An Omics-Guided Investigation of a Hospital Outbreak Caused by bla_NDM-1-Producing Pseudocitrobacter faecalis

Carbapenemase-producing Enterobacterales (CPE) pose a major healthcare challenge. We report the first hospital outbreak of Pseudocitrobacter faecalis carrying bla_NDM-1 using an omics-based approach. Short- and long-read sequencing enabled genomic epidemiological investigation to track its spread, characterize its resistome, and analyze the genomic context of bla_NDM-1. Additionally, we developed and implemented a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method for rapid outbreak isolate typing using protein biomarkers. Our investigation identified 2 independent bla_NDM-1-producing clonal clusters of multidrug- and carbapenem-resistant P faecalis circulating for over 3 years, carrying bla_NDM-1 either chromosomally or on a plasmid. MALDI-TOF MS spectra analysis revealed candidate protein markers corresponding to genomic clusters, with 1 predicted biomarker applicable for rapid typing. Pseudocitrobacter faecalis is an emerging CPE taxon requiring hospital surveillance. Early whole genome sequencing unexpectedly revealed 2 intertwined clones with independent carbapenemase acquisition routes. Cluster-specific markers enabled rapid typing, serving as proof of concept for validating proteomics in future surveillance.