Vancomycin-intermediate Methods: Three pairs of VSSA/VISA clinical isolates have been isolated from persistent bloodstream infections during prolonged antibiotic therapy. Clinical pairs were compared for different features: i) biofilm formation ability using the crystal violet staining method (mature biofilm) and the Biofilm test based on measurement of superparamagnetic microbeads mobility trapped by biofilm (early biofilm), ii) cytotoxicity and immune response by quantifying lactate dehydrogenase (LDH) and Interleukin(IL)-6 release and iii) intracellular bacterial persistence using in vitro “lysostaphin protection” infection model of human osteoblasts.Aim
Method
Intracellular persistence of S. aureus is believed to be one of the major mechanisms leading to bone and joint infection (BJI) chronicity and relapses. Despite its poor intracellular activity, daptomycin (DAP) is increasingly used in the treatment of staphylococcal BJI. The well-known in vitro synergy of daptomycin with various betalactam antibiotics consequently led us to investigate whether these combinations enhance the activity of daptomycin against the intracellular reservoir of methicillin-susceptible (MSSA) and -resistant (MRSA) Osteoblastic MG63 cells were infected for 2h with MSSA strain or its isogenic MRSA. After killing the remaining extracellular bacteria with lysostaphin, infected cells were then incubated for 24h with DAP, oxacillin (OXA) or ceftaroline (CPT) alone or in combination, at the intraosseous concentrations reached with standard human therapeutic doses. Intracellular bacteria were then quantified by plating cell lysates. Minimum inhibitory concentrations (MICs) of these molecules alone and in combination were determined using the checkerboard method at pH7, but also at pH5 to mimic intracellular conditions.Aim
Method
