Pseudomonas aeruginosabiofilm formation and slime excretion on antibiotic-loaded bone cement

Abstract

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. 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. 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. 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.