DITHIOTHREITOL AND SONICATION TO IMPROVE SENSITIVITY OF SYNOVIAL FLUID CULTURE: AN IN VITRO STUDY

Introduction & Aim Synovial fluid culture plays a pivotal role for diagnosis of prosthetic joint infections. However, its sensitivity is often low, resulting in failure to grow the pathogen. The presence of biofilm-like aggregates in synovial fluids, as recently shown, could significantly affect the yield of culture. Therefore we hypothesized that sensitivity of synovial fluid culture could benefit from treatments favouring bacterial release from biofilm-like aggregate. At this purpose, we compared the efficacy of sonication and Dithiothreitol in improving bacterial recovery from an in vitro model of infected synovial fluid. Methods Synovial fluids collected from patients with suspected prosthetic joint infection were pooled and U.V sterilized. Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa previously isolated from PJI were used to inoculate pooled synovial fluid. After incubation at 37°C for 72 hours to allow formation of biofilm like aggregates, each sample was divided in 3 aliquots that were treated with 1. Sonication for 5 min at 30 kHz in a water bath at 37°C; 2. Dithiothreitol 0.1% for 15 min under horizontal shaking; 3. Vortexing for 1 min (control treatment). After serial dilutions, 100 µL were seeded onto agar plates for colony counts. Analysis with confocal laser microscopy after dying with Alive Dead staining was performed to assess for the presence of aggregates and the effects of each treatment. Results Treatments of synovial fluids with sonication and dithiothreitol allowed recovering of higher bacterial counts in comparison with vortexing. Statistically significant differences were observed when dithiothreitol treatment was compared with the control one (mean counts 8.35 vs 7.30 log CFU/mL, respectively), while no differences were observed between sonication and control treatment (8.05 vs 7.30 log CFU/mL, respectively) or sonication and dithiothreitol treatment. Confocal laser microscopy confirmed the ability of the tested strains to aggregate in synovial fluids and the disaggregating activity of sonication and dithiothreitol. Conclusions Use of disaggregating strategies for treatment of biofilm may significantly improve bacterial recovery from synovial fluids, thus implementing diagnostic sensitivity of prosthetic joint infections. Since this study was performed on few strains, further large studies on clinical samples are needed to confirm the present data.