Comprehensive In Vitro Evaluation of Antimicrobial Combinations Against Extensively Drug- Resistant Acinetobacter baumannii: Planktonic and Biofilm Perspectives

Introduction: Acinetobacter baumannii is a major cause of healthcare-associated infections due to its multidrug resistance (MDR), biofilm-forming capacity, and environmental persistence. Resistance is exacerbated in biofilm-associated cells, requiring combination therapies for effective treatment. Objectives: To evaluate the in vitro antimicrobial activity and synergistic potential of clinically relevant antibiotics—doxycycline, meropenem, polymyxin B, amikacin, and gentamicin—against XDR A. baumannii isolates, with a focus on biofilm-associated resistance. Methods: Twenty clinical isolates were collected from two hospitals. Susceptibility testing was performed by broth microdilution. The presence of the blaOXA-23 gene was confirmed via qPCR. Checkerboard and time-kill assays assessed synergy. Biofilm formation on polyurethane was evaluated using crystal violet, MTT, MBEC assays, and scanning electron microscopy. Clonality was analyzed using Fourier-transform infrared spectroscopy (FTIR). Results: All isolates carried blaOXA-23 and were resistant to carbapenems. High resistance rates to gentamicin (85%) and amikacin (95%) were observed. Polymyxin B retained 100% activity. Doxycycline showed 45% susceptibility (MIC ≤1 mg/L). Synergy was detected in seven doxycycline-aminoglycoside combinations. In biofilm models, strong biomass formation persisted despite antimicrobial exposure. Doxycycline + gentamicin showed >2 log₁₀ CFU/cm² reduction in viable biofilm cells and enhanced killing in time-kill assays. FTIR identified 15 clonal clusters, indicating potential horizontal transmission. Conclusion: Doxycycline in combination with gentamicin demonstrated promising synergistic activity against XDR A. baumannii , including biofilm-associated cells. However, biofilm tolerance remains a therapeutic barrier. These findings support the consideration of combination therapies and biofilm-targeted evaluations in managing MDR infections. Further in vivo studies are warranted.