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Pseudomonas aeruginosa was cultivated at low growth rates under iron-limiting conditions on acrylic tiles. Biofilm cells exhibited increased tobramycin resistance compared with that of planktonic cells, and in old biofilms were more resistant than were cells in young biofilms. However, on suspension of the biofilm bacteria, glycocalyx-mediated resistance was lost.

An in vitro chemostat system in which Pseudomonas aeruginosa can be cultivated at a slow growth rate and under iron limitation conditions was used to study the susceptibilities of sessile bacteria of mucoid and nonmucoid P. aeruginosa strains to tobramycin and piperacillin. Planktonic cells of both mucoid and nonmucoid P. aeruginosa strains were susceptible to tobramycin and piperacillin. None of the cells was found to be viable after 2 h of exposure to 200 ,ug of piperacillin plus 10 ,g of tobramycin per ml. Young sessile bacteria were slightly more resistant to piperacillin or tobramycin than the planktonic cells were. However, eradication of young sessile bacteria could be achieved with a combination of piperacillin and tobramycin. None of these young bio!ldm bacteria were found to be viable after a 2-h exposure to 200 ,g of piperacillin plus 10 ,ug of tobramycin per ml. Old sessile bacteria were very resistant to these antibiotics. Eradication of old sessile bacteria could not be achieved with either tobramycin (200 jg/ml) or piperacillin (200 ,ug/ml) alone.Combination of higher concentrations of tobramycin with piperacillin resulted in an enhancement of killing of the old sessile bacteria. Exposure of old sessile bacteria to 200 ,Ig of piperacillin plus 100 " of tobramycin per ml resulted in the reduction of the viable count to approximately 0.02%. The data suggest that the eradication of bioffilm-associated infections is best carried out as early as possible. Enhanced activities against the sessile bacteria were achieved when higher concentrations of aminoglycosides were combined with P-lactam antibiotics.

Mucoid Pseudomonas aeruginosa isolated from a patient with cystic fibrosis was cultivated at a slow growth rate (D = 0.05 h) under iron limitation in a chemostat. Biofilm was allowed to form on acrylic tiles. The kinetics of the biofilm formation was then investigated. The population of sessile bacteria reached 1.5 x 10(9) cells/cm2 on day 5 and remained relatively constant throughout the study (day 7). The population of planktonic cells in the chemostat reached 4 x 10(9) on day 1 and stayed fairly constant throughout. Planktonic cells were very sensitive to tobramycin. They were killed by exposure to 10 mg/l tobramycin within 2 h. Young biofilm cells of P. aeruginosa (day 2 of colonization) were found to be more resistant. Approximately 40% of the adherent cells remained viable after exposure to 10 micrograms tobramycin/ml for 5 h. An increase in the concentration of tobramycin to 20 mg/l resulted in an enhancement of the killing of young biofilm bacteria and approximately 1.5% of them remained viable after exposure to this concentration of antibiotic for 5 h. Old biofilm bacteria, examined at day 7, were the most resistant and 15% of the cells were found to be viable after they were exposed to 200 mg/l of tobramycin for 5 h. When either young or old biofilm cells of mucoid P. aeruginosa were scraped from the tiles to produce a planktonic cell suspension they were sensitive to 5 mg/l tobramycin.