Development of a novel targeting system for lethal photosensitization of antibiotic-resistant strains of Staphylococcus aureus

Abstract

Light-activated antimicrobial agents (photosensitizers) are promising alternatives to antibiotics for the treatment of topical infections. To improve efficacy and avoid possible damage to host tissues, targeting of the photosensitizer to the infecting organism is desirable, and this has previously been achieved using antibodies and chemical modification of the agent. In this study we investigated the possibility of using a bacteriophage to deliver the photosensitizer tin(IV) chlorin e6 (SnCe6) to Staphylococcus aureus. SnCe6 was covalently linked to S. aureus bacteriophage 75, and the ability of the conjugate to kill various strains of S. aureus when exposed to red light was determined. Substantial kills of methicillin- and vancomycin-intermediate strains of S. aureus were achieved using low concentrations of the conjugate (containing 1.5 microg/ml SnCe6) and low light doses (21 J/cm2). Under these conditions, the viability of human epithelial cells (in the absence of bacteria) was largely unaffected. On a molar equivalent basis, the conjugate was a more effective bactericide than the unconjugated SnCe6, and killing was not growth phase dependent. The conjugate was effective against vancomycin-intermediate strains of S. aureus even after growth in vancomycin. The results of this study have demonstrated that a bacteriophage can be used to deliver a photosensitizer to a target organism, resulting in enhanced and selective killing of the organism. Such attributes are desirable in an agent to be used in the photodynamic therapy of infectious diseases.

FIG. 1.

Lethal photosensitization of MRSA-16 with phage-SnCe6 conjugate in the presence and absence of divalent metal ions. To suspensions of MRSA-16 in 0.85% saline with either 10 mM CaCl₂ or 10 mM EDTA, an equal volume of either saline (L−S−), phage suspension (3.8 × 10⁸ PFU), SnCe6, or phage-SnCe6 (1.5 μg/ml) was added. The SnCe6 and phage-SnCe6 samples were exposed to 21 J/cm² HeNe laser light. Numbers above bars represent percent kills relative to the L−S− sample, to which only saline was added. Error bars indicate standard deviations.

FIG. 2.

Comparison of the effectiveness of SnCe6 and phage-SnCe6 conjugate for the lethal photosensitization of MRSA. SnCe6 (circles) or phage-SnCe6 (squares) was added to suspensions of MRSA-16 in 0.85% NaCl-10 mM CaCl₂ at the concentrations indicated and exposed to 21 J/cm² HeNe laser light. Error bars indicate standard deviations.

FIG. 3.

Lethal photosensitization of MRSA-16 in different growth phases, using phage-SnCe6. To suspensions of MRSA-16 in 0.85% saline, an equal volume of either saline (L−S−), phage suspension (final concentration = 1.46 × 10⁷ PFU/ml), or phage-SnCe6 (final concentration equivalent to 1.5 μg/ml of SnCe6) was added. The phage-SnCe6-containing suspensions were exposed to 21 J/cm² HeNe laser light. Numbers above bars represent percent kills relative to the L−S− suspension, to which only saline was added. exp, exponential phase; stat, stationary phase. Error bars indicate standard deviations.

FIG. 4.

Relative susceptibilities of MRSA and human epithelial cells to lethal photosensitization. To either human epithelial cells (Hep2 cells) or EMRSA-16, SnCe6 or phage-SnCe6 conjugate was added to give a final concentration of 5 μg/ml, and the cultures were then exposed to 21 J/cm² of laser light. To L−S− samples, only saline was added and the suspensions were not exposed to laser light. Error bars indicate standard deviations.

FIG. 5.

Effect of growth in vancomycin on the killing of vancomycin-intermediate strains Mu3 (top) and Mu50 (bottom). The organisms were grown in medium with no added vancomycin and then passaged through media containing increasing vancomycin in a stepwise fashion. To bacterial suspensions in 0.85% NaCl-10 mM CaCl₂, 0.85% NaCl (L−S−) or phage-SnCe6 (L+S+) was added and exposed to laser light. Numbers above the bars show the percent kills achieved. The limit of detection was 400 CFU/ml. Error bars indicate standard deviations.