RNA sequencing data.

Klebsiella pneumoniae is becoming increasingly difficult to treat as multidrug-resistant (MDR) strains become more prevalent. The formation of biofilm heightens this threat by embedding bacterial cells in a polysaccharide-rich matrix that limits antibiotic penetration. Here we dissect the anti-biofilm bovine host-defense cathelicidin peptide fragment bac7 (1–35), exploring its anti-biofilm mechanism, evaluating its ability to curb colonization of the vital organs by hypervirulent K. pneumoniae, and testing its breadth of activity against diverse clinical isolates. Transcriptomic profiling revealed that bac7 (1–35) simultaneously compromises the bacterial membrane and inhibits ribosomal function, a dual assault that precipitates rapid biofilm collapse and blocks bacterial spread. Further, bac7 (1–35) eradicated the strongest biofilms produced by MDR clinical isolates in the Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) diversity panel. Although bac7 (1–35) kills bacterial cells via a cytosolic mechanism, membrane interaction profiles varied among MRSN isolates, correlating with differential peptide translocation. In a delayed-treatment murine skin-abscess model, bac7 (1–35) halted in vivo colonization of the vital organs by the hypervirulent strain NTUH-K2044. Collectively, these results delineate a multifaceted mode of action for bac7 (1–35) and underscore its therapeutic promise against biofilm-associated MDR K. pneumoniae infections.