Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar

Background: Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar. Methods: MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution. Results: The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982-1.000), P