Early and mature <i>Achromobacter xylosoxidans</i> biofilm in cystic fibrosis and non-cystic fibrosis isolates: dynamics and response to clinically relevant antibiotics.

Achromobacter xylosoxidans is an opportunistic pathogen in both cystic fibrosis (CF) and non-CF patients, in whom biofilm formation contributes to bacterial persistence and antibiotic tolerance. This study aimed to characterize early and mature biofilm formation in 57 clinical A. xylosoxidans isolates using complementary and physiologically relevant approaches and to compare biofilm phenotypes according to isolate origin (CF/non-CF). Early adhesion was assessed using the Biofilm Ring Test®, mature biofilm viable biomass was quantified under static conditions by colony-forming units counts, and biofilm dynamics were analyzed in a continuous-flow microfluidic system. The effects of five clinically relevant antibiotics (trimethoprim-sulfamethoxazole, piperacillin-tazobactam, meropenem, imipenem, and cefiderocol) were evaluated under dynamic conditions at sub-inhibitory concentrations (0.5 × Minimum Inhibitory Concentration (MIC)) and on preformed biofilm at inhibitory concentrations (10 × MIC). Non-CF isolates displayed faster early adhesion than CF isolates, whereas mature biofilm biomass was comparable between groups. If early adhesion did not predict mature biofilm biomass, dynamic biofilm coverage under flow conditions correlated with static mature biofilm levels. Sub-inhibitory antibiotic concentrations failed to prevent initial adhesion and elicited three distinct responses: biofilm formation enhancement (piperacillin-tazobactam, meropenem, imipenem), no effect (trimethoprim-sulfamethoxazole), or biofilm reduction (cefiderocol). Exposing mature biofilm to 10 × MIC identified trimethoprim-sulfamethoxazole and cefiderocol as the most effective agents in biofilm biomass reduction, whereas carbapenems and piperacillin-tazobactam were less effective. These findings provide new insights into A. xylosoxidans biofilm biology and may help guide therapeutic strategies for infections caused by this emerging, increasingly drug-resistant pathogen.