Background Klebsiella pneumoniae (Kpn) is an important cause of neonatal sepsis in sub-Saharan Africa. Kpn are intrinsically resistant to penicillins and frequently resistant to gentamicin and 3rd-generation cephalosporins, key agents in the treatment of neonatal sepsis. Such infections can be prevented by effective infection prevention and control (IPC), but the most effective IPC packages for resource-limited healthcare settings are incompletely defined. Methods We recruited a cohort of 94 neonates admitted to a Malawian neonatal unit, alongside their mothers, and utilised single colony whole genome sequencing and post-enrichment metagenomics to determine the most important transmission routes of Extended-Spectrum Beta-Lactamase producing (ESBL) Kpn. These data were analysed at the level of ST and pairwise SNP distance and combined with statistical models to infer transmission. Findings ESBL-Kpn rapidly colonised neonates; female sex, receipt of oxygen, caesarean delivery and antibiotic use increased colonisation risk. STs causing invasive infection and stool colonisation were temporally related to those found in the ward. There was greatest circulation of ESBL-Kpn between compartments that are in contact with neonatal stool (neonate stool, mothers' hands