Members of the archaeal order Methanomassiliicoccales are methanogens mainly associated with animal digestive tracts. However, environmental members remain poorly characterized as no representatives not associated with a host have been cultivated so far. In this study, metabarcoding screening combined with quantitative PCR analyses on a collection of diverse non-host-associated environmental samples revealed that Methanomassiliicoccales were very scarce in most terrestrial and aquatic ecosystems. Relative abundance of Methanomassiliicoccales and substrates/products of methanogenesis were monitored during incubation of environmental slurries. A sediment slurry enriched in Methanomassiliicoccales was obtained from a freshwater sample. It allowed the reconstruction of a high-quality metagenome-assembled genome (MAG) corresponding to a new candidate species, for which we propose the name of Candidatus 'Methanomassiliicoccus armoricus MXMAG1'. Comparison of the annotated genome of MXMAG1 with the published genomes and MAGs from Methanomassiliicoccales belonging to the 2 known clades ('free-living'/non-host-associated environmental clade and 'host-associated'/digestive clade) allowed us to explore the putative physiological traits of Candidatus 'M. armoricus MXMAG1'. As expected, Ca. 'Methanomassiliicoccus armoricus MXMAG1' had the genetic potential to produce methane by reduction of methyl compounds and dihydrogen oxidation. This MAG encodes for several putative physiological and stress response adaptations, including biosynthesis of trehalose (osmotic and temperature regulations), agmatine production (pH regulation), and arsenic detoxication, by reduction and excretion of arsenite, a mechanism that was only present in the 'free-living' clade. An analysis of co-occurrence networks carried out on environmental samples and slurries also showed that Methanomassiliicoccales detected in terrestrial and aquatic ecosystems were strongly associated with acetate and dihydrogen producing bacteria commonly found in digestive habitats and which have been reported to form syntrophic relationships with methanogens.

Red mark syndrome (RMS) is a fish disease caused by the infection of Rickettsial agents, especially affecting rainbow trout (Oncorhynchus mykiss). The disease is prevalent in many countries in Europe (France, Switzerland, Italy, and Slovenia), South America (Chile), North America (USA), and even Asia (Japan). However, it has not been reported in Korean aquaculture. In February 2019, rainbow trout presenting red spot lesions with swollen features on the lateral side of their body were observed at a hatchery in Korea. Fishes showing those clinical signs were fry weighing 25 ± 5 g. Moreover, the fish showing the red spot lesions were found dead, which suggests an outbreak of a mortality-causing disease. The symptoms were similar to those of RMS, and we identified the presence of Rickettsia-like organisms associated with this disease using polymerase chain reaction (PCR), sequencing, histopathologic examination, and transmission electron microscopy. The distinct features of this infection, compared to that in previous reports, were that RMS occurred in small-sized fish and accompanied mortality. Additionally, the presence of the Rickettsia agent was accompanied with outbreak of the disease. Therefore, this is the first report of RMS outbreak in rainbow trout fisheries in Korea.

Land use disrupts the ecosystem functioning of freshwater systems and significantly affects trophic state. Consequently, biodiversity is severely affected by changes to the ecosystem. Microbial eukaryotes (i.e., protists) play an essential role in ecosystem functioning, contributing to biogeochemical processes, nutrient cycling, and food webs. Protist composition is a useful biological quality parameter for monitoring aquatic ecosystems and determining aquatic system health. In this study, we investigated the effects of land usage and trophic state on the communities of microbial eukaryotes in the New River (Belize, C.A.). Land use and trophic state both significantly affected protist community compositions, with impacted and mesotrophic sampled sites having higher biodiversity when compared to other sites. Autotrophic organisms dominated indirectly impacted and eutrophic sites, while impacted and mesotrophic sites had proportional ratios of autotrophic and heterotrophic organisms. Our study highlights the significant effects of trophic gradients on protistan community composition, even at the local scales.

