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Abstract Background Multidrug-resistant (MDR) Acinetobacter baumannii in hospitals contribute to high mortality rates and entail expensive infection control procedures. Conventionally, species and resistance determination of MDR bacteria is performed by mass spectrometry and labor-intensive phenotypical assays in clinical microbiology laboratories. Some institutions also apply time-consuming and expensive whole genome sequencing for in-depth resistance and transmission analysis. In contrast, faster and more cost-efficient MDR typing can be achieved by on-site application of real-time PCR-based point-of-care tests (POCTs) that target species- and resistance-specific genes and enable rapid screening of large sample sizes. Methods To construct a real-time PCR-based POCT system for species and resistance determination of MDR A. baumannii, we selected two species-specific genes (rpoB, OXA-51-like) and 18 resistance genes found in MDR A. baumannii (OXA-23-like, OXA-24/40-like, OXA-51-like, OXA-58-like, OXA-143-like, mcr, ADC, NDM, GIM, VIM, IMP, GES, PER, TEM, SHV, VEB, CTX-M, KPC) as targets. For each gene, one to six sets of primers and fluorophore-labeled probes that amplify conserved genetic regions were designed and their functionality was tested in vitro. Parallel, a fully automated system comprising microfluidic chips and a bench-top analyzer able to perform DNA extraction and real-time PCR starting from swab eluate was developed. Results All oligonucleotides tested so far reliably detected 100 to 100,000 copies of DNA extracted from MDR bacteria in single- and multiplex experiments in vitro. Tests with the microfluidic system showed that lysis of different bacterial species could be accomplished via sonication on a membrane located on the microfluidic chip. In addition, chip implementation of all PCR reagents like lyophilized master mix as well as primers and probes was successful and can be stored on chip at least one year. So far, the system delivered detectable fluorescence signals within one hour when performing test runs with bacteria eluted from swabs. Conclusions A real-time PCR-based Point-of-Care screening platform was developed for the detection of antibiotic-resistant A. baumannii from various patient sample materials starting directly from swabs, which detects colonization/infection and at the same time the relevant resistance genes within about an hour.