BACKGROUND:The worldwide emergence and clonal spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is of great concern. The aim of this nationwide study was to investigate the prevalence of CRAB isolates in Serbia and to characterize underlying resistance mechanisms and their genetic relatedness. METHODS:Non-redundant clinical samples obtained from hospitalized patients throughout Serbia were included in the prospective, observational, multicenter study conducted from January to June 2018. Samples were initially screened for the presence of Acinetobacter baumannii-calcoaceticus (Acb) complex using conventional bacteriological techniques. Acb complexes recovered from clinical samples obtained from inpatients with confirmed bacterial infections were further evaluated for the presence of A. baumannii. Identification to the species level was done by the detection of the blaOXA-51 gene and rpoB gene sequence analysis. Susceptibility testing was done by disk diffusion and broth microdilution method. CRAB isolates were tested for the presence of acquired carbapenemases (blaOXA-24-like, blaOXA-23-like,blaOXA-58-like, blaOXA-143-like, blaIMP, blaVIM, blaGIM, blaSPM, blaSIM, blaNDM) by PCR. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS:Acb complex was isolated in 280 out of 2401 clinical samples (11.6%). Overall, A. baumannii was identified in 237 out of 280 Acb complex (84.6%). CRAB prevalence was found to be 93.7% (237/222). The MIC50/MIC90 for imipenem and meropenem were 8/> 32 μg/mL and 16/> 32 μg/mL, respectively. Although susceptibility was high for colistin (95.7%; n = 227) and tigecycline (75.1%; n = 178), ten isolates (4.3%) were classified as pandrug-resistant. The following carbapenemases-encoding genes were found: 98 (44.2%) blaOXA-24-like, 76 (34.5%) blaOXA-23-like, and 7 (3.2%) blaNDM-1. PFGE analysis revealed six different clusters. MLST analysis identified three STs: ST2 (n = 13), ST492 (n = 14), and ST636 (n = 10). Obtained results evaluated that circulating CRAB clones in Serbia were as follows: blaOXA66/blaOXA23/ST2 (32.4%), blaOXA66/blaOXA23/blaOXA72/ST2 (2.7%), blaOXA66/blaOXA72/ST492 (37.8%), and blaOXA66/blaOXA72/ST636 (27.1%). CONCLUSION:This study revealed extremely high proportions of carbapenem resistance among A. baumannii clinical isolates due to the emergence of blaOXA-72, blaOXA-23, and blaNDM-1 genes among CRAB isolates in Serbia and their clonal propagation.