Monitoring intracellular replication dynamics unveils high proportion of non-replicating antibiotic-tolerant Staphylococcus aureus inside osteoblasts

Therapeutic failures and relapses are critical challenges in Staphylococcus aureus bone and joint infections. These issues may stem, in part, from the incomplete eradication of S. aureus residing within osteoblasts, the bone-forming cells, despite recommended antibiotic treatment. However, the mechanisms underlying intraosteoblastic S. aureus survival remain poorly understood. Here, we used automated real-time fluorescence microscopy at the single-host-cell level to monitor the intracellular replication dynamics of clinical S. aureus strains and their survivors of rifampicin treatment in MG-63 osteoblast cell line. S. aureus replication dynamics was heterogeneous both within and across strains, while survival to rifampicin treatment was uniformly characterized by a non-replicative phenotype. Surprisingly, rifampicin killed less than 0.3 log of intraosteoblastic S. aureus , and only during the early phase of infection. The majority of S. aureus that survived rifampicin treatment remained non-replicative intracellularly after rifampicin withdrawal, yet they retained the capacity to regrow on agar following release from host cells. This high proportion of non-replicative antibiotic-tolerant S. aureus inside osteoblasts may contribute to the high rates of therapeutic failures in bone and joint infections.