Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections

Abstract

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYO^Sa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYO^Sa Because of the mode of action of PYO^Sa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYO^Sa; none were observed in the 13 clinical isolates tested. PYO^Sa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYO^Sa and antibiotics raise issues that must be addressed before PYO^Sa is employed clinically. Despite the maintenance of the phage, PYO^Sa does not clear populations of S. aureus Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise-resurrection dynamics of PYO^Sa and S. aureus Critically for phage therapy, our experimental results suggest that treatment with PYO^Sa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.

Keywords: Staphylococcus aureus; phage therapy; population dynamics.

Fig. 1.

Small-colony variants and the population dynamics of PYO^Sa and S. aureus Newman in serial transfer culture. (A) Wild-type S. aureus and small-colony variants visualized. (B) Changes in densities of S. aureus Newman and PYO^Sa in three independent (red, green, blue) serial transfer experiments diluted 1/100 in fresh media daily. (C) Changes in densities of S. aureus Newman and PYO^Sa in serial transfer cultures initiated two small-colony variants isolated from the fifth transfer of the cultures with PYO^Sa and S. aureus Newman in B. (D) Changes in densities of S. aureus Newman and PYO^Sa in serial transfer cultures initiated with PYO^Sa and equal densities of cultures derived from small colonies and the ancestral S. aureus Newman. (E) Changes in densities of S. aureus and PYO^Sa in serial transfer cultures initiated with PYO^Sa and single colonies of evolved (wild-type colony morphology) bacteria isolated from the fifth transfer of the cultures in B. (F) Changes in the ratio of bacteria and phage at time 0 and 24 h. Wild-type S. aureus Newman (W), small-colony variants (S), and evolved bacteria (E). Three independent replicas.

Fig. 2.

Joint action of bacteriostatic antibiotics and PYO^Sa. The concentrations of these antibiotics are 10 μg/mL. (A) The ratio of the change in density of S. aureus after 24 h of exposure to antibiotics (blue), antibiotics plus phage (purple, green, orange, refers to order of addition as explained in the text) or phage alone (red). Hash red, the density of S. aureus recovered was below the detection limit, ∼10² cells per mL. (B) The ratio of the change in the density of PYO^Sa after 24 h of confronting wild-type S. aureus in combination with antibiotics or alone.

Fig. 3.

The joint action of bactericidal antibiotics and PYO^Sa. Concentrations of the different antibiotics in μg/mL (RIF 0.02, OXA 3, CIP 0.5, VAN 8, DAP 64, KAN 46), corresponding to minimum bacteriocidal concentrations. (A) The ratio of the change in density of S. aureus after 24 h of exposure to antibiotics, antibiotics and phage, or phage alone. Hash red, the density of S. aureus recovered was below the detection limit, ∼10² per mL. (B) The ratio of the change in the density of PYO^Sa after 24 h of confronting S. aureus Newman in combination with antibiotics or alone.

Fig. 4.

Changes in the densities of bacteria and phage in serial transfer cultures treated with antibiotics alone or in combination with phages. (A) Bacteria treated with antibiotics alone. (B) Bacterial densities in cultures containing both the antibiotic and the phage. (C) Phage densities in cultures containing both the antibiotic and the phage. Mean and SEs of three independent experiments. Row 1: Treatment with RIF and DAP, 0.02 and 64 µg/mL, respectively. Row 2: Treatment with KAN and OXA, 46 and 3 µg/mL, respectively. Row 3: Treatment with CIP and VAN, 0.5 and 8 µg/mL, respectively.