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

Acinetobacter baumannii, a member of the A. baumannii–calcoaceticus complex (ABC), is a major cause of hospital-acquired infections. Differentiating A. baumannii 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 A. baumannii 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 A. baumannii within 1 h, with recovery rates of 72.5% in buffer and 55.7% in clinical sputum specimens. Specificity was confirmed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and non-A. baumannii strains, with no significant cross-reactivity. The detection limit was approximately 3.4 × 103 CFU/mL. All captured clinical isolates harbored the blaOXA-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 A. baumannii, offering strong potential for clinical diagnostics and point-of-care testing.