Characterization, genomics, and applications of the <i>Cronobacter sakazakii</i> bacteriophage Csp-D17

<i>Cronobacter sakazakii</i> 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 <i>Caudoviricetes</i> within the family <i>Ackermannviridae</i>. 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 <i>C. sakazakii</i> (36.4%). The frequency of insensitive mutations in <i>C. sakazakii</i> D17 to phage Csp-D17 was 3.8 × 10^-7. High-dose phage treatment was more effective in inhibiting the growth of <i>C. sakazakii</i> D17 in the powdered infant formula model, which was correlated with its short latency period, high lysis amount<i>,</i> 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<i>,</i> 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 <i>C. sakazakii</i>.IMPORTANCEThis study significantly advances the field of food safety biocontrol by identifying and characterizing Csp-D17, a novel lytic phage specifically targeting <i>Cronobacter sakazakii</i>. 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 <i>C. sakazakii</i> 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.