Unveiling the overlooked small-sized microbiome in river ecosystems

Enriching microorganisms using a 0.22-μm pore size is a general pretreatment procedure in river microbiome research. However, it remains unclear the extent to which this method loses microbiome information. Here, we conducted a comparative metagenomics-based study on microbiomes with sizes over 0.22 μm (large-sized) and between 0.22 μm and 0.1 μm (small-sized) in a subtropical river. Although the absolute concentration of small-sized microbiome was about two orders of magnitude lower than that of large-sized microbiome, sequencing only large-sized microbiome resulted in a significant loss of microbiome diversity. Specifically, the microbial community was different between two sizes, and 347 genera were only detected in small-sized microbiome. Small-sized microbiome had much more diverse viral community than large-sized fraction. The viruses had abundant ecological functions and were hosted by 825 species of 169 families, including pathogen-related families. Small-sized microbiome had distinct antimicrobial resistance risks from large-sized microbiome, showing an enrichment of eight antibiotic resistance gene (ARG) types as well as the detection of 140 unique ARG subtypes and five enriched risk rank I ARGs. Draft genomes of five major resistant pathogens having diverse ecological and pollutant-degrading functions were only assembled in small-sized microbiome. These findings provide novel insights into river ecosystems, and highlight the overlooked small-sized microbiome in the environment.