Emodin Combined with Multiple-Low-Frequency, Low-Intensity Ultrasound To Relieve Osteomyelitis through Sonoantimicrobial Chemotherapy

Treatment of osteomyelitis is still challenging, as conventional antibiotic therapy is limited by the emergence of resistant strains and the formation of biofilms. Sonoantimicrobial chemotherapy (SACT) is a novel therapy of low-frequency and low-intensity ultrasound (LFLIU) combined with a sonosensitizer. Therefore, in our study, a sonosensitizer named emodin (EM) was proposed to be combined with LFLIU to relieve acute osteomyelitis caused by methicillin-resistant Staphylococcus aureus (MRSA) through antibacterial and antibiofilm effects. The efficiencies of different intensities of ultrasound, including single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 3 times for 5 min at 4-h intervals), against bacteria and biofilms were compared, contributing to developing the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) had significant combined bactericidal and antibiofilm effects, with EM+M-LFLIU in particular exhibiting superior antibiofilm performance. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of the bacterial membrane and cell wall, and downregulate the expression of genes involved in oxidative stress, membrane wall synthesis, and bacterial virulence, as well as that of other related genes (agrB, pbp3, sgtB, gmk, zwf, and msrA). In vivo studies, micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis due to MRSA infection. Our work proffers an original approach to bacterial osteomyelitis treatment that weakens drug-resistant bacteria and suppresses and degrades biofilm formation through SACT, which may provide new prospects for clinical treatment. IMPORTANCE Antibiotic therapy is the first choice for clinical treatment of osteomyelitis, but the formation of bacterial biofilms and the emergence of many drug-resistant strains also create an urgent need to find an alternative treatment to effectively eliminate the infection. Recently, LFLIU has come to be considered a safe and promising method of debridement and antibacterial therapy. In this study, we found that ultrasound and EM have a significant combined antibacterial effect in vivo and in vitro, which may play an antibacterial role by stimulating the production of ROS, destroying the bacterial cell wall, and inhibiting the expression of related genes. Our study expands the body of knowledge on the antibacterial effect of drugs—specifically emodin (EM)—through combined physiotherapy. If successfully integrated into clinical practice, these methods may reduce the burden of high concentrations of drugs needed to treat bacterial biofilms and avoid the growing resistance of bacteria to antibiotics.