Characterization, genomics, and applications of the Cronobacter sakazakii bacteriophage Csp-D17

Cronobacter sakazakii is an opportunistic pathogen that can contaminate infant formula, resulting in meningitis and neurological disorders in infants. Bacteriophages have been consistently identified as effective inhibitors of pathogenic bacteria. In this study, a lytic phage, Csp-D17, was isolated from environmental wastewater, and its characterization, genomics, and application were analyzed. Transmission electron microscopy revealed that the phage belonged to the order Caudoviricetes within the family Ackermannviridae. The phage Csp-D17 exhibited notable stability across varied thermal and pH conditions, with an optimal multiplicity of infection of 0.01 and a lysis capacity of 40 plaque-forming units per infected cell. Evaluation of its host range revealed lytic activity against 4 out of 11 tested strains of C. sakazakii (36.4%). The frequency of insensitive mutations in C. sakazakii D17 to phage Csp-D17 was 3.8 × 10^-7. High-dose phage treatment was more effective in inhibiting the growth of C. sakazakii D17 in the powdered infant formula model, which was correlated with its short latency period, high lysis amount, and low frequency of insensitive mutations in the host bacteria. The results demonstrated that phage Csp-D17 was not lysogenic. Furthermore, the genomic study of phage Csp-D17 revealed that the genome structure of this phage was a double-stranded DNA molecule with a length of 143,081 base pairs, a guanine + cytosine content of 46.42%, 233 open reading frames (the average gene length is 553 base pairs), and it does not contain any genes related to virulence or lysogenicity. Furthermore, the genetic function, expression mechanism, and phylogenetic diversity of Csp-D17 were analyzed by genomic comparison. This study provides a theoretical basis for developing novel biocontrol agents based on phage control of C. sakazakii.IMPORTANCEThis study significantly advances the field of food safety biocontrol by identifying and characterizing Csp-D17, a novel lytic phage specifically targeting Cronobacter sakazakii. Through comprehensive morphological, physiological, and whole-genomic analyses, Csp-D17 is rigorously confirmed as a distinct and well-defined viral agent. The demonstration that Csp-D17 can serve as a potent biocontrol agent against C. sakazakii in powdered infant formula highlights its direct practical application in mitigating a critical pathogen responsible for severe neonatal infections. This work not only contributes a new tool to the antimicrobial arsenal but also provides a foundational framework for developing phage-based strategies to enhance the safety of food products, particularly those intended for vulnerable populations.