Research

Background: It is generally known that E. coli form biofilms clinically; however the optimal conditions for biofilm formation in vitro are debated, specifically, the role of nutrient content. In nutrient rich media, biofilm forms at the air-liquid interface, whereas minimal media forms more consistent biofilm throughout wells.
 
Methods: We evaluated biofilm positive (BF+) control (MG1655), a biofilm negative (BF-) strain and 3 clinical strains of E. coli. Biofilm formation assays were performed using modified Naves et al. 2008/2010 methods. M9 minimal media supplemented with 0.8% glucose and 0.5% casamino acids (sM9) and Mueller Hinton Broth (MHB) were tested. Strains were grown overnight in Luria-Bertani broth with 5g/L glucose at 37°C. Overnight cultures were diluted 1:100 in media, and aliquots of 130uL were placed in wells of non-treated polystyrene microtiter plates (Costar 3370). All plates were incubated at 30°C without shaking. Biofilm was determined after 24h, 48h and/or 48h with replenishment. Replenished plates were incubated 24h, drained of media, washed once with sterile water (sH2O), replaced with fresh media, and incubated an additional 24h. To measure biofilm formation, all wells were washed once with sH2O and dried 20 min; adherent bacteria were stained with 0.1% crystal violet for 20 min. Excess dye was removed by rinsing four times with sH2O then dried for 1h. Adherent stain was re-solubilized using 95% ethanol. Biofilm adherence was determined by reading optical density (OD) at 570nm, media control wells were subtracted from all results. Each strain was tested under each condition in eight replicates four times. Average OD was compared by t test.
Related People: Kerry L. LaPlante
1 University of Rhode Island, College of Pharmacy, Kingston, RI
2 Providence Veterans Affairs Medical Center, Providence, RI
3 Warren Alpert Medical School of Brown University, Providence, RI