Artificial Pseudomonas aeruginosa biofilms and confocal laser scanning microscopic analysis

Bacterial biofilms may be formed at various sites, including mucous membranes, teeth, and infectious lesions. To elucidate the structure and the function of biofilms, artificial biofilms of mucoid-type Pseudomonas aeruginosa organisms (strain PT1252) were made by centrifuging the organisms onto the surface of a coverglass and culturing further in broth media supplied continuously (45 ml/h). The biofilm structure at 4, 8, 12, and 24 h was visualized with fluorescent staining (SYTO9, propidium iodide [PI], and/or fluorescein isothiocyanate-concanavalin A [FITC-ConA]) by confocal laser scanning microscopy (CLSM). It was clearly demonstrated that the number of bacteria (10(4)--10(6)/ml) could be estimated by their fluorescence intensity. Sectional analysis of each biofilm layer (1-microm thickness) made it possible to demonstrate the three-dimensional development of biofilms, and revealed that the biofilms were 9 microm in height after 12 h. The live and dead organisms were differentiated by SYTO9 and PI, respectively, in situ in biofilms, and about 13% of the organisms were dead in 12-h-old biofilms. When 12-h-old biofilms were exposed to ciprofloxacin at minimum bactericidal concentration (6.26 microg/ml) for 90 min, all the organisms were killed, but some organisms (11 +/- 1.3%; n = 3) in 24-h-old biofilms with thicker and denser structure were still alive after exposure for 120 min. These results indicate that the CLSM analysis of artificial biofilms was useful for elucidating bacterial functions in biofilms, and may lead to a new quantitative system for estimating the bactericidal efficacy of antibacterial drugs in biofilms.