Combination of bacteriophages and vancomycin in a co-delivery hydrogel for localized treatment of fracture-related infections

Abstract

Fracture-related infections (FRIs), particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA), are challenging to treat. This study designed and evaluated a hydrogel loaded with a cocktail of bacteriophages and vancomycin (1.2 mg/mL). The co-delivery hydrogel showed 99.72% reduction in MRSA biofilm in vitro. The hydrogel released 54% of phages and 82% of vancomycin within 72 h and maintained activity for eight days, in vivo the co-delivery hydrogel with systemic antibiotic significantly reduced bacterial load by 0.99 log10 CFU compared to controls, with active phages detected in tissues at euthanasia (2 × 10³ PFU/mL). No phage resistance was detected in the phage treatment groups, and serum neutralization resulted in only a 20% reduction in phage count. In this work, we show that a phage-antibiotic co-delivery system via CMC hydrogel is a promising adjunct to systemic antibiotic therapy for MRSA-induced FRI, highlighting its potential for localized, sustained delivery and improved treatment outcomes.

Fig. 1. SEM image and bacterial count determined for GE-MRSA15.

Twenty-four-hour biofilm was formed on porous glass beads (PGB) by incubation in 1 mL medium inoculated with 1:100 dilution from a one-time use bacterial glycerol stock. A SEM image of bead in GE-MRSA15, magnifications × 2000; B Bacterial counts were determined by RT-qPCR after 24 h of GE-MRSA15 biofilm exposure to either phage cocktail (1:1 CUB_GE-MRSA15_R14 and CUB_MRSA-COL_R23, 10⁷ PFU/mL), vancomycin (VAN at 0.5, 1, 10 and 100 times the MIC), or the combination of both. Growth control (GC) refers to GE-MRSA15 biofilm not exposed to antimicrobials. MIC_VAN = 1 µg/mL. The dots correspond to biological replicates (n = 3 per tested condition) and the bars represent the mean and standard error. VAN vancomycin.

Fig. 2. Stability and release profiles of phages and vancomycin in co-delivery hydrogel.

A Stability of phages/vancomycin in co-delivery hydrogel. The red solid line represented the titer of phages in co-delivery hydrogel and should be read on the left Y-axis. The blue dashed line illustrated the stability of vancomycin in co-delivery hydrogel and should be read on the right Y-axis. The measurements were taken at different time points for 8 days. B, C The release profile of phage cocktail (CUB_GE-MRSA15_R14 and CUB_MRSA-COL_R23) and vancomycin in co-delivery hydrogel at 37 °C. The green solid line represents the drug concentration observed in the supernatant at specific timepoints and should be read on the left Y-axis. The orange dashed line illustrates the cumulative release profile up to the specific timepoints indicated and should be read on the right Y-axis. The measurements were taken at different time points for 72 h. The data are presented as means ± SD of results and error bars represent standard deviation. Experiments were performed with biological triplicates (n = 3 per tested condition).

Fig. 3. Quantitative bacteriological evaluation of tissues after treatment.

Soft tissue (A), implant (B), bone (C) and summary (D). They were shown for the Ctrl (n = 7), Sys group (n = 5), cGel group (n = 5) and cGel+Sys group (n = 7). All animals received treatment for five days after revision surgery except Ctrl that receive NaCl. Each symbol represents data from a single mouse. The data were presented as mean ± standard deviation of results and error bars represent standard deviation, and statistical significance was determined using a Kruskal-Wallis test followed by Tukey posttest (*p < 0.05 and **p < 0.01). CFU colony forming units.

Fig. 4. Evaluation of phage therapy against MRSA: distribution, resistance, and neutralization.

A The presence of phages against GE-MRSA15 in soft tissue, implant, bone and their summary at euthanasia. The total titer of the two phages was reported due to the indistinguishability between CUB_GE-MRSA15_R14 and CUB_MRSA-COL_R23 by plaque morphology. Samples for which no phage was recovered are plotted as 1 on the x-axis. B The efficiency of plating (EOP) for the isolates retrieved after euthanasia of the animals against phage cocktail. The isolates from soft tissue, bone, and implant were harvested for phage susceptibility tests. Each symbol represents data from a single colony collected from mice. C The percentage of neutralized phage particles. Each symbol represents data the mean for triplicate samples from one mouse. The data were presented as mean ± standard deviation of results and error bars represent standard deviation, and statistical significance was determined using a student’s t-test (*p < 0.05). PFU, plaque-forming units.

Fig. 5. Scheme representing the formulation of co-delivery hydrogel and mouse study design.

A The 3% w/v co-delivery hydrogel was formulated by mixing CUB_GE-MRSA15_R14, CUB_MRSA-COL_R23, and vancomycin with CMC powder at room temperature. B The mice were randomly assigned to one of four treatment groups. cGel group: a single dose of locally administered co-delivery hydrogel; cGel+Sys group: a single dose of locally administered co-delivery hydrogel and systemic subcutaneous vancomycin; Sys group: subcutaneous vancomycin (100 mg/kg) administration for five days; and Ctrl group: only received 0.3 mL of 0.9% NaCl solution. C The timeline of the animal experiment. After osteotomy, plate, and screw osteosynthesis, mice were inoculated with GE-MRSA15 and observed for five days and subsequently underwent revision surgery and treatment on day 5. From day 10 to day 13, mice did not receive any treatment. All mice were euthanized on day 13, and tissues were collected for analysis. VAN: vancomycin, NS: normal saline (0.9% NaCl). This figure was created with Biorender (biorender.com).