Isolation and characterization of new phage vB_CtuP_A24 and application to control Cronobacter spp. in infant milk formula and lettuce

Abstract

Cronobacter spp. are opportunistic pathogenic bacteria that cause severe diseases in neonates and infants. Bacteriophages are novel antibacterial agents with a potential to control this pathogen. In the current study, a novel lytic Cronobacter phage, vB_CtuP_A24, was isolated from a river in Guangzhou, China. The phage was characterized by a short, non-contractile tail and a long head and identified as a new member of the family Podoviridae. Complete genome sequence analysis of this phage indicated that its genome contained 75,106 bp of DNA, an average GC content of 44.05%, and 108 predicted open reading frames (ORFs). The annotated ORFs were associated with phage structure, packaging, host lysis, DNA manipulation, transcription, and additional functions. Genes responsible for antibiotic resistance, virulence, and toxic effects were not present. Cronobacter phage vB_CtuP_A24 is a novel lytic phage that can lyse five Cronobacter spp. It was stable over a wide range of temperatures (25–60 °C) and pH values (pH 4–11) and displayed a short latent period (approximately 10 min) and a large burst size (500 plaque-forming units (PFUs)/cell). In Luria-Bertani (LB) broth, phage A24 effectively inhibited the growth of C. dublinensis cro280B, C. sakazakii 465G, and C. malonaticus cro695W up to 9, 10, and 12 h, respectively, while in infant milk formulas, it inhibited two strains, C. sakazakii 465G and C. malonaticus cro695W, up to 24 h at 37 °C, with maximum reduction levels of approximately 5.12 ± 3.95 and 7.38 ± 3.03 log10 colony-forming unit (CFU)/mL, respectively. In lettuce, the reduction of all three strains was highly significant at 25 °C. However, the growth of C. dublinensis cro280B and C. sakazakii 465G were not significantly inhibited at 4 °C. In conclusion, Cronobacter spp. phage vB_CtuP_A24, which has lytic ability against five Cronobacter species, stability under different environments, and shows potential as a promising biocontrol agent against Cronobacter spp. in food production.