Plumbagin enhances antimicrobial and anti-biofilm capacities of chlorhexidine against clinical <i>Klebsiella pneumoniae</i> while reducing resistance mutations

With the widespread misuse of disinfectants, the clinical susceptibility of <i>Klebsiella pneumoniae</i> (<i>K. pneumoniae</i>) to chlorhexidine (CHX) has gradually diminished, posing significant challenges to clinical disinfection and infection control. <i>K. pneumoniae</i> employs overexpression of efflux pumps and the formation of thick biofilms to evade the lethal effects of CHX. Plumbagin (PLU) is a natural plant extract that enhances membrane permeability and reduces proton motive force. In this study, we elucidated the synergistic antimicrobial activity of PLU in combination with CHX, effectively reducing the MIC of CHX against <i>K. pneumoniae</i> to 1 µg/mL and below. Crucially, through crystal violet staining and confocal laser scanning microscopy live/dead staining, we discovered that PLU significantly enhances the anti-biofilm capability of CHX. Mechanistically, experiments involving membrane permeability, alkaline phosphatase leakage, reactive oxygen species, and RT-qPCR suggest that the combination of PLU and CHX improves the permeability of bacterial inner and outer membranes, promotes bacterial oxidative stress, and inhibits oqxA/B efflux pump expression. Furthermore, we conducted surface disinfection experiments on medical instruments to simulate clinical environments, demonstrating that the combination effectively reduces bacterial loads by more than 3 log₁₀ CFU/mL. Additionally, results from resistance mutation frequency experiments indicate that combined treatment reduces the generation of resistant mutants within the bacterial population. In summary, PLU can serve as an adjuvant, enhancing the anti-biofilm capability of CHX and reducing the occurrence of resistance mutations, thereby extending the lifespan of CHX.IMPORTANCEAs disinfectants are extensively and excessively utilized worldwide, clinical pathogens are progressively acquiring resistance against these substances. However, high concentrations of disinfectants can lead to cross-resistance to antibiotics, and concurrent use of different disinfectants can promote bacterial resistance mutations and facilitate the horizontal transfer of resistance genes, which poses significant challenges for clinical treatment. Compared with the lengthy process of developing new disinfectants, enhancing the effectiveness of existing disinfectants with natural plant extracts is important and meaningful. CHX is particularly common and widely used compared with other disinfectants. Meanwhile, <i>Klebsiella pneumoniae</i>, as a clinically significant pathogen, exhibits high rates of resistance and pathogenicity. Previous studies and our data indicate a significant decrease in the sensitivity of clinical <i>K. pneumoniae</i> to CHX, highlighting the urgent need for novel strategies to address this issue. In light of this, our research is meaningful.