Aeromonas hydrophila are ubiquitous in the environment and are highly distributed in aquatic habitats. They have long been known as fish pathogens but are opportunistic human pathogens. Aeromonas spp. have persisted through food-processing safeguards and have been isolated from fresh grocery vegetables, dairy, beef, pork, poultry products and packaged ready-to-eat meats, thus providing an avenue to foodborne illness. A beta-hemolytic, putative Escherichia coli strain collected from diarrheic neonatal pigs in Oklahoma was subsequently identified as A. hydrophila, and designated CVM861. Here we report the whole-genome sequence of A. hydrophila CVM861, SRA accession number, SRR12574563; BioSample number, SAMN1590692; Genbank accession number SRX9061579. The sequence data for CVM861 revealed four Aeromonas-specific virulence genes: lipase (lip), hemolysin (hlyA), cytonic enterotoxin (ast) and phospholipid-cholesterolacyltransferase (GCAT). There were no alignments to any virulence genes in VirulenceFinder. CVM861 contained an E. coli resistance plasmid identified as IncQ1_1__M28829. There were five aminoglycoside, three beta-lactam, and one each of macrolide, phenicol, sulfonamide, tetracycline and trimethoprim resistance genes, all with over 95% identity to genes in the ResFinder database. Additionally, there were 36 alignments to mobile genetic elements using MobileElementFinder. This shows that an aquatic pathogen, rarely considered in human disease, contributes to the resistome reservoir and may be capable of transferring resistance and virulence genes to other more prevalent foodborne strains such as E. coli or Salmonella in swine or other food production systems.

Events of groundwater recharge are associated with changes in the composition of aquifer microbial communities but also abiotic conditions. Modification in the structure of the community can be the result of different environmental condition favoring or hindering certain taxa, or due to the introduction of surface-derived taxa. Yet, in both cases, the local hydrogeochemical settings of the aquifer is likely to affect the amount of variation observed. Therefore, in our study, we used 16S rRNA gene sequencing to assess how microbial communities change in response to snowmelt and the potential connectivity between subsurface and surface microbiomes in two distinct aquifers located in the region of Vaudreuil-Soulanges (Québec, Canada). At both sites, we observed an increase in groundwater level and decrease in temperature following the onset of snow melt in March 2019. Bacterial community composition of each aquifer was significantly different (p < 0.05) between samples collected prior and after groundwater recharge. Furthermore, microbial source tracking results suggested a low contribution of surface environments to the groundwater microbiome except for in the months associated with recharge (March 2019 and April 2019). Overall, despite differences in soil permeability between both sites, the period of snow melt was followed by important changes in the composition of microbial communities from aquifers.

Aquifer systems are composed of water flowing from surface recharge areas, to the subsurface and back to the surface in discharge regions. Groundwater habitats harbor a large microbial biomass and diversity, potentially contributing to surface aquatic ecosystems. Although this contribution has been widely studied in marine environments, very little is known about the connection between underground and surface microbial communities in freshwater settings. Therefore, in this study, we used amplicon sequencing to analyze the archaeal, bacterial, and eukaryotic community diversity and structure in groundwater and surface water samples, spanning the vast regions of the Laurentides and Lanaudières in the Quebec province (Canada). Our results show significant differences between subsurface and surface taxa; with more fungi, Amoebozoa, and chemolithoautotrophic prokaryotes involved in nitrogen-, sulfur-, and iron-cycling dominating the underground samples; while algae, ciliates, methanogens, and Actinobacteria dominate the surface discharge waters. Microbial source tracking suggested that only a small portion of the microbial communities in the groundwater contributed to the surface discharge communities. However, many taxa were shared between both habitats, with a large range of functional diversity, likely explaining their survival in both subsurface and surface water ecosystems.

