Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis

Abstract

Orthopedic device-related infection (ODRI) caused by <i>Staphylococcus aureus</i>, especially methicillin-resistant <i>S. aureus</i> (MRSA) biofilm may lead to persist infection and severe inflammatory osteolysis. Previous studies have demonstrated that both isobavachalcone and curcumin possess antimicrobial activity, recent studies also reveal their antiosteoporosis, anti-inflammation, and immunoregulatory effect. Thus, this study aims to investigate whether the combination of isobavachalcone and curcumin can enhance the anti-<i>S. aureus</i> biofilm activity of gentamicin and alleviate inflammatory osteolysis <i>in vivo</i>. EUCAST and a standardized MBEC assay were used to verify the synergy between isobavachalcone and curcumin with gentamicin against planktonic <i>S. aureus</i> and its biofilm <i>in vitro</i>, then the antimicrobial and immunoregulatory effect of cocktail therapy was demonstrated in a femoral ODRI mouse model <i>in vivo</i> by μCT analysis, histopathology, quantification of bacteria in bone and myeloid-derived suppressor cell (MDSC) in bone marrow. We tested on standard MSSA ATCC25923 and MRSA USA300, 5 clinical isolated MSSA, and 2 clinical isolated MRSA strains and found that gentamicin with curcumin (62.5-250 μg/ml) and gentamicin with isobavachalcone (1.56 μg/ml) are synergistic against planktonic MSSA, while gentamicin (128 μg/ml) with curcumin (31.25-62.5, 250-500 μg/ml) and gentamicin (64-128 μg/ml) with isobavachalcone (1.56-12.5 μg/ml) exhibit synergistic effect against MSSA biofilm. Results of further study revealed that cocktail of 128 μg/ml gentamicin together with 125 μg/ml curcumin +6.25 μg/ml isobavachalcone showed promising biofilm eradication effect with synergy against USA300 biofilm <i>in vitro</i>. Daily intraperitoneal administration of 20 mg/kg/day isobavachalcone, 20 mg/kg/day curcumin, and 20 mg/kg/day gentamicin, can reduce inflammatory osteolysis and maintain microarchitecture of trabecular bone during orthopedic device-related MRSA infection in mice. Cocktail therapy also enhanced reduction of MDSC M1 polarization in peri-implant tissue, suppression of MDSC amplification in bone marrow, and Eradication of USA300 biofilm <i>in vivo</i>. Together, these results suggest that the combination of isobavachalcone and curcumin as adjuvants administrated together with gentamicin significantly enhances its antimicrobial effect against <i>S. aureus</i> biofilm, and can also modify topical inflammation in ODRI and protect bone microstructure <i>in vivo</i>, which may serve as a potential treatment strategy, especially for <i>S. aureus</i> induced ODRI.