Construction and characterization of a chimeric Salmonella phage P6-UPF2 via homologous replacement of the phage P6 tail spike protein Gp9.

With over 2,600 Salmonella serotypes identified, no single natural or existing bacteriophage can cover this diversity, making customized phages a promising strategy for precise control. In this study, the previously characterized phage P6 served as the recipient, and the broad-spectrum phage UPF_BP1 (94.91% nucleotide identity with P6) served as the donor. Two fragments (b1 and b2) from the donor's tail spike protein gene were individually recombined into the Gp9 locus of P6, generating two chimeric phages: P6-UPF1 and P6-UPF2. Host range analysis showed that P6-UPF1 exhibited no alteration in host spectrum against the bacterial strains tested, whereas P6-UPF2 retained activity against S. Enteritidis, S. Typhimurium, and S. Bovismorbificans, and gained activity against S. Muenster and S. Newlands, with preliminary evidence of genetic stability. Competitive adsorption inhibition assays showed that UPF-b2 significantly suppressed the adsorption of P6-UPF2 to S. Typhimurium, S. Muenster, and S. Newlands (P < 0.001), whereas P6-b1 significantly inhibited the adsorption of P6-UPF2 to S. Enteritidis (P < 0.001). The results identify, for the first time, the UPF-b2 and P6-b1 fragments within the Gp9 tail spike protein of P22-like phages as host range determinants for S. Typhimurium, S. Muenster, S. Newlands, and S. Enteritidis, respectively. Biological characterization indicated that P6-UPF2 exhibited an optimal multiplicity of infection of 0.1, a latent period of 10 min, a burst period of 90 min, and a burst size of up to 104 PFU/cell, along with stability observed under tested conditions to variations in temperature and pH. These findings provide an experimental basis for the rational design of phages based on receptor-binding proteins, and the constructed phages were shown to exhibit a broader host range against the Salmonella serovars tested.