High-resolution metagenomic reconstruction of the freshwater spring bloom.

BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

The combined impact of low temperatures and shifting phosphorus availability on the competitive ability of cyanobacteria.

In freshwater systems, cyanobacteria are strong competitors under enhanced temperature and eutrophic conditions. Understanding their adaptive and evolutionary potential to multiple environmental states allows us to accurately predict their response to future conditions. To better understand if the combined impacts of temperature and nutrient limitation could suppress the cyanobacterial blooms, a single strain of Microcystis aeruginosa was inoculated into natural phytoplankton communities with different nutrient conditions: oligotrophic, eutrophic and eutrophic with the addition of bentophos. We found that the use of the bentophos treatment causes significant differences in prokaryotic and eukaryotic communities. This resulted in reduced biodiversity among the eukaryotes and a decline in cyanobacterial abundance suggesting phosphorus limitation had a strong impact on the community structure. The low temperature during the experiment lead to the disappearance of M. aeruginosa in all treatments and gave other phytoplankton groups a competitive advantage leading to the dominance of the eukaryotic families that have diverse morphologies and nutritional modes. These results show cyanobacteria have a reduced competitive advantage under certain temperature and nutrient limiting conditions and therefore, controlling phosphorus concentrations could be a possible mitigation strategy for managing harmful cyanobacterial blooms in a future warmer climate.

Untargeted Plasma Metabolomics and Gut Microbiome Profiling Provide Novel Insights into the Regulation of Platelet Reactivity in Healthy Individuals.

BACKGROUND: Considerable variation exists in platelet reactivity to stimulation among healthy individuals. Various metabolites and metabolic pathways influence platelet reactivity, but a comprehensive overview of these associations is missing. The gut microbiome has a strong influence on the plasma metabolome. Here, we investigated the association of platelet reactivity with results of untargeted plasma metabolomics and gut microbiome profiling. METHODS: We used data from a cohort of 534 healthy adult Dutch volunteers (the 500 Functional Genomics study). Platelet activation and reactivity were measured by the expression of the alpha-granule protein P-selectin and the binding of fibrinogen to the activated integrin αIIbß3, both in unstimulated blood and after ex vivo stimulation with platelet agonists. Plasma metabolome was measured using an untargeted metabolic profiling approach by quadrupole time-of-flight mass spectrometry. Gut microbiome data were measured by shotgun metagenomic sequencing from stool samples. RESULTS: Untargeted metabolomics yielded 1,979 metabolites, of which 422 were identified to play a role in a human metabolic pathway. Overall, 92/422 (21.8%) metabolites were significantly associated with at least one readout of platelet reactivity. The majority of associations involved lipids, especially members of eicosanoids, including prostaglandins and leukotrienes. Dietary-derived polyphenols were also found to inhibit platelet reactivity. Validation of metabolic pathways with functional microbial profiles revealed two overlapping metabolic pathways ("alanine, aspartate, and glutamate metabolism" and "arginine biosynthesis") that were associated with platelet reactivity. CONCLUSION: This comprehensive overview is an resource for understanding the regulation of platelet reactivity by the plasma metabolome and the possible contribution of the gut microbiota.

Spatio-temporal insights into microbiology of the freshwater-to-hypersaline, oxic-hypoxic-euxinic waters of Ursu Lake.

