We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n ؍ 41) and Acinetobacter baumannii (n ؍ 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of <2 and >2 g/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.T he increasing occurrence of infections due to multidrug-resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae led to the revival of old and neglected antibiotics that may remain active, such as polymyxins (polymyxin B and colistin) (1, 2). Colistin is increasingly being used as a last-resort treatment option for infections caused by MDR organisms (2, 3), particularly carbapenem-resistant (CR) Gramnegative bacteria (4). However, during the last years, increasing colistin resistance emerged worldwide, especially among Klebsiella pneumoniae and A. baumannii isolates, further limiting treatment options (5-7). In Europe, the evolving colistin resistance is more pronounced in southern countries (notably Greece, Romania, and Italy) (8-10).Rapid and reliable colistin susceptibility testing (ST) is needed in routine clinical laboratories to allow appropriate therapeutic decision-making. Thus far, few studies have assessed the performance of colistin ST methods, displaying controversial results, and thus, the most accurate one is still challenging (11).Disk diffusion, commonly used in many clinical laboratories, yielded high error rates compared to MIC-based methods and is considered unreliable for the detection of colistin resistance (12)(13)(14). Among commercial methods, gradient diffusion strips are convenient tests for determining colistin MICs, but their performance is not well established. Some studies demonstrated very good correlations between the results of Etest (bioMérieux, Marcy l'Etoile, France) and broth microdilution (BMD) or agar dilution (AD) methods for colistin , while other reports questioned the reliability of Etest (18,19). Another gradient diffusion test, MIC test strip (MTS) (Liofilchem SRL, Italy), has not been evaluated for colistin ST to the best of our k...