Five glucose concentrations were tested (Fig. 3). The biofilm cultures showed an increased sensitivity to ampicillin when the initial glucose concentration was at least 1 g/L. The shift in kanamycin tolerance was observed between initial glucose concentrations

of 1 and 5 g/L. It should be noted that LB media contains trace concentrations of sugar but the quantities are not significant enough to support measurable growth in CBL0137 price respiration negative E. coli [20]. Figure 3 Effect of glucose concentration on antibiotic tolerance of wild-type E. coli K-12 biofilm cultures. Cultures were grown as biofilms for 6 hours before being transferred to antibiotic treatment plates for 24 hours. LB medium was supplemented with varying amounts of glucose indicated below each bar ranging from 0-10 g/L. Reported cfu/biofilm data was determined after treatment. Black bars = control, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. SIS3 research buy cfu = colony forming unit. The effect of glucose on antibiotic tolerance was expanded to test other common hexoses found in the human diet including the glucose isomer fructose, the more reduced sorbitol, and the more oxidized gluconate. All tested hexoses had effects analogous

to glucose and made the biofilm cultures more susceptible to ampicillin (Fig. 4). Experiments also examined media augmented with the carbohydrate selleck inhibitor glycerol or the organic acid succinic acid. The presence of glycerol produced an ampicillin tolerance response similar to the hexose grown cultures AMP deaminase and a kanamycin response similar to the LB only cultures. Cultures grown on succinic acid supplemented medium had antibiotic tolerances analogous to the LB only cultures. Figure 4 Effect of nutritional environment on antibiotic tolerance of wild-type E. coli biofilm cultures. Cells were grown as biofilms for 6 hours before being transferred to

treatment plates for 24 hours. All cultures were grown at 37°C in LB medium with or without an additional carbon source. All carbon source supplements were added at 10 g/L, the succinic acid solution was pH adjusted to 6.8 before being added to medium. Reported cfu/biofilm data was determined after treatment. Black bars = control, dark gray bars = kanamycin (100 ug/ml) challenge, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit. E. coli strains unable to utilize glucose were constructed by sequential deletion of the ptsG, ptsM, glk, and gcd genes using the KEIO gene knock-out library and P1 transduction methods (see materials and methods). The glucose negative cultures did not respond to glucose perturbations; antibiotic tolerance did not change significantly between the presence and absence of glucose (Fig. 5).