Distinct epigenetic signatures of classical and hypervirulent Klebsiella pneumoniae

Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.