New approaches for molecular typing in Gram-positive pathogens

Antimicrobial resistance (AMR) represents one of the most severe global health threats of the 21st century. Vancomycin-resistant enterococci (VRE), primarily Enterococcus faecium and E. faecalis, are significant nosocomial pathogens that cause difficult-to-treat infections with high mortality rates in immunocompromised patients. Precise and rapid diagnostics are essential for infection control and targeted therapy. This dissertation addresses these challenges through the development and evaluation of innovative molecular methods for the detection and high-resolution typing of VRE. The first part of the study involved the design and optimization of a DNA microarray assay. Based on bioinformatic analyses using the ConsensusPrime pipeline, three generations of microarrays were developed, covering up to 327 target genes including resistance, virulence, and species-specific markers. In tests involving over 180 clinical isolates and 220 strains from Germany and Romania, the system demonstrated significantly higher discriminatory power than traditional Multilocus Sequence Typing (MLST). By combining microarray data with Next-Generation Sequencing (Illumina, Nanopore) and implementing a novel, image-based hexadecimal nomenclature system, regional differences in VRE populations were accurately identified. The second part of the work focused on rapid clinical diagnostics using a multiplex real-time PCR and recombinase polymerase amplification assays. These assays allow for the simultaneous identification of species and key resistance genes (vanA, vanB) directly from clinical colonies without extensive sample preparation. With a sensitivity of up to 100% and high cost-efficiency, the method offers significant potential for routine clinical diagnostics. In summary, this work provides critical advancements for the epidemiological monitoring and rapid identification of VRE. The developed tools contribute significantly to global strategies against the spread of AMR.