Summary Statement. Conventional culture techniques have poor sensitivity for detecting bacteria growing in biofilms, which can result in under-diagnosis of infections. Sonication of biofilm colonised orthopaedic biomaterials can render bacteria in biofilm more culturable, thereby improving diagnosis of orthopaedic implant infections. Introduction. Prosthetic joint infection (PJI) is a potentially devastating complication in arthroplasty. Biofilm formation is central to PJI offering protection to the contained bacteria against host defence system and antimicrobials. Orthopaedic biomaterials generally have a proclivity to biofilm colonisation. Conventional culture technique has a low sensitivity for detecting bacteria in biofilm. Sonication can disrupt bacteria biofilms aggregations and dislodge them from colonised surfaces, rendering them culturable and consequently improve the diagnosis of otherwise culture-negative PJI. We investigated the effect of ultrasonication on biofilms adherent to poylmethylmethacrylate PMMA cement. Method. Identical PMMA cement beads were aseptically prepared using 7mm bead templates. Each sample comprised of two beads and with multiple replicates made for each sample. Two proficient biofilm forming strains of Staphylococcus epidermidis (5179-R1 and 1457) were used for the experiments. Each set of cement sample was immersed in Brain Heart Infusion broth inoculated with a pre-culture of the chosen bacteria strains (final concentration approximately 4 × 10. 6. CFU/ml). All samples were then incubated for 24 hours at 37°C to allow for biofilm growth and colonisation of the cement surfaces, as well as for biofilm maturity. After incubation, each sample was washed twice with sterile phosphate buffer saline (PBS) to remove non-adherent and loosely adherent bacteria. The cement beads were transferred to a fresh sterile bottle at each stage of the experiment, while ensuring the maintenance of asepsis. After the final wash, 10ml of sterile PBS was added to the cement beads and each sample was sonicated for varying periods: 0min, 5min, 10min, 20min and 40min. Sonicate fluid were collected after each period of sonication, with which culture plates were inoculated for the purpose of viable bacteria counting. Results. The optimum sonication period was between 5min and10 min. The mean pre-sonication CFU/ml were 4.7 × 10. 5. and 8.3 × 10. 5. for bacteria strains 5179-R1 and 1457 respectively, while the mean CFU/ml after 10min of sonication were 1.4 × 10. 7. and 0.74 × 10. 7. for bacteria strains the respective bacteria strains. Discussion / Conclusion. Our study showed a significant increase (almost 100 fold) in bacteria culture yield following sonication. We were also able to demonstrate that the optimum duration for sonication (using comparable sonicators) was approximately 10min. Sonication was able to completely remove adherent bacteria from the surfaces of our cement samples allowing them to be cultured. Our result suggests that sonication of bone cement can be instrumental in improving the diagnosis of biofilm associated PJI

We have designed a prospective study to evaluate the usefulness of prolonged incubation of cultures from sonicated orthopaedic implants. During the study period 124 implants from 113 patients were processed (22 osteosynthetic implants, 46 hip prostheses, 54 knee prostheses, and two shoulder prostheses). Of these, 70 patients had clinical infection; 32 had received antibiotics at least seven days before removal of the implant. A total of 54 patients had sonicated samples that produced positive cultures (including four patients without infection). All of them were positive in the first seven days of incubation. No differences were found regarding previous antibiotic treatment when analysing colony counts or days of incubation in the case of a positive result. In our experience, extending incubation of the samples to 14 days does not add more positive results for sonicated orthopaedic implants (hip and knee prosthesis and osteosynthesis implants) compared with a conventional seven-day incubation period.

Cite this article: Bone Joint J 2013;95-B:1001–6.