Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

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, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC 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.

Exploring the relationship between metal(loid) contamination rate, physicochemical conditions, and microbial community dynamics in industrially contaminated urban soils.

Increasing metal(loid) contamination in urban soils and its impact on soil microbial community have attracted considerable attention. In the present study, the physicochemical parameters and the effects of twelve metal(loid) pollution on soil microbial diversity, their ecotoxic effects, and human health risk assessment in urban soils with different industrial background were studied in comparison with an unpolluted forest soil sample. Results showed that urban soils were highly contaminated, and metal(loid) contamination significantly influenced structure of the soil microbial communities. In all samples the bacterial community was dominated by Proteobacteria, and on the level of phyla characteristic differences were not possible to observe between polluted and control sampling sites. However, clear differences emerged at class and genus level, where several rare taxa disappeared from contaminated urban soils. Simper test results showed that there is 71.6 % bacterial OTU and 9.5 % bacterial diversity dissimilarity between polluted and control samples. Ratio of Patescibacteria, Armatimonadetes, Chlamydiae, Fibrobacteres, and Gemmatimonadetes indicated a significant (p < 0.05) positive correlation with soil Zn, Cr, Pb, Sn, Cu, Mn content, suggest that metal(loid)s strongly influence the structure of microbial community. In contrast, the presence of metal(loid) contamination in urban soils has been found to significantly reduce the population of Archaeal communities. This can be attributed to the depletion of organic matter caused by contamination that reached a minimum of 0.5 m/m% for nitrate and 0.9 m/m% for total organic carbon. The values of urban soil pH were close to neutral, ranging from 5.9 to 8.3. The findings of ecotoxicology test are alarming, as all the studied urban soil sites were cytotoxic to soil microorganisms, and in one site metal(loid) contamination reached genotoxic level. Moreover, all the metal(loid) contaminated sites pose severe and persistent health risk to children, highlighting the urgent need for effective measures to mitigate metal(loid) pollution in urban areas.

The Impact of Anti-Inflammatory Drugs on the Prokaryotic Community Composition and Selected Bacterial Strains Based on Microcosm Experiments.

Non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly recognized as potential environmental contaminants that may induce toxicity in aquatic ecosystems. This 3-week microcosm experiment explores the acute impacts of NSAIDs, including diclofenac (DCF), ibuprofen (IBU), and acetylsalicylic acid (ASA), on bacterial communities using a wide range of these substances (200-6000 ppm). The results showed that the NSAID-treated microcosms had higher cell count values than control samples, though the diversity of microbial communities decreased. The isolated heterotrophic bacteria mostly belonged to Proteobacteria, particularly Klebsiella. Next-generation sequencing (NGS) revealed that NSAIDs altered the structure of the bacterial community composition, with the proportion of Proteobacteria aligning with the selective cultivation results. Bacteria had higher resistance to IBU/ASA than to DCF. In DCF-treated microcosms, there has been a high reduction of the number of Bacteroidetes, whereas in the microcosms treated with IBU/ASA, they have remained abundant. The numbers of Patescibacteria and Actinobacteria have decreased across all NSAID-treated microcosms. Verrucomicrobia and Planctomycetes have tolerated all NSAIDs, even DCF. Cyanobacteria have also demonstrated tolerance to IBU/ASA treatment in the microcosms. The archaeal community structure was also impacted by the NSAID treatments, with Thaumarchaeota abundant in all microcosms, especially DCF-treated microcosms, while Nanoarchaeota is more typical of IBU/ASA-treated microcosms with lower NSAID concentrations. These results indicate that the presence of NSAIDs in aquatic environments could lead to changes in the composition of microbial communities.

Effect of Climate on Bacterial and Archaeal Diversity of Moroccan Marine Microbiota.

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 ß-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.

Microbiological investigations of two thermal baths in Budapest, Hungary. Report: effect of bathing and pool operation type on water quality.

