Ambroxol hydrochloride as an antibiofilm agent synergizes with tetracycline antibiotics against mature biofilms of multidrug-resistant Klebsiella pneumoniae

Multidrug-resistant <i>Klebsiella pneumoniae</i> (MDR-KP) is a major pathogen responsible for hospital-acquired infections, associated with high morbidity and mortality. Biofilm formation plays a key role in the pathogenicity of MDR-KP and contributes significantly to its antibiotic resistance, substantially impairing the effectiveness of antimicrobial therapies. To enhance the efficacy of existing antibiotics, this study investigates a biofilm-targeting synergistic strategy inspired by the structural similarity between sputum and biofilm matrices. In this study, 87 clinical isolates of MDR-KP were initially screened for biofilm-forming capacity, and strong biofilm producers were selected to establish an <i>in vitro</i> model for systematic evaluation of the anti-biofilm efficacy of six mucolytic agents. Ambroxol hydrochloride (ABH) emerges as the optimal effective, disrupting biofilm structure at 0.7 mg/mL and achieving 50 % clearance within 8 h. ABH enhanced the anti-biofilm activity of tetracycline and doxycycline <i>in vitro</i>, reducing their IC₅₀ values by 98.9 % and 98.6 %, respectively, against preformed biofilms of MDR-KP compared to monotherapy. Additionally, the excellent physical and chemical compatibility between ABH and tetracycline or doxycycline provides a stable basis for <i>in vivo</i> co-administration. <i>In vivo</i>, the combination alleviates pulmonary inflammation, reduces bacterial load and inflammatory factor levels, and shows no tissue toxicity. In conclusion, ABH combined with tetracycline antimicrobials enhanced their efficacy against MDR-KP infections, especially biofilm-associated infections, in both <i>in vitro</i> and <i>in vivo</i> models, and possessed a favorable physicochemical compatibility and safety profile. These findings suggested that ABH-tetracycline therapy could represent a translationally promising and effective strategy for combating clinical MDR-KP infections.