Ursu Lake is located in the Middle Miocene salt deposit of Central Romania. It is stratified, and the water column has three distinct water masses: an upper freshwater-to-moderately saline stratum (0-3 m), an intermediate stratum exhibiting a steep halocline (3-3.5 m), and a lower hypersaline stratum (4 m and below) that is euxinic (i.e. anoxic and sulphidic). Recent studies have characterized the lake's microbial taxonomy and given rise to intriguing ecological questions. Here, we explore whether the communities are dynamic or stable in relation to taxonomic composition, geochemistry, biophysics, and ecophysiological functions during the annual cycle. We found: (i) seasonally fluctuating, light-dependent communities in the upper layer (≥0.987-0.990 water-activity), a stable but phylogenetically diverse population of heterotrophs in the hypersaline stratum (water activities down to 0.762) and a persistent plate of green sulphur bacteria that connects these two (0.958-0.956 water activity) at 3-3.5 to 4 m; (ii) communities that might be involved in carbon- and sulphur-cycling between and within the lake's three main water masses; (iii) uncultured lineages including Acetothermia (OP1), Cloacimonetes (WWE1), Marinimicrobia (SAR406), Omnitrophicaeota (OP3), Parcubacteria (OD1) and other Candidate Phyla Radiation bacteria, and SR1 in the hypersaline stratum (likely involved in the anaerobic steps of carbon- and sulphur-cycling); and (iv) that species richness and habitat stability are associated with high redox-potentials. Ursu Lake has a unique and complex ecology, at the same time exhibiting dynamic fluctuations and stability, and can be used as a modern analogue for ancient euxinic water bodies and comparator system for other stratified hypersaline systems.

Author Correction: Mud volcanoes and the presence of PAHs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mud volcanoes and the presence of PAHs.

A mud volcano (MV) is a naturally hydrocarbon-spiked environment, as indicated by the presence of various quantities of PAHs and aromatic isotopic shifts in its sediments. Recurrent expulsion of various hydrocarbons consolidates the growth of hydrocarbonoclastic bacterial communities in the areas around MVs. In addition to the widely-known availability of biologically malleable alkanes, MVs can represent hotbeds of polyaromatic hydrocarbons (PAHs), as well - an aspect that has not been previously explored. This study measured the availability of highly recalcitrant PAHs and the isotopic signature of MV sediments both by GC-MS and δ13C analyses. Subsequently, this study highlighted both the occurrence and distribution of putative PAH-degrading bacterial OTUs using a metabarcoding technique. The putative hydrocarbonoclastic taxa incidence are the following: Enterobacteriaceae (31.5%), Methylobacteriaceae (19.9%), Bradyrhizobiaceae (16.9%), Oxalobacteraceae (10.2%), Comamonadaceae (7.6%) and Sphingomonadaceae (5.5%). Cumulatively, the results of this study indicate that MVs represent polyaromatic hydrocarbonoclastic hotbeds, as defined by both natural PAH input and high incidence of putative PAH-degrading bacterial OTUs.

Investigating the potential use of an Antarctic variant of Janthinobacterium lividum for tackling antimicrobial resistance in a One Health approach.

The aim of this paper is to describe a new variant of Janthinobacterium lividum - ROICE173, isolated from Antarctic snow, and to investigate the antimicrobial effect of the crude bacterial extract against 200 multi-drug resistant (MDR) bacteria of both clinical and environmental origin, displaying various antibiotic resistance patterns. ROICE173 is extremotolerant, grows at high pH (5.5-9.5), in high salinity (3%) and in the presence of different xenobiotic compounds and various antibiotics. The best violacein yield (4.59 ± 0.78 mg·g-1 wet biomass) was obtained at 22 °C, on R2 broth supplemented with 1% glycerol. When the crude extract was tested for antimicrobial activity, a clear bactericidal effect was observed on 79 strains (40%), a bacteriostatic effect on 25 strains (12%) and no effect in the case of 96 strains (48%). A very good inhibitory effect was noticed against numerous MRSA, MSSA, Enterococci, and Enterobacteriaceae isolates. For several environmental E. coli strains, the bactericidal effect was encountered at a violacein concentration below of what was previously reported. A different effect (bacteriostatic vs. bactericidal) was observed in the case of Enterobacteriaceae isolated from raw vs. treated wastewater, suggesting that the wastewater treatment process may influence the susceptibility of MDR bacteria to violacein containing bacterial extracts.

Draft Genome Sequence of Janthinobacterium sp. Strain ROICE36, a Putative Secondary Metabolite-Synthesizing Bacterium Isolated from Antarctic Snow.

