Contrary effects of increasing temperatures on the spread of antimicrobial resistance in river biofilms.

River microbial communities regularly act as the first barrier of defense against the spread of antimicrobial resistance genes (ARGs) that enter environmental microbiomes through wastewater. However, how the invasion dynamics of wastewater-borne ARGs into river biofilm communities will shift due to climate change with increasing average and peak temperatures remains unknown. Here, we aimed to elucidate the effects of increasing temperatures on the naturally occurring river biofilm resistome, as well as the invasion success of foreign ARGs entering through wastewater. Natural biofilms were grown in a low-anthropogenic impact river and transferred to artificial laboratory recirculation flume systems operated at three different temperatures (20°C, 25°C, and 30°C). After 1 week of temperature acclimatization, significant increases in the abundance of the naturally occurring ARGs in biofilms were detected at higher temperatures. After this acclimatization period, biofilms were exposed to a single pulse of wastewater, and the invasion dynamics of wastewater-borne ARGs were analyzed over 2 weeks. After 1 day, wastewater-borne ARGs were able to invade the biofilms successfully with no observable effect of temperature on their relative abundance. However, thereafter, ARGs were lost at a far increased rate at 30°C, with ARG levels dropping to the initial natural levels after 14 days. Contrary to the lower temperatures, ARGs were either lost at slower rates or even able to establish themselves in biofilms with stable relative abundances above natural levels. Hence, higher temperatures come with contrary effects on river biofilm resistomes: naturally occurring ARGs increase in abundance, while foreign, invading ARGs are lost at elevated speeds.IMPORTANCEInfections with bacteria that gained resistance to antibiotics are taking millions of lives annually, with the death toll predicted to increase. River microbial communities act as a first defense barrier against the spread of antimicrobial resistance genes (ARGs) that enter the environment through wastewater after enrichment in human and animal microbiomes. The global increase in temperature due to climate change might disrupt this barrier effect by altering microbial community structure and functions. We consequently explored how increasing temperatures alter ARG spread in river microbial communities. At higher temperatures, naturally occurring ARGs increased in relative abundance. However, this coincided with a decreased success rate of invading foreign ARGs from wastewater to establish themselves in the communities. Therefore, to predict the effects of climate change on ARG spread in river microbiomes, it is imperative to consider if the river ecosystem and its resistome are dominated by naturally occurring or invading foreign ARGs.

Molecular Typing Reveals Environmental Dispersion of Antibiotic-Resistant Enterococci under Anthropogenic Pressure.

As a consequence of global demographic challenges, both the artificial and the natural environment are increasingly impacted by contaminants of emerging concern, such as bacterial pathogens and their antibiotic resistance genes (ARGs). The aim of this study was to determine the extent to which anthropogenic contamination contributes to the spread of antibiotic resistant enterococci in aquatic compartments and to explore genetic relationships among Enterococcus strains. Antimicrobial susceptibility testing (ampicillin, imipenem, norfloxacin, gentamycin, vancomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole) of 574 isolates showed different rates of phenotypic resistance in bacteria from wastewaters (91.9-94.4%), hospital effluents (73.9%), surface waters (8.2-55.3%) and groundwater (35.1-59.1%). The level of multidrug resistance reached 44.6% in enterococci from hospital effluents. In all samples, except for hospital sewage, the predominant species were E. faecium and E. faecalis. In addition, E. avium, E. durans, E. gallinarum, E. aquimarinus and E. casseliflavus were identified. Enterococcus faecium strains carried the greatest variety of ARGs (blaTEM-1, aac(6')-Ie-aph(2″), aac(6')-Im, vanA, vanB, ermB, mefA, tetB, tetC, tetL, tetM, sul1), while E. avium displayed the highest ARG frequency. Molecular typing using the ERIC2 primer revealed substantial genetic heterogeneity, but also clusters of enterococci from different aquatic compartments. Enterococcal migration under anthropogenic pressure leads to the dispersion of clinically relevant strains into the natural environment and water resources. In conclusion, ERIC-PCR fingerprinting in conjunction with ARG profiling is a useful tool for the molecular typing of clinical and environmental Enterococcus species. These results underline the need of safeguarding water quality as a strategy to limit the expansion and progression of the impending antibiotic-resistance crisis.

Municipal Wastewaters Carry Important Carbapenemase Genes Independent of Hospital Input and Can Mirror Clinical Resistance Patterns.

