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

Abstract

Case 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 intracellular S. aureus in human osteocytes remains largely unknown. Rifampicin, vancomycin, levofloxacin, ofloxacin, amoxicillin, oxacillin, doxycycline, linezolid, gentamicin and tigecycline were assessed for their MIC and minimum bacterial 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. Five antibiotics reached clinically relevant concentrations above their respective MIC and were tested in osteocyte infection models. Osteocytes were treated at 1, 4 and 10x the MIC for 1 and 7 days immediately following infection (acute model), or after 14 days of infection (chronic model). The effectivity of each antibiotic on intracellular bacteria was measured in terms of colony forming unit (CFU) reduction, small colony variant (SCV) formation and specific bacterial mRNA expression change. Only 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. This indicates that S. aureus adapts phenotypically during intracellular infections of osteocyte-like cells, adopting a reversible quiescent state and is protected against antibiotics even at 10x MIC. Thus, new therapeutic approaches are necessary to cure S. aureus intracellular infections in osteocytes.