Whole-genome sequencing investigation of a neonatal intensive care unit outbreak involving colonization and infection Klebsiella grimontii ST566 and Klebsiella michiganensis ST35

The Klebsiella oxytoca complex (KoC) comprises opportunistic pathogens capable of causing outbreaks in neonatal intensive care units (NICUs). We investigated a NICU KoC outbreak involving colonized and infected preterm infant in the NICU of a Chinese tertiary hospital to characterize its genomic features and infer potential transmission routes. KoC isolates were recovered from rectal and oral swabs collected from infants in the NICU. All isolates underwent whole-genome sequencing, multilocus sequence typing, single-nucleotide polymorphism-based analysis, and phylogenetic reconstruction. Virulence-associated genes and antimicrobial resistance determinants were annotated, and putative transmission pathways were inferred by integrating genomic and epidemiological data. A total of 102 KoC isolates were obtained from 28 preterm infants with low or very low birth weight. Among them, 21 developed clinical signs consistent with infection; one had microbiologically confirmed bacteremia and two were diagnosed with necrotizing enterocolitis. The outbreak involved two highly clonal lineages: 86 Klebsiella grimontii isolates belonging to ST566 and 15 Klebsiella michiganensis isolates belonging to ST35. One additional K. michiganensis ST98 isolate was genetically distant from the ST35 cluster and was therefore not considered part of the main ST35 transmission lineage. Isolates within each major lineage were nearly identical, differing by 0–2 SNPs. Comparative analysis of publicly available genomes showed that ST566 and ST35 were uncommon or underrepresented among currently available KoC genomes. All outbreak isolates carried iron acquisition-associated genes, including yersiniabactin-related ybtS and fyuA and enterobactin-related entA and entB , as well as tilivalline/tilimycin-associated toxin genes. Spatiotemporal and bed-movement data suggested possible contact-mediated transmission and environmental contamination. Most isolates were susceptible to the tested antimicrobials, although a possible resistant subpopulation was observed in one patient. This NICU outbreak involved two genetically distinct KoC clones. Genomic and epidemiological evidence supported recent clonal spread, highlighting the importance of species-level identification, genomic surveillance, and strict infection prevention measures in vulnerable neonatal populations.