Phage Cell Wall Binding Domain for Gram-Negative Bacteria and Its Application in Detection of Acinetobacter baumannii

The cell wall binding domain (CBD) of endolysin plays an irreplaceable role in the propagation cycle of phages as it can specifically bind with the cell wall of target bacteria. Although it is a promising agent for recognizing bacteria, this domain is usually found in the endolysin of phages infecting Gram-positive bacteria, hindering its application for the detection of Gram-negative bacteria. Herein, we isolated a highly virulent phage termed as Abp18 for infecting Acinetobacter baumannii (A. baumannii), and found that its endolysin has a CBD termed as CBD18 for binding Gram-negative bacteria. This CBD was acquired using an Escherichia coli (E. coli) expression system, which can bind with 16 of the 18 tested A. baumannii strains with a recognition rate of approximate 89%. In accordance with the result of the turbidity reduction experiment, CBD18 has no lytic activity and thus can be used as a capture agent for A. baumannii. Furthermore, its fusion body with green fluorescent protein was acquired by fusion expression technology, which can be adopted as a high-performance signal probe for tracing A. baumannii. These two proteins were used to establish a sandwich method for detecting this pathogen with a linear range of 4.0 × 10² to 4.0 × 10⁸ CFU/mL and a detection limit of 1.7 × 10² CFU/mL. The practicability of the CBD-based method was validated by detecting A. baumannii spiked in diverse types of real samples, with recoveries of 89-120%. The pioneering study paves an avenue for the usage of phage CBD in recognizing Gram-negative bacteria.