A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor

Abstract

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Keywords: Clostridioides difficile (Clostridium difficile); S-layer; SlpA; bacteriophage; phage receptor; phage therapy.

FIG 1

Phage particle morphology as visualized using TEM for (a) ΦCD418, (b) ΦCD2301, (c) ΦCD1801, and (d) ΦCD08011. Measurements represent the mean ± standard deviation (SD) of values of 5 individual phage particles.

FIG 2

Graphical representation of phage genomes. (a) ΦCD1801, (b) ΦCD08011, (c) ΦCD418, and (d) ΦCD2301. Assembled using CLC Genomics Workbench and manually annotated using Artemis, BLAST, UniProt, and pfam. Genes of particular importance are discussed in the main body of text. Notably, the genomes contain integrase genes indicating their temperate nature. Proteins making up the capsids and tails, including the base plate proteins, were also identified.

FIG 3

Heat map of C. difficile sensitivity to bacteriophage infection. Phage host range was determined using standard plaque assays for phages ΦCD08011, ΦCD2301, ΦCD418, and ΦCD1801. Efficiency of plating (EOP) values are depicted for each infection study represented by color. Magnolia, resistant strain; yellow, EOP of <0.1; orange, EOP 0.1 to 1; red, EOP of >1.

FIG 4

Phage binding is SLCT dependent in RT 012. The capacity for CD630 to bind to ΦCD1801 was assessed by means of a phage-binding assay with wild-type CD630 and CD630 harnessing plasmid-borne SLC H2/6 (pJK002), SLC 6 (pJK018), and SLC 2 (pJK023) under the control of a tetracycline inducible promoter. Following coincubation with wild-type or plasmid-bearing CD630, the titer of free ΦCD1801 particles was assessed through a plaque assay using the propagating strain CD18101 as an indicator. Data represent the mean ± SD of 3 biological replicates *, P < 0.05 according to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test.