“REAL-TIME” QUANTITATIVE MONITORING OF STAPHYLOCOCCAL BIOFILM FORMATION ON BONE GRAFTS AND BONE SUBSTITUTES. A COMPARISON BETWEEN MICROCALORIMETRY AND A STANDARD METHOD

Abstract

Background: Bacteria form biofilms on the surface of orthopaedic devices, causing persistent infections. Monitoring biofilm formation on bone grafts and bone substitutes is challenging due to heterogeneous surface characteristics. We analyzed various bone grafts and bone substitutes regarding their propensity for in-vitro biofilm formation caused by S. aureus and S. epidermidis.

Methods: Beta-tricalciumphosphate (β-TCP, Chro-nOsTM), processed human spongiosa (TutoplastTM) and PMMA (EndobonTM) were investigated. PE was added as a growth control. As test strains S. aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) were used. Test materials were incubated with defined bacterial solution (105 colony-forming units (cfu)/ml) at 37°C for 24 h without shaking. After 24 h, the test materials were removed and washed 3 times in PBS, followed by a standardised sonication protocol (Trampuz et al. 2007, NEJM). The resulting sonication fluid was plated in aliquots of 100μl onto aerobe blood agar with 5% sheep blood and incubated at 37°C with 5% CO2 for 24 h. Bacterial counts were enumerated and expressed as cfu/ml. Sonicated samples were transferred to a microcalorimeter (TA Instrument) and heat flow at 37°C was continuously monitored over a 24h period with a precision of 0.0001°C and a sensitiviy of 200μW. All experiments were performed in triplicates to calculate the mean ± standard deviation. ANOVA analysis was used for statistical calculations.

Results: For S. aureus bacterial counts (log10 cfu/ sample) were significantly higher (p< 0.001) for the porous (β-TCP 7.67 ± 0.17, Tutoplast 7.65 ± 0.15) than for the solid samples (PMMA 6.12 ± 0.18, PE 5.17 ± 0.22). Bacterial density (log10 cfu/surface) was 10^1–10^2 times higher for the S. epidermidis than for the S. aureus. In calorimetry the shape of the heat flow curves was characteristic for the individual strain and was not influenced by the test materials. The time to detection (TTD) was shortest for β-TCP for both strains and TTD was always shorter for S. aureus than S. epidermidis with corresponding material. Cfu/sample calculated from the calorimetric data was concordant with the standard culturing method.

Conclusion: Our results demonstrate biofilm formation with both strains on all tested materials. The calorimetry in all cases was able to detect quantitatively the amount of biofilm. Further studies are needed to see whether calorimetry is a suitable tool also to monitor approaches to prevent and treat infections associated with bone grafts and bone substitutes.