The researchers isolated WAH2 from wastewater collected at a food-factory treatment facility in Guangzhou, China, using B. cereus strain BY118LC as the propagation host. This strain had been recovered from Chinese sausages and was resistant to several tested antibiotics, including penicillin, amoxicillin, cefoxitin, tiamulin, ceftiofur, sulfamethoxazole-trimethoprim, and tigecycline. It also carried the enterotoxin, emetic-toxin, cytolysin, and other virulence determinants previously associated with the isolate. After enrichment and three rounds of plaque purification, WAH2 produced clear, uniform, circular plaques averaging 1.78 ± 0.18 mm in diameter. Transmission electron microscopy showed an icosahedral capsid measuring 88.28 ± 2.11 nm and a contractile tail 199.44 ± 6.62 nm long, a morphology consistent with a myovirus.

WAH2 had a narrow but experimentally productive lytic spectrum. Spot assays detected lysis in seven of the 51 B. cereus strains tested, equivalent to 13.7% of the panel, and all seven produced visible plaques in double-layer agar cultures. Their efficiency of plating relative to BY118LC ranged from 0.013 to 0.564. The susceptible isolates belonged to three sequence types—ST2818, ST2821, and ST395—suggesting that the phage could infect more than one genetic lineage even though it covered a minority of the panel. WAH2 did not form lysis zones against the representative non-target bacteria tested, including Staphylococcus aureus, Staphylococcus borealis, Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Escherichia coli, and Salmonella Typhimurium. The test panel did not include other members of the B. cereus group, additional foodborne Bacillus species, or lactic acid bacteria, leaving the phage’s full specificity and ecological safety uncertain.

Whole-genome analysis placed WAH2 in the genus Bastillevirus of the family Herelleviridae. Its 158,991-base-pair genome was a linear, double-stranded DNA molecule with a GC content of 37.96%. Annotation identified 284 open reading frames, of which 71 received functional assignments and 213 were classified as hypothetical proteins. The assigned genes were organized into modules associated with replication and regulation, structure and assembly, packaging, and lysis, while six tRNA genes were also detected. The genome shared 92.47% nucleotide identity over 92% coverage with Bacillus phages OTooleKemple52 and Kamfam. Intergenomic comparisons placed WAH2 near those phages and Bastille, and analysis of the large terminase subunit supported its assignment as a novel species within Bastillevirus. No antibiotic-resistance genes, virulence factors, or lysogeny-related elements were identified, although the absence of such sequences does not by itself establish safety in every application setting.

The phage’s replication characteristics were favorable for biological control. The best phage yield occurred at a multiplicity of infection, or MOI, of 0.0001, with a titer of 1.6 × 10^8 plaque-forming units per milliliter. A one-step growth experiment identified a latent period of 30 minutes and a burst size of 208 plaque-forming units per infected cell. Adsorption was rapid: approximately 44.05% of WAH2 particles attached within five minutes, rising to 78.89% after 20 minutes. WAH2 also retained infectivity from 4°C to 45°C, in 2% to 10% sodium chloride, and from pH 5 to 11. Titer fell progressively after exposure to 55°C, and the phage was completely inactivated at 65°C and above; it was also fully inactivated at pH 2 and 13, with smaller reductions at pH 3, 4, and 12. These laboratory stability limits define the environmental conditions under which the virus remained active.

In broth culture, WAH2 produced a clear dose-dependent inhibition of BY118LC. Growth was completely suppressed throughout the 36-hour observation period at MOIs of 1,000, 100, and 10. At MOIs of 1 and 0.1, the bacteria showed some initial growth before proliferation stopped, with no regrowth detected by the end of the assay. The phage also prevented biofilm formation in 96-well plates over 24 hours. At MOIs ranging from 1,000 to 0.1, the measured biofilm biomass decreased by 0.57 to 0.66 optical-density units relative to the controls. It also acted on established biofilms: after 48 hours of biofilm formation on stainless-steel coupons, 12 hours of treatment with 10^7 or 10^8 plaque-forming units per milliliter reduced biomass by 1.07 ± 0.04 and 0.99 ± 0.07 units, respectively. Viable biofilm-associated cells fell by 3.84 ± 0.06 and 3.68 ± 0.19 log10 colony-forming units per milliliter.

