Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which sources of transmission can be challenging to elucidate. We describe the use of whole genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Methods: Ceftriaxone-resistant Klebsiella spp. and Escherichia coli isolated from neonates (or their mothers) and the

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... al environment were included. Short-read (Illumina) and long-read (MinION, Oxford Nanopore Technologies) sequencing was used to confirm species taxonomy, define antimicrobial resistance genes and determine phylogenetic relationships using single nucleotide polymorphism (SNP) profiling. Results: A total of 27 organisms (16 patient-derived and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from baby bath drains and multiple detergent dispensing bottles were either identical or closely related by SNP comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk-expressing equipment. No new cases were identified once the detergent bottles were removed and the baby baths decommissioned. The investigation further revealed unrecognized environmental reservoirs of ST131 ESBL-producing E. coli that were closely related to neonatal strains (diverging by <30 SNPs). Conclusions: Environmental reservoirs may be an important source in outbreaks of multi-drug resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.