Novel phage vB_CsaM_2375H2 effectively controls Cronobacter sakazakii biofilms and contamination in powdered infant formula

Abstract

Background

Cronobacter sakazakii (C. sakazakii), a significant foodborne pathogen in powdered infant formula (PIF), forms resilient biofilms that confer resistance to disinfectants, antibiotics and environmental stresses. Phages represent a promising alternative for controlling bacterial contamination in food.

Aims and Scope

This study reports the isolation and characterisation of a novel Straboviridae phage, vB_CsaM_2375H2 (hereafter 2375H2), and evaluates its efficacy in reducing C. sakazakii biofilm and its bactericidal activity against the pathogen in PIF.

Method

The phage 2375H2 was isolated using C. sakazakii cro2375w as the host bacterium. Morphological and biological characteristics were performed via TEM, genome sequencing and bioinformatics. Host range analysis, multiplicity of infection (MOI, the ratio of phage particles to bacterial cells), one-step growth curve, thermal and pH stability were determined using double-layer agar assays. Biofilm removal was evaluated with a crystal violet-based method, and antibacterial activity in PIF was assessed by plate counting.

Major Findings

Phage 2375H2 exhibits a typical myovirus morphology with an icosahedral head and a contractile tail. Host range analysis against 68 strains revealed its specificity to the Cronobacter genus, lysing 13 strains. It has an optimal MOI of 0.01, a latent period of 20 min, and maintains stability under temperatures of 25–60°C and pH 4–11. Genomic classification places it within the Pseudotevenvirus genus, with no virulence or antibiotic resistance genes detected. Biofilm assays demonstrated that phage treatment at 10⁶ and 10⁷ PFU/mL reduced biofilm biomass by 61.11% and 62.55% after 8 h, respectively. Moreover, in powdered infant formula, 10¹⁰ PFU/mL of phage achieved a 99% bactericidal rate within 6 h.

Scientific Implications

Phage 2375H2 achieved >60% eradication of preformed C. sakazakii biofilms and eliminated 99% of pathogens in PIF. These findings establish phage 2375H2 as a promising biocontrol agent against C. sakazakii contamination in food systems.