Minimum biofilm eradication concentration.

MBEC was determined by a modification of the broth microdilution assay. Biofilm was formed by incubating bacterial suspensions overnight at 35°C, ambient air, in wells of a microtiter plate using tryptic soy broth containing 1% glucose as growth medium. After removal of the growth media and rinsing the biofilms formed on the bottom of the microtiter wells with sterile phosphate-buffered saline (PBS), antibiotics were added in MH-F broth in 2-fold serial dilutions (30). Following overnight incubation and PBS wash, fresh broth without antibiotics was added. The biofilms were disintegrated by sonication, and the resulting suspensions were transferred to a fresh microtiter plate and incubated overnight. The MBEC was determined as the lowest concentration of antimicrobial agent resulting in no growth evident by visual assessment.