Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

Abstract

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. To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. In vitro experimental study. Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100000 cells/wells in triplicate) for 48hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n=15) at various doses (25-0.39mg/mL in complete DMEM media) for 24hours 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 72hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. 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>25mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. 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.