FIGURE 1.

Colistin MIC determination by the broth microdilution method in CAMHB for the clinical reference strain K279a. (A) The optical density at 550 nm (white diamond) and CFU/ml (black triangle) in each well of the microtiter plate was measured after incubation at 37°C. Values are means of four replicates and error bars indicate the standard deviation. Under the graph, the photograph of triplicate MIC determinations in a 96-well microdilution plate using the colorimetric indicator resazurin shows the purple/pink transition zone (boxed) between colistin concentrations of 32 and 2 mg/L. Colistin concentrations are in serial twofold dilutions starting at 256 mg/L. The colistin MIC, taken as the lowest concentration that inhibited 80% of growth as compared to the control well without antibiotic (dashed line in the graph), was 16 mg/L. (B) Effect of colistin concentration on the attachment of the cells to the surface of the microtiter wells and on cell survival in the formed biofilm. Total biofilm formation relative to bacterial growth in each well is shown as a bar graph (Y axis on the left) and the viability of the biofilm determined by resazurin staining as a line graph (Y axis on the right). Fluorescence units are expressed as relative to the biofilm formed in each well of the microtiter plate. For each experiment values are means of four replicates and error bars indicate the standard deviation. The significant differences between biofilm formation values were calculated using one-way ANOVA with Dunnett’s multiple comparison test. Statistically significant increases with respect to untreated control are indicated: ^∗∗P < 0.01 and ^∗∗∗P < 0.001.