Exploring the Antibiotic Effects in Bacterial Biofilms by Epifluorescence and Scanning Electron Microscopy

Biofilm bacteria are more resistant to antibiotics than their free-living counterparts. For development of effective antimicrobial therapies, it is crucial to understand the interactions between antibiotics and biofilm cells. This work evaluates the killing and removal effects of ciprofloxacin in Escherichia coli biofilms using a combination of epifluorescence and scanning electron microscopy (SEM). When ciprofloxacin was applied to 24 h-old biofilms, two distinct phases were observed. In phase 1 (corresponding to the initial 3 h after application), only 4 % of sessile cells were killed and no removal from the surface was observed. Phase 2 comprises two stages, in the first (between 3 and 4.5 h of antibiotic exposure) about 70 % of the bacterial cells were removed but 60 % of the remaining cells were still alive. In the second stage, it was possible to attain complete cell inactivation (after 7.5 h) although 10 % of the initial cells remained attached to the surface. SEM micrographs show that antibiotic-treated cells have filamentous forms when compared to untreated cells