RESUMO
Epilithic biofilms are ubiquitous in large river environments and are crucial for biogeochemical processes, but their community structures and functions remain poorly understood. In this paper, the seasonal succession in the morphological structure and the taxonomic composition of an epilithic bacterial biofilm community at a polluted site of the Danube River were followed using electron microscopy, high-throughput 16S rRNA gene amplicon sequencing and multiplex/taxon-specific PCRs. The biofilm samples were collected from the same submerged stone and carried out bimonthly in the littoral zone of the Danube River, downstream of a large urban area. Scanning electron microscopy showed that the biofilm was composed of diatoms and a variety of bacteria with different morphologies. Based on amplicon sequencing, the bacterial communities were dominated by the phyla Pseudomonadota and Bacteroidota, while the most abundant archaea belonged to the phyla Nitrososphaerota and Nanoarchaeota. The changing environmental factors had an effect on the composition of the epilithic microbial community. Critical levels of faecal pollution in the water were associated with increased relative abundance of Sphaerotilus, a typical indicator of "sewage fungus", but the composition and diversity of the epilithic biofilms were also influenced by several other environmental factors such as temperature, water discharge and total suspended solids (TSS). The specific PCRs showed opportunistic pathogenic bacteria (e.g. Pseudomonas spp., Legionella spp., P. aeruginosa, L. pneumophila, Stenotrophomonas maltophilia) in some biofilm samples, but extended spectrum ß-lactamase (ESBL) genes and macrolide resistance genes could not be detected.
RESUMO
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.