The draft genome assembly of Janthinobacterium sp. strain ROICE36 has 207 contigs, with a total genome size of 5,977,006 bp and a G+C content of 62%. Preliminary genome analysis identified 5,363 protein-coding genes and a total of 7 secondary metabolic gene clusters (encoding bacteriocins, nonribosomal peptide-synthetase [NRPS], terpene, hserlactone, and other ketide synthases).

Investigating antibiotics, antibiotic resistance genes, and microbial contaminants in groundwater in relation to the proximity of urban areas.

Groundwater is an essential public and drinking water supply and its protection is a goal for global policies. Here, we investigated the presence and prevalence of antibiotic residues, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and microbial contamination in groundwater environments at various distances from urban areas. Antibiotic concentrations ranged from below detection limit to 917 ng/L, being trimethoprim, macrolide, and sulfonamide the most abundant antibiotic classes. A total of eleven ARGs (aminoglycoside, ß-lactam, chloramphenicol, Macrolide-Lincosamide-Streptogramin B - MLSB, sulfonamide, and tetracycline), one antiseptic resistance gene, and two MGEs were detected by qPCR with relative abundances ranging from 6.61 × 10-7 to 2.30 × 10-1 copies/16S rRNA gene copies. ARGs and MGEs were widespread in the investigated groundwater environments, with increased abundances not only in urban, but also in remote areas. Distinct bacterial community profiles were observed, with a higher prevalence of Betaproteobacteria and Bacteroidetes in the less-impacted areas, and that of Firmicutes in the contaminated groundwater. The combined characteristics of increased species diversity, distinct phylogenetic composition, and the possible presence of fecal and/or pathogenic bacteria could indicate different types of contamination. Significant correlations between ARGs, MGEs and specific taxa within the groundwater bacterial community were identified, revealing the potential hosts of resistance types. Although no universal marker gene could be determined, a co-selection of int1, qacEΔ1 and sulI genes, a proxy group for anthropogenic pollution, with the tetC, tetO, tetW resistance genes was identified. As the tet group was observed to follow the pattern of environmental contamination for the groundwater samples investigated in this study, our results strongly support the proposal of this group of genes as an environmental tracer of human impact. Overall, the present study investigated several emerging contaminants in groundwater habitats that may be included in monitoring programs to enable further regulatory and protection measures.

Microbial Composition and Diversity Patterns in Deep Hyperthermal Aquifers from the Western Plain of Romania.

A limited number of studies have investigated the biodiversity in deep continental hyperthermal aquifers and its influencing factors. Here, we present the first description of microbial communities inhabiting the Pannonian and Triassic hyperthermal aquifers from the Western Plain of Romania, the first one being considered a deposit of "fossilized waters," while the latter is embedded in the hydrological cycle due to natural refilling. The 11 investigated drillings have an open interval between 952 and 3432 m below the surface, with collected water temperatures ranging between 47 and 104 °C, these being the first microbial communities characterized in deep continental water deposits with outflow temperatures exceeding 80 °C. The abundances of bacterial 16S rRNA genes varied from approximately 105-106 mL-1 in the Pannonian to about 102-104 mL-1 in the Triassic aquifer. A 16S rRNA gene metabarcoding analysis revealed distinct microbial communities in the two water deposits, especially in the rare taxa composition. The Pannonian aquifer was dominated by the bacterial genera Hydrogenophilus and Thermodesulfobacterium, together with archaeal methanogens from the Methanosaeta and Methanothermobacter groups. Firmicutes was prevalent in the Triassic deposit with a large number of OTUs affiliated to Thermoanaerobacteriaceae, Thermacetogenium, and Desulfotomaculum. Species richness, evenness, and phylogenetic diversity increased alongside with the abundance of mesophiles, their presence in the Triassic aquifer being most probably caused by the refilling with large quantities of meteoric water in the Carpathian Mountains. Altogether, our results show that the particular physico-cheminal characteristics of each aquifer, together with the water refilling possibilities, seem to determine the microbial community structure.

