Abstract
The rapid spread of antibiotic resistance genes (ARGs) in the microbiota is a major global public health concern. While the viruses infecting prokaryotic microbes (called phages) are known as important mediators of the spread of ARGs, our knowledge of ARGs of those viruses infecting eukaryotic microbes is still extremely limited. To address this knowledge gap, we systematically explored ARGs of nucleocytoplasmic large DNA viruses (NCLDVs; also called giant viruses) that probably infect all major eukaryotic microbial lineages. Analysis of 1,416 NCLDV genomes including those of almost all currently available cultured isolates and high-quality metagenome-assembled genomes from diverse habitats across the globe revealed that 43.7% of them carried ARGs, which was approximately 23 times higher than that for phage genomes. Remarkably, two NCLDVs isolated from a > 30,000-year-old permafrost sample were found to harbor ARGs. A total of 14 ARG types were encoded by NCLDVs, covering nearly half of all known ARG types. Trimethoprim, polymyxin and rifampin were the major ARG types of NCLDVs, and antibiotic target alteration was their predominant antibiotic resistance mechanism. Phylogenies of the three most abundant NCLDV-encoded ARGs hinted that NCLDVs acquired ARGs not only from eukaryotes but also from prokaryotes and phages. A significant interdependence between the presence of ARGs and insertion sequences (ISs) in NCLDV genomes was observed, indicating the involvement of ISs in the acquisition of ARGs by NCLDVs. Minimum inhibitory concentration experiments with two Escherichia coli strains carrying trimethoprim resistance genes of NCLDVs provided the first evidence that NCLDV-encoded ARGs can confer an antibiotic resistance phenotype. Taken together, these findings underscore the overlooked importance of taking into account NCLDVs to obtain a comprehensive understanding of the spread of antibiotic resistance within the whole microbiota.