Evaluating the safety, pharmacokinetics and efficacy of phage therapy in treating fracture-related infections with multidrug-resistant Staphylococcus aureus: intravenous versus local application in sheep

Abstract

Background: Fracture-related infections (FRI), particularly those caused by antibiotic resistant Staphylococcus aureus, present significant clinical challenges due to the formation of biofilm on the implanted device, and reduced options for conventional antibiotic treatment. Bacteriophage (phage) therapy (PT) offers a targeted approach to managing such infections, however, evidence for pharmacokinetics and optimal route of administration is limited for FRI. This study aimed to evaluate safety, phage distribution kinetics, phage neutralization, and antibacterial efficacy after intravenous or local administration in a sheep model.

Methods: The study was conducted in two phases: Phase 1 assessed the safety and distribution of two successive rounds of intravenous and local phage administration in non-infected sheep, while Phase 2 evaluated the therapeutic efficacy of intravenous versus local phage administration in combination with intravenous vancomycin in treating MRSA-induced FRI (tibial osteotomy with plate fixation). The specific pathogen and phage used in the sheep were both taken from a human FRI patient treated with PT. Phage neutralization and phage distribution were the primary outcomes measured in both phases of the sheep study.

Results: Both intravenous and local phage administration were well-tolerated in non-infected sheep. Phages were cleared rapidly from circulation after intravenous administration, with no phage detected after 240 minutes. Phage neutralization increased during PT, peaking at 99.9% in non-inoculated sheep by the end of the second phage treatment (day 50). In infected sheep, phage neutralization levels reached a maximum of 99.9% earlier (day 13), with no significant differences between intravenous and local administration. The bacterial load was not significantly changed by PT, either IV or locally applied.

Conclusions: PT is a safe adjunct to antibiotic treatment for FRI, however, phage neutralization developed rapidly and was accelerated in infected hosts. Further research is required to optimize phage selection, dosing, and delivery methods to enhance its therapeutic potential as an adjunct to conventional antibiotic therapy, particularly in the face of challenges such as rapid clearance and phage neutralization.

Keywords: MRSA; Staphylococcus aureus; administration; bacteriophage; fracture related infection; neutralization; osteomyelitis; pharmacokinetics.

Figure 1

Overview of study design. Phased experimental protocol for administering phage therapy (PT) and vancomycin (Van) in sheep, depicting both non-infection and infection phases. The green box describes three treatments, including phage intravenous administration, phage local administration, and vancomycin intravenous administration. The red box is Phase 1 (Non-infection): Groups receive treatments by specific administration routes, Group 1 received intravenous phage administration, and Group 2 received phage instillation through a drainage tube (local). Groups 1 and 2 initially received 10 days of PT (1st round), followed by a subsequent 28-day no-treatment interval. This is then succeeded by a second 10 days of PT (2nd round), concluding on Day 50. The blue box is Phase 2 (Infection): The sheep included in this phase received osteotomy, plate and screw osteosynthesis, and were infected with MRSA MSI-003 at day 0. Revision surgery was performed at day 21 after which they received a combination of intravenous phage and intravenous vancomycin for group 3, a combination of local phage and intravenous vancomycin for group 4, and intravenous vancomycin only for group 5. These treatments continued for 10 days, followed by a two-day wash-out period, leading up to a final evaluation through euthanasia and sample collection at day 33. The orange box is outcome measures, including the general condition of the sheep, the distribution of phage within the body, the presence of phage in tissues and organs, the bacterial burden in infected areas, and the level of phage neutralization.

Figure 2

Phage distribution in serum after the first and the second round of intravenous administration. Measurements were taken at 5, 30, 60, 120, 240, and 360 minutes. The titer of phage administered intravenously and locally was 10⁹ PFU/mL for both. PFU, Plaque-forming unit. The data were presented as mean ± standard deviation of results, and statistical significance was determined using a one-way ANOVA test followed by Tukey posttest, *p < 0.05. The data for the local administration are not shown in this figure as phage titers were below the detectable limit. The detection limit of the assay used to quantify active phage was 10 PFU/mL.

Figure 3

Phage neutralization between intravenous and local administrations. The black solid line represented the percentage of phage neutralization and should be read on the left Y-axis. The grey dashed line represented the phage titer of phage neutralization and should be read on the right Y-axis. PT was administered over two 10-day treatment periods separated by a 28-day no-treatment period. Higher neutralization values indicated reduced phage activity. The data are presented as mean ± standard deviation, and statistical significance was determined using a student’s t-test (*p < 0.05). The detection limit of the assay used to quantify active phage was 10 PFU/mL.

Figure 4

Bacterial load in soft tissue, osteotomy, implant, and total bacterial load. Sheep were treated with either vancomycin alone, intravenous phage therapy combined with intravenous vancomycin, or local phage therapy combined with intravenous vancomycin. Data represent the mean ± standard deviation, and statistical significance was determined using a Kruskal-Wallis test followed by Tukey post test. CFU, colony forming units.

Figure 5

Phage distribution in plasma after intravenous administration and local administration. Measurements were taken at 5, 30, 60, 120, 240, and 360 minutes on days 1, 5, and 10 post-PT. The titer of phage administered intravenously and locally was10⁹ PFU/mL. The data were presented as mean ± standard deviation of results and error bars represent standard deviation, and statistical significance was determined using a one-way ANOVA test followed by Tukey posttest. The detection limit of the assay used to quantify active phage was 10 PFU/mL.

Figure 6

Phage neutralization between intravenous and local administrations over a 13-day period. Error bars indicate the standard deviation of the measurements. The black solid line represented the percentage of phage neutralization and should be read on the left Y-axis. The grey dashed line represented the phage titer of phage neutralization and should be read on the right Y-axis. 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). The detection limit of the assay used to quantify active phage was 10 PFU/mL.