Highly Specific Bacteriophage-Affinity Strategy for Rapid Separation and Sensitive Detection of Viable Pseudomonas...

A virulent bacteriophage highly specific to Pseudomonas aeruginosa (P. aeruginosa) was isolated from hospital sewage using a lambda bacteriophage isolation protocol. The bacteriophage, named as PAP1, was used to functionalize tosyl-activated magnetic beads to establish a bacteriophage-affinity strategy for separation and detection of viable P. aeruginosa. Recognition of the target bacteria by tail fibers and baseplate of the bacteriophage led to capture of P. aeruginosa onto the magnetic beads. After a replication cycle of about 100 min, the progenies lysed the target bacteria and released the intracellular adenosine triphosphate. Subsequently, firefly luciferase-adenosine triphosphate bioluminescence system was used to quantitate the amount of P. aeruginosa. This bacteriophage-affinity strategy for viable P. aeruginosa detection showed a linear range of 6.0 × 10² to 3.0 × 10⁵ CFU mL^-1, with a detection limit of 2.0 × 10² CFU mL^-1. The whole process for separation and detection could be completed after bacteria capture, bacteriophage replication, and bacteria lysis within 2 h. Since the isolated bacteriophage recognized the target bacteria with very high specificity, the proposed strategy did not show any signal response to all of the tested interfering bacteria. Furthermore, it excluded the interference from inactivated P. aeruginosa because the bacteriophage could replicate only in viable cells. The proposed strategy had been applied for detection of P. aeruginosa in glucose injection, human urine, and rat plasma. In the further work, this facile bacteriophage-affinity strategy could be extended for detection of other pathogens by utilizing virulent bacteriophage specific to other targets.