Artikel
Evaluation of the activity of bacteriophages against Staphylococcus aureus in implant-associated bone infections
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Veröffentlicht: | 26. Oktober 2021 |
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Gliederung
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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. Here, we are aimed to evaluate bacteriophage to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella.
Methods: The S. aureus bacteriophages were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) using double layer agar method to visualize the plaque formation. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages and S. aureus EDCC 5055 (1X107 CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37oC and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analyzing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well U-bottom plate for 24 hours. The eradication of biofilm was analyzed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts with 109 PFU/well, followed by CFU analysis at 2h, 4h and 24h time points. In addition, we have analyzed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Further experiments are in progress to prove the effect of bacteriophages to clear in vivo biofilm on implant material using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis.
Results and Conclusion: The results showed that bacteriophage were able to kill the planktonic S. aureus, eradicate biofilm and have effect on the intracellular survival of S. aureus inside osteoblasts. Treatment of bacteriophages in combination with gentamicin and rifampicin showed synergistic with reduction of S. aureus survival in osteoblasts. Further, experiments with the treatment of in vivo biofilm insect model G. mellonella could show the efficiency of bacteriophages to treat implant-associated bone infections.