ESTABLISHMENT OF A CHRONIC ORTHOPAEDIC INFECTION MODEL WITH MATURE BIOFILM FORMATION USING AN IN VITRO-IN VIVO HYBRID APPROACH

Introduction Chronic biomaterial-associated infections (BAI), particularly those involving biofilm formation, represent a significant clinical challenge. Despite intensive experimental research, few in vivo models with chronic biofilm formation are available, mainly using vertebrates. These models are often costly and subject to strict animal welfare regulations. Most simple non-vertebrates in vivo models, such as Galleria mellonella , has rarely been investigated to study chronic infections due to their limited lifetime. Aim The general aim of this study was to establish a chronic orthopaedic infection model with mature biofilm formation using an in vitro-in vivo approach with the CDC biofilm reactor (CBR) and G. mellonella . Methods Three orthopaedic-related S. aureus strains were selected to form biofilms on the surface of Kirschner wires (K-wires) over an incubation period of up to 7 days using either a static (microplate model) or a dynamic flow system (CBR; BioSurface Technologies). After biofilm maturation periods of 1 and 7 days, these implants were washed and then used: (i) to characterize the biofilms (quantitative cultures, SEM, 3D-CLSM, and gene expression of biofilm markers), (ii) to assess in vitro rifampicin susceptibility (EUCAST), and (iii) to be introduced into G. mellonella larvae for an additional 5 days. Rifampicin larval administration (80 µg/g) was assessed as a surrogate marker for biofilm maturation using Kaplan-Meier analysis. Results The data demonstrated that dynamic flow maturation in the CBR for 7 days with subsequent implantation into G. mellonella larvae led to mature biofilm formation with drastically decreased rifampicin effectiveness. Furthermore, unlike biofilms formed under static conditions, dynamic flow conditions significantly increased biofilm formation after 7 days in vitro . This approach demonstrated an increase of 2-4 log 10 CFU on K-wires and thicker biofilms, partly due to enhanced secretion of extracellular matrix components, before introduction into the larvae. Conclusions Overall, we established an in vitro - in vivo hybrid method to mimic chronic orthopaedic implant infection with mature biofilm formation. The use of a high shear flow device to initiate strong biofilm formation and maturation on orthopaedic device surfaces prior to introduction into larvae proved essential. This high-throughput and cost-effective method could contribute to assessing new treatment strategies targeting chronic biofilm infections.