Exploring the anti-biofilm effect of darobactin B and colistin in static and dynamic environments

The increasing challenges of treating biofilm-associated infections highlight the importance to develop new options against biofilm-embedded bacteria. In this study, we evaluated darobactin B-a promising pre-clinical antibiotic candidate targeting the outer membrane protein BamA-against <i>Pseudomonas aeruginosa</i> biofilms. First, a static biofilm assay was used for primary screening and showed that darobactin B reduced the viable cells within a biofilm by six orders of magnitude compared to the untreated control. Subsequently, a microfluidic model was conducted to allow real-time monitoring of biofilm development, treatment, and potential regrowth under controlled hydrodynamic conditions. In this assay, the reference compound colistin exhibited strong activity after single-dose administration. Although less effective after single dosing, darobactin B showed notable anti-biofilm activity by killing 85%-97% of biofilm cells and suppressing biofilm recovery for at least 48 h when applied repeatedly once daily for 5 days. When administered repeatedly in the multi-dose regimen, darobactin B caused biofilm disruption and cell detachment, leading to ~40% surface coverage reduction. Our findings highlight the potential of darobactin B as an anti-biofilm agent.IMPORTANCEBacterial biofilms pose serious healthcare challenges, contributing to chronic and device-related infections. Biofilm-embedded bacteria are highly resistant to conventional antibiotics and lead to growing reliance on last-resort drugs, thus underscoring the need for new therapeutic approaches. This study shows that darobactin B consistently disrupts <i>Pseudomonas aeruginosa</i> biofilms and delays regrowth. The multi-dose microfluidic assay provides a flexible platform for real-time evaluation of biofilms, which may support the optimization of treatment regimens.