A. baumannii is an opportunistic pathogen and a major cause of various community-acquired infections. Strains of this species can be resistant to multiple antimicrobial agents, leaving limited therapeutic options, also lacking in methods for accurate and prompt diagnosis. In this context, AbTJ, a novel phage that infects A. baumannii MDR-TJ, was isolated and characterized, together with its two tail fiber proteins. Morphological analysis revealed that it belongs to Podoviridae family. Its host range, growth characteristics, stability under various conditions, and genomic sequence, were systematically investigated. Bioinformatic analysis showed that AbTJ consists of a circular, double-stranded 42670-bp DNA molecule which contains 62 putative open reading frames (ORFs). Genome comparison revealed that the phage AbTJ is related to the Acinetobacter phage Ab105-1phi (No. KT588074). Tail fiber protein (TFPs) gp52 and gp53 were then identified and confirmed as species-specific proteins. By using a combination of bioluminescent methods and magnetic beads, these TFPs exhibit excellent specificity to detect A. baumannii. The findings of this study can be used to help control opportunistic infections and to provide pathogen-binding modules for further construction of engineered bacteria of diagnosis and treatment.

Antibiotic resistance has become a serious threat to global health. It frequently emerges in real‐world environments such as biofilms, where antibiotic gradients impose selective pressures that drive bacterial adaptation. However, the mechanisms underlying resistance development remain poorly understood, largely due to challenges in experimentally controlling confounding parameters. Here, we report a 3D microfluidic platform that enables real‐time monitoring of bacterial dynamics under well‐defined antibiotic gradients, along with programmable cell retrieval to identify key factors in resistance development. We show that motile Escherichia coli migrated randomly within the gradients regardless of antibiotic class (i.e., gentamicin, ciprofloxacin, and ampicillin). Resistance emerged only when a critical mass of bacteria sustained migration through the gradient for a sufficiently duration, revealing a previously unrecognized migration‐duration threshold for adaptive evolution. Whole‐genome resequencing revealed gene mutations in the resistant cells, including mutations in ATP synthase‐related genes (i.e., atpG , atpD , and atpF ). A trade‐off between resistance and cell growth was also observed. Similar phenomena were observed in other motile bacteria species, including Serratia marcescens . Overall, we demonstrate that cell motility is a critical determinant of resistance development in heterogeneous antibiotic landscapes. Targeting cell motility and growth could offer effective strategies to combat antibiotic resistance in motile bacteria.