The Vibrio genus includes bacteria widely distributed in aquatic habitats and the infections caused by these bacteria can affect a wide range of hosts. They are able to adhere to numerous surfaces, which can result in biofilm formation that helps maintain them in the environment. The involvement of the biofilm lifestyle in the virulence of Vibrio pathogens of aquatic organisms remains to be investigated. Vibrio harveyi ORM4 is a pathogen responsible for an outbreak in European abalone Haliotis tuberculata populations. In the present study, we used a dynamic biofilm culture technique coupled with laser scanning microscopy to characterize the biofilm formed by V. harveyi ORM4. We furthermore used RNA-seq analysis to examine the global changes in gene expression in biofilm cells compared to planktonic bacteria, and to identify biofilm- and virulence-related genes showing altered expression. A total of 1565 genes were differentially expressed, including genes associated with motility, polysaccharide synthesis, and quorum sensing. The up-regulation of 18 genes associated with the synthesis of the type III secretion system suggests that this virulence factor is induced in V. harveyi ORM4 biofilms, providing indirect evidence of a relationship between biofilm and virulence.

Phenacetin, an antipyretic and analgesic drug, poses a serious health risk to both humans and aquatic organisms, which is of concern since this micropollutant is frequently detected in various aquatic environments. However, rare pure bacterial cultures have been reported to degrade phenacetin. Therefore, in this study, the novel phenacetin-degrading strain PNT-23 was isolated from municipal wastewater and identified as a Rhodococcus sp. based on its morphology and 16S rRNA gene sequencing. The isolated strain could completely degrade 100 mg/L phenacetin at an inoculum concentration of OD600 1.5 within 80 h, utilizing the micropollutant as its sole carbon source for growth. Strain PNT-23 exhibited optimal growth in LB medium at 37 °C and a pH of 7.0 with 1% NaCl, while the optimal degradation conditions in minimal medium were 30 °C and a pH of 7.0 with 1% NaCl. Two key intermediates were identified during phenacetin biodegradation by the strain PNT-23: N-acetyl-4-aminophenol and 4-aminophenol. This study provides novel insights into the biodegradation of phenacetin using a pure bacterium culture, expands the known substrate spectra of Rhodococcus strains and presents a potential new candidate for the microbial removal of phenacetin in a diverse range of environments.

Two CXC chemokines in Nile tilapia (On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1-10 μg strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro.

Scuticociliatosis is an invasive external or systemic infection caused by ciliated protozoa, mainly those within the subclass Scuticociliatia (scuticociliates). Many scuticociliates are fish pathogens, including Miamiensis avidus, Philasterides dicentrarchi, Pseudocohnilembus persalinus, and Uronema marinum. Our previous study showed that hemolysis-related genes derived from bacteria through horizontal gene transfer (HGT) may contribute to virulence in P. persalinus. Hemorrhagic lesions are a common feature of scuticociliatosis, but it is not known whether other scuticociliates also have bacteria-derived hemolysis-related genes. In this study, we constructed a high-quality macronuclear genome of another typical pathogenic scuticociliate, U. marinum. A total of 105 HGT genes were identified in this species, of which 35 were homologs of hemolysis-related genes (including hemolysin-like genes) that had previously been identified in P. persalinus. Sequencing of an additional five species from four scuticociliate families showed that bacteria-derived hemolysis-related genes (especially hemolysin-like genes) are widely distributed in scuticociliates. Based on these findings, we suggest that hemolysin-like genes may have originated before the divergence of scuticociliates.

