Complete Genome Sequence of a Shiga Toxin-Converting Bacteriophage, Escherichia Phage Lys12581Vzw, Induced from an Outbreak Shiga Toxin-Producing Escherichia coli Strain.

Although numerous Shiga toxin (Stx)-producing Escherichia coli (STEC) strains have been sequenced, genomic information on Stx-converting phages, highly related to the primary virulence factors of STEC, is scarce. Here, we report the complete genome sequence of a Stx-converting phage induced from an outbreak STEC O145 strain.

ANNOUNCEMENT

Shiga toxin (Stx)-producing Escherichia coli (STEC), as one of the major foodborne pathogens, has been widely associated with multiple foodborne outbreaks in the United States (1, 2). Stx-converting phages, carrying the major virulence genes of STEC, are usually induced from STEC strains and have been related to the emergence of new STEC strains through the transfer of stx genes to other bacteria (3, 4). However, there is a limited number of complete whole-genome sequences of Stx-converting phages. In this study, the complete genome sequence of a Stx2-converting phage isolated from an outbreak STEC O145 strain is described.

The Stx-converting phage Lys12581Vzw was induced from E. coli O145:H28 (RM12581), associated with the U.S. romaine lettuce outbreak in 2010 (5), by using mitomycin C (0.5 μg/ml) in LB broth at 37°C overnight. Phage purification was performed using a double-layer plaque assay against E. coli strain WG5 (ATCC 700078) from a single plaque picking, followed by CsCl gradient concentration to get rid of bacterial DNA and debris. The phage DNA was extracted using a phage DNA extraction kit (Norgen Biotek, Ontario, Canada). The DNA library (2 × 250 bp) was constructed using a TruSeq Nano DNA library prep kit (Illumina, San Diego, CA) and was subsequently sequenced on an Illumina MiSeq sequencer, resulting in 6,779,298 paired-end reads. The raw sequence reads were quality filtered and trimmed using FastQC and Trimmomatic (6), with the average quality set at Q30. De novo assembly of the remaining quality reads was performed using SPAdes version 3.13.0 on the KBase server (7, 8) with the minimum contig length set to >10,000 bp. The resulting contig was annotated using both Prokka (9) and RAST server (10) pipelines with default settings. The annotations were subsequently compared and curated with UniProt (11) and BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins), with the identity and query coverage greater than 95%, using Geneious (version 11.0.4). The packaging mechanisms and genome termini were determined using PhageTerm (12). tRNAs were predicted using the tRNAscan-SE search server (13). ResFinder (version 3.0) and VirulenceFinder were used to screen for antibiotic resistance genes and virulence genes (14, 15).

Phage Lys12581Vzw, belonging to the family Podoviridae, has double-stranded DNA with a genome size of 62,668 bp (3,257× coverage) and an average G+C content of 50.1%. The phage has a Mu-like packaging mechanism; the phage genome lacks obvious termini and carries pieces of the host sequences. There are 81 open reading frames (ORFs) predicted, of which 45 are annotated with functional proteins, such as DNA replication and modification (restriction endonuclease, DNA primase, and methylase), membrane-related proteins (lysis proteins, membrane proteins, and cell division inhibitors), antirepressors, virion structures, integrases, Shiga toxin, and tRNAs. Most functional ORFs contribute to the virulence and propagation of Stx-converting phages. None of the ORFs contain antibiotic resistance genes. The whole-genome sequence of Lys12581Vzw shares 95% average nucleotide identity with two published Stx2-converting phages—Enterobacteria phage VT2-Sakai (GenBank accession number NC_000902) and bacteriophage 933W (GenBank accession number AF125520)—induced from different E. coli O157:H7 strains. This study provides valuable insights into the diversity of Stx-converting phages which are associated with the evolution of STEC strains.

Data availability.

The complete genome sequence of Escherichia phage Lys12581Vzw has been deposited in GenBank under the accession number MN067333. The sequencing reads have been deposited under the BioSample accession number SAMN12086808. The version of the phage genome described in this paper is the first version.