A Burkholderia phage selects for attenuated virulence and antimicrobial hypersensitivity through increased outer membrane permeability

Burkholderia gladioli is an opportunistic pathogen with intrinsic antimicrobial resistance and is therefore a compelling target for phage therapy (PT), yet bacteriophages infecting this species remain largely uncharacterized. Here, we show that the functionally lytic (FL), lipopolysaccharide (LPS)-binding myovirus KS12, originally isolated against Burkholderia cenocepacia , suppresses B. gladioli growth in vitro and infection-associated mortality in Galleria mellonella . KS12 selects for resistant subpopulations carrying mutations in the O -antigen biosynthesis and export pathways that compromise outer membrane (OM) integrity, resulting in attenuated virulence in vivo and hypersensitivity to human serum, antimicrobial peptides, and polymyxins. Consistent with this trade-off, KS12 and colistin interact synergistically to substantially reduce bacterial densities, suggesting that predation by KS12 may impose an evolutionary trap on B. gladioli . Furthermore, comparative analyses indicate that outer membrane permeability correlates strongly with colistin susceptibility across Gram-negative pathogens, implying that antivirulence steering with LPS-binding phages could provide a strategy to sensitize intrinsically resistant pathogens to antibiotics of last resort. Although KS12 particles were inactivated by innate humoral immunity, they did not appear to be degraded by or stimulate pro-inflammatory responses in phagocytes, indicating that the antibacterial activity of this phage is not driven by direct immunostimulation. Together, these results identify KS12 as a promising phage targeting B. gladioli and highlight the potential of LPS-binding phages to steer bacterial populations toward attenuated virulence and hypersensitivity, thereby yielding more clinically tractable phenotypes.