Optimization of Fermentation Conditions for Antarctic Bacteria and Investigation of Its Antimicrobial Mechanism Against Klebsiella pneumoniae

<i>Klebsiella pneumoniae</i> is the second-most common opportunistic pathogen in clinical practice and has developed resistance to potent antibacterial drugs such as carbapenems. Therefore, developing safe and effective strategies for the prevention and treatment of <i>K. pneumoniae</i> infections remains a critical challenge. In this study, a strain named Tie-10 isolated from Antarctic samples demonstrated potent antibacterial activity against <i>K. pneumoniae</i>, which was subsequently identified as <i>Bacillus nakamurai</i>. The fermentation medium and culture conditions were systematically optimized through single-factor experiments, orthogonal array testing, and response surface methodology. The optimal medium composition was determined to be beef extract, peptone, and KNO₃. The culture conditions included a time of 24 h, temperature of 37 °C, pH of 7.0, and bottling volume of 80 mL. Antagonistic experiments demonstrated that the crude extract of <i>B. nakamurai</i> Tie-10 exhibited significant inhibitory activity against <i>K. pneumoniae</i>. The alkaline protease (AKP) assay demonstrated that the crude extract effectively disrupted the cellular integrity of <i>K. pneumoniae</i>, a finding further corroborated by scanning electron microscopy (SEM) analysis. Furthermore, the crude extract significantly inhibited extracellular protease secretion in <i>K. pneumoniae</i>, downregulated the expression of virulence-associated genes, and effectively disrupted biofilm formation. The study presented innovative strategies for the management and containment of <i>K. pneumoniae</i> infections.