Integration of metagenome-assembled genomes with clinical isolates expands the genomic landscape of gut-associated Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic pathogen causing diseases ranging from gastrointestinal disorders to severe liver abscesses. While clinical isolates of K. pneumoniae have been extensively studied, less is known about asymptomatic variants colonizing the human gut across diverse populations. Developments in genome-resolved metagenomics have offered unprecedented access to metagenome-assembled genomes (MAGs), expanding the known bacterial diversity within the gut microbiome. Here we analysed 656 human gut-derived K. pneumoniae genomes (317 MAGs, 339 isolates) from 29 countries to investigate the population structure and genomic landscape of gut-associated lineages. Over 60% of MAGs were found to belong to new sequence types, highlighting a large uncharacterized diversity of K. pneumoniae missing among sequenced clinical isolates. In particular, integrating MAGs nearly doubled gut-associated K. pneumoniae phylogenetic diversity, and uncovered 86 MAGs with >0.5% genomic distance compared to 20,792 Klebsiella isolate genomes from various sources. Pan-genome analyses identified 214 genes exclusively detected among MAGs, with 107 predicted to encode putative virulence factors. Notably, combining MAGs and isolates revealed genomic signatures linked to health and disease and more accurately classified disease and carriage states compared to isolates alone. These findings showcase the value of metagenomics to understand pathogen evolution and diversity with implications for public health surveillance strategies.