CRISPR-Cas9 modified bacteriophage for treatment of Staphylococcus aureus induced osteomyelitis and soft tissue infection

Abstract

Osteomyelitis, or bone infection, is often induced by antibiotic resistant Staphylococcus aureus strains of bacteria. Although debridement and long-term administration of antibiotics are the gold standard for osteomyelitis treatment, the increase in prevalence of antibiotic resistant bacterial strains limits the ability of clinicians to effectively treat infection. Bacteriophages (phages), viruses that in a lytic state can effectively kill bacteria, have gained recent attention for their high specificity, abundance in nature, and minimal risk of host toxicity. Previously, we have shown that CRISPR-Cas9 genomic editing techniques could be utilized to expand temperate bacteriophage host range and enhance bactericidal activity through modification of the tail fiber protein. In a dermal infection study, these CRISPR-Cas9 phages reduced bacterial load relative to unmodified phage. Thus we hypothesized this temperate bacteriophage, equipped with the CRISPR-Cas9 bactericidal machinery, would be effective at mitigating infection from a biofilm forming S. aureus strain in vitro and in vivo. In vitro, qualitative fluorescent imaging demonstrated superiority of phage to conventional vancomycin and fosfomycin antibiotics against S. aureus biofilm. Quantitative antibiofilm effects increased over time, at least partially, for all fosfomycin, phage, and fosfomycin-phage (dual) therapeutics delivered via alginate hydrogel. We developed an in vivo rat model of osteomyelitis and soft tissue infection that was reproducible and challenging and enabled longitudinal monitoring of infection progression. Using this model, phage (with and without fosfomycin) delivered via alginate hydrogel were successful in reducing soft tissue infection but not bone infection, based on bacteriological, histological, and scanning electron microscopy analyses. Notably, the efficacy of phage at mitigating soft tissue infection was equal to that of high dose fosfomycin. Future research may utilize this model as a platform for evaluation of therapeutic type and dose, and alternate delivery vehicles for osteomyelitis mitigation.

Therapeutics and delivery vehicles for local treatment of osteomyelitis

“…35,[37][38][39][40] In vivo, phage has also shown significant clearance of soft tissue infections. 41,42 In one abscess study, 10 9 PFU of bacteriophage M Sa eradicated infection in 97% of mice, when treated immediately after inoculation of 10 8 CFU of S. aureus intravenously. 41 The same treatment administered 10 days after intravenous (IV) delivery of 5 × 10 6 CFU S. aureus resulted in clearance in all phage treated mice 10 days after treatment, whereas all nontreated mice remained infected through 20 days.…”

“…45 In addition, since phages die without a bacterial host, efficient, and precise delivery to the site of infection is critical for bactericidal activity. 34,40,42 In one recent study, alginate hydrogel was used to deliver CRISPR-Cas9 bacteriophage to S. aureus infected femora. Although the surrounding soft tissue infection was reduced, bacterial load in the bone was unchanged, presumably due to insufficient localization of phage at the bony site.…”

“…Although the surrounding soft tissue infection was reduced, bacterial load in the bone was unchanged, presumably due to insufficient localization of phage at the bony site. 42 Recent research has focused on engineering hydrogels, powders, nanoparticles, and implant coatings for delivery and prolonged bioavailability of phages. 34,35,46,47 Long term, another hurdle will be reliable generation and storage of phages needed for widespread commercialization.…”

“…95 Against some strains, Ag may be more beneficial at higher doses, in conjunction with other materials, or when utilized in a silver ion state. [95][96][97] 3 | DELIVERY VEHICLES Some natural materials that constitute hydrogels include, but are not limited to: alginate, 42,98,99 chitosan, 100 silk, 94 gelatin, 101,102 and keratin. 103 Alternatively, synthetic materials can be used, including:…”

“…101,109 Hydrogels are generally biodegradable and may be cleared by the body in as early as 2 to 6 weeks. 101,105,109 Hydrogels have been used as carriers for antibiotics, 103,105,108,110,111 bacteriophages, 34,42,47,106 and proteins, 42,49,71,74 as well as nanoparticles and microparticles containing therapeutics. 94,105,110 One of the advantages of hydrogels is the ability to tailor their properties including porosity, viscosity, elasticity, degree of swelling, and rate of drug elution.…”