Staphylococcus aureus adopts revertible intracellular viable-but-non-culturable phenotypes in chronic osteocyte infections

Abstract

Osteomyelitis associated with periprosthetic joint infection (PJI) is a serious and growing complication of orthopaedic joint replacement surgery, with a failure to cure rate within 2-years of between 15-46%. PJI is a severe chronic infectious disease with a 20% recurrence rate and culture-negativity in 7-39% of cases, of which 14-27% are PCR-positive, findings that remain poorly explained. Osteocytes, the most numerous bone cell type, have been identified as potential long-term intracellular reservoirs for Staphylococcus aureus, shielding it from antimicrobial treatment. Here we demonstrate that in chronic intra-osteocytic infections, S. aureus consistently adopts a viable-but-non-culturable (VBNC) state, capable of spontaneous host cell escape and reversion to active growth. Whole-genome sequencing identified single nucleotide polymorphisms (SNPs) in gene pairs between clones. Two revertant clones exhibited SNPs in srrB and murA2, and five exhibited SNPs in garR and upstream of tatB. While the functional importance of these changes is uncertain, they suggest that diverse genetic mechanisms underly entrance into and exit from the intracellular VBNC state. These findings provide a compelling explanation for culture-negative, PCR-positive osteomyelitis and have direct implications for the diagnosis and treatment of PJI.