Hidden reservoir of highly adaptable multi-host plasmids that propagate antibiotic genes in healthy human populations.

Plasmids are key vectors for disseminating antibiotic resistance genes, yet their diversity and dynamics in the healthy human gut microbiome remain largely unexplored. Using fecal metagenomes from two cohorts (n = 498 samples), we constructed a comprehensive atlas of the healthy human gut plasmidome. We observed a polarization: while 97.4% of 19 151 plasmid clusters exhibited low prevalence (<5%), we identified 17 plasmid clusters that were detected in >30% of individuals. Among these, the plasmid pGut1 emerged as a paradigm of a stealth vector. Prevalent globally (>50% in independent cohorts), pGut1 possesses a minimal 4-kb conserved backbone ensuring stability and a hypervariable region acting as a "plug-and-play" module. We documented 40 distinct cargo inserts, including multiple antibiotic resistance genes such as cfr(C), erm(B), and aphA, across individuals, within individuals over time, and even within single fecal samples- validated by single-cell and long-read Nanopore sequencing. Screening of 2.3 million bacterial genomes revealed pGut1 in 93 strains across 49 genera and 2 phyla, including pathogenic Clostridioides difficile and three distinct Salmonella enterica strains. This pattern suggests potential repeated cross-species transmission events, equipping diverse pathogens with new antibiotic resistance genes. Our study exposes a hidden reservoir of highly adaptable, multi-host plasmids like pGut1 silently propagating antibiotic resistance genes in healthy populations. These plasmids, pre-adapted for cross-boundary dissemination, may pose a threat by fueling the emergence of multidrug-resistant pathogens.