ROLE OF BACTERIAL DNA IN TRIGGERING MACROPHAGE ACTIVATION BY A USA300 ISOLATE OF COMMUNITY-ACQUIRED METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS EXPOSED TO DIFFERENT CLASSES OF ANTIBIOTICS.

Abstract

Background Infections caused by community-acquired, methicillin-resistant strains of Staphylococcus aureus (CA-MRSA) have become a major problem in children and adults. We recently reported that either of two clinical CA-MRSA isolates exposed to daptomycin (vs vancomycin) stimulated less macrophage tumor necrosis factor (TNF) secretion. We hypothesized that this difference might result from reduced release of bacterial products (such as bacterial DNA) from CA-MRSA isolates exposed to daptomycin (versus vancomycin). Purpose We sought to determine whether bacterial DNA contributed to the up-regulation of macrophage TNF production in response to a live CA-MRSA isolate exposed to vancomycin, daptomycin, clindamycin, or linezolid. Methods RAW 264.7 murine macrophages were stimulated for 16 to 18 hours with 107 cfu/mL of a well-characterized CA-MRSA isolate (USA300) in the presence of either vancomycin (20 mg/L), daptomycin (20 mg/L), clindamycin (20 mg/L), or linezolid (10 mg/L). In parallel, cells were preincubated for 1 hour prior to bacterial challenge with chloroquine (2.5 mg/L), a selective inhibitor of the bacterial DNA/Toll-like receptor 9 (TLR9) pathway. After stimulation, cell supernatants were collected and assayed for TNF concentration by ELISA. Results As we have previously reported, macrophages exposed to the CA-MRSA isolate in the presence of daptomycin (vs vancomycin) secreted significantly less TNF; clindamycin and linezolid were intermediate. Interestingly, preincubation of RAW cells with chloroquine led to substantial decreases in TNF secretion in response to the CA-MRSA isolate exposed to any of the four antibiotics. Furthermore, the magnitude of inhibition was consistently greater (70% vs 20-30%) in cells exposed to the SA isolate in the presence of daptomycin (compared with vancomycin, linezolid, or clindamycin). Conclusions Our data suggest that bacterial DNA plays an important role in the stimulation of TNF secretion by macrophages exposed to this virulent USA300 CA-MRSA isolate in the presence of any of the four antibiotics tested. Additional studies are needed to define the importance of bacterial DNA in stimulating the inflammatory response to staphylococcal infections.