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Using an experimental in vitro culture system, we investigated the effect of clarithromycin on biofilm formation by a leucine-requiring Pseudomonas aeruginosa mutant strain (HU1). Biofilm formation on celldesks in a chemically defined medium was assessed by viable cell count as well as by measurement of glycocalyx production and scanning electron-microscopic observation. Cells proliferated exponentially until day 3 and remained stationary afterwards. The amount of glycocalyx, simultaneously semiquantified, showed a linear increase from day 1 to day 12. Scanning electron microscopy revealed firm biofilms on day 5. Three different concentrations of Clarithromycin (CAM) (minimum inhibitory concentration MIC 64 microg/ml) were added continuously at the early and late phases of biofilm formation, and the antibiofilm effect of CAM was evaluated by the changes in cell count and glycocalyx production. CAM was effective on biofilms at 100 microg/ml but neither at 1 nor at 10 microg/ml. It is suggested that glycocalyx production started following bacterial multiplication and continued even after the cells had entered the stationary phase to form mature biofilms. No antibiofilm effect of CAM was observed at sub-MIC., (Copyright 2000 S. Karger AG, Basel)

The growth rate of biofilm bacteria of a leucine-requiring mutant Pseudomonas aeruginosa HU1 was regulated by the leucine concentration in a chemically-defined medium. The semiquantitative measurement of glycocalyx and scanning electron microscopy revealed that the kinetics of HU1-biofilm formation were dependent on the incubation time and the leucine concentration in the medium. The effect of the growth rate of biofilm cells on their susceptibility to antimicrobial agents, three beta-lactams and four fluoroquinolones, was evaluated. beta-Lactams showed weak bactericidal activity to biofilm cells; the activity was greater in younger biofilm cells growing in high concentrations of leucine. Fluoroquinolones revealed strong bactericidal activity to biofilm bacteria regardless of the growth rate. The following is suggested: the bactericidal action of beta-lactams against biofilm cells is affected by the cell growth rate, while that of fluoroquinolones is considerably greater and independent on the growth rate.

In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method. Bacteria used were a leucine-requiring mucoid mutant. Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days. At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured. PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25%. Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM. This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.

The growth rate of biofilm cells of a leucine-requiring mutant Pseudomonas aeruginosa HU1 was regulated by the leucine concentration in the chemically defined medium, and the effect of the growth rate of biofilm cells on the antimicrobial activities of the antimicrobial agents piperacillin (PIPC), imipenem (IPM) and ofloxacin (OFLX) were evaluated. PIPC showed little effect on the biofilm bacteria regardless of the leucine concentration in the medium. IPM showed weak bactericidal activity to biofilm cells; activity was greater in younger biofilm cells growing in high concentrations of leucine. On the other hand, OFLX revealed strong bactericidal activity to biofilm bacteria regardless of the growth rate. Our data suggest that the bactericidal action of antimicrobial agents to biofilm bacteria is different from that to planktonic bacteria. The bioassay using mutants with regulated growth is useful for the evaluation of the efficacy of antimicrobial agents against biofilm bacteria.