Vibrio cholerae non-O1, non-O139 bacteria are natural inhabitants of aquatic ecosystems and have been sporadically associated with human infections. They mostly lack the two major virulence factors of toxigenic V. cholerae serogroups O1 and O139 strains, which are the causative agent of cholera. Non-O1, non-O139 strains are found in water bodies, sediments, and in association with other aquatic organisms. Occurrence of these bacteria in fecal specimens of waterfowl were reported, and migratory birds likely contribute to the long-distance transfer of strains. We investigated four V. cholerae non-O1, non-O139 isolates for phenotypic traits and by whole genome sequencing (WGS). The isolates were recovered from organs of domestic ducks with serious disease symptoms. WGS data revealed only a distant genetic relationship between all isolates. The isolates harbored a number of virulence factors found in most V. cholerae strains. Specific virulence factors of non-O1, non-O139 strains, such as the type III secretion system (TTSS) or cholix toxin, were observed. An interesting observation is that all isolates possess multifunctional autoprocessing repeats-in-toxin toxins (MARTX) closely related to the MARTX of toxigenic El Tor O1 strains. Different primary sequences of the abundant OmpU proteins could indicate a significant role of this virulence factor. Phenotypic characteristics such as hemolysis and antimicrobial resistance (AMR) were studied. Three isolates showed susceptibility to a number of tested antimicrobials, and one strain possessed AMR genes located in an integron. Knowledge of the environmental occurrence of V. cholerae non-O1, non-O139 in Germany is limited. The source of the infection of the ducks is currently unknown. In the context of the 'One Health' concept, it is desirable to study the ecology of V. cholerae non-O1, non-O139, as it cannot be excluded that the isolates possess zoonotic potential and could cause infections in humans.

Fungi are ecologically important in several ecosystem processes, yet their community composition, ecophysiological roles, and responses to changing environmental factors in historical sediments are rarely studied. Here we explored ancient fungal DNA from lake Lielais Svētiņu sediment throughout the Holocene (10.5 kyr) using the ITS metabarcoding approach. Our data revealed diverse fungal taxa and smooth community changes during most of the Holocene with rapid changes occurring in the last few millennia. More precisely, plankton parasitic fungi became more diverse from the Late Holocene (2-4 kyr) which could be related to a shift towards a cooler climate. The Latest Holocene (~2 kyr) showed a distinct increase in the richness of plankton parasites, mycorrhizal, and plant pathogenic fungi which can be associated with an increased transfer rate of plant material into the lake and blooms of planktonic organisms influenced by increased, yet moderate, human impact. Thus, major community shifts in plankton parasites and mycorrhizal fungi could be utilized as potential paleo-variables that accompany host-substrate dynamics. Our work demonstrates that fungal aDNA with predicted ecophysiology and host specificity can be employed to reconstruct both aquatic and surrounding terrestrial ecosystems and to estimate the influence of environmental change.

The haloarchaeon Halorubrum lacusprofundi is among the few polyextremophilic organisms capable of surviving in one of the most extreme aquatic environments on Earth, the Deep Lake of Antarctica (-18 °C to +11.5 °C and 21-28%, w/v salt content). Hence, H. lacusprofundi has been proposed as a model for biotechnology and astrobiology to investigate potential life beyond Earth. To understand the mechanisms that allow proteins to adapt to both salinity and cold, we structurally (including X-ray crystallography and molecular dynamics simulations) and functionally characterized the β-galactosidase from H. lacusprofundi (hla_bga). Recombinant hla_bga (produced in Haloferax volcanii) revealed exceptional stability, tolerating up to 4 M NaCl and up to 20% (v/v) of organic solvents. Despite being cold-adapted, hla_bga was also stable up to 60 °C. Structural analysis showed that hla_bga combined increased surface acidity (associated with halophily) with increased structural flexibility, fine-tuned on a residue level, for sustaining activity at low temperatures. The resulting blend enhanced structural flexibility at low temperatures but also limited protein movements at higher temperatures relative to mesophilic homologs. Collectively, these observations help in understanding the molecular basis of a dual psychrophilic and halophilic adaptation and suggest that such enzymes may be intrinsically stable and functional over an exceptionally large temperature range.

