The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, bla_TEM with intermediate abundance and bla_OXA-48like, bla_{CTX-M-1 group}, bla_{CTX-M-9 group} and bla_KPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

In the present study 12 water samples of five sampling sites (Tatabánya, Dandár, Szentendre, Szent Flórián and Ciprián groundwaters) known as nutrient-depleted aquatic environments were studied using amplicon sequencing (NGS) and cultivation techniques. Diversity indices and cell counts were determined to assess the species richness in relation to the cell counts within the samples, and the oligocarbophile growth capability of the isolated bacteria was tested in microtiter plates. Altogether, 55 bacterial phyla were identified from the samples by amplicon sequencing. The microbial communities of the different sampling times of the same sites did not differ significantly. Patescibacteria and Proteobacteria were present in all samples. Ciprián sample was dominated by Bacteroidetes, while in Dandár sample a high ratio of Chloroflexi was detected. Rokubacteria and WOR-1 dominated Szent Flórián sample and Tatabánya had a high number of Epsilonbacteraeota. Nine archaeal phyla were also detected; the samples were characterized by the presence of unclassified archaea and Nanoarchaeota, among them Woesearchaeia, as the most dominant. Crenarchaeota and Altiarchaeota were detected in high ratios in Dandár water samples. Among Thaumarchaeota the family Nitrosopumilaceae, and orders of Nitrosotaleales and Nitrososphaerales appeared in Szent Flórián and Tatabánya samples. Key organisms of the different biogeochemical cycles were discovered in these nutrient-depleted environments: methanogenic archaea, methanotrophic bacteria, ammonia oxidizer, nitrate reducers, diazotrophs, sulfate reducers, and sulfur oxidizer. Diversity indices and cell counts of the samples show negative correlation in case of bacteria and positive in case of archaea in Ciprián sample. The high diversity indices in Szentendre samples are connected to low cell counts, most probably due to the vulnerability of the groundwaters to the external environment factors which lead to the infiltration of soil microbes and contaminants to the water. The isolated bacteria were affiliated into four phyla, most of them belonging to Proteobacteria (59%) followed by Actinobacteria (21%), Firmicutes (17%) and Verrucomicrobia (1%). The members of the facultative chemolithotrophic genera of Sphingobium, Sphingomonas, Sphingopyxis were characterizing only Szentendre, Szent Flórián and Tatabánya samples. Only 10% of the isolated species showed an obligate oligocarbophile character. From the samples, a high number of novel bacterial taxa were cultivated. As a conclusion, our results confirmed the predominance of unclassified and unknown taxa in subsurface water, pointing to the importance and necessity of further studies to characterize these microbial populations.

The Moroccan coast is characterized by a diversity of climate, reflecting a great richness and diversity of fauna and flora. By this, marine microbiota plays a fundamental role in many biogeochemical processes, environmental modifications, and responses to temperature changes. To date, no exploration by high-throughput techniques has been carried out on the characterization of the Moroccan marine microbiota. The objective of this work is to study the diversity and metabolic functions of MMM from the Moroccan coast (Atlantic and Mediterranean) according to the water source (WS) and the type of climate (CT) using the approach high-throughput sequencing of the 16SrRNA gene. Four water samples of twelve sampling sites from the four major climates along the Moroccan coastline were collected, and prokaryotic DNA was extracted. V4 region of 16S rRNA gene was amplified, and the product PCR was sequenced by Illumina Miseq. The β-diversity and α-diversity indices were determined to assess the species richness and evenness. The obtained results were analyzed by Mothur and R software. A total of twenty-eight Bacterial phyla and twelve Archaea were identified from the samples. Proteobacteria, Bacteroidetes, and Cyanobacteria are the three key bacterial phyla, and the Archaeal phyla identified are: Euryarchaeota, Nanoarchaeaeota, Crenarchaeota, Hydrothermarchaeota, Asgardaeota, Diapherotrites, and Thaumarchaeota in the Moroccan coastline and the four climates studied. The whole phylum are involved in marine biogeochemical cycles, and through their functions they participate in the homeostasis of the ocean in the presence of pollutants or stressful biotic and abiotic factors. In conclusion, the obtained results reported sufficient deepness of sequencing to cover the majority of Archaeal and Bacterial genera in each site. We noticed a strong difference in microbiota diversity, abundance, and taxonomy inter- and intra-climates and water source without significant differences in function. To better explore this diversity, other omic approaches can be applied such as the metagenomic shotgun, and transcriptomic approaches allowing a better characterization of the Moroccan marine microbiota and to understand the mechanisms of its adaptation and its impacts in/on the ecosystem.

