| Summary: | <i>Acinetobacter baumannii</i> (<i>A.</i> <i>baumannii</i>) is a major cause of severe nosocomial infections worldwide. The emergence of infections associated with <i>A.</i> <i>baumannii</i> poses a significant health risk in Germany. <i>A. baumannii</i> is part of the ACB complex and is difficult to distinguish from other species phenotypically, necessitating its reliable identification. The current study analyzed 89 <i>A. baumannii</i> strains from human and non-human origins by matrix-assisted laser desorption/ionization (MALDI–TOF) and PCR detection of intrinsic <i>bla</i><sub>OXA-51-like</sub> carbapenemase, <i>bla</i><sub>OXA-23-like</sub>, <i>bla</i><sub>OXA-24-like</sub>, <i>bla</i><sub>OXA-58-like</sub>, and IS<i>Aba</i> 1 genes. Whole-genome sequencing (WGS) was applied for species confirmation and strain type determination. Combining the molecular detection of the intrinsic <i>bla</i><sub>OXA-51-like</sub> carbapenemase gene together with MALDI–TOF with a score value of >2.300 proved to be a suitable tool for <i>A. baumannii</i> identification. WGS data for all of the sequenced strains confirmed the identity of all <i>A. baumannii</i> strains. The Pasteur scheme successfully assigned 79.7% of the strains into distinct STs, while the Oxford scheme succeeded in allocating only 42.7% of isolates. Multilocus sequence typing (MLST) analysis based on the Pasteur scheme identified 16 STs. ST/241 was the most prevalent in samples from non-human origin, whereas ST/2 was predominant in human samples. Furthermore, eight isolates of non-human origin were allocated to seven new STs (ST/1410, ST/1414, ST/1416, ST/1417, ST/1418, ST/1419, and ST/1421). Ten isolates from non-human origin could not be typed since new alleles were observed in the loci Pas_<i>cpn</i>60, Pas_<i>rpo</i>B, and Pas_<i>glt</i>A. MLST analysis based on the Pasteur scheme was more appropriate than the Oxford scheme for the current group of <i>A. baumannii.</i>
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