Discovery of Shiga Toxin-Producing Escherichia coli (STEC)-Specific Bacteriophages From Non-fecal Composts Using Genomic Characterization

Abstract

Composting is a complex biodegradable process that converts organic materials into nutrients to facilitate crop yields, and, if well managed, can render bactericidal effects. Majority of research focused on detection of enteric pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in fecal composts. Recently, attention has been emphasized on bacteriophages, such as STEC-specific bacteriophages, associated with STEC from the fecal-contaminated environment because they are able to sustain adverse environmental condition during composting process. However, little is known regarding the isolation of STEC-specific bacteriophages in non-fecal composts. Thus, the objectives were to isolate and genomically characterize STEC-specific bacteriophages, and to evaluate its association with STEC in non-fecal composts. For bacteriophage isolation, the samples were enriched with non-pathogenic E. coli (3 strains) and STEC (14 strains), respectively. After purification, host range, plaque size, and phage morphology were examined. Furthermore, bacteriophage genomes were subjected to whole-genome sequencing using Illumina MiSeq and genomic analyses. Isolation of top six non-O157 and O157 STEC utilizing culture methods combined with PCR-based confirmation was also conducted. The results showed that various STEC-specific bacteriophages, including vB_EcoM-Ro111lw, vB_EcoM-Ro121lw, vB_EcoS-Ro145lw, and vB_EcoM-Ro157lw, with different but complementary host ranges were isolated. Genomic analysis showed the genome sizes varied from 42kb to 149kb, and most bacteriophages were unclassified at the genus level, except vB_EcoM-Ro111lw as FelixO1-like viruses. Prokka predicted less than 25% of the ORFs coded for known functions, including those essential for DNA replication, bacteriophage structure, and host cell lysis. Moreover, none of the bacteriophages harbored lysogenic genes or virulence genes, such as stx or eae. Additionally, the presence of these lytic bacteriophages was likely attributed to zero isolation of STEC and could also contribute to additional antimicrobial effects in composts, if the composting process was insufficient. Current findings indicate that various STEC-specific bacteriophages were found in the non-fecal composts. In addition, the genomic characterization provides in-depth information to complement the deficiency of biological features regarding lytic cycle of the new bacteriophages. Most importantly, these bacteriophages have great potential to control various serogroups of STEC.

Keywords: STEC-specific bacteriophages; Shiga toxin-producing E. coli (STEC); complementary host range; non-fecal composts; whole genome sequencing.

FIGURE 1

The morphology of plaques formed by phage vB_EcoM-Ro111lw (A1), phage vB_EcoM-Ro121lw (A2), phage vB_EcoS-Ro145lw (A3), and phage vB_EcoM-Ro157lw (A4) on their host strains of STEC O111, O121, O145, and O157, respectively, using plaque assay, and the transmission electron microscopy images of phage vB_EcoM-Ro111lw (B1), phage vB_EcoM-Ro121lw (B2), phage vB_EcoS-Ro145lw (B3), and phage vB_EcoM-Ro157lw (B4).

FIGURE 2

Whole genome comparison of Escherichia phage vB_EcoM-Ro111lw (A), Escherichia phage vB_EcoM-Ro121lw (B), Escherichia phage vB_EcoS-Ro145lw (C), and Escherichia phage vB_EcoM-Ro157lw (D) with their reference genomes from NCBI database using EasyFig. A gray-scaled shaded area indicates the different levels of sequence similarity between two phage sequences. The arrow shapes indicate the annotated ORFs in the genome, and the unshared ORFs are highlighted as red arrows.

FIGURE 3

Circular genome map of the phages vB_EcoM-Ro111lw (A), vB_EcoM-Ro121lw (B), vB_EcoS-Ro145lw (C), and vB_EcoM-Ro157lw (D). The outer circle represents genes as indicated by the arrows. Predicted functional categories of the genes were determined according to annotation and are represented with different colors. The central graph in blue shows genomic GC content variation.

FIGURE 4

Phylogenetic comparison based on Clustal Omega alignment of the genes coding for tail fiber (A) and endolysin/lysozyme (B) from the newly isolated phages (red underline) and other reference phage genomes obtained from NCBI database. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree.