Isolation, characterization, and application of a novel, lytic phage vB_SalA_KFSST3 with depolymerase for the control of Salmonella and its biofilm on cantaloupe under cold temperature

Abstract

This study investigated the efficacy of a novel Salmonella phage with depolymerase activity to control S. Typhimurium (ST) and its biofilm on cantaloupes, for the first time, under simulated cold temperature. vB_SalA_KFSST3 forming a halo zone was isolated and purified from a slaughterhouse with a final concentration of 12.1 ± 0.1 log PFU/mL. Based on the morphological and bioinformatics analyses, vB_SalA_KFSST3 was identified as a novel phage belonging to the family Ackermannviridae. Before employing the phage on cantaloupe, its genetic characteristics, specificity, stability, and bactericidal effect were investigated. Genetic analyses confirmed its safety and identified endolysin and two depolymerase domains possessing antibiofilm potential. In addition, the phage exhibited a broad specificity with great efficiencies toward five Salmonella strains at 4 °C, 22 °C, and 37 °C, as well as stable lytic activity over a wide range of pHs (3 to 11) and temperatures (-20 °C to 60 °C). The optimal multiplicity of infection (MOI) and exposure time of phage were determined to be 100 and 2 h, respectively, based on the highest bacterial reduction of ∼2.7 log CFU/mL. Following the formation of ST biofilm on cantaloupe at 4 °C and 22 °C, the cantaloupe was treated with phage at an MOI of 100 for 2 h. The antibiofilm efficacy of phage was evaluated via the plate count method, confocal laser scanning microscopy, and scanning electron microscopy (SEM). The initial biofilm population at 22 °C was significantly greater and more condensed than that at 4 °C. After phage treatment, biofilm population and the percentage of viable ST in biofilm were reduced by ∼4.6 log CFU/cm² and ∼90% within 2 h, respectively, which were significantly greater than those at 22 °C (∼2.0 log CFU/cm² and ∼45%) (P < 0.05). SEM images also confirmed more drastic destruction of the cohesive biofilm architecture at 4 °C than at 22 °C. As a result of its cold temperature-robust lytic activity and the contribution of endolysin and two depolymerases, vB_SalA_KFSST3 demonstrated excellent antibiofilm efficacy at cold temperature, highlighting its potential as a promising practical biocontrol agent for the control of ST and its biofilm.

Keywords: Antibiofilm efficacy; Cantaloupe; Cold temperature; Depolymerase; Salmonella.