Glacier-fed stream biofilms harbour diverse resistomes and biosynthetic gene clusters

Background: Antimicrobial resistance (AMR) is a universal phenomenon whose origins lay in natural ecological interactions such as competition within niches, within and between micro- to higher-order organisms. However, the ecological and evolutionary processes shaping AMR need to be better understood in view of better antimicrobial stewardship. Resolving antibiotic biosynthetic pathways, including biosynthetic gene clusters (BGCs), and corresponding antimicrobial resistance genes (ARGs) may therefore help in understanding the inherent mechanisms. However, to study these phenomena, it is crucial to examine the origins of AMR in pristine environments with limited anthropogenic influences. In this context, epilithic biofilms residing in glacier-fed streams (GFSs) are an excellent model system to study diverse, intra- and inter-domain, ecological crosstalk. Results: We assessed the resistomes of epilithic biofilms from GFSs across the Southern Alps (New Zealand) and the Caucasus (Russia) and observed that both bacteria and eukaryotes encoded twenty-nine distinct AMR categories. Of these, beta-lactam, aminoglycoside, and multidrug resistance were both abundant and taxonomically distributed in most of the bacterial and eukaryotic phyla. AMR-encoding phyla included Bacteroidota and Proteobacteria among the bacteria, alongside Ochrophyta (algae) among the eukaryotes. Additionally, BGCs involved in the production of antibacterial compounds were identified across all phyla in the epilithic biofilms. Furthermore, we found that several bacterial genera (Flavobacterium, Polaromonas, etc.) including representatives of the superphylum Patescibacteria encode both ARGs and BGCs within close proximity of each other, thereby demonstrating their capacity to simultaneously influence and compete within the microbial community. Conclusions: Our findings highlight the presence and abundance of AMR in epilithic biofilms within GFSs. Additionally, we identify their role in the complex intra- and inter-domain competition and the underlying mechanisms influencing microbial survival in GFS epilithic biofilms. We demonstrate that eukaryotes may serve as AMR reservoirs owing to their potential for encoding ARGs. We also find that the taxonomic affiliation of the AMR and the BGCs are congruent. Importantly, our findings allow for understanding how naturally occurring BGCs and AMR contribute to the epilithic biofilms mode of life in GFSs. Importantly, these observations may be generalizable and potentially extended to other environments which may be more or less impacted by human activity.

Diversity of Aerobic Anoxygenic Phototrophs and Rhodopsin-Containing Bacteria in the Surface Microlayer, Water Column and Epilithic Biofilms of Lake Baikal

The diversity of aerobic anoxygenic phototrophs (AAPs) and rhodopsin-containing bacteria in the surface microlayer, water column, and epilithic biofilms of Lake Baikal was studied for the first time, employing pufM and rhodopsin genes, and compared to 16S rRNA diversity. We detected pufM-containing Alphaproteobacteria (orders Rhodobacterales, Rhizobiales, Rhodospirillales, and Sphingomonadales), Betaproteobacteria (order Burkholderiales), Gemmatimonadetes, and Planctomycetes. Rhodobacterales dominated all the studied biotopes. The diversity of rhodopsin-containing bacteria in neuston and plankton of Lake Baikal was comparable to other studied water bodies. Bacteroidetes along with Proteobacteria were the prevailing phyla, and Verrucomicrobia and Planctomycetes were also detected. The number of rhodopsin sequences unclassified to the phylum level was rather high: 29% in the water microbiomes and 22% in the epilithon. Diversity of rhodopsin-containing bacteria in epilithic biofilms was comparable with that in neuston and plankton at the phyla level. Unweighted pair group method with arithmetic mean (UPGMA) and non-metric multidimensional scaling (NMDS) analysis indicated a distinct discrepancy between epilithon and microbial communities of water (including neuston and plankton) in the 16S rRNA, pufM and rhodopsin genes.

