Genomic taxonomy reveals distinct evolutionary architectures within the Enterobacter cloacae complex

The Enterobacter cloacae complex is a major nosocomial pathogen group, yet its taxonomy remains poorly resolved below the species level, obscuring the evolutionary drivers of virulence and antimicrobial resistance. Here we show, by analyzing 254 clinical isolates from Taiwan alongside 6,493 public genomes, that a genome-resolved taxonomic framework corrects taxonomic assignments for 54.9% of public genomes, primarily by resolving Enterobacter hormaechei into its five subspecies. Subspecies-level resolution reveals two distinct evolutionary patterns. Virulence determinants and intrinsic resistance genes are strongly associated with subspecies identity and remain globally conserved, including near-universal carriage of the salmochelin locus in E. hormaechei subsp. steigerwaltii and strict linkage of chromosomal AmpC β-lactamases to specific species. By contrast, acquired carbapenemases show pronounced geographic variation independent of taxonomy. These findings demonstrate that subspecies represent the fundamental biological and epidemiological units within the Enterobacter cloacae complex and highlight the importance of subspecies-resolved genomics for diagnostics, antimicrobial stewardship, and global surveillance.