Background Intra‐articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re‐evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. Objectives To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. Study design In vitro experimental study. Methods Chondrocytes and synovial cells were harvested from three horses and plated on 24‐well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25‐0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. Results Antibiotics from different antimicrobial classes expressed dose‐dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. Main limitations Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. Conclusions Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence‐based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.

Septic arthritis causes significant morbidity and mortality in veterinary and human clinical practice and is increasingly complicated by multidrug-resistant infections. Intra-articular (IA) antibiotic administration achieves high local drug concentrations but is considered off-label usage, and appropriate doses have not been defined. Using an equine joint model, we investigated the effects of amikacin injected at three different doses (500, 125, and 31.25 mg) on the immune and cartilage responses in tibiotarsal joints. Synovial fluid (SF) was sampled at multiple time points over 24 h, the cell counts determined, and amikacin concentrations measured by liquid chromatography-mass spectrometry. Cytokine concentrations and collagen degradation products in SF were measured by ELISA and multiplex immunoassays. The mean amikacin concentrations in SF were greater than or equal to the minimum inhibitory concentration (MIC) (0.004 mg/ml) for most common equine joint pathogens at all time points tested to 24 h for all three amikacin doses evaluated. The inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) increased significantly in SF in the highest amikacin dose group, despite the fact that increases in SF cell counts were not observed. Similarly, the biomarkers of cartilage type II collagen cleavage (C2C and C12C) were increased in SF following amikacin injection. Mechanistically, we further demonstrated using in vitro studies that chondrocytes and synoviocytes killed by exposure to amikacin underwent apoptotic cell death and were phagocytosed by macrophages in a non-inflammatory process resembling efferocytosis. Neutrophils and T cells were susceptible to amikacin cytotoxicity at clinically relevant doses, which may result in blunting of cellular inflammatory responses in SF and account for the lack of increase in total nucleated cell counts following amikacin injection. In summary, decisions on whether to inject cytotoxic antibiotics such as aminoglycosides intra-articularly and what doses to use should take into account the potential harm that antibiotics may cause and consider lower doses than those previously reported in equine practice.

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