Chronic Staphylococcus aureus infection of osteocytes promotes an intracellular viable-but-non-culturable phenotype

Introductory paragraphInfectious osteomyelitis is a potentially severe disease with an increased incidence of 10% over the last 10 years (1, 2) and a 20% recurrence rate creating chronic infections (3). Diagnosis and pathogen identification remain challenging, with culture-negative results in 7-39% of cases (4-6), of which 14-27% are PCR-positive from drained fluids (7). Of recent intensive research focus, osteomyelitis associated with Prosthetic Joint Infection (PJI) is a serious and growing complication of orthopaedic joint replacement surgery, with a failure to cure rate within 2-years internationally of between 15-46% (8, 9). Osteocytes act as potential long-term reservoirs for intracellular Staphylococcus aureus, protecting it against the immune responses and antimicrobial treatment (10-15). However, the intra-osteocytic adaptation mechanisms by S. aureus remain understudied, and it is unknown why so many infections are culture-negative but PCR-positive. Here, we found that in chronic intra-osteocytic infections, S. aureus consistently adopts a viable but non-culturable (VBNC) state. Whole genome sequencing of parental and a revertant strain indicated that initially, S. aureus cells possessing TarP and a complete T7SS dominate the bacterial community but these cell numbers declined with genetic rearrangements seemingly linked with the shift to a VBNC state. The subsequent acquisition of a single nucleotide polymorphism (SNP) in srrB was associated with resuscitation to a culturable state, suggesting at least one mechanism underlying entrance and exit from the intracellular VBNC state.