Core lipopolysaccharide-specific phage SSU5 as an Auxiliary Component of a Phage Cocktail for Salmonella biocontrol

Abstract

Salmonella spp. are among the major food-borne pathogens that cause mild diarrhea to severe bacteremia. The use of bacteriophages to control various food-borne pathogens, including Salmonella, has emerged as a promising alternative to traditional chemotherapy. We isolated the Siphoviridae family phage SSU5, which can infect only rough strains of Salmonella. The blocking of SSU5 adsorption by periodate treatment of host Salmonella cells and spotting and adsorption assays with mutants that contain various truncations in their lipopolysaccharide (LPS) cores revealed that the outer core region of the LPS is a receptor of SSU5. SSU5 could infect O-antigen (O-Ag)-deficient Salmonella mutants that developed by challenging of O-Ag-specific phages, and consequently, it delayed the emergence of the phage-resistant Salmonella population in broth culture when treated together with phages using O-Ag as a receptor. Therefore, these results suggested that phage SSU5 would be a promising auxiliary component of a phage cocktail to control rough strains of Salmonella enterica serovar Typhimurium, which might emerge as resistant mutants upon infection by phages using O-Ag as a receptor.

FIG 1

Transmission electron microscopy images of phage SSU5. SSU5 was negatively stained with 2% uranyl acetate and observed by TEM. The scale bar is at the bottom right corner of each image.

FIG 2

Effect of periodate and proteinase K treatments on SSU5 adsorption. An SSU5 adsorption assay was performed with the periodate- or proteinase K-treated ΔrfbP mutant strain. The untreated ΔrfbP cells in LB broth were used as the control (untreated cells). An SSU5 titer in cell-free LB broth was considered the 100% control (LB), and the residual phage titer in each sample after 15 min of adsorption at 37°C is represented by relative percentages. The means with SDs for three independent experiments are shown.

FIG 3

SSU5 adsorbed to the outer core OS of Salmonella LPS. (A) Schematic representation of the LPS structure of S. Typhimurium. Carbohydrates are shown as hexagons, and the relevant genes involved in the biosynthesis of various residues of LPS are indicated below the line connecting the hexagon. The mutation of each gene caused the elimination of the left residues marked with gene names on the structure (rfbP, undecaprenyl-phosphate galactosephosphotransferase; rfaJ, LPS 1,2-glycosyltransferase; rfaI, LPS 1,3-galactosyltransferase; rfaG, glucosyltransferase I; rfaF, ADP-heptose-LPS heptosyltransferase; rfaC, LPS heptosyltransferase I; kdtA, 3-deoxy-d-manno-octulosonic acid transferase). Abbreviations: Kdo, 3-deoxy-d-manno-octulosonic acid; Hep, heptose; Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; Rha, rhamnose; P, phosphate; PPEtN, pyrophosphorylethanolamine. (B) SSU5 spotting assay with mutants with truncations in the LPS core OS. Serially diluted (10-fold) SSU5 lysates were spotted on the lawns of indicated Salmonella mutants. The phage titer (PFU/ml) used for each spot is indicated in the grids at the bottom right corner. (C) Assay of adsorption of SSU5 to various core OS mutants. The adsorption constant (k) was calculated as described in the text. The means with SD of three independent assays are represented.

FIG 4

SSU5 infects Salmonella mutants that lost their O-Ag and are resistant to O-Ag-specific phages. (A) Serially diluted SSU5 lysate was spotted onto the lawns of isolated P22H5- or SSU14-resistant mutants (indicated as P22-R[number] or SSU14-R[number], respectively). Phage titers used are the same as for Fig. 3B. (B) DOC-PAGE of LPS extracted from P22H5- or SSU14-resistant mutants. LPSs that were extracted by the hot-phenol extraction method were electrophoresed on a 15% polyacrylamide slab gel. Gels were stained using the Pro-Q Emerald 300 lipopolysaccharide gel stain kit prior to visualization under UV light. Lanes 1 and 11, Salmonella LPS standard (purchased from Sigma); lanes 2 and 12, WT LT2(c); lane 3, S. Typhimurium KCTC 1925 (see Table 1); lanes 4 and 13, ΔrfbP; lanes 5 to 10, P22-R1 to -R6 in panel A; lanes 14 to 19, SSU14-R1 to -R6 in panel A.

FIG 5

Bacterial challenge assay with SSU5, SSU14, or the phage cocktail, which consisted of both phages. WT LT2(c) cultures at the early exponential growth phase were infected by phages at an MOI of 1 (black arrow), and the OD₆₀₀ was measured every hour to monitor the bacterial growth. Instead of phages lysate, SM buffer was added to the LT2(c) culture for use as a negative control. The means with SD of three independent experiments are shown.