A Phage Endolysin-Derived Binding Protein Targeting the LysM Motif Enables Highly Specific Identification of <i>Acinetobacter baumannii</i>

<i>Acinetobacter baumannii</i>, a member of the <i>A. baumannii</i>-<i>calcoaceticus</i> complex (ABC), is a major cause of hospital-acquired infections. Differentiating <i>A. baumannii</i> from other ABC species remains challenging. In this study, we developed a novel diagnostic platform utilizing the cell wall-binding domain (CBD) of an endolysin from <i>A. baumannii</i> phage Φ1656-2. The CBD was expressed as a recombinant protein (AbCD) and conjugated to epoxy magnetic beads (eMB) to form the AbCD-eMB complex. This complex enabled rapid, selective capture of <i>A. baumannii</i> within 1 h, with recovery rates of 72.5% in buffer and 55.7% in clinical sputum specimens. Specificity was confirmed against <i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, <i>Klebsiella pneumoniae</i>, and non-<i>A. baumannii</i> strains, with no significant cross-reactivity. The detection limit was approximately 3.4 × 10³ CFU/mL. All captured clinical isolates harbored the <i>bla</i>_OXA-51-like gene. Functional analysis and molecular docking identified the Lysin motif (LysM)-containing peptidoglycan-binding domain as the bacterial receptor for AbCD. The AbCD-eMB complex remained stable at 4 °C for up to one month. This study demonstrates that the AbCD-eMB platform provides a culture-independent, rapid, and highly specific diagnostic approach for <i>A. baumannii</i>, offering strong potential for clinical diagnostics and point-of-care testing.