Differences in Temperature and Water Chemistry Shape Distinct Diversity Patterns in Thermophilic Microbial Communities.

This report describes the biodiversity and ecology of microbial mats developed in thermal gradients (20 to 65°C) in the surroundings of three drillings (Chiraleu [CH], Ciocaia [CI], and Mihai Bravu [MB]) tapping a hyperthermal aquifer in Romania. Using a metabarcoding approach, 16S rRNA genes were sequenced from both DNA and RNA transcripts (cDNA) and compared. The relationships between the microbial diversity and the physicochemical factors were explored. Additionally, the cDNA data were used for in silico functionality predictions, bringing new insights into the functional potential and dynamics of these communities. The results showed that each hot spring determined the formation of distinct microbial communities. In the CH mats (40 to 53°C), the abundance of Cyanobacteria decreased with temperature, opposite to those of Chloroflexi and ProteobacteriaEctothiorhodospira, Oscillatoria, and methanogenic archaea dominated the CI communities (20 to 65°C), while the MB microbial mats (53 to 65°C) were mainly composed of Chloroflexi, Hydrogenophilus, Thermi, and Aquificae Alpha-diversity was negatively correlated with the increase in water temperature, while beta-diversity was shaped in each hot spring by the unique combination of physicochemical parameters, regardless of the type of nucleic acid analyzed (DNA versus cDNA). The rank correlation analysis revealed a unique model that associated environmental data with community composition, consisting in the combined effect of Na+, K+, HCO3-, and PO43- concentrations, together with temperature and electrical conductivity. These factors seem to determine the grouping of samples according to location, rather than with the similarities in thermal regimes, showing that other parameters beside temperature are significant drivers of biodiversity.IMPORTANCE Hot spring microbial mats represent a remarkable manifestation of life on Earth and have been intensively studied for decades. Moreover, as hot spring areas are isolated and have a limited exchange of organisms, nutrients, and energy with the surrounding environments, hot spring microbial communities can be used in model studies to elucidate the colonizing potential within extreme settings. Thus, they are of great importance in evolutionary biology, microbial ecology, and exobiology. In spite of all the efforts that have been made, the current understanding of the influence of temperature and water chemistry on the microbial community composition, diversity, and abundance in microbial mats is limited. In this study, the composition and diversity of microbial communities developed in thermal gradients in the vicinity of three hot springs from Romania were investigated, each having particular physicochemical characteristics. Our results expose new factors that could determine the formation of these ecosystems, expanding the current knowledge in this regard.

Hypersaline sapropels act as hotspots for microbial dark matter.

Present-day terrestrial analogue sites are crucial ground truth proxies for studying life in geochemical conditions close to those assumed to be present on early Earth or inferred to exist on other celestial bodies (e.g. Mars, Europa). Although hypersaline sapropels are border-of-life habitats with moderate occurrence, their microbiological and physicochemical characterization lags behind. Here, we study the diversity of life under low water activity by describing the prokaryotic communities from two disparate hypersaline sapropels (Transylvanian Basin, Romania) in relation to geochemical milieu and pore water chemistry, while inferring their role in carbon cycling by matching taxa to known taxon-specific biogeochemical functions. The polyphasic approach combined deep coverage SSU rRNA gene amplicon sequencing and bioinformatics with RT-qPCR and physicochemical investigations. We found that sapropels developed an analogous elemental milieu and harbored prokaryotes affiliated with fifty-nine phyla, among which the most abundant were Proteobacteria, Bacteroidetes and Chloroflexi. Containing thirty-two candidate divisions and possibly undocumented prokaryotic lineages, the hypersaline sapropels were found to accommodate one of the most diverse and novel ecosystems reported to date and may contribute to completing the phylogenetic branching of the tree of life.

Abundance of antibiotics, antibiotic resistance genes and bacterial community composition in wastewater effluents from different Romanian hospitals.

