Genus-wide comparative genomics identifies the Cupriavidus gilardii species complex as a high-AMR-burden lineage

The environmentally versatile genus Cupriavidus is increasingly recognized as an underappreciated reservoir of antimicrobial resistance (AMR) and a source of multidrug-resistant opportunistic infections, yet its species diversity, resistome structure, and clinically relevant lineages remain poorly resolved. Here, we analyzed 476 high-quality Cupriavidus genomes, including the complete genome of a newly sequenced multidrug-resistant clinical isolate, C. gilardii WM02, to resolve lineage-level AMR patterns within a genus-wide genome-based framework. Genome delineation resolved 75 genome-defined species, revealing extensive underrecognized diversity and taxonomic inconsistency. Pan-genome analysis indicated an open, flexible gene repertoire, whereas resistome profiling revealed strong lineage-specific variation dominated by low-prevalence determinants. Phylogenetic mapping identified the C. gilardii species complex as a high-AMR-burden lineage. Conserved resistance loci, including soxR , mexC – mexD , aac(3)-IV , and emrE , suggest early establishment and lineage-associated retention. In contrast, mcr-5.1 displayed variable copy numbers, chromosomal and plasmid backgrounds, and remodeled transposon contexts, indicating recurrent mobilization and local stabilization of this colistin resistance determinant. Virulence profiling revealed an additional type III secretion system within this complex. 16S rRNA amplicon meta-analysis indicated human-associated enrichment signals for C. gilardii and C. cauae . These findings provide a genomic framework for prioritizing underrecognized opportunistic Cupriavidus lineages in AMR surveillance.