were positively correlated with fungal richness (P < 0.05). The bacterial rare taxa had a significant correlation with the abundance taxa (P < 0.05). The relative abundances of Bacteroidetes, Actinobacteria, and Chytridiomycota microbial phyla were significantly different among different order streams (P < 0.05). Using the neutral community model, we found that the fungal community structure was significantly shaped by hydro-chemical properties, while the bacterial community structure was largely regulated by stochastic processes. Our findings suggest that variations in microbial community structure in subtropical headwaters are largely shaped by the water temperature and dissolved oxygen.