Isolation and characterization of bacteriophages as potential agents against Shiga toxin – producing Escherichia coli (STEC) strains

Authors: QUINTELA, IRWIN - University Of Maine; Liao, Yen-Te; VALENTINE, DON - University Of Maine; ZHANG, RONG - University Of Maine; ANDERSON, GARY - University Of Maine; DELOS REYES, BENILDO - Texas Tech University; Nguyen, Kimberly; Wu, Vivian

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 2/23/2017
Publication Date: 2/23/2017
Citation: Quintela, I.A., Liao, Y., Valentine, D., Zhang, R., Anderson, G., Delos Reyes, B.G., Nguyen, K.M., Wu, V.C. 2017. Isolation and characterization of bacteriophages as potential agents against Shiga toxin – producing Escherichia coli (STEC) strains. Meeting Proceedings. . 2017 USDA-ARS/FSIS Annual Food Safety Meeting. NCTC, Shepherdstown, WV. Section Z, P12.

Interpretive Summary:

Technical Abstract: Shiga – toxin producing Escherichia coli (STEC) is a significant group of foodborne pathogens that can cause mild diarrhea to serious human illnesses. The gastrointestinal tracts of cattle and other ruminants are the primary reservoirs of STEC strains and may co-harbor bacteriophages as part of its microbiota. Bacteriophages are bacterial predators that can act as important limiting factors of bacterial populations in the environment. The aim of this study was to isolate and characterize STEC – specific bacteriophages and investigate their potentials against STEC serogroups. The isolation of bacteriophage was conducted from twenty-two cow manure samples (10 g/sample) and pure culture strains representing seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157), Salmonella spp. and Listeria monocytogenes. Enrichment, isolation using plaque assays, purification by double layer agar method, lysate centrifugation (4000 x g, 15 mins), filtration and spot testing were sequentially performed prior to the characterization. Plaque formation, lysis time, host susceptibility and specificity against STEC and non – STEC strains, and multiplicity of infection (MOI) were determined. Furthermore, morphological features were viewed using transmission electron microscopy (TEM) and the presence of virulence factors encoding genes (stx1 and stx2) was examined using conventional PCR to facilitate precise and more comprehensive characterization. Twenty – one bacteriophages were isolated from cow manures samples (n=22) – all of which were specific to various STEC serogroups but not to non – STEC strains. These cow manure samples were collected from five different sites. The isolates formed pin – point plaques (< 1 mm), and had a lysis time of <20 mins as determined by plaque assays and spectrophotometric data analysis. Morphologically, the bacteriophage isolates had icosahedral head and sheathed – tail ultrastructures that closely described the members of Myoviridae. Sampling sites with high prevalence of STEC bacteriophages showed less isolates and/or absence of various STEC strains indicating that endemic bacteriophages may have direct negative impact on its natural bacterial host population due to predation and inherent lytic life - cycle. Molecular characterization showed selected phages were devoid of stx1 or stx2 gene; only one isolate generated an amplicon (stx1). Stx – negative bacteriophages do not pose threats of possible horizontal virulence genes transfer, therefore can be potentially utilized as biocontrol agents against STEC strains. This study established a protocol for isolating bacteriophages from environmental samples, specifically showing the potentials of lytic bacteriophages that are devoid of virulent genes (stx negative) as natural biocontrol agents against STEC strains. However, further studies of bacteriophage – STEC lysogenic properties should be explored to better understand the induction of stx genes in the natural environment and the mechanisms behind Shiga toxin production.