Genomic Characterization of Two Shiga Toxin-Converting Bacteriophages Induced From Environmental Shiga Toxin-Producing Escherichia coli

Abstract

Shiga toxin (Stx), encoded by stx genes located in prophage sequences, is the major agent responsible for the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and is closely associated with the development of hemolytic uremic syndrome (HUS). Although numerous Stx prophage sequences have been reported as part of STEC bacterial genomes, the information about the genomic characterization of Stx-converting bacteriophages induced from STEC strains is relatively scarce. The objectives of this study were to genomically characterize two Stx-converting phages induced from environmental STEC strains and to evaluate their correlations with published Stx-converting phages and STEC strains of different origins. The Stx1-converting phage Lys8385Vzw and the Stx2-converting phage Lys19259Vzw were induced from E. coli O103:H11 (RM8385) and E. coli O157:H7 (RM19259), respectively. Whole-genome sequencing of these phages was conducted on a MiSeq sequencer for genomic characterization. Phylogenetic analysis and comparative genomics were performed to determine the correlations between these two Stx-converting phages, 13 reference Stx-converting phages, and 10 reference STEC genomes carrying closely related Stx prophages. Both Stx-converting phages Lys8385Vzw and Lys19259Vzw had double-stranded DNA, with genome sizes of 50,953 and 61,072 bp, respectively. Approximately 40% of the annotated coding DNA sequences with the predicted functions were likely associated with the fitness for both phages and their bacterial hosts. The whole-genome-based phylogenetic analysis of these two Stx-converting phages and 13 reference Stx-converting phages revealed that the 15 Stx-converting phages were divided into three distinct clusters, and those from E. coli O157:H7, in particular, were distributed in each cluster, demonstrating the high genomic diversity of these Stx-converting phages. The genomes of Stx-converting phage Lys8385Vzw and Lys19259Vzw shared a high-nucleotide similarity with the prophage sequences of the selected STEC isolates from the clinical and environmental origin. The findings demonstrate the genomic diversity of Stx-converting phages induced from different STEC strains and provide valuable insights into the dissemination of stx genes among E. coli population via the lysogenization of Stx-converting phages.

Keywords: Shiga toxin-producing Escherichia coli; Stx-converting bacteriophages; comparative genomics; genetic diversity; virulence gene transfer.

FIGURE 1

Circular genome maps of Stx1-converting phage Lys8385Vzw (A) and Stx2-converting phage Lys19259Vzw (B) using GCview server. The rings from inside out represent GC skew (green and pink), GC content (brown), and CDSs (blue). tRNAs (red) are only detected in Stx2-converting phage Lys19259Vzw (B). The functions of the annotated CDSs are listed in Supplementary Tables S3, S4.

FIGURE 2

Maximum likelihood phylogenetic analysis of two Stx-converting phages, Lys8385Vzw and Lys19259Vzw, and 13 reference Stx-converting phages from the NCBI database. Different line colors represent different clusters. The phages highlighted with blue font were sequenced in this study. The bacterial strains next to each phage are the hosts from which the Stx-converting phages were induced. Phage morphologies for the Siphoviridae and Podoviridae families are indicated with red and green color bars, respectively. The circle with orange or blue color indicates the stx gene encoded in the Stx-converting phages.

FIGURE 3

Whole-genome comparison of the Stx-converting phages Lys8385Vzw (A) and Lys19259Vzw (B) with the phylogenetically related Stx-converting phages 1717 and 933W, respectively, using BLASTn and visualization with EasyFig. Arrows represent annotated CDSs of each phage genome (top: phages Lys8385Vzw and Lys19259Vzw; bottom: phages 1717 and 933W), and biological functions of the CDSs are coded with different colors in the legend. Regions of sequence similarity, ranging from 70 to 100%, are connected by a gray-scale shaded area.

FIGURE 4

Pairwise comparison of the Stx-converting phages Lys8385Vzw (A) and Lys19259Vzw (B) with the corresponding Stx prophage sequences obtained from the bacterial genomes of E. coli RM8385 and E. coli RM19259, respectively, using the PHASTER web server. Arrows represent annotated CDSs, and the biological functions of the CDSs for the Stx-converting phages Lys8385Vzw and Lys19259Vzw (top) are coded with different colors in the legend. All CDSs are coded with purple for the Stx prophages (bottom). Regions of sequence similarity, ranging from 70 to 100%, are connected by a gray-scale shaded area.

FIGURE 5

Genomic relationships between the Stx-converting phages Lys8385Vzw (A) and Lys19259Vzw (B) with five similar Stx prophages within STEC reference genomes. The color key coding for five circular maps of the bacterial sequence fragments with high sequence identity to the Stx-converting phage genomes is illustrated on the right, and the gradients of each color indicate the sequence identity, ranging from 50 to 100%, between Lys8385Vzw and its closely related phages (A) and between Lys19259Vzw and its closely related phages (B). The CDSs with functions from the phages Lys8385Vzw and Lys19259Vzw are displayed in the outermost ring with red color. The BLASTn comparison of the phage sequences was generated using BLAST Ring Image Generator (BRIG) with a minimum nucleotide sequence identity of 50%.