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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #337472

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

Title: Biotinylation of environmentally isolated Shiga toxin-producing Escherichia coli (STEC) – specific bacteriophages for biosensor and biocontrol applications

item QUINTELA, IRWIN - University Of Maine
item Wu, Vivian

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/18/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Like common bacteriophages, Shiga toxin-producing Escherichia coli (STEC) bacteriophages are viruses that recognize and bind to specific bacterial host (STEC) for propagation. They co-exist with STEC hosts, which cause epidemic food and waterborne illnesses, but may act as host populations limiting factor via predation and lytic life cycle. To facilitate the application of STEC bacteriophages for numerous diagnostics and biocontrol use, biotin which provides proteins chemical flexibility such as efficient affinity labeling and strong linking capacity may be utilized. The aim of this study was to develop biotinylated STEC-specific bacteriophages that were isolated from environment for biosensor and biocontrol applications. Bacteriophages specific to various STEC strains (O26, O45, O103, O121, O157 and O179) were isolated from cattle feces and irrigation water. In sequential steps, enrichment, isolation using plaque assays, purification by a double layer agar method, filtration, centrifugation and spot testing were conducted. Morphological characterization using transmission electron microscope (TEM) was performed. Bacteriophage heads were decorated with biotin by incubating purified lysates (> 8 log PFU/ml titer) with 20-fold molar excess of biotin. Lysates were then dialyzed using buffer exchange and desalting columns to remove unbound biotin. To confirm successful biotinylation, streptavidin-conjugated nanocrystals (1µM, 1 hr) were allowed to bind to biotinylated heads and viewed under TEM. To estimate biotin incorporation level, colorimetric assay (absorbance at 500 nm) using 4'-hydroxyazobenzene-2-carboxylic acid (HABA) reagent was performed. Plaque assays were conducted to ensure the viability of biotinylated bacteriophages. Results showed that the isolated bacteriophages had icosahedral head (50 x 65 nm) and sheathed-tail (150 nm) ultrastructures (Myoviridae). The heavy concentration of streptavidin-conjugated nanocrystals around the icosahedral head observed under TEM confirmed successful occurrence of biotinylation. The number of incorporated biotin was approximated 500 per bacteriophage head. The titer level did not significantly decrease (> 8 log PFU/ml) based on pre-and post-biotinylation viability assays. This study established a groundwork for inexpensive chemical modification of selected morphological features of bacteriophages specific to six major STEC serogroups including O157. Biotinylated bacteriophages offer opportunities for biosensor development and biocontrol due to its stability, potential compatibility with biosensing platforms and specificity to bacterial pathogens.