Isolation and characterization of novel species Bacillus Phage CM1 to control milk contamination

Bacillus cereus is a foodborne pathogen. The increasing prevalence of antibiotic resistance among B. cereus isolates has highlighted the need for alternative therapeutic strategies. Phage therapy has emerged as a promising approach. In this study, a novel lytic bacteriophage targeting B. cereus was isolated and characterized. The stability of the phage was evaluated under different pH levels, temperatures, and salinity. Multiplicity of infection (MOI) and adsorption rate were determined. Whole-genome sequencing was performed to analyze genomic features. Host range and bacterial sensitivity to phage analyses were conducted using B. cereus isolates from food samples and other bacterial species. In addition, anti-biofilm activity of the phage was evaluated and a food challenge test was performed. One hundred isolates were recovered from different food sources. The highest resistance was observed for gentamicin (97%). The isolated phage having a tail length of 142 ± 3 nm and a head of 48 ± 2 nm in diameter, named Bacillus phage CM1, belonged to the Caudoviricetes . pH values either above 10 or close to 6 affected the phage stability. The phage titer decreased gradually with an increased NaCl concentration from 1% to 35%. Before and beyond the optimum temperature of 30 °C, the titer of the phage was decreased. It showed an optimal MOI of 1 and an adsorption rate of 88.7% within 35 min. Host range analysis indicated that the phage was specific to B. cereus . Bacterial sensitivity to phage demonstrated lytic activity against 69% of the B. cereus isolates. Furthermore, the phage significantly reduced biofilm biomass by B. cereus . In food challenge test the titer of the bacterium was significantly decreased in the treatment groups receiving the mixture of the bacterium and the phage ( P < 0.05). Whole-genome sequencing revealed a double-stranded DNA genome of 156,598 bp with a GC content of 39.7%, and no resistance or virulence genes were detected. Furthermore, depolymerase and lysin related domains in tail-associated proteins were detected. Due to its strong favorable properties, Bacillus phage CM1 appears to be a promising and safe candidate biocontrol agent against B. cereus in food-related settings.