Background: Shiga toxin-producing Escherichia coli (STEC) continually challenge the food safety system worldwide. Bacteriophages demonstrate great potential as a biocontrol agent for STEC in the food supply chain. Naturally occurring phages are ubiquitous in cattle and their environment, regulating the diversity of STEC via unknown mechanisms. Hypothesis: We hypothesized that endogenous phages within the same genera undergo alterations in their tail genes to enhance their ability to infect various serogroups of STEC. Objective: This study characterized the lytic activity and genomics of six STEC-infecting phages isolated from commercial feedlots in Alberta and elucidated the function of tail proteins in host recognition and attachment. Methods and Results: Overall, the six phages (e.g. AHF125, AXO103A/B/C, AXO45B, and AXO26A) exhibited broad host range and strong lytic activity against STEC serogroups O26, O45, O103, O145 and O157 in the laboratory. The estimated adsorption rate of these phages ranged from 5.2×10^10 to 2.2×10^9 ml/min. Taxonomically, they belonged to the Tequintavirus genus of the Markadamsvirinae subfamily, containing 106 – 109 kb dsDNA (155 – 173 coding sequences, 22-24 tRNAs). Comparative genomics revealed that the genomes of the phages had a similarity of 86–89%. However, their L-shaped tail fibers (LTFs) and receptor binding proteins (RBP), which are known to be responsible for host attachment varied (e.g. as low as 24% identity of LtfA of phage AXO45B to that of reference phage AKFV33) at the amino acid level. Unlike the typical Ltf structure of Tequinatavirus, Ltfs of STEC phages consisted of 2–3 gene products, namely LtfA and LtfB, with or without a hypothetical protein orf between these gene products. To determine the function of these unique Ltf and RBP toward host adsorption, Ltfs and RBP of O157-infecting phage were cloned, expressed, and purified for use in a phage adsorption inhibition assay. In the presence of LtfA (≥0.05 mg/ml) and orf136 (≥0.1 mg/ml), 66 – 82% of phage failed to attach, a higher proportion (p < 0.001) than that of the protein-free control (~ 0.2 % unabsorbed phages after 15 min). This observation suggests that LtfA and orf136 are bound to the same bacterial receptors as the parental phages, preventing them from initiating adsorption. Conclusions: Environmental Tequintavirus was efficacious against multiple clinically important STEC serogroups. Variations of genes encoding for Ltfs and RBP from Tequintavirus may be responsible for STEC infectivity.

Keywords: Shiga toxin-producing Escherichia coli, bacteriophages, Host range, phage adsorption, Tequintavirus, L-shaped tail fiber, receptor binding protein.