The spatiotemporal variation of several carbapenemase-encoding genes (CRGs) was investigated in the influent and effluent of municipal WWTPs, with or without hospital sewage input. Correlations among gene abundances, bacterial community composition, and wastewater quality parameters were tested to identify possible predictors of CRGs presence. Also, the possible role of wastewaters in mirroring clinical resistance is discussed. The taxonomic groups and gene abundances showed an even distribution among wastewater types, meaning that hospital sewage does not influence the microbial diversity and the CRG pool. The bacterial community was composed mainly of Proteobacteria, Firmicutes, Actinobacteria, Patescibacteria, and Bacteroidetes. Acinetobacter spp. was the most abundant group and had the majority of operational taxonomic units (OTUs) positively correlated with CRGs. This agrees with recent reports on clinical data. The influent samples were dominated by blaKPC, as opposed to effluent, where blaIMP was dominant. Also, blaIMP was the most frequent CRG family observed to correlate with bacterial taxa, especially with the Mycobacterium genus in effluent samples. Bacterial load, blaNDM, blaKPC, and blaOXA-48 abundances were positively correlated with BOD5, TSS, HEM, Cr, Cu, and Fe concentrations in wastewaters. When influent gene abundance values were converted into population equivalent (PE) data, the highest copies/1 PE were identified for blaKPC and blaOXA-48, agreeing with previous studies regarding clinical isolates. Both hospital and non-hospital-type samples followed a similar temporal trend of CRG incidence, but with differences among gene groups. Colder seasons favored the presence of blaNDM, blaKPC and blaOXA-48, whereas warmer temperatures show increased PE values for blaVIM and blaIMP. IMPORTANCE Wastewater-based epidemiology has recently been recognized as a valuable, cost-effective tool for antimicrobial resistance surveillance. It can help gain insights into the characteristics and distribution of antibiotic resistance elements at a local, national, and even global scale. In this study, we investigated the possible use of municipal wastewaters in the surveillance of clinically relevant carbapenemase-encoding genes (CRGs), seen as critical antibiotic resistance determinants. In this matter, our results highlight positive correlations among CRGs, microbial diversity, and wastewater physical and chemical parameters. Identified predictors can provide valuable data regarding the level of raw and treated wastewater contamination with these important antibiotic resistance genes. Also, wastewater-based gene abundances were used for the first time to observe possible spatiotemporal trends of CRGs incidence in the general population. Therefore, possible hot spots of carbapenem resistance could be easily identified at the community level, surpassing the limitations of health care-associated settings.

Wastewaters, with or without Hospital Contribution, Harbour MDR, Carbapenemase-Producing, but Not Hypervirulent Klebsiella pneumoniae.

Carbapenemase-producing Klebsiella pneumoniae (CPKP) isolated from influent (I) and effluent (E) of two wastewater treatment plants, with (S1) or without (S2) hospital contribution, were investigated. The strains belonged to the Kp1 phylogroup, their highest frequency being observed in S1, followed by S2. The phenotypic and genotypic hypervirulence tests were negative for all the strains tested. At least one carbapenemase gene (CRG), belonging to the blaKPC, blaOXA-48, blaNDM and blaVIM families, was observed in 63% of CPKP, and more than half co-harboured two to four CRGs, in different combinations. Only five CRG variants were observed, regardless of wastewater type: blaKPC-2, blaNDM-1, blaNDM-6, blaVIM-2, and blaOXA-48. Sequence types ST258, ST101 and ST744 were common for both S1 and S2, while ST147, ST525 and ST2502 were found only in S1 and ST418 only in S2. The strains tested were multi-drug resistant (MDR), all being resistant to beta-lactams, cephalosporins, carbapenems, monobactams and fluoroquinolones, followed by various resistance profiles to aminoglycosides, trimethoprim-sulphamethoxazole, tigecycline, chloramphenicol and tetracycline. After principal component analysis, the isolates in S1 and S2 groups did not cluster independently, confirming that the antibiotic susceptibility patterns and gene-type profiles were both similar in the K. pneumoniae investigated, regardless of hospital contribution to the wastewater type.

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.

Contrasting taxonomic stratification of microbial communities in two hypersaline meromictic lakes.