Lumpfish is utilized as a cleaner fish to biocontrol sealice infestations in Atlantic salmon farms. Aeromonas salmonicida, a Gram-negative facultative intracellular pathogen, is the causative agent of furunculosis in several fish species, including lumpfish. In this study, lumpfish were intraperitoneally injected with different doses of A. salmonicida to calculate the LD50. Samples of blood, head-kidney, spleen, and liver were collected at different time points to determine the infection kinetics. We determined that A. salmonicida LD50 is 102 CFU per dose. We found that the lumpfish head-kidney is the primary target organ of A. salmonicida. Triplicate biological samples were collected from head-kidney, spleen, and liver pre-infection and at 3- and 10-days post-infection for RNA-sequencing. The reference genome-guided transcriptome assembly resulted in 6246 differentially expressed genes. The de novo assembly resulted in 403,204 transcripts, which added 1307 novel genes not identified by the reference genome-guided transcriptome. Differential gene expression and gene ontology enrichment analyses suggested that A. salmonicida induces lethal infection in lumpfish by uncontrolled and detrimental blood coagulation, complement activation, inflammation, DNA damage, suppression of the adaptive immune system, and prevention of cytoskeleton formation.

A microcosm designed for culturing aquatic phototrophic biofilms on artificial substrata was used to perform experiments with microphytobenthos sampled during summer toxic outbreaks of Ostreopsis cf. ovata along the Middle Tyrrhenian coast. This dynamic approach aimed at exploring the unique and complex nature of O. cf. ovata bloom development in the benthic system. Epibenthic assemblages were used as inocula for co-cultures of bloom organisms on polycarbonate slides at controlled environmental conditions. Biofilm surface adhesion, growth, and spatial structure were evaluated along with shifts in composition and matrix production in a low disturbance regime, simulating source habitat. Initial adhesion and substratum colonisation appeared as stochastic processes, then community structure and physiognomy markedly changed with time. Dominance of filamentous cyanobacteria and diatoms, and dense clusters of Amphidinium cf. carterae at the mature biofilm phases, were recorded by light and confocal microscopy, whilst O. cf. ovata growth was visibly limited in the late culture phases. Life-form strategies, competitiveness for resources, and possibly allelopathic interactions shaped biofilm structure during culture growth. HPLC (High Performance Liquid Chromatography) analysis of exopolysaccharidic matrix revealed variations in sugar total amounts and composition. No toxic compounds were detected in the final communities tested by LC-MS (Liquid Chromatography- Mass Spectrometry) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time OF Flight Mass Spectroscopy) techniques.

The release of a wide variety of persistent chemical contaminants into wastewater has become a growing concern due to their potential health and environmental risks. While the toxic effects of these pollutants on aquatic organisms have been extensively studied, their impact on microbial pathogens and their virulence mechanisms remains largely unexplored. This research paper focuses on the identification and prioritization of chemical pollutants that increase bacterial pathogenicity, which is a public health concern. In order to predict how chemical compounds, such as pesticides and pharmaceuticals, would affect the virulence mechanisms of three bacterial strains (Escherichia coli K12, Pseudomonas aeruginosa H103, and Salmonella enterica serovar. Typhimurium), this study has developed quantitative structure-activity relationship (QSAR) models. The use of analysis of variance (ANOVA) functions assists in developing QSAR models based on the chemical structure of the compounds, to predict their effect on the growth and swarming behavior of the bacterial strains. The results showed an uncertainty in the created model, and that increases in virulence factors, including growth and motility of bacteria, after exposure to the studied compounds are possible to be predicted. These results could be more accurate if the interactions between groups of functions are included. For that, to make an accurate and universal model, it is essential to incorporate a larger number of compounds of similar and different structures.

Biofilms are a ubiquitous formation of microbial communities found on surfaces in aqueous environments. These structures have been investigated as biomonitoring indicators for stream heath, and here were used for the potential use in forensic sciences. Biofilm successional development has been proposed as a method to determine the postmortem submersion interval (PMSI) of remains because there are no standard methods for estimating the PMSI and biofilms are ubiquitous in aquatic habitats. We sought to compare the development of epinecrotic (biofilms on Sus scrofa domesticus carcasses) and epilithic (biofilms on unglazed ceramic tiles) communities in two small streams using bacterial automated ribosomal intergenic spacer analysis. Epinecrotic communities were significantly different from epilithic communities even though environmental factors associated with each stream location also had a significant influence on biofilm structure. All communities at both locations exhibited significant succession suggesting that changing communities throughout time is a general characteristic of stream biofilm communities. The implications resulting from this work are that epinecrotic communities have distinctive shifts at the first and second weeks, and therefore the potential to be used in forensic applications by associating successional changes with submersion time to estimate a PMSI. The influence of environmental factors, however, indicates the lack of a successional pattern with the same organisms and a focus on functional diversity may be more applicable in a forensic context.

