Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: Implications for the microbiological diagnosis of implant associated infection

PLoS One. 2020 Apr 8;15(4):e0231389. doi: 10.1371/journal.pone.0231389. eCollection 2020.


The diagnosis of implant-associated infections is hampered due to microbial adherence and biofilm formation on the implant surface. Sonication of explanted devices was shown to improve the microbiological diagnosis by physical removal of biofilms. Recently, chemical agents have been investigated for biofilm dislodgement such as the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the activity of chemical methods for biofilm dislodgement to sonication in an established in vitro model of artificial biofilm. Biofilm-producing laboratory strains of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 53278) were used. After 3 days of biofilm formation, porous glass beads were exposed to control (0.9% NaCl), sonication or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting, isothermal microcalorimetry and scanning electron microscopy. Recovered colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% NaCl for biofilms of S. epidermidis (6.3 and 6.1 vs. 6.0 log10 CFU/mL, S. aureus (6.4 and 6.3 vs. 6.3 log10 CFU/mL), E. coli (5.2 and 5.1 vs. 5.1 log10 CFU/mL and P. aeruginosa (5.1 and 5.2 vs. 5.0 log10 CFU/mL, respectively). In contrast, with sonication higher CFU counts were detected with all tested microorganisms (7.5, 7.3, 6.2 and 6.5 log10 CFU/mL, respectively) (p <0.05). Concordant results were observed with isothermal microcalorimetry and scanning electron microscopy. In conclusion, sonication is superior to both tested chemical methods (EDTA and DTT) for dislodgement of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilms. Future studies may evaluate potential additive effect of chemical dislodgement to sonication.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / drug effects
  • Bacteria / isolation & purification*
  • Bacterial Load / methods
  • Biofilms / drug effects*
  • Calorimetry
  • Chelating Agents / pharmacology*
  • Dithiothreitol / pharmacology
  • Edetic Acid / pharmacology
  • Escherichia coli / isolation & purification
  • Escherichia coli / physiology
  • Humans
  • Microscopy, Electron, Scanning
  • Prosthesis-Related Infections / diagnosis*
  • Prosthesis-Related Infections / microbiology
  • Pseudomonas aeruginosa / isolation & purification
  • Pseudomonas aeruginosa / physiology
  • Reducing Agents / chemistry
  • Reducing Agents / pharmacology*
  • Sodium Chloride / pharmacology
  • Sonication*
  • Staphylococcus aureus / isolation & purification
  • Staphylococcus aureus / physiology
  • Staphylococcus epidermidis / isolation & purification
  • Staphylococcus epidermidis / physiology


  • Chelating Agents
  • Reducing Agents
  • Sodium Chloride
  • Edetic Acid
  • Dithiothreitol

Grants and funding

This work was funded by PRO-IMPLANT Foundation (, a non-profit organization supporting research, education, global networking and care of patients with bone, joint or implant-associated infection. The funding had no influence on the data analysis or interpretation of the results.