SPECIAL ISSUE ARTICLE An in vitro biofilm model of Staphylococcus aureus infection of bone E. Sweeney 1 , A.M. Lovering 2 , K.E. Bowker 2 , A.P. MacGowan 2 and S.M. Nelson 3 1 School of Life Sciences, University of Warwick, Coventry, UK 2 Bristol Centre for Antimicrobial Research & Evaluation, North Bristol NHS Trust, Department of Infection Sciences, Southmead Hospital, Westbury-on-Trym, Bristol, UK 3 Department of Applied Sciences, University of the West of England, Bristol, UK Significance and Impact of the Study: The majority of studies of antibiotic efficacy in the treatment of chronic osteomyelitis are carried out in animals. We developed an in vitro model of Staphylococcus aur- eus infection of bone to evaluate the ability of antibiotics to eradicate mature biofilms on surfaces anal- ogous to necrotic bone. The results demonstrated the difficulties which occur in osteomyelitis treatment, with only very high concentrations of antibiotic able to penetrate the bone sufficiently to reduce bacterial survival whilst still failing to eradicate biofilms. This model could be of use in initial screening of novel compounds intended for use in the treatment of osteomyelitis. Keywords biofilm, daptomycin, gentamicin, in vitro model, osteomyelitis, Staphylococcus aureus. Correspondence Shona Nelson, Department of Applied Sciences, University of the West of England, Bristol, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK. E-mail: shona.nelson@uwe.ac.uk 2018/2136: received 9 November 2018, revised 15 January 2019 and accepted 7 February 2019 doi:10.1111/lam.13131 Abstract Chronic osteomyelitis is difficult to treat, with biofilm growth and the diffusion barrier to antibiotics presented by bone contributory factors. The aim of this study was to develop and evaluate an in vitro model of osteomyelitis. A bioluminescent strain of Staphylococcus aureus was grown in bone blocks made from bovine femur. Light output was insufficient for detection of bacterial cells within bone by 24 h and viable counting of crushed bone blocks was used to determine bacterial survival. Challenge of 72 h biofilms with gentamicin and daptomycin for 24 h demonstrated that only concentrations of 10 times the clinical peak serum target levels (100 mg l À1 gentamicin and 1000 mg l À1 daptomycin) resulted in significant reductions in cell viability compared to controls. Once daily dosing over 7 days resulted in ≥3 log reductions in cell numbers by 48 h. Thereafter no significant reduction was achieved, although emergence of resistance was suppressed. Determination of antibiotic concentration in bone blocks over 7 days indicated that neither agent was able to consistently reach levels in bone of >10% of the original dose. The model was, therefore, able to demonstrate the challenges posed by biofilm growth on and within bone. Introduction Osteomyelitis, microbial infection of cortical and cancel- lous bone, can occur in any bone in the human body (Beck-Broichsitter et al. 2015). It may be acquired exoge- nously via direct inoculation of micro-organisms through trauma or surgery, or endogenously via haematogenous spread of micro-organisms (Walter et al. 2012) or con- tiguous spread from, for example diabetic foot ulcers (Malhotra et al. 2014). With population ageing and concomitant increases in orthopaedic intervention and prosthesis implantation, as well as an increasing diabetic population (WHO 2016), the incidence of osteomyelitis is likely to increase. Osteomyelitis is inherently difficult to diagnose and treat. In its chronic form, bone necrosis and microbial biofilm formation limit delivery of antibiotics to, and within, the infection site (Cook et al. 2015). Relapse of infection is common and surgical debridement of the infected site (Tuchscherr et al. 2016) and pro- longed antimicrobial chemotherapy add to the already Letters in Applied Microbiology 68, 294--302 © 2019 The Society for Applied Microbiology 294 Letters in Applied Microbiology ISSN 0266-8254