In Hungary, which is famous for its thermal baths, according to the regulations, waters are investigated in hygienic aspects with standard cultivation methods. In the present study, two thermal baths were investigated (the well and three different pool waters in both) using cultivation methods, taxon-specific polymerase chain reactions (PCRs), multiplex PCRs and next-generation amplicon sequencing. Mainly members of the natural microbial community of the well waters and bacteria originating from the environment were detected but several opportunistic pathogenic taxa, e.g., Pseudomonas aeruginosa, P. stutzeri, Acinetobacter johnsoni, Acinetobacter baumanni, Moraxella osloensis, Microbacterium paraoxydans, Legionella spp., Stenotrophomonas maltophilia and Staphylococcus aureus were revealed by the applied methods. Pools with charging-unloading operation had higher microscopic cell counts, colony-forming unit (CFU) counts, number of cocci, P. aeruginosa and S. aureus compared to the recirculation systems. Bacteria originating from human sources (e.g., skin) were identified in the pool waters with less than 1% relative abundance, and their presence was sporadic in the pools. Comparing the microbiological quality of the pools based on the first sampling time and the following four months' period it was revealed that recirculation operation type has better water quality than the charging-unloading pool operation from a hygienic point of view.

Whole genome sequence analysis of Cupriavidus campinensis S14E4C, a heavy metal resistant bacterium.

Cupriavidus sp. are model organisms for heavy metal(loid) resistance and aromatic compound's degradation studies and these characteristics make them a perfect candidate for biotechnological purposes. Bacterial strain S14E4C (identified as Cupriavidus campinensis) was isolated from a playground by enrichment method in a 0.25 mM containing medium. The analysis revealed that this bacterium is able to tolerate high concentrations of heavy metal(loid)s: Cd up to 19.5 mM, Pb to 9 mM, Hg to 5.5 mM and As to 2 mM in heavy metal(loid) salt containing nutrient medium. The whole genome data and analysis of the type strain of C. campinensis CCUG:44526T have not been available so far, thus here we present the genome sequencing results of strain S14E4C of the same species. Analysis was carried out to identify possible mechanisms for the heavy metal resistance and to map the genetic data of C. campinensis. The annotation pipelines revealed that the total genome of strain S14E4C is 6,375,175 bp length with a GC content of 66.3% and contains 2 plasmids with 295,460 bp (GC content 59.9%) and 50,483 bp (GC content 63%). In total 4460 coding sequences were assigned to known functions and 1508 to hypothetical proteins. Analysis proved that strain S14E4C is having gene clusters such as czc, mer, cus, chr, ars to encode various heavy metal resistance mechanisms that play an important role to survive in extreme environments.

Isolation and characterisation of porcine epidemic diarrhoea virus in Hungary - Short communication.

Porcine epidemic diarrhoea virus (PEDV) is an emerging enteropathogen, causing great economic losses in the pig industry. After many years of quiescence, PEDV was detected in Hungary in 2016 with a recombination in its S gene. In order to determine the extent of this change, an attempt was made to isolate the recombinant PEDV. This study was extended with a variety of samples collected from three separate farms with newly identified PEDV in 2018. The recombinant PEDV from 2016 was isolated successfully along with three viruses from 2018, and one isolate from the new cases was used for whole genome determination. Whole genome sequence alignment revealed the highest identity with recombinant Hungarian and Slovenian PEDV within the low-pathogenic European viruses. This suggests that these recombinant PEDV are circulating in this area and may spread to other parts of the continent.

Frequency of diarrhoea-associated viruses in swine of various ages in Hungary.

Enteric viral diseases of swine are one of the most frequent disorders causing huge economic losses in pork production. After the reappearance of an emerging enteropathogen, porcine epidemic diarrhoea virus (PEDV) in Hungary in 2016, an extensive survey was initiated in an attempt to identify diarrhoea-related porcine viruses, including adeno-, astro-, boca-, calici-, circo-, corona-, kobu-, rota- and Torque teno viruses. A total of 384 faecal samples collected during a twoyear period from diarrhoeic and asymptomatic pigs of various ages in 17 farms were screened by conventional and real-time PCR methods. Half of the samples contained at least one examined virus with the dominance of kobuvirus (55.1%) followed by bocaviruses (33.2%) and rotavirus groups A and C together (20.9%), while coronaviruses including PEDV were not found in this set of samples. Statistical analysis showed a highly significant difference (P < 0.0001) in the frequency of single infections compared to mixed ones with the exception of weaned pigs, in which group additionally most viruses were detected. The results of this study suggest that the complexity of this disease may vary with age, which makes the prevention of diarrhoea a challenge, especially in weaned pigs.