Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection

Abstract

Background: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy.

Results: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection.

Conclusions: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.

Figure 1

Schematic and PCR analysis of parent and recombinant endolysin-deficient phage P954. Alignment of primers to the (a) parent phage DNA template and (b) recombinant phage DNA template, in which the cat gene had been inserted into the endolysin gene (LYS). (c) Endolysin forward and reverse primers yield a 750-bp PCR product of the parent phage P954 and 2400-bp product of the recombinant phage P954. (d) The holin forward primer and endolysin reverse primer yield a 1000-bp PCR product with parent phage P954 and 2650-bp product of the recombinant phage P954. Both PCR panels include lane 1: PCR buffer (negative control); lane 2: parent phage P954 lysogen B7, lane 3: molecular weight marker (λ/HindIII-EcoRI); lane 4: recombinant phage P954 lysogen H10.

Figure 2

Mitomycin C induction of parent and endolysin-deficient phage P954 lysogens. (a) Growth profiles of the parent (B7) and endolysin-deficient (H10) phage P954 lysogens after Mitomycin C induction showing absorbance of cultures at 600 nm. The graph is representative of two experiments. The error bars represent mean plus standard deviation (n = 3) (b) Phage release into the culture medium from parent (B7) and endolysin-deficient (H10) phage P954 lysogens after Mitomycin C induction. The graph is representative of 2 experiments.

Figure 3

Complementation with heterologous endolysin gene for enrichment of endolysin-deficient phage P954. Ten-fold serial dilutions of endolysin-deficient phage P954 (5 × 10¹⁰ PFU/ml) spotted on (a) S. aureus RN4220 lawn and (b) complementing host pGMB540/S. aureus RN4220, which expresses a heterologous endolysin. Plaque assay of enriched endolysin-deficient phage P954 on (c) non-complementing host S. aureus RN4220 and (d) complementing host pGMB540/S. aureus RN4220.

Figure 4

Bactericidal activity of parent and lysis-deficient phage P954. Bactericidal activity of parent and lysis-deficient phage P954 (10 MOI equivalent) on eight clinical isolates of MRSA (B910, B954, B9053, B9194, B9195) and MSSA (B911, B9007, B9030). Phage resistant isolate indicated with asterix (*). The error bars represent standard deviation (n = 3, single experiment).

Figure 5

In vivo efficacy of endolysin-deficient phage P954. Survival of mice challenged with clinical MRSA isolate (B911). Groups 1-3 were challenged with MRSA (5 × 10⁷ cells per mouse). Groups 4-6 were not challenged with MRSA and served as controls. The following treatments were administered: groups 1 and 4 (25 mM Tris-HCl, pH 7.5); groups 2 and 5 (two doses of endolysin-deficient phage P954, 200 MOI); groups 3 and 6 (two doses of chloramphenicol, 50 mg/kg).