Antibiotics at Clinical Concentrations Show Limited Effectivity Against Acute and Chronic Intracellular S. aureus Infections in Osteocytes

Abstract

ObjectivesCase numbers of osteomyelitis are rising and chronic infections remain difficult to cure. While it is known that the major pathogen Staphylococcus aureus can persist intracellularly in osteocytes, the effectivity of antibiotics against this condition remains largely unknown. We sought to determine if current clinically utilised antibiotics were capable of clearing an intracellular osteocyte S. aureus infection. MethodsRifampicin, vancomycin, levofloxacin, ofloxacin, amoxicillin, oxacillin, doxycycline, linezolid, gentamicin and tigecycline were assessed for their MIC and minimum bactericidal concentrations (MBC) against 11 S. aureus clinical isolates and the reference strain ATCC 25923, at pH 5.0 and 7.2 to mimic lysosomal and cytoplasmic environments, respectively. Those antibiotics whose bone achievable concentration was commonly above their respective MICs for the strains tested were further assayed in a human osteocyte infection model under either acute or chronic conditions. Osteocyte-like cells were treated at 1, 4 and 10x the MIC for 1 and 7 days following infection (acute model), or after 14 days of infection (chronic model). The intracellular effectivity of each antibiotic was measured in terms of colony forming unit (CFU) reduction, small colony variant (SCV) formation and bacterial mRNA expression change. ResultsOnly rifampicin, levofloxacin and linezolid reduced intracellular CFU numbers significantly in the acute model. The effect was larger after 7 days compared to 1 day of treatment. However, no treatment reduced the quantity of bacterial mRNA, nor prevented non-culturable bacteria from returning to a culturable state. DiscussionThese findings indicate that S. aureus adapts phenotypically during intracellular infection of osteocytes, adopting a reversible quiescent state which is protected against antibiotics, even at 10x their MIC. Thus, new therapeutic approaches are necessary to cure S. aureus intracellular infections in osteocytes.