WAH2 did not directly kill dormant B. cereus spores or measurably interfere with their germination. In saline, viable spore counts changed little in either phage-treated or untreated samples during 12 hours, and the optical-density response used to assess germination did not differ significantly between the groups. The apparent effect emerged after spores germinated: in nutrient-rich broth, control spores germinated and grew to stationary phase in about 10 hours, whereas none of the phage-treated groups showed an increase in optical density during the 12-hour assay. Counts obtained before and after heat treatment also indicated that WAH2 prevented the outgrowth of newly germinated cells rather than eliminating dormant spores. The result demonstrates a limit of this lytic strategy: it cannot replace measures directed at resistant spores and instead acts once germination supplies metabolically active hosts.

In food-matrix experiments, the researchers inoculated sterile UHT whole milk, rice powder, and pork with BY118LC and treated them with WAH2 at MOIs of 100, 1,000, or 10,000. At 25°C, bacterial counts in untreated controls rose rapidly, while phage-treated counts declined in a dose-dependent manner. In milk, bacteria became undetectable within three hours at MOIs of 10,000 and 1,000 and within six hours at an MOI of 100. In rice, the corresponding detection times were three, six, and 24 hours. At the highest dose, pork bacteria were undetectable after 12 hours; at 72 hours, MOIs of 1,000 and 100 had produced reductions of 6.68 and 1.66 log10 colony-forming units per gram. No treated group showed bacterial regrowth during the 72-hour period. These were controlled laboratory matrices inoculated with one strain, rather than naturally contaminated foods with diverse microbial communities.

WAH2 remained effective under refrigeration, although killing was generally slower at 4°C. In milk, bacteria became undetectable within 12 hours at an MOI of 10,000 and within 24 hours at an MOI of 1,000; the lowest dose produced a 1.14 log10 reduction after 72 hours. In rice, detectable bacteria disappeared by 48 hours at the highest dose, while reductions of 1.80 and 0.59 log10 colony-forming units per milliliter were measured at MOIs of 1,000 and 100 after 72 hours. In pork, the corresponding 72-hour reductions were 2.12, 0.85, and 0.15 log10 colony-forming units per gram. No regrowth was detected in any treated food group. WAH2 remained infectious for up to 120 hours in tryptic soy broth and UHT whole milk at both temperatures, with smaller titer losses at 4°C. No phage-resistant survivors were recovered in the resistance assays, so their frequency remained below the assay’s detection limit in the tested matrices.

The results support further evaluation of WAH2, not immediate deployment as a universal food-safety intervention. Its principal strength is sustained lytic activity: the phage replicated efficiently, remained active across a substantial range of temperature, pH, and salt conditions, reduced established biofilms, and prevented regrowth in the tested milk, rice, and pork systems. Its principal weakness is host coverage, since only 13.7% of the B. cereus panel was susceptible. That narrow range could be an advantage in a precisely defined formulation, but it may also require complementary phages for genetically diverse food isolates. The experiments also used sterilized or surface-treated foods, one bacterial strain, and a single phage exposure; they did not test naturally contaminated products, repeated dosing, commercial processing conditions, or evolution of resistance over longer periods. WAH2 is therefore a technically promising and genomically well-characterized biocontrol candidate whose practical value will depend on broader host-range testing, validation in real foods, and assessment of resistance dynamics.

Subject of Research: Characterization and food biocontrol potential of the lytic phage WAH2 against Bacillus cereus

Article Title: A novel lytic phage with potent efficacy against Bacillus cereus , its biofilms and spores, and biocontrol potential in food matrices

Article References: Wan, P., He, Z., Liu, W., Huang, H., Chen, H., Lin, S., Xiong, W., & Zeng, Z. (2026). A novel lytic phage with potent efficacy against Bacillus cereus, its biofilms and spores, and biocontrol potential in food matrices. Current Research in Food Science, Article 101603. https://doi.org/10.1016/j.crfs.2026.101603

Image Credits: AI Generated

DOI: 10.1016/j.crfs.2026.101603

Keywords: bacteriophage WAH2, Bacillus cereus, food biocontrol, lytic phage, bacterial biofilms, foodborne pathogen, spore outgrowth, milk preservation, refrigerated food, phage resistance, novel, lytic