Isolation, characterization, and in vitro efficacy of phage Citro-6 against Citrobacter freundii-induced bovine...

Bovine mastitis is a prevalent infectious disease in dairy herds worldwide, resulting in substantial economic losses. Citrobacter freundii has emerged as an opportunistic pathogen associated with mastitis, and its increasing antibiotic resistance poses significant therapeutic challenges.

In this study, a novel lytic phage, Citro-6, was isolated from dairy farm sewage and fecal samples using C. freundii Z6 as the host bacterium. The phage was characterized through morphological and genomic analyses. Its host range, stability under varying temperature and pH conditions, and effects on bacterial adhesion, invasion, cytotoxicity, and pro-inflammatory cytokine expression in bovine mammary epithelial cells were evaluated in vitro.

Morphological and genomic analyses identified Citro-6 as a member of the class Caudoviricetes. It encodes a complete endolysin sequence with an intact catalytic domain and lacks virulence or antibiotic resistance genes. Citro-6 exhibited broad lytic activity, lysing 83.3% of tested Citrobacter strains, and remained stable at temperatures up to 45 °C and across a pH range of 5.0 to 9.0. In cellular assays, Citro-6 treatment significantly reduced bacterial adhesion and invasion by approximately 1.66 and 1.19 log units, respectively. Furthermore, it effectively attenuated pathogen-induced cytotoxicity, reducing cell death by approximately 75.2% at 8 h post-infection compared with the Z6-infected group. Citro-6 also prevented morphological cellular damage and suppressed the overexpression of pro-inflammatory cytokines, resulting in decreases in IL-1β and TNF-α concentrations of approximately 37.1% and 47.6%, respectively, relative to the infected control.

These findings demonstrate that phage Citro-6 is a promising biocontrol candidate against C. freundii-induced bovine mastitis; however, further in vivo evaluation is warranted to confirm its therapeutic potential.