Effect of Sonication on the Disruption of Gram-Negative Bacterial Biofilms Associated with Prosthetic Joint Infections (PJI) at Different Maturation Stages: An In Vitro Study

Background/Objectives: Periprosthetic joint infections (PJIs) remain one of the most challenging complications after arthroplasties due to the ability of pathogens to form biofilms on implant surfaces. Although staphylococci predominate, Gram-negative bacilli, have increasingly been associated with more aggressive clinical courses and diagnostic failure. This study aimed to evaluate the structural characteristics and maturation of E. coli and P. aeruginosa biofilms and to assess the effectiveness of a standardized sonication protocol in disrupting these biofilms and releasing viable cells. Methods: Biofilms of E. coli (ATCC 25922) and P. aeruginosa (ATCC 53278) were grown on polyethylene catheter segments for 24, 48, and 72 hours. Morphological and structural features were assessed by scanning electron microscopy. A standardized sonication protocol was then applied to evaluate its ability to disrupt the extracellular polymeric matrix. Viability of released cells was confirmed by culturing aliquots of the sonication fluid on BHI agar. Biofilms were produced in triplicate for each time point. Results: Both species formed increasingly dense and structured biofilms over time. Mature biofilms exhibited markedly thicker EPS layers compared to 24-h biofilms. P. aeruginosa developed highly complex, multilayered matrices, while E. coli produced characteristic but less elaborate biofilm structures. Sonication consistently disrupted immature and mature biofilms of both organisms, fragmenting the matrix and releasing individual or small clusters of bacterial cells. Cultures from the sonication fluid demonstrated that bacterial cells remained viable following the procedure. Conclusions: The standardized sonication protocol effectively disrupted Gram-negative biofilms at different maturation stages and released viable microorganisms, reinforcing its value as a complementary diagnostic tool for PJIs, especially in chronic or low-grade infections where conventional culture methods show reduced sensitivity.