Glacier-Fed Stream Biofilms Harbor Diverse Resistomes and Biosynthetic Gene Clusters

Abstract

Antimicrobial resistance (AMR) is a universal phenomenon the origins of which lay in natural ecological interactions such as competition within niches, within and between micro- to higher-order organisms. To study these phenomena, it is crucial to examine the origins of AMR in pristine environments, i.e., 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. 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, biosynthetic gene clusters (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, Superphylum Patescibacteria) encode both atimicrobial resistance genes (ARGs) and BGCs within close proximity of each other, demonstrating their capacity to simultaneously influence and compete within the microbial community. Our findings help unravel how naturally occurring BGCs and AMR contribute to the epilithic biofilms mode of life in GFSs. Additionally, we report that eukaryotes may serve as AMR reservoirs owing to their potential for encoding ARGs. Importantly, these observations may be generalizable and potentially extended to other environments that may be more or less impacted by human activity. IMPORTANCE Antimicrobial resistance is an omnipresent phenomenon in the anthropogenically influenced ecosystems. However, its role in shaping microbial community dynamics in pristine environments is relatively unknown. Using metagenomics, we report the presence of antimicrobial resistance genes and their associated pathways in epilithic biofilms within glacier-fed streams. Importantly, we observe biosynthetic gene clusters associated with antimicrobial resistance in both pro- and eukaryotes in these biofilms. Understanding the role of resistance in the context of this pristine environment and complex biodiversity may shed light on previously uncharacterized mechanisms of cross-domain interactions.

Keywords: antimicrobial resistance; biosynthetic gene clusters; cross-domain interactions; freshwater ecosystems; glacier-fed streams; metagenomics.

FIG 1

Epilithic biofilms in GFSs harbor a diverse resistome. (a) Relative abundance of 29 AMR categories within 21 epilithic biofilms collected from four New Zealand Southern Alps (SA) and four Russian Caucasus (CU) GFSs. (b) Bar plots depicting the relative abundance of bacteria and eukaryotes encoding ARGs. (c) Phylum-level representation of the AMR abundances across bacteria and eukaryotes. Size of the closed circle indicates the normalized relative abundance (RNum_Gi; see Materials and Methods), and the color represents individual phyla.

FIG 2

Phylogenetic analyses of putative eukaryotic ARGs. (a) Phylogenetic tree depicting the relatedness and potential homology between ARG sequences from the reference database, i.e., CARD, with the TETA (tetracycline) resistance gene sequence retrieved from GFS eukaryotes. (b) Tree built using the ERM (MLS phenotype) resistance genes retrieved from the eukaryotes and CARD databases. Group in both figures indicates the origins of the sequence, i.e., CARD database or eukaryote.

FIG 3

Biosynthetic gene clusters indicate the resistome potential. (a) Heatmap depicting the overall abundance of BGCs identified across bacterial and eukaryotic MAGs. The respective phyla are listed on the left while the colored legend represents the taxonomic order. (b) In-depth characterization of the “antibacterial” BGCs found within all phyla and orders across medium-to-high quality MAGs. (c) Alluvial plots depicting the taxa where both BGCs and AMR were found adjacently on the same contig. Colours indicate the genera associated with the MAGs. (d) Genome plots indicating the location of the ARGs in relation to the biosynthetic gene clusters.