(M-483) The stability and diffusion of bacteriophages in hydrogels for skin and soft tissue infections

Conventional antibiotics are often ineffective in treating skin and soft tissue infections (SSTIs), leading to prolonged healing times and an increased risk of bacterial resistance. In recent years, bacteriophage (or phage) antimicrobials have emerged as a promising alternative for treating SSTIs. Phages are beneficial because they not only target and kill bacteria but also do so without damaging the beneficial skin microbiota. Bacteria are also less likely to develop resistance to phages over time. Hydrogels are three-dimensional structures of hydrophilic polymer chains and are one of the most common wound dressings. Hydrogels are beneficial because they form hydrogen bonds between the water and phage proteins, which is important for the stabilization of phages. There is currently a lack of defined methods for testing the stability, and diffusion of bacteriophages embedded within hydrogels, which makes it challenging to evaluate the potential of this combination therapy. Therefore, we have devised techniques for assessing the stability and diffusion of bacteriophage-hydrogel systems against bacterial biofilms commonly found in SSTIs using precursory system with a Pseudomonas aeruginosa PAO1 Krylov strain specific bacteriophage, ϕKMV, and a non-ionic, thermoreversbile polymer, Pluronic F-127 (PF-127).

For our tests, we have employed a variety of traditional and novel methods to measure the diffusion of phages from hydrogels. We initially tested the stability of phage in different conditions, including the polymer itself, a range of pH buffer solutions, and clinical wound exudate by incubating overnight at 37°C and using the double layer agar (DLA) method to measure the phage titer. Rheological analysis of hydrogel is completed to identify the mechanical properties and how they are affected by the introduction of phage and phage buffer salts. Simple diffusion assays in which the polymer is placed in a test tube with buffer atop was used to see how the phage can diffuse out of the polymer without a barrier. Transwell inserts (0.4 μm) were used to measure the diffusion of phage across a barrier and to compare different concentrations of PF-127 (20% vs 30%) and the effect this can have on phage diffusion. A rapid hydrogel-based phage susceptibility test was also used in which phage release from a polymer was measured in LB media and bacteria using an absorbance plate reader to determine how bacterial concentration in the wells decreased over time. This experiment is to be repeated using a range of multiplicities of infection (MOIs) and antibiotic loaded hydrogels to determine how these compare to this hydrogel phage system. Finally, Confocal Laser Scanning Microscopy (CLSM) will be employed to check if the phage is evenly distributed throughout the polymer.

We found that our ϕKMV phage is stable in 20% PF-127 for a timespan ranging from 24 hours to 2 months. The phage is also stable in phage buffer solutions with pH range of 4.0-10.0 and clinical wound exudate. The zone of inhibition of the phage in hydrogel is not significantly different than that of the phage in buffer, however both are significantly greater than that of buffer or 20% PF-127 alone.  In a simple diffusion assay, ϕKMV phage in 20% PF-127 exhibits a substantial reduction in diffusion compared to control. In a transwell assay, ϕKMV phage in 20% PF-127 was not significantly different from ϕKMV phage in 30% PF-127, but both were significantly different than control and had a greater diffusion capability than the phage in buffer alone. In a rapid hydrogel-based phage susceptibility test, ΦKMV phage in PF-127 demonstrates a diminished rate of phage release initially and is eventually comparable to control.

Overall, the study contributes to filling the current knowledge gap in testing the stability and diffusion of bacteriophage-hydrogel systems, providing valuable insights for evaluating the potential of this combination therapy in treating SSTIs. Further research and optimization of this approach may pave the way for more effective and sustainable treatments for skin and soft tissue infections, potentially mitigating the challenges associated with antibiotic resistance and improving healing outcomes.