Antimicrobial resistance represents a growing and significant public health threat, which requires a global response to develop effective strategies and mitigate the emergence and spread of this phenomenon in clinical and environmental settings. We investigated, therefore, the occurrence and abundance of several antibiotics and antibiotic resistance genes (ARGs), as well as bacterial community composition in wastewater effluents from different hospitals located in the Cluj County, Romania. Antibiotic concentrations ranged between 3.67 and 53.05 µg L-1, and the most abundant antibiotic classes were ß-lactams, glycopeptides, and trimethoprim. Among the ARGs detected, 14 genes confer resistance to ß-lactams, aminoglycosides, chloramphenicol, macrolide-lincosamide-streptogramin B (MLSB) antibiotics, sulfonamides, and tetracyclines. Genes encoding quaternary ammonium resistance and a transposon-related element were also detected. The sulI and qacEΔ1 genes, which confer resistance to sulfonamides and quaternary ammonium, had the highest relative abundance with values ranging from 5.33 × 10-2 to 1.94 × 10-1 and 1.94 × 10-2 to 4.89 × 10-2 copies/16 rRNA gene copies, respectively. The dominant phyla detected in the hospital wastewater samples were Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Among selected hospitals, one of them applied an activated sludge and chlorine disinfection process before releasing the effluent to the municipal collector. This conventional wastewater treatment showed moderate removal efficiency of the studied pollutants, with a 55-81% decrease in antibiotic concentrations, 1-3 order of magnitude lower relative abundance of ARGs, but with a slight increase of some potentially pathogenic bacteria. Given this, hospital wastewaters (raw or treated) may contribute to the spread of these emerging pollutants in the receiving environments. To the best of our knowledge, this study quantified for the first time the abundance of antibiotics and ARGs in wastewater effluents from different Romanian hospitals.

Exploring the Role of Coliform Bacteria in Class 1 Integron Carriage and Biofilm Formation During Drinking Water Treatment.

This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacEΔ1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.

Structure, mineralogy, and microbial diversity of geothermal spring microbialites associated with a deep oil drilling in Romania.

Modern mineral deposits play an important role in evolutionary studies by providing clues to the formation of ancient lithified microbial communities. Here we report the presence of microbialite-forming microbial mats in different microenvironments at 32°C, 49°C, and 65°C around the geothermal spring from an abandoned oil drill in Ciocaia, Romania. The mineralogy and the macro- and microstructure of the microbialites were investigated, together with their microbial diversity based on a 16S rRNA gene amplicon sequencing approach. The calcium carbonate is deposited mainly in the form of calcite. At 32°C and 49°C, the microbialites show a laminated structure with visible microbial mat-carbonate crystal interactions. At 65°C, the mineral deposit is clotted, without obvious organic residues. Partial 16S rRNA gene amplicon sequencing showed that the relative abundance of the phylum Archaea was low at 32°C (<0.5%) but increased significantly at 65°C (36%). The bacterial diversity was either similar to other microbialites described in literature (the 32°C sample) or displayed a specific combination of phyla and classes (the 49°C and 65°C samples). Bacterial taxa were distributed among 39 phyla, out of which 14 had inferred abundances >1%. The dominant bacterial groups at 32°C were Cyanobacteria, Gammaproteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Thermi, Actinobacteria, Planctomycetes, and Defferibacteres. At 49°C, there was a striking dominance of the Gammaproteobacteria, followed by Firmicutes, Bacteroidetes, and Armantimonadetes. The 65°C sample was dominated by Betaproteobacteria, Firmicutes, [OP1], Defferibacteres, Thermi, Thermotogae, [EM3], and Nitrospirae. Several groups from Proteobacteria and Firmicutes, together with Halobacteria and Melainabacteria were described for the first time in calcium carbonate deposits. Overall, the spring from Ciocaia emerges as a valuable site to probe microbes-minerals interrelationships along thermal and geochemical gradients.