Exploring the IS-capades of Klebsiella pneumoniae: insertion sequences drive metabolic loss in obscure sub-lineages

<b>Introduction.</b> <i>Klebsiella pneumoniae</i> is an opportunistic pathogen that causes a wide spectrum of infections within healthcare settings and the community. Four <i>K. pneumoniae</i> sub-lineages, defined using core gene multi-locus sequence types, are known to cause distinct infections of the nasal and/or upper respiratory passages: SL91 and SL10031 (also referred to as subspecies <i>ozaenae</i>), SL10032 (subspecies <i>rhinoscleromatis</i>) and SL82. These sub-lineages have also demonstrated reduced carbon source utilization, which, in other species, has been linked with high loads of insertion sequences (ISs).<b>Methods.</b> We performed comparative genomics, analysed IS composition and loads and constructed genome-scale metabolic models for available public sequences from these four sub-lineages. These were then compared with other sub-lineages from the wider <i>K. pneumoniae</i> population.<b>Results.</b> The four focal sub-lineages displayed significantly higher IS loads (median range, 88-120 per genome) than other <i>K. pneumoniae</i> sub-lineages (median range, 12-73). Notably, each <i>K. pneumoniae</i> sub-lineage had unique IS profiles, consistent with distinct evolutionary trajectories of IS acquisition and expansion. Across sub-lineages, higher IS loads were inversely associated with the number of metabolic model genes per genome (R²=0.16; <i>P</i><0.001), as well as predicted aerobic substrate utilization for phosphorus sources (R²=0.39; <i>P</i><0.001), as per a second-degree polynomial regression model (<i>n</i>=1,664 genomes). Additionally, the four IS-dense sub-lineages displayed a combination of convergent, sub-lineage-specific substrate utilization losses, including the parallel loss of 3-phospho-d-glycerate, d-glycerate-2-phosphate and phosphoenolpyruvate utilization as carbon/phosphorus sources. Finally, inspection of IS insertion sites demonstrated frequent and non-destructive insertion next to transcriptional, carbohydrate and amino acid metabolism genes.<b>Conclusions.</b> IS accumulation in <i>K. pneumoniae</i> was significantly associated with reduced metabolic substrate usage, consistent with an inverse relationship between IS load and metabolic capacity. Despite these losses, the affected lineages still demonstrate substantial metabolic breadth, consistent with early-stage, ongoing reductive evolution.