Antimicrobial resistance is a major public health concern restricted not only to healthcare settings but also to veterinary and environmental ones. In this study, we analyzed, by whole genome sequencing (WGS) the resistome, mobilome and virulome of 12 multidrug-resistant (MDR) marine strains belonging to Shewanellaceae and Vibrionaceae families collected at aquaculture centers in Italy. The results evidenced the presence of several resistance mechanisms including enzyme and efflux pump systems conferring resistance to beta-lactams, quinolones, tetracyclines, macrolides, polymyxins, chloramphenicol, fosfomycin, erythromycin, detergents and heavy metals. Mobilome analysis did not find circular elements but class I integrons, integrative and conjugative element (ICE) associated modules, prophages and different insertion sequence (IS) family transposases. These mobile genetic elements (MGEs) are usually present in other aquatic bacteria but also in Enterobacteriaceae suggesting their transferability among autochthonous and allochthonous bacteria of the resilient microbiota. Regarding the presence of virulence factors, hemolytic activity was detected both in the Shewanella algae and in Vibrio spp. strains. To conclude, these data indicate the role as a reservoir of resistance and virulence genes in the environment of the aquatic microbiota present in the examined Italian fish farms that potentially might be transferred to bacteria of medical interest.

Microbes are considered to have a global distribution due to their high dispersal capabilities. However, our knowledge of the way geographically distant microbial communities assemble after dispersal in a new environment is limited. In this study, we examined whether communities would converge because similar taxa would be selected under the same environmental conditions, or would diverge because of initial community composition, after artificial dispersal. To this aim, a microcosm experiment was performed, in which the temporal changes in the composition and diversity of different prokaryoplankton assemblages from three distant geographic coastal areas (Banyuls-sur-Mer in northwest Mediterranean Sea, Pagasitikos Gulf in northeast Mediterranean and Woods Hole, MA, USA in the northwest Atlantic), were studied. Diversity was investigated using amplicon pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA. The three assemblages were grown separately in particle free and autoclaved Banyuls-sur-mer seawater at 18 °C in the dark. We found that the variability of prokaryoplankton community diversity (expressed as richness, evenness and dominance) as well as the composition were driven by patterns observed in Bacteria. Regarding community composition, similarities were found between treatments at family level. However, at the OTU level microbial communities from the three different original locations diverge rather than converge during incubation. It is suggested that slight differences in the composition of the initial prokaryoplankton communities, resulted in separate clusters the following days even when growth took place under identical abiotic conditions.

Plastic pollution has become one of the most critical environmental issues, as rapidly increasing production, compounded by persistence of plastic wastes in the environment, are outpacing efforts to keep ecosystems plastic-free. A switch to plastics more amenable to microbial attack is one of several possible responses. Against this background, the current study describes the isolation, enumeration and polyphasic characterization of plastic-degrading bacteria present in Norwegian terrestrial and aquatic habits. It shows that these bacteria are present in relatively high numbers, and that plastic-degrading capabilities are found in several taxa, most especially Streptomyces. Some isolates wereable to degrade several plastics. Notably, a Rhodococcus sp. and a Streptomyces sp. degraded, respectively, four and six of the eight plastics investigated and a number of other polymers relevant for plastic blends. The paper also has a methodological aspect, presenting various approaches for assaying plastic-degrading properties and a PCR/sequencing-based approach for the identification of potential polyethylene terephthalate-degrading genes. A candidate gene was detected in several Streptomyces isolates. The study shows that Norwegian environments are a rich source of bacteria with the ability to degrade bioplastics possibly representing a natural remediation capacity, as well as a potential source of useful enzymes.