DOI: 10.6084/m9.figshare.31231733.v1
title: High-resolution molecular typing of vancomycin-resistant <i>Enterococcus faecium</i> from Romania and Bavaria: combining enhanced DNA microarray and next generation sequencing

description: Antimicrobial resistance poses a significant challenge for infection control, requiring the development of accurate and high-throughput diagnostic techniques. We expanded and optimized an existing DNA microarray platform for the molecular characterization of vancomycin-resistant <i>Enterococcus</i> (VRE) by incorporating resistance, virulence, species-specific, and typing markers. The enhanced microarray allows for the simultaneous analysis of up to 96 strains, providing detailed genetic profiles of clinical isolates. VRE strains from Romania and Bavaria, Germany, were analyzed, and the results were compared to those obtained using traditional typing methods, such as multilocus sequence typing (MLST). Next-generation sequencing (NGS) was used in parallel to validate the microarray findings and explore genomic relationships. The microarray revealed considerable genetic diversity and potential epidemiological linkages among isolates. A novel hexadecimal-based nomenclature system was introduced for standardized and scalable strain classification. Comparative analysis demonstrated that the array profiles provided greater discriminatory power and practical resolution than MLST. Receiver operating characteristic (ROC) curve analysis of 187 target genes in 220 isolates gave diagnostic sensitivity and specificity of 100%. This integrated approach offers a cost-effective, rapid, and adaptable global VRE surveillance and infection control tool. It provides a practical alternative to conventional typing systems and facilitates early detection of outbreaks and emerging clones. Enterococci isolates were cultured, and high-quality genomic DNA was extracted, biotin-labeled, and hybridized to a custom microarray for vancomycin-resistant enterococci (VRE) for simultaneous target detection. Sequencing was performed using Illumina and Nanopore platforms, followed by genome assembly, multilocus sequence typing (MLST), and phylogenetic analysis. Receiver operating characteristic (ROC) analysis assessed hybridization performance, and a standardized hexadecimal coding system was applied to array profiles for reproducible strain identification. The study expanded an existing DNA microarray for vancomycin-resistant enterococci (VRE) by incorporating additional typing, resistance, and virulence markers, enabling comprehensive molecular characterization of clinical VRE strains.The developed microarray allows simultaneous high-throughput genotyping of up to 96 samples, providing detailed hybridization profiles and enabling the identification of array clusters and strain types.Application of the microarray to VRE strains from Romania and Bavaria, Germany, revealed both genetic diversity and potential epidemiological linkages among clinical isolates.Receiver operating characteristic (ROC) curve analysis of 195 target genes demonstrated high diagnostic sensitivity and specificity, validating the microarray’s accuracy and reliability in detecting resistance and virulence determinants.A novel hexadecimal-based nomenclature system for array profiles was introduced, facilitating integration of new strains and improving strain identification, particularly as array profiles did not reliably match traditional multilocus sequence typing (MLST) for <i>E. faecium.</i>The microarray system can be adapted to include new resistance genes, integrated with other diagnostic platforms, and expanded globally to improve epidemiological surveillance and infection control.Combining microarray analysis with next-generation sequencing (NGS) provided complementary insights into the diversity and resistance mechanisms of VRE.Overall, the study presents a reliable, efficient, and comprehensive microarray-based platform for molecular characterization of VRE, supporting infection control and epidemiological research. The study expanded an existing DNA microarray for vancomycin-resistant enterococci (VRE) by incorporating additional typing, resistance, and virulence markers, enabling comprehensive molecular characterization of clinical VRE strains. The developed microarray allows simultaneous high-throughput genotyping of up to 96 samples, providing detailed hybridization profiles and enabling the identification of array clusters and strain types. Application of the microarray to VRE strains from Romania and Bavaria, Germany, revealed both genetic diversity and potential epidemiological linkages among clinical isolates. Receiver operating characteristic (ROC) curve analysis of 195 target genes demonstrated high diagnostic sensitivity and specificity, validating the microarray’s accuracy and reliability in detecting resistance and virulence determinants. A novel hexadecimal-based nomenclature system for array profiles was introduced, facilitating integration of new strains and improving strain identification, particularly as array profiles did not reliably match traditional multilocus sequence typing (MLST) for <i>E. faecium.</i> The microarray system can be adapted to include new resistance genes, integrated with other diagnostic platforms, and expanded globally to improve epidemiological surveillance and infection control. Combining microarray analysis with next-generation sequencing (NGS) provided complementary insights into the diversity and resistance mechanisms of VRE. Overall, the study presents a reliable, efficient, and comprehensive microarray-based platform for molecular characterization of VRE, supporting infection control and epidemiological research.