Hypersaline meromictic lakes are extreme environments in which water stratification is associated with powerful physicochemical gradients and high salt concentrations. Furthermore, their physical stability coupled with vertical water column partitioning makes them important research model systems in microbial niche differentiation and biogeochemical cycling. Here, we compare the prokaryotic assemblages from Ursu and Fara Fund hypersaline meromictic lakes (Transylvanian Basin, Romania) in relation to their limnological factors and infer their role in elemental cycling by matching taxa to known taxon-specific biogeochemical functions. To assess the composition and structure of prokaryotic communities and the environmental factors that structure them, deep-coverage small subunit (SSU) ribosomal RNA (rDNA) amplicon sequencing, community domain-specific quantitative PCR and physicochemical analyses were performed on samples collected along depth profiles. The analyses showed that the lakes harbored multiple and diverse prokaryotic communities whose distribution mirrored the water stratification patterns. Ursu Lake was found to be dominated by Bacteria and to have a greater prokaryotic diversity than Fara Fund Lake that harbored an increased cell density and was populated mostly by Archaea within oxic strata. In spite of their contrasting diversity, the microbial populations indigenous to each lake pointed to similar physiological functions within carbon degradation and sulfate reduction. Furthermore, the taxonomy results coupled with methane detection and its stable C isotope composition indicated the presence of a yet-undescribed methanogenic group in the lakes' hypersaline monimolimnion. In addition, ultrasmall uncultivated archaeal lineages were detected in the chemocline of Fara Fund Lake, where the recently proposed Nanohaloarchaeota phylum was found to thrive.

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.

Spatial and temporal distribution of archaeal diversity in meromictic, hypersaline Ocnei Lake (Transylvanian Basin, Romania).

Saline, meromictic lakes with significant depth are usually formed as a result of salt mining activity. Ocnei Lake is one of the largest Transylvanian (Central Romania) neutral, hypersaline lake of man-made origin. We aimed to survey the seasonal dynamics of archaeal diversity in the water column of Ocnei Lake by employing microbiological methods as well as molecular techniques based on the sequence analysis of the 16S rRNA gene. We found that archaeal diversity in the water column increased with depth and salinity, with 8 OTUs being detected in the epilimnion compared to 21 found in the chemocline, and 32 OTUs in the monimolimnion. Down to 3.5 m depth, the archaeal community was markedly dominated by the presence of an unclassified archaeon sharing 93% sequence identity to Halogeometricum spp. At the chemocline, the shift in archaeal community composition was associated with an increase in salinity, the main factor affecting the vertical distribution of archaeal assemblages. It appears that the microoxic and hypersaline monimolimnion is populated by several major haloarchaeal taxa, with minor fluctuations in their relative abundances throughout all seasons. The culturable diversity was reasonably correlated to the dominant OTUs obtained by molecular methods. Our results indicate that Ocnei Lake represents a relatively stable extreme habitat, accommodating a diverse and putatively novel archaeal community, as 30% of OTUs could not be classified at the genus level.

Archaeal and bacterial diversity in two hot spring microbial mats from a geothermal region in Romania.

The diversity of archaea and bacteria was investigated in two slightly alkaline, mesophilic hot springs from the Western Plain of Romania. Phylogenetic analysis showed a low diversity of Archaea, only three Euryarchaeota taxa being detected: Methanomethylovorans thermophila, Methanomassiliicoccus luminyensis and Methanococcus aeolicus. Twelve major bacterial groups were identified, both springs being dominated by Cyanobacteria, Chloroflexi and Proteobacteria. While at the phylum/class-level the microbial mats share a similar biodiversity; at the species level the geothermal springs investigated seem to be colonized by specific consortia. The dominant taxa were filamentous heterocyst-containing Fischerella, at 45 °C and non-heterocyst Leptolyngbya and Geitlerinema, at 55 °C. Other bacterial taxa (Thauera sp., Methyloversatilis universalis, Pannonibacter phragmitetus, Polymorphum gilvum, Metallibacterium sp. and Spartobacteria) were observed for the first time in association with a geothermal habitat. Based on their bacterial diversity the two mats were clustered together with other similar habitats from Europe and part of Asia, most likely the water temperature playing a major role in the formation of specific microbial communities that colonize the investigated thermal springs.

Methodological constraints in the molecular biodiversity study of a thermomineral spring cyanobacterial mat: a case study.

The biodiversity of a specific cyanobacterial mat associated to a thermomineral spring from the Western Plain of Romania was investigated. Light and electron microscopy, together with molecular tools (denaturing gradient gel electrophoresis-DGGE, automated ribosomal intergenic spacer analysis-ARISA and amplified ribosomal DNA restriction analysis-ARDRA), based on 16S rDNA and 16S-23S internal transcribed spacer markers were used. Based on the partial 16S rRNA fragments sequenced, eight cyanobacterial taxons were identified, all belonging to the Oscillatoriales order, Phormidium and Leptolyngbya being dominant. A significant difference was observed, in comparison with the morphological approach. In certain conditions, DGGE can provide misleading information due to multiple melting domains in the same sequence, to multiple rrn operons in the same genome and due to unspecific hybridization among closely related sequences. This can lead to an overestimated species abundance which can cause incorrect description of the microbial community investigated. Additional techniques, such as ARISA and ARDRA, can improve the microbial biodiversity studies, thus providing optimal results.