Phage vB_KpnM_NB cocktail synergizing with amikacin in inhibiting persister cells of Klebsiella pneumoniae

Abstract

ABSTRACT The emergence of multidrug-resistant Klebsiella pneumoniae (KPN) and antibiotic-tolerant persister cells poses a significant challenge to existing anti-infection therapies. Given the urgent need for sustainable alternatives to antibiotics, phage cocktails are emerging as a promising alternative to control K. pneumoniae infections. We isolated three lytic phages vB_KpnM_NB (1–3) from Ningbo environmental samples, classified them into the Drexlerviridae family, and determined the biological characteristics of two representative phages. Genomic analysis confirmed that these phages are closely related and lack resistance and virulence genes, ensuring biosafety. Subsequently, a stable KPN persister model was established using amikacin, with a biphasic killing pattern observed during treatment. At a multiplicity of infection of 10, the phage cocktail eliminated 99.00% of persister cells, while individual phages were less effective. The phage cocktail also inhibited persister-derived biofilm formation, showing improved results when combined with amikacin. This combination significantly reduced capsule polysaccharide production in persisters, weakening the outer membrane barrier. These findings demonstrate that the phage cocktail-amikacin combination effectively targets planktonic cells, persister cells, and biofilms, providing a promising strategy against persisters and recurrent K. pneumoniae infections. IMPORTANCE This study fills the critical gap in understanding how phage cocktails synergize with amikacin against K. pneumoniae persister cells. By constructing a highly specific phage vB_KpnM_NB cocktail, establishing a stable persister model, and performing in vitro bactericidal and biofilm assays, we demonstrate that the cocktail effectively eliminates planktonic cells, persisters, and biofilms. We clarify the core synergistic mechanism: inhibiting capsular polysaccharide synthesis, improving phage adsorption, and disrupting the bacterial outer membrane barrier. These findings provide experimental evidence for the prevention and control of multidrug-resistant and carbapenem-resistant K. pneumoniae persister infections, establishing a safe and effective phage-antibiotic combination therapy. The results are crucial for addressing antibiotic tolerance and controlling chronic, recurrent infections. They hold significant theoretical and translational value for the treatment of refractory infections in clinical settings and offer new insights into the development of novel antimicrobial strategies. This study fills the critical gap in understanding how phage cocktails synergize with amikacin against K. pneumoniae persister cells. By constructing a highly specific phage vB_KpnM_NB cocktail, establishing a stable persister model, and performing in vitro bactericidal and biofilm assays, we demonstrate that the cocktail effectively eliminates planktonic cells, persisters, and biofilms. We clarify the core synergistic mechanism: inhibiting capsular polysaccharide synthesis, improving phage adsorption, and disrupting the bacterial outer membrane barrier. These findings provide experimental evidence for the prevention and control of multidrug-resistant and carbapenem-resistant K. pneumoniae persister infections, establishing a safe and effective phage-antibiotic combination therapy. The results are crucial for addressing antibiotic tolerance and controlling chronic, recurrent infections. They hold significant theoretical and translational value for the treatment of refractory infections in clinical settings and offer new insights into the development of novel antimicrobial strategies.