Development and assessment of a high-throughput biofilm and biomass testing platform.

Abstract

To develop and evaluate a simple platform technology for developing static biofilms in a 96-well microtitre plate for various downstream applications. The technology allows monitoring of growth rate, biofilm formation and quantifying biofilm biomass by using crystal violet (CV) and safranin O (SO) staining over seven-day time periods for pathogens including clinical isolates most commonly associated with hard-to-treat wound infections. A total of 157 bacteria including Acinetobacter, Enterobacter, Klebsiella, Pseudomonas and Staphylococcus spp. were used in the study. Bacterial growth was measured at 600nm optical density (OD). Biofilm formation was monitored and assessed quantitatively with CV at 570nm and SO staining at 492nm for one-, two-, three- and seven-day incubation periods. Bacterial growth rate and static biofilm biomass in the 96-well plates varied for various strains tested. Both CV and SO staining showed similar results in the biomass, with SO assay displaying more reproducible data throughout the study. Most of the strains were metabolically active even at the seven-day incubation period. Microbial adherences of all bacterial strains on the plastic surface was assessed with CV staining: 28 Acinetobacter, 17 Staphylococcus, 12 Pseudomonas and four Enterobacter strains were strong biofilm producers. Moderate biofilm-producing strains included 27 Staphylococcus, 14 Acinetobacter, eight Pseudomonas and three Enterobacter. Weak biofilm-producing strains included: 33 Staphylococcus, six Enterobacter, two Pseudomonas and one Acinetobacter. Only one Pseudomonas aeruginosa strain did not develop biofilm. Our results demonstrate the feasibility of using 96-well microtitre plates as a high-throughput platform for quantitative measurement and assessment of biofilm development over time. Studying microbial adherence or biofilm biomass generated on various surfaces using a high-throughput system could provide valuable information for in vitro testing and developing therapeutics for biofilm infections. Employing the biofilm testing platform described in this study makes it possible to simultaneously develop different biofilms formed by specific pathogens, and study potential association between the quantity of bacterial biomass and strength of a biofilm formed by specific wound pathogens. In addition, the described testing approach could provide an optimal model for standardised and high-throughput screening of candidate antibiofilm therapeutics.