Gunn, N. J.; Zelmer, A. R.; Kidd, S. P.; Solomon, L. B.; Yang, D.; Roscioli, E.; Atkins, G. J.

Background: Staphylococcus aureus is a major causative pathogen of osteomyelitis. Intracellular infection of osteocytes and other bone cell types have been reported, with the bacteria able to survive and persist for an extended period, despite application of gold-standard clinical interventions. The mechanisms, by which intracellular S. aureus persists through antibiotic therapy are unknown. In this study we investigated if antibiotic-mediated dysregulation of autophagy is a contributing factor. Methods: Human osteocyte-like cells were exposed to combinations of rifampicin, vancomycin and modulators of autophagy, in the presence or absence of S. aureus. Intracellular bacterial growth characteristics were assessed through CFU analysis, viable DNA abundance and the rate of escape into antibiotic-free medium, in parallel with measures of host cell autophagic flux. Results: Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of the intracellular bacterial community concomitant with increased absolute bacterial DNA levels. Low dose vancomycin alone increased both measures but this was reversed at higher doses. Both antibiotics significantly inhibited autophagic flux. Whilst the modulation of autophagic flux affected bacterial culturability, this did not affect viable bacterial DNA levels. Conclusions: Autophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth phenotype of intracellular S. aureus. Whilst rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this was not explanative of the paradoxical response of S. aureus to antibiotic treatment. As such, whilst rifampicin and vancomycin had off-target effects on autophagy, similar to those of S. aureus, these were not causally linked to the antibiotic-mediated pressure towards persistent infection.