Two Novel Single-Chain Variable Fragments, EB211 and EB279, EB211 and EB279, Exert Antibacterial Activity Against Acinetobacter baumannii by Destabilizing the Outer Membrane

Introduction: Acinetobacter baumannii is notorious for its high resistance levels, and the development of clinically effective antimicrobial agents is an urgent medical challenge.Single-chain variable fragments (scFvs) that exhibit antibacterial properties against challenging pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, have the potential to improve therapeutic strategies significantly.Their unique ability to function independently of the host immune system makes scFvs a highly promising option for effective treatment.In our previous studies, we identified two human scFvs (EB211 and EB279) that showed direct growth inhibition activity against A. baumannii strains in vitro and therapeutic effectiveness in immunocompromised mice with pneumonia caused by an extensively drugresistant A. baumannii strain.In the present study, we endeavored to demonstrate how EB211 and EB279 could inhibit the growth of A. baumannii.Methods: A. baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains were individually incubated with the scFv in the presence of a high concentration of magnesium (MgSO 4 ; 20 mM).Epitope mapping and immunoblotting were conducted to identify A. baumannii proteins likely bound by EB211 and EB279.Results: It was found that EB211 and EB279, similar to colistin sulfate, lost their activity in the presence of magnesium.Moreover, immunoblotting revealed that EB211 and EB279 might bind the OprD family outer membrane porin and TonB family C-terminal domain protein, respectively.Conclusion: EB211 and EB279 elicit direct growth inhibitory activity against A. baumannii without needing immune cells or complements, which could be helpful for immunocompromised patients.