A Quest of Great Importance-Developing a Broad Spectrum Escherichia coli Phage Collection

Abstract

Shigella ssp. and enterotoxigenic Escherichia coli are the most common etiological agents of diarrheal diseases in malnourished children under five years of age in developing countries. The ever-growing issue of antibiotic resistance and the potential negative impact of antibiotic use on infant commensal microbiota are significant challenges to current therapeutic approaches. Bacteriophages (or phages) represent an alternative treatment that can be used to treat specific bacterial infections. In the present study, we screened water samples from both environmental and industrial sources for phages capable of infecting E. coli laboratory strains within our collection. Nineteen phages were isolatedand tested for their ability to infect strains within the ECOR collection and E. coli O157:H7 Δstx. Furthermore, since coliphages have been reported to cross-infect certain Shigella spp., we also evaluated the ability of the nineteen phages to infect a representative Shigella sonnei strain from our collection. Based on having distinct (although overlapping in some cases) host ranges, ten phage isolates were selected for genome sequence and morphological characterization. Together, these ten selected phages were shown to infect most of the ECOR library, with 61 of the 72 strains infected by at least one phage from our collection. Genome analysis of the ten phages allowed classification into five previously described genetic subgroups plus one previously underrepresented subgroup.

Keywords: Escherichia coli; Shigella ssp.; bacteriophage; host range; phage therapy.

Figure 1

Hierarchical clustering of isolated phages depending on their host range; 72 ECOR strains, E. coli O157:H7 Δstx strain and S. sonnei 53G strain; green box represents successful phage infection and black box - no infection.

Figure 2

TEM morphologies of selected phages. Each phage name is indicated in the upper left corner of the picture while the scale is mentioned in the lower right. The star signs indicate the position of long tail fibers of the phages, while the arrow shapes in the JK16 picture point out the globular structures.

Figure 3

Comparison of the tail fiber genes of the T-even phages; the percent values of the similarity between the structural proteins were acquired by BLASTp; blue arrow represents the gp34 proximal fiber, orange–gp35 tail fiber hinge, pink–gp36 small distal tail fiber subunit, green–gp37 large distal tail fiber subunit and purple–gp38 tail fiber adhesin. The dark blue arrow present in the pseudo-T-evens indicates the Dc5 ORF. Different tones of grayscale represent different range of protein similarity between the phages. Absence of shaded regions is indicative of ORFs with no shared similarity. All-against-all BlastP analysis was first undertaken to identify the isolates with the most similarity and they are placed in order of similarity in the figure above. In this comparison, both phages isolated in this paper and reference phages (RB49, wV7 and RB69) are compared. Percentage similarity values presented indicate the similarity of the proteins between the two neighboring isolates.

Figure 4

Genome maps of JK16 phage and its close relatives, vB_Eco_swan01 and pSf-1. The putative functional regions are indicated with the colors described above. ORF sharing amino acid identity are linked by grey shading. The degree of sequence sharing is described by the shading scale in the bottom of the figure. The ORFs of JK16 phage that have been identified as structural components using ESI-MS/MS are outlined with a thick line.