A simple solid media assay for detection of synergy between bacteriophages and antibiotics

Abstract

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

Keywords: antibiotics; bacteriophages; cooperativity; solid media; synergy.

Fig 1

Workflow for phage–antibiotic cooperativity assays.

Fig 2

Model with parameters predictive of experimental results. Prediction of antibiotic (e.g., vancomycin) and phage (e.g., Ben) profiles based on different potential interactions. Concentration contour plots for representative antibiotic (C_α = 1.5 µg/mL and D_α = 1 x 10⁻⁶ cm²/s) and phage (C_β = 1.2 × 10⁻² µg/mL and D_β = 5 x 10⁻⁸ cm²/s). (A) No interaction, (B) additive, (C) synergistic “medium” k = 1e6; (D) synergistic “high” k = 1e12; (E) antagonistic q = 1. Assuming MBC = 0.1 µg/mL (red). Panels A’–E’ and A”–E” represent magnifications of portions of the panels shown in panels A–E, respectively.

Fig 3

Summary heat map of all used combinations of bacteria, phage, and antibiotics evaluated for cooperativity. K-values were calculated for each experiment based on data of three biological replicates. (A) E. faecium and E. faecalis strains using phages Ben, Bop, Bob, or PL with antibiotics ampicillin, vancomycin, or linezolid. (B) Stenotrophomonas maltophilia strains using phages KB824, 2ϕ2, or ANB28 with antibiotics ceftazidime or levofloxacin.

Fig 4

Solid media cooperativity assays for VRE. Each specimen was tested with vancomycin (vertical strip) and a phage (horizontal strip). E. faecium EF98PII (VRE) with phage Bop is demonstrated in panels A–D, where A–C represent three separate replicates of the cooperativity assay, and panel D represents the control plate with a vertical vancomycin strip (left), blank strip (middle), phage strip (right), antibiotic disk (bottom), and phage spot (top). E. faecalis V587 (VRE) with phage Bop is demonstrated in panels E–H, where panels E–G represent separate replicates and panel H represents the control plate.

Fig 5

Comparison of experimental results and model predictions. (A) E. faecium EF98PII (VRE) treated with vancomycin (vertical strip) and phage Bop (horizontal strip). This resulted in a synergistic profile that extended 1.0 cm from the leading edge of the vertical zone of inhibition. (B) E. faecalis V587 (VRE) treated with vancomycin (vertical strip) and phage Bop (horizontal strip). This resulted in a synergistic profile that extended 2.5 cm from the leading edge of the vertical zone of inhibition. Model predictions for E. faecium EF98PII (A’) and E. faecalis V587 (B’) showed similar synergistic profile extensions and dimensions when the synergy coefficient was adjusted from medium synergy (k = 1e6) to high synergy (k = 1e16).

Fig 6

Solid media cooperativity assays for VRE. Each specimen was tested with linezolid (vertical strip) and a phage (horizontal strip). E. faecium NYU with phage Bob is demonstrated in panels A–D, where A–C represent three separate replicates of the cooperativity assay, and panel D represents the control plate with a vertical linezolid strip (left), blank strip (middle), phage strip (right), antibiotic disk (bottom), and phage spot (top). E. faecalis B3286 with phage PL is demonstrated in panels E–H, where panels E–G represent separate replicates and panel H represents the control plate.

Fig 7

Solid media cooperativity assays for VRE. Each specimen was tested with ampicillin (vertical strip) and a phage (horizontal strip). E. faecium NYU with phage Bob is demonstrated in panels A–D, where A–C represent three separate replicates of the cooperativity assay, and panel D represents the control plate with a vertical ampicillin strip (left), blank strip (middle), phage strip (right), antibiotic disk (bottom), and phage spot (top). E. faecalis Yi-6 with phage Bop is demonstrated in panels E–H, where panels E–G represent separate replicates and panel H represents the control plate.

Fig 8

Solid media cooperativity assays for STM. Each specimen was tested with ceftazidime (vertical strip) and a phage (horizontal strip). STM B28B with phage KB824 is demonstrated in panels A–C, which represent three separate replicates of the cooperativity assay. Panel D represents the control plate with a vertical ceftazidime strip (left), blank strip (middle), phage strip (right), antibiotic disk (bottom), and phage KB824 spot (top). STM B28S with phage KB824 is demonstrated in panels E–G, which represent separate replicates. Panel H represents the control plate with a vertical ceftazidime strip (left), blank strip (middle), phage strip (right), antibiotic disk (bottom), and phage KB824 spot (top).