Quantitative determination of endotoxins released by bacterial biofilms

Residual endotoxins, commonly associated with bacterial biofilms colonizing reusable medical devices have been associated with pyrogenic reactions in patients. We have used a quantitative, sensitive and reproducible kinetic c hromogenic adaptation of the Limulus Amebocyte LS-sate assay to assess endo toxin recovery from an in-vitro bacterial biofilm. The 'recovery method' wa s based on a combination of physical treatment (vortexing and sonication) a nd chemical treatment (immersion in recovery solution). Five recovery solut ions were investigated. The recovered endotoxin was greater when the biofil m was treated with a 1% SDS solution. The sensitive and reproducible method we have developed should allow the recovery and measurement of biofilm bac terial endotoxins on implanted and colonized medical devices. Moreover, the amount of endotoxin was sufficient (> 1000 endotoxin units/cm(2) of substr ate) to enable a substantial reduction by sterilization processes, the effi ciency of which on biofilm endotoxins has yet to be proven. (C) 1999 The Ho spital Infection Society.