Objective: Biofilm microorganisms are known to have a much higher tolerance to antimicrobials compared to their planktonic equivalents. Therefore, traditional antimicrobial susceptibility testing may not extrapolate to clinical treatment of infections of biofilm origin, and as a result, there is a need to not only develop antimicrobials with antibiofilm activity, but also suitable in vitro testing methods for their evaluation. In this study, we report on a novel method of antibiofilm testing using a thermo-reversible matrix (poloxamer 407), coupled with live/dead staining of bacteria cultured from the matrix.
Method: Pseudomonas aeruginosa (NCIMB 8626) was cultured in medium containing poloxamer 407 at 37°C for 24 hours to generate biofilms. The preparation was cooled to liquefy the poloxamer and allow recovery of the biofilm cells, which were then stained with SYTO9 to determine viability following exposure to four antimicrobials: polyhexanide, octenadine dihydrochloride, povidone-iodine and silver carbonate. Over an 8-minute time period, fluorescence levels were spectrophotometrically measured and compared with bacterial controls, cultured in the absence of poloxamer and without antimicrobial.
Results: Untreated cells showed no reduction in viability over this period. Importantly, planktonic cells were more susceptible to test agents compared with those of a 'biofilm' phenotype cultured in poloxamer. Antibiofilm activity was evident for all of the test agents, with highest relative activity seen with octenadine dihydrochloride.
Conclusion: In summary, a novel and relatively rapid approach to screen compounds for antibiofilm activity has been described. The method uses standard laboratory equipment and can be readily adapted to test a wide range of microorganisms and other antibiofilm compounds.
Declaration of interest: This research was, in part, supported by Advanced Medical Solutions in the form of a Knowledge Transfer Project. Mr J. Nosworthy was employed by Advanced Medical Solutions. There are no other conflicts of interests to declare.
Keywords: Pseudomonas aeruginosa; SYTO9; antibiofilm agents; biofilm modelling; poloxamer.