A Novel Virulent Bacteriophage against Yersinia enterocolitica: Biological Characterization and Biocontrol Efficacy in Food Models

To isolate and characterize a bacteriophage targeting Yersinia enterocolitica and to evaluate its potential as a biocontrol agent for food applications.

The phage was isolated and purified using the double-layer agar plate method. Morphological observation was conducted by transmission electron microscopy (TEM). The genome was extracted for whole-genome sequencing and subsequent bioinformatics analysis. The host range was determined using the spot test method and the double-layer agar assay. The optimal multiplicity of infection (MOI), one-step growth kinetics, and stability were evaluated. The in vitro antibacterial efficacy was determined by monitoring the optical density at 600 nm (OD_{600 nm}) during culture in tryptone soya broth (TSB). The ability to inhibit and remove the biofilm of Y. enterocolitica was quantified using crystal violet staining. The phage efficacy against Y. enterocolitica in milk and on the surface of lettuce and pork stored at 4 or 25 ℃ was assessed by plate counting.

A novel virulent phage, designated Yersinia phage vB＿Yep＿15, was isolated. TEM revealed a short-tailed morphology typical of the Autographiviridae family with an icosahedral head of 54.80 nm × 52.50 nm. Genomic analysis classified it into the genus Teseptimavirus（the subfamily Studiervirinae）, and no virulence, antibiotic resistance, or tRNA-related genes were detected. Host range analysis showed that the phage lysed 8（30.8%）out of 26 tested strains, including 6 isolates from pigs and 2 isolates from Tibetan wild donkeys. Its optimal MOI was 0.01, with a latent period of 20 minutes and a burst size of 204 PFU/cell. The phage activity remained stable at pH 4–11 and 4–37 ℃. In vitro antibacterial assays showed significant bacterial growth inhibition within 120 minutes at an MOI of 100, with no resuscitation observed over 480 minutes (ΔOD_{600 nm} < 0.02). The biofilm removal rate was 40.8%, 25.6%, 38.4%, and 53.4% at MOIs of 0.1, 1, 10, and 100, respectively, demonstrating that the phage effectively removed biofilms formed by the host bacterium. Furthermore, it significantly reduced Y. enterocolitica counts in milk and on lettuce and pork surfaces at both storage temperatures, indicating good application prospects.

The virulent phage vB＿Yep＿15 exhibits favorable biological characteristics, potent biofilm eradication ability, and significant antibacterial efficacy against Y. enterocolitica on diverse food surfaces under both refrigeration and ambient conditions. This study provides a basis for the development and application of phage-based formulations targeting Y. enterocolitica in food safety biocontrol.