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Updated: Aug 18, 2026
Microtiter Dish Biofilm Formation Assay
Published on: January 30, 2011
Pseudomonas aeruginosa biofilm formation and slime excretion on antibiotic-loaded bone cement
Daniëlle Neut1, Johannes G E Hendriks, Jim R van Horn
1Department of Biomedical Engineering, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Background:
Infection is an infrequent but serious complication of prosthetic joint surgery. These infections will usually not clear until the implant is removed and re-implantation has a high failure rate, especially when Pseudomonas aeruginosa is involved.
Material And Methods:
We examined Pseudomonas aeruginosa biofilm formation on plain and gentamicin-loaded bone cement with confocal scanning laser microscopy (CSLM). Two different stains were applied in order to visualize and quantify the distribution of bacterial cells and extracellular polymeric substances (slime) from the bone cement surface to the top of the biofilm. Staining with LIVE/DEAD viability stain differentiated between live and dead bacteria within the biofilm, and slime production was evaluated after staining with Calcofluor white.
Results:
CSLM showed that the biofilm was a nonuniform structure of variable thickness, with differences in local bacterial cell and slime densities. Incorporation of gentamicin in bone cement resulted in a 44% reduction in bacterial viability, while the slime density increased significantly. In addition, conventional plate counting showed the development of small-colony variants on gentamicin-loaded bone cement with a decreased sensitivity for gentamicin (MIC: 8 m/L), as compared with normal-sized colonies taken from plain and gentamicin-loaded bone cement (MIC: 3 m/L). The enhanced slime production on antibiotic-loaded bone cement, together with the formation of small-colony variants, resulted in decreased susceptibility to antibiotics--probably concomitant with the onset of persistent and relapsing infections.
Interpretation:
In the clinical situation, our findings help to explain the frequent re-implantation failure of joint replacements infected with P. aeruginosa when the procedure has been performed using antibiotic-loaded bone cement.
Insights
Pseudomonas aeruginosa prosthetic joint infections are difficult to treat. Gentamicin-loaded bone cement reduces bacterial viability but increases slime and promotes antibiotic-resistant small-colony variants, hindering treatment.
Area of Science:
- Orthopedic Surgery
- Infectious Diseases
- Biomaterials Science
Background:
- Prosthetic joint infections are a serious complication, particularly those caused by Pseudomonas aeruginosa.
- Re-implantation after infection has a high failure rate, especially with P. aeruginosa.
Purpose of the Study:
- To investigate Pseudomonas aeruginosa biofilm formation on plain and gentamicin-loaded bone cement.
- To analyze the impact of gentamicin on bacterial viability, slime production, and antibiotic susceptibility.
Main Methods:
- Confocal scanning laser microscopy (CSLM) was used to visualize biofilm structure.
- LIVE/DEAD viability stain and Calcofluor white were used to assess bacterial cells and extracellular polymeric substances (slime).
- Conventional plate counting determined bacterial sensitivity to gentamicin.
Main Results:
- Gentamicin-loaded bone cement reduced bacterial viability by 44% but significantly increased slime density.
- Small-colony variants with decreased gentamicin sensitivity (MIC: 8 µg/mL) developed on gentamicin-loaded cement.
- Enhanced slime production and small-colony variants led to decreased antibiotic susceptibility.
Conclusions:
- Increased slime and small-colony variants on gentamicin-loaded bone cement contribute to persistent and relapsing infections.
- Findings explain high re-implantation failure rates in P. aeruginosa-infected joint replacements using antibiotic-loaded cement.
