Location: Produce Safety and Microbiology ResearchTitle: Genomic Characterization of a T4-Like Bacteriophage Isolated from Surface Water in a Produce-Growing Area in Salinas, California as a Potential Biocontrol Agent for Shiga Toxin-Producing Escherichia coli Author
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/28/2019
Publication Date: N/A
Technical Abstract: Shiga toxin-producing E. coli (STEC) has been associated with numerous produce outbreaks, causing severe illnesses, around the world. Due to the killing effect and fast replication, a lot of lytic phages have been isolated and characterized to control E. coli O157:H7. However, the number of phages specific to STEC non-O157 and concurrently characterized using whole genome sequencing is relatively low. Thus, the objective of this research was to characterize a newly-isolated phage as a potential antimicrobial agent simultaneously against STEC O45 and O157. Phage Sa45lw was isolated from surface water collected in a produce-growing area. The sample was enriched with E. coli ATCC13706 strain in tryptic soy broth supplemented with 10% calcium chloride at 37'C overnight. After purification and CsCl treatment, the concentrated phage was subjected to TEM and whole genome sequencing. Phage DNA library was constructed and sequenced on Illumina MiSeq for genomic analyses. Biocontrol potential of the phage was examined by host range assay, efficacy of plating (EOP), and in vitro antimicrobial study against the serogroups of O157 and top six non-O157 STEC strains using spectrophotometer. The results show that Sa45lw is a myophage containing a double-stranded DNA of 167,353 bp and is highly related to T4-like phages, sharing 97% average nucleotide identity to Shigella phage Sf23 and E. coli O157 typing phage. The Sa45lw contains 11 tRNA and 263 open reading frames (ORFs), including 121 annotated proteins with functions of virion structures, DNA replication and packaging, and cell lysis. The RecA-like recombinant protein, associated with strict lytic cycle, of Sa45lw shares 99% identity of the amino acid sequences to Escherichia phage wV7, which is lytic against E. coli O157:H7. Additionally, the baseplate wedge subunit and tail pin (ORF_43) of Sa45lw shares 100% identity of the amino acid sequence to Escherichia phage wV7. No lysogenic or virulence genes were found. Furthermore, in vitro analysis shows that after 18-h incubation at 37'C, Sa45lw treatment (MOI of 100) against STEC cultures resulted in 71% and complete suppression of O157 and O45 strains, respectively. The findings suggest that Sa45lw has the genomic characteristics containing tRNA and RecA-like recombinant protein, both contributing to the strict lytic effect of the phage With absence of genes coding for lysogenic and virulence factors, this phage has the potential as a biocontrol agent for STEC O45 and O157.