Application of next-generation sequencing to investigate a Serratia marcescens outbreak in a neonatal intensive care unit

Introduction: Nosocomial infections in Neonatal Intensive Care Units (NICU) are a major concern due to the vulnerability of premature and immunocompromised infants. Serratia marcescens is an opportunistic pathogen often involved in these infections, contributing significantly to morbidity and mortality. Integrating Next-Generation Sequencing (NGS) into infection control programs can enhance detection, surveillance, and prevention efforts. This study aimed to develop a mapping-based pipeline for strain typing and phylogenetic analysis of nosocomial infections, enabling detailed comparison of microbial genomes. Methods: A retrospective study describing the outbreak was conducted on 18 S. marcescens strains from 14 patients and 2 from environmental swabs collected in the NICU of the University Hospital of Udine between 2023 and 2024. Genomic DNA was extracted and libraries were prepared using the FX DNA Library Preparation Kit (Qiagen). Whole Genome Sequencing (WGS) was performed using an Illumina MiSeq platform (2 × 300 bp paired-end). Data analysis was carried out with CLC Genomic Workbench (Qiagen), using a custom-optimized pipeline for sequence typing (ST). The bioinformatics workflow was developed and validated in-house to ensure accurate SNP-based phylogenetic analysis. Results: WGS revealed phylogenetic relationships among strains. Six isolates showed close genetic relatedness. Identical genotypes were detected in strains from patient blood samples, rectal swabs, and environmental sources, suggesting potential transmission links. Conclusions: NGS offers detailed insights into the molecular epidemiology of infections and colonization in the NICU. The genomic data generated can support real-time, evidence-based refinement of infection control strategies, contributing to improved patient safety and outbreak prevention.