In this study, a Gram-stain-positive, non-motile, oxidase- and catalase-negative, rod-shaped, bacterial strain (SG_E_30_P1^T) that formed light yellow colonies was isolated from a groundwater sample of Sztaravoda spring, Hungary. Based on 16S rRNA phylogenetic and phylogenomic analyses, the strain was found to form a distinct linage within the family Microbacteriaceae . Its closest relatives in terms of near full-length 16S rRNA gene sequences are Salinibacterium hongtaonis MH299814 (97.72 % sequence similarity) and Leifsonia psychrotolerans GQ406810 (97.57 %). The novel strain grows optimally at 20–28 °C, at neutral pH and in the presence of NaCl (1–2 w/v%). Strain SG_E_30_P1^T contains MK-7 and B-type peptidoglycan with diaminobutyrate as the diagnostic amino acid. The major cellular fatty acids are anteiso-C_15 : 0, iso-C_16 : 0 and iso-C_14 : 0, and the polar lipid profile is composed of diphosphatidylglycerol and phosphatidylglycerol, as well as an unidentified aminoglycolipid, aminophospholipid and some unidentified phospholipids. The assembled draft genome is a contig with a total length of 2 897 968 bp and a DNA G+C content of 65.5 mol%. Amino acid identity values with it closest relatives with sequenced genomes of <62.54 %, as well as other genome distance results, indicate that this bacterium represents a novel genus within the family Microbacteriaceae . We suggest that SG_E_30_P1^T (=DSM 111415^T=NCAIM B.02656^T) represents the type strain of a novel genus and species for which the name Antiquaquibacter oligotrophicus gen. nov., sp. nov. is proposed.

In the present study, water samples from 8 sites known as: Taploca, Nagy-borvíz, Piricske (located in Romania) and Szent Jakab, Kiskút, Kossuth Lajos, Polányi kút and Berzsenyi (located in Balaton highland in Hungary) and characterized with low nutrient content, were studied using cultivation independent methods. Diversity indices and cell counts were determined to assess the species richness in relation to the cell counts within the samples. Next generation sequencing was used to reveal the existing microbial community, and taxon specific PCR was used to detect the presence of some species with hygienic. 18 bacterial phyla above a ratio of 2% were identified in addition to 13 archaeal phyla using amplicon sequencing.Nagy-borvíz water sample showed a unique presence of Chloroflexi, Bacteroidota, Desulfobacterota, Acidobacteriota and Actinobacteriota. Piricske water sample showed mostly Caldisericota and Spirochaetota. Taploca water sample contained a unique presence of Planctomycetota and Myxococcota at the same time with Szent Jakab water sample which showed an additional presence of Chloroflexi, Berzsenyi contained Campylobacterota at the same time with Polányi kút which had important fractions of Nitrospirota and Desulfobacterota. Finally, Kossuth Lajos water sample was dominated by Fusobacteriota, mainly members of Hypnocyclicus genus.Piricske and especially Nagy-borvíz water samples contained important fractions of Altiarchaeota, moreover, Piricske contained high fraction of Euryarchaeota phylum -most of them belong to Methanobacteriaceae family.Nitrosopumilaceae dominated Taploca and Kiskút in addition to their important presence in the rest of the samples. Diversity indices and cell count showed in general that cell count values are tending to be lower when the diversity is higher. The results of taxon specific PCR reactions showed that water treatment would be essential in some water samples.