Tests for Biofilm Susceptibility

TESTS  FOR  BIOFILM  SUSCEPTIBILITY   

Biofilms present is an additional problem to antimicrobial testing, as the biofilm may be resistant to more than 1000 times the MIC concentration of antimicrobial. Antimicrobial testing of biofilms under standardized conditions has really only become available since 1999 and the development of the MBEC Assay (Calgary Biofilm Device). Previous techniques for biofilm susceptibility testing suffered from a lack of replicates, as in the case of flow cell technology, or the need for continuous pumping of fluids and bacteria that presented a leakage and contamination risk that was not tolerable in a diagnostic laboratory. Forming biofilms directly in 96-well plates provided replicate numbers, but in this assay system the initial inoculum could not be calculated and the efficacy of treatment was based not on viable cell counts but on a dye absorbance assay that could be measuring a change in extracellular matrix rather than a change in viable bacterial cell number. The MBEC assay placed pegs protruding from the lid of the plate into each well of a 96-well plate. Shear force created by gyration of the plate initiated bacterial adhesion to the peg and biofilm formation, the density of which could be determined by sonication of the biofilm back into a suspension culture and enumeration of viable cell number by standard plate counts. The peg-borne biofilms could then be used as a biofilm inoculation in a standard 96-well MIC assay, only in this case the susceptibility of a biofilm rather than a planktonic population would be determined. Following antimicrobial exposure bacteria would again be sonicated from the pegs and counted to determine the biofilm MIC (BMIC), biofilm bactericidal concentration (BMBC) and biofilm eradication concentration (MBEC) in a highly standardized and reproducible assay based on existing MIC technology. Mycobacteria and fungi can be assayed using a similar format allowing the biofilm susceptibility of these organisms to be tested.

a)   Synergy Biofilm Assays

The reduced susceptibility of biofilms to antimicrobials often results in the effective in vitro drug concentration to far exceed a safe or achievable dose. Combinations of drugs or drugs and other cofactors are proving to be more effective against biofilms than single drug therapies. Synergies in biofilm testing have been defined on formulas based on the American Society for Microbiology standards. The calculation of synergy as defined in Harrison et al. (2008) where the mathematical definition of synergy was based on the sum of the fraction bactericidal concentration (FBC) value or FBC index for each combination of antimicrobial agents. Therefore in a two-component assay of agents A + B synergy would be defined as follows: