Abstract: ObjectiveTo evaluate the reliability of Vitek 2 AST-N335 card for determining the susceptibility of Acinetobacter bauman-nii（AB) to cefoperazone/sulbactam. MethodsA total of 318 non-repeated clinical isolates of AB collected in 2023were tested for antimicrobial susceptibility to cefoperazone/sulbactam using broth microdilution（BMD)，the AST-N335 card, and the Kirby-Bauer (K-B) disk diffusion method. Using BMD as the reference method, the reliability of AST-N335 card was assessed, and the accuracy of K-B disk diffusion method as the confirmatory test was validated. ResultsCompared with BMD,the susceptibility testing of 318 AB strains to cefoperazone/sulbactam using the AST-N335 card showed categorical agrement（CA) of 87.8% (279/318),very major error（VME)of 6.0%(19/318)，major error(ME)of 0%(O/318)，and minor error（mE)of 1.9%(6/318)，which fall outside of the acceptable error range. In contrast,the K-B method achieved CA of 99.4%（316/318),VME of 0%,ME of 0.3%(1/318)，and mE of O.3% (1/318)，all within acceptable limits.Of these,the errors with AST-N335 card occurred within the minimum inhibitory concentration （MIC) range of 8-32 pg/mL. Using BMD as the reference method, further analysis was performed on the 171 AB strains with AST-N335 card MIC values of 8-32 pg/mL for cefoperazone/sulbactam. It was revealed that at MIC of 32pg/mL,the CA was 0%；at MIC of 16 pg/mL,CA was5.3% (1/19)and VME rate was 84.2% (16/19)，both of which substantially exceeded acceptable error ranges. At MIC of 8 pg/mL,the CA was 94.9% (131/138)and VME was 2.2% (3/138),both approaching the acceptable ranges. ConclusionThe results obtained with Vitek 2 AST-N335 card in determining for cefoperazone/sulbactam are unreliable when the MIC values fall within the range of 8-32pg/mL,which leads to an underestimation of the resistance rate to cefoperazone/sulbac-tam.This issue requiresurgent attention inboth laboratoriesand clinical practice.The K-B diskdiffusion method could serve asasup- plementary verification approach in routine laboratories.