Method of evaluating effects of antibiotics on bacterial biofilm

Antibiotics are generally not effective against organisms in exopolysaccharide biofilms. A simple method of studying the effect of antibiotics on bacteria in established biofilms is reported. Escherichia coli ATCC 25922 cells grown overnight at 37 degrees C on Mueller-Hinton agar were suspended in buffer and dispensed on 0.5-cm2 catheter disks. The disks were incubated for 1 h at 37 degrees C, washed, transferred to petri dishes containing 20 ml of broth, and incubated at 37 degrees C for 20 to 22 h, at which time thick biofilms were established. Disks were washed, placed in broth or broth containing antibiotic, and incubated at 37 degrees C for 4 h. The disks were removed, and viable counts were determined. This process was repeated at other selected time intervals (e.g., 8 and 24 h). Viable bacterial counts decreased from 10(3) to 10(4) CFU/cm2 in 24 h with 400 micrograms of amdinocillin or cefamandole per ml. A combination containing 400 micrograms of each antibiotic per ml decreased the viable counts to an undetectable level (less than 100 CFU/cm2) in 24 h. Other antibiotics and organisms were also examined in this system.