Experimental phage therapy against Staphylococcus aureus in mice

Abstract

The present study describes a bacteriophage (M(Sa)) active against Staphylococcus aureus, including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with S. aureus A170 (10(8) CFU/mouse), phage (10(9) PFU) rescued 97% of the mice; when applied to nonlethal (5 x 10(6) CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage M(Sa), delivered inside macrophages by S. aureus, kills the intracellular staphylococci in vivo and in vitro. The phage can also prevent abscess formation and reduce the bacterial load and weight of abscesses. These results suggest a potential use of the phage for the control of both local and systemic human S. aureus infections.

FIG. 1.

Phages W^Sa and M^Sa are serologically distinct. (A) MαM^Sa antibodies react with the mutant phage M^Sa but not with the wild phage W^Sa. The peak of phage W^Sa coincides with the peak of the negative control. (B) RαW^Sa antibodies react with the heterologous antigen (phage M^Sa) more strongly than with the homologous one (phage W^Sa).

FIG. 2.

Phage M^Sa given concurrently with S. aureus A170 rescues 93% (29/30 mice) of mice artificially infected with a lethal dose of the pathogen. (A) Mice infected with S. aureus A170 (10⁸ CFU/mouse). (B) Mice infected with S. aureus (10⁸ CFU/mouse) and treated immediately after with phage M^Sa (10⁹ PFU/mouse).

FIG. 3.

In vivo bactericidal activity of phage M^Sa. (A) Fluorescence microscopy pictures of kidney cells recovered from mice 4 days after intravenous infection with 10⁸ CFU of GFP-expressing S. aureus. (B) Kidney cells from mice infected concurrently with 10⁸ CFU of GFP-expressing S. aureus and 10⁹ PFU of phage M^Sa. Cells were counterstained with 4′,6′-diamino-2-phenylindole (DAPI). (Left) Cells analyzed with a 340- to 380-nm filter (DAPI). (Center) Cells analyzed with a 450- to 490-nm filter (GFP). (Right) Overlay. Magnification, ×1,000 (oil immersion).

FIG. 4.

In vivo phage replication occurs when the bacterial density reaches a threshold of approximately 10⁴ CFU/ml. Mice were treated concurrently with 10⁸ CFU S. aureus A170 and 10⁹ PFU phage M^Sa. The organs were dissected in saline, diluted in distilled water, and plated. In the bloodstream, bacteria did not reach the threshold for phage replication. The bacterial density thresholds were approximately the same for the various organs.

FIG. 5.

Expression levels of IL-6 and TNF-α genes in the kidneys of mice treated with phage M^Sa alone, S. aureus alone, or both. Expression levels were measured by real-time RT-PCR 24 h after treatment. Expression levels of the IL-6 and TNF-α genes are given relative to the average levels measured in three control mice. Results refer to three independent experiments, with each one carried out in triplicate. Significant results (at a P value of <0.001) are marked with asterisks.

FIG. 6.

Phage M^Sa is active against the methicillin-resistant strain S. aureus A352. (A) Mice infected with S. aureus A352 (10⁸ CFU/mouse). (B) Mice infected with S. aureus A352 (10⁸ CFU/mouse) and treated immediately after with phage M^Sa (10⁹ PFU/mouse).

FIG. 7.

Using mouse sterilization as a criterion for measuring phage activity, M^Sa was shown to be active when administered 10 days after infection. Mice received 5 × 10⁶ CFU S. aureus A170 and, 10 days later, 10⁹ PFU phage M^Sa. The experiment included 20 phage-treated and 20 untreated animals.

FIG. 8.

Effect of phage M^Sa on abscess formation. (A) One single dose of phage M^Sa, given concurrently with S. aureus, inhibits abscess formation. (B) One single dose of phage M^Sa, given 4 days after S. aureus, cannot prevent abscess formation but significantly reduces the bacterial load and the weight of abscesses. (C) Multiple doses of phage M^Sa, given 4 days after S. aureus, are more effective than a single dose in reducing the bacterial load and the weight of abscesses. Significant results (at a P value of <0.001) are marked with asterisks.

FIG. 9.

M^Sa kills intracellular S. aureus A170. (A) Numbers of live bacteria recovered in the 0- to 48-h time interval from mouse peritoneal macrophages (10⁵/well) infected with S. aureus A170 (10⁴ CFU/well) and subsequently treated with phage M^Sa alone (▪), infected with S. aureus A170 preincubated with phage M^Sa (⧫), or left untreated (▴). (B) Flow cytometric analysis of dead and live intracellular staphylococci recovered from mouse peritoneal macrophages infected with S. aureus A170 and subsequently treated for 48 h with S. aureus A170 preincubated with phage M^Sa. (C) Flow cytometric analysis of dead and live intracellular staphylococci recovered from mouse peritoneal macrophages infected with S. aureus A170 and subsequently treated for 48 h with phage M^Sa. When used concurrently, propidium iodide stains dead bacteria and SYTO9 stains the bacteria which are alive.