Molecular and Physio-Morphological Characterization of Novel Bacteriophages Targeting Diverse Strains of Biofilm-forming Shiga Toxigenic Escherichia coli

Introduction: Shiga toxin-producing Escherichia coli (STEC) has emerged as a major group of foodborne pathogens with ruminants as host-reservoirs. Implicated in approximately 73,000 illnesses each year in the United States, human infections by STEC have been associated with a variety of foods. It is therefore crucial to devise effective control strategies. Use of target-specific and ubiquitous bacteriophages can be an attractive alternative to control STECs in the food industry. 

Purpose: Molecular and physio-morphological characterization of STEC-infecting bacteriophages isolated from beef cattle operations in Oklahoma. 

Methods: Bacteriophages specifically targeting biofilm-forming STEC (O157:H7, O121, O111, O103, O26, O145, O45) were isolated from environmental samples. Morphological features of isolated phages were examined under transmission electron microscope (TEM).  In vitro inhibition assays were performed to measure the degree of STEC-lysis caused by phages. Viability of selected bacteriophages was tested at various pH and temperature ranges. One-step growth-kinetics assays were performed to measure the eclipse-period, latent-period, and burst-size of each phage. Phage adsorption-rate and optimal lysis time was also determined. Bacteriophage DNA was extracted using phenol-chloroform method and digested using restriction enzyme. The digested fragment was cloned and transformed into E. coli XO-1-Blue and sequenced subsequently. 

Results: Several phages (n=52) showing inhibition towards the STEC serotypes were isolated. Phages were classified into Myoviridae, Siphoviridae, or Tectiviridae family, using TEM. All phages were resistant to temperature of up to 60°C and survived acidic (2, 5) and alkaline (7, 9, 11) pH ranges. Phages had a short latent-period (~20 mins), with large burst-size (41-67 virons/infecting cell), indicating high lytic activity. Genome sequencing revealed that the tail proteins of isolated phages were closely related to enteric phages specific to Salmonella, Vibrio, Shigella, and Yersinia.

Significance: Characterization of selected bacteriophages, targeting diverse strains of biofilm-forming STECs, would help with the development of effective bio-control strategies with potential uses in the food industry.