gms | German Medical Science

Objectives: Bacteriophages are the viruses that infect and replicate inside the bacteria and finally release from the host bacteria through lysis. The lytic activity of bacteriophages has been exploited as antimicrobial to treat bacterial infections. Staphylococcus aureus predominantly causes implant-associated bone infections with biofilm formation on surface of implant and internalization of osteoblasts. Recently, we demonstrated the in vitro efficacy of bacteriophageagainst planktonic S. aureus, in vitro S. aureus biofilm as well as S. aureus internalized in an osteoblastic cell line. Whereas, Galleria mellonella infection model, single administration of phage failed to show an improvement in survival rate but appeared to show a not statistically significant enhanced effect with gentamicin or rifampicin. Here, we are aimed to evaluate multiple dose of bacteriophage treatment in implant associated larvae infectionmodel.

Methods: The S. aureus bacteriophages were provided from D&D Pharma GmbH and propagated on the S. aureus EDCC 5055 strain. G. mellonella larvae were ordered from Flotex Evergreen (Augsburg, Germany) and maintained on artificial diet in an incubator at 30°C. After infection, G. mellonella were maintained at 37°C. For the infectionprocess, implants were pre-incubated in bacterialgrowth culture medium with 1 × 10⁶ CFU/mL S. aureus EDCC 5055 for 30 min while shaking at 150 rpm. Later, the implants were washed with 10 mL PBS and implanted on the rear side in the abdomen ofthe larvae by piercing the cuticle of the larvae usingthe sharp edge of the K-wire. To determine the effect of bacteriophages on bacterial survival in the early-stage implant biofilm infection model, The larvae were treated with 10⁹ PFU/larvae were injected on day 1 and it is repeated on every day for 5 days. To check the synergistic effect of gentamicin, antibiotic was injected everyday after phage treatment every day. To check the effect of bacteriophage therapy, bacterial burden in larvae tissue and on implant material were measured on 2^nd day.

Results and conclusion: The results showed that single dose ofbacteriophage treatment didn't improve the survival of G. mellonella larvae. Treatment with multiple doses of bacteriophages, the survival of the larvae (56 %) was better compared to single dose (43 %)and along with gentamicin showed significant increased larvae survival (83%). Bacterial burden analysis also showed significant reduction with multiple doses of phages along with gentamicin treatment. The combined effect of phages and gentamicin treatment resulted in more than 3 log reduction of bacterial burden in larvae tissue as well as on the implant surface. Thus, combination of phages and antibiotics could be used to treat life devastating implant associated infections. Further, G. mellonella larvae model could be used as alternative in vivo model to evaluate the treatment effects against implant associated infections.