Biological characterization of novel Escherichia coli O157:H7 phages and their bacteriostatic effects in milk and pork

Qinghai Ren ^1,2,3 Zhiwei Wang ¹ Yichen Ge ^1* Yucui Huang ^1* Shengliang Cao ¹ Wei Zhang ^2* Chunxue Liu ^3* Yubao Li ^1*

• ¹ Liaocheng University, Liaocheng, Shandong Province, China
• ² Nanjing Agricultural University, Nanjing, Jiangsu Province, China
• ³ Anyou Biotechnology Group Co., Ltd., Taicang, China

Foodborne bacteria, particularly Escherichia coli (E. coli) O157:H7, are significant contributors to foodborne illnesses, with antibiotic overuse exacerbating the issue through the emergence of multidrug-resistant strains. This study investigated the potential of E. coli phages in food safety, examining their biological traits and bacteriostatic properties. Two phages (vB_EcoP_SD2, vB_EcoP_SD6) of E. coli O157:H7 were isolated from slaughterhouse sewage and characterized for morphology, genomic composition, phage phylogenetic tree, optimal multiplicity of infection (MOI), one-step growth curve, thermal and pH stability and antibacterial efficacy. The optimal MOIs of vB_EcoP_SD2 and vB_EcoP_SD6 was 0.1 and 0.01, and temperature range for maintaining activity was 4℃ to 55℃.The host range of vB_EcoP_SD2 and vB_EcoP_SD6 was 65% (13/20) and 55% (11/20), which was partially complementary to each other (75%,15/20).Notably, vB_EcoP_SD2displayed a latent period of 10 minutes, a burst period of 80 minutes, and a burst volume of 80 PFU per cell, while vB_EcoP_SD6 had a burst volume of 10 PFU per cell.Comprehensive whole-genome analysis confirmed two phages has no presence of pathogenic factors or resistance genes. Genomic comparisons suggest vB_EcoP_SD2 and vB_EcoP_SD6 respectively constituted a novel member of a new genus, Justusliebigvirus genus and Kayfunavirus genus which genome respectively was found to be149066 bp,40202bp long with an average GC content of 37.5% and 49.8%. The phages effectively inhibited host bacteria in LB broth for at least 6 hours and showed promise in inhibiting bacteria in milk and pork, which indicated that the two phages exhibited a favorable bacteriostatic effect on milk and pork within the first 6 hours under the optimal MOI. In the milk bacteriostasis experiment, vB_EcoP_SD2 could reduce bacteria by 3.16×10 4 CFU/mL, and vB_EcoP_SD6 could reduce bacteria by 1.05×10 4 CFU/mL. Phage vB_EcoP_SD2 decreased bacteria by 1.14×10 4 CFU/mL, and vB_EcoP_SD6 decreased bacteria by 2.04×10 3 CFU/mL in the pork. There was no disparity in bacteriostatic effect of different MOI within the first 6 hours, but bacteriostatic effect of all groups still remained different from that of the control group. This study indicates the two phages possess excellent biological characteristics , thereby providing a theoretical foundation for the subsequent development of natural fungicides.