Hydromorphologic Sorting of In-Stream Nitrogen Uptake Across Spatial Scales

Nitrogen (N) uptake is a key process in stream ecosystems that is mediated mainly by benthic microorganisms (biofilms on different substrata) and has implications for the biogeochemical fluxes at catchment scale and beyond. Here, we focused on the drivers of assimilatory N uptake, especially the effects of hydromorphology and other environmental constraints, across three spatial scales: micro, meso and reach. In two seasons (summer and spring), we performed whole-reach 15N-labelled ammonium injection experiments in two montane, gravel-bed stream reaches with riffle–pool sequences. N uptake was highest in epilithic biofilms, thallophytes and roots (min–max range 0.2–545.2 mg N m−2 day−1) and lowest in leaves, wood and fine benthic organic matter (0.05–209.2 mg N m−2 day−1). At the microscale, N uptake of all primary uptake compartments except wood was higher in riffles than in pools. At the mesoscale, hydromorphology determined the distribution of primary uptake compartments, with fast-flowing riffles being dominated by biologically more active compartments and pools being dominated by biologically less active compartments. Despite a lower biomass of primary uptake compartments, mesoscale N uptake was 1.7–3.0 times higher in riffles than in pools. At reach scale, N uptake ranged from 79.6 to 334.1 mg N m−2 day−1. Highest reach-scale N uptake was caused by a bloom of thallopyhtes, mainly filamentous autotrophs, during stable low discharge and high light conditions. Our results reveal the important role of hydromorphologic sorting of primary uptake compartments at mesoscale as a controlling factor for reach-scale N uptake in streams.

Assessing arsenic bioremediation potential of epilithic biofilms affected by acid-mine drainage

<p>Arsenic is a toxic but naturally abundant metalloid that globally leads to contamination in groundwater and soil, exposing millions of people to cancer and other arsenic-related diseases. In several areas in Northern Italy arsenic in soil and water exceeds law limits (20 mg kg<sup>-1</sup> and 10 mg L<sup>-1</sup>, respectively), due to both the mineralogy of bedrock and former mining activities. The Rio Rosso stream, located in the Anzasca Valley (Piedmont) is heavily affected by an acid mine drainage originated from an abandoned gold mine. Arsenic, together with other heavy metals, is transferred by the stream to the surrounding area. The stream is characterized by the presence of an extensive reddish epilithic biofilm at the opening of the mine and on the whole contaminated waterbed.</p> <p>The aim of this study was to characterize the mechanisms allowing the biotic fraction of this biofilm to cope with extreme arsenic concentrations. The composition and functionality of the microbial communities constituting the epilithic biofilms sampled in the close proximity and downstream the mine were unraveled by 16S rRNA genes and shotgun Illumina sequencing in relation to the extreme physico-chemical parameters. In parallel, autotrophic and heterotrophic microbial populations were characterized <em>in vivo</em> by enrichment cultivation and isolated strains were tested for their ability to perform arsenic redox transformation.</p> <p>Preliminary analyses indicated that the biofilm accumulated arsenic in the order of 6 · 10<sup>3</sup> mg kg<sup>-1</sup>, in contrast to 0.14 mg L<sup>-1</sup>, measured in the surrounding water. The main chemical parameter affecting the composition of the microbial community was the pH, being 2 next to the mine and 6.7 in the downstream sampling point. In both sampling sites iron- and sulfur-cycling microorganisms were retrieved by both cultivation and molecular methods. However, the diversity of the microbial community living next to the mine was significantly lower with respect to the community developed downstream. In the latter, autotrophic <em>Cyanobacteria</em> belonging to the species <em>Tychonema</em> were the dominant taxa. A complete arsenic cycle was shown to occur, with heterotrophic bacteria mainly responsible for arsenate reduction and autotrophic bacteria performing arsenite  oxidation.</p> <p>These observations indicate that the epilithic biofilm living in the Rio Rosso stream represents a peculiar ecosystem where microorganisms cope with metalloid toxicity likely using diverse mechanisms. Such microbial metabolic properties might be exploited in bioremediation strategies applied in arsenic-contaminated environments.</p>