en Anti-microbial agents, resistance and treatment protocols Anti-microbial agents, resistance and treatment protocols Original article Original article <b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Introduction:</span></span></span></b><i><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times=""> Acinetobacter baumannii</span></span></span></i><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times=""> 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 <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i>, 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. I<span style="color:black">n our previous studies, w</span>e identified two human scFvs (EB211 and EB279) that showed direct growth inhibition activity against <i>A. baumannii</i> strains <i>in vitro </i><span style="color:black">and<i> </i>therapeutic effectiveness in immunocompromised mice with pneumonia caused by an extensively drug-resistant <i>A. baumannii </i>strain. In the present study, we endeavored to demonstrate how EB211 and EB279 could inhibit the growth of <i>A. baumannii</i>. <b>Methods:</b> <i>A. baumannii</i>,<i> </i></span><i>Klebsiella pneumoniae</i>, and<i> Pseudomonas aeruginosa </i><span style="color:black">strains were individually incubated with the scFv in the presence of a high concentration of magnesium </span>(MgSO₄; 20 mM)<span style="color:black">. Epitope mapping and immunoblotting were conducted to identify <i>A. baumannii</i> proteins likely bound by EB211 and EB279.&nbsp;<b>Results:</b> 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.<b> Conclusion: </b>EB211 and EB279 elicit direct growth inhibitory activity against <i>A. baumannii</i> without needing immune cells or complements, which </span>could be helpful for immunocompromised patients.</span></span></span><br> &nbsp; Acinetobacter baumannii, Antibacterial agents, Monoclonal antibodies, Single-chain nariable fragments, Magnesium 299 311 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-608-1&slc_lang=en&sid=1 Eilnaz Basardeh Eilnaz8676@gmail.com 0009-0001-5884-4279 No Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran Farzaneh Nazari farzanehnazari14294@gmail.com 0009-0004-8906-2167 No Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran Abolfazl Fateh fatehab7679@gmail.com 0000-0002-2043-8523 No Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran Seyed Davar Siadat sia5452@gmail.com 0000-0002-6892-5603 No Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran Akbar Oghalaie Oghaak14596@gmail.com 0000-0003-2135-8143 No Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Masoumeh Azizi masiA1567@gmail.com No Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Fatemeh Rahimi Jamnani Rahimi@pasteur.ac.ir 0000-0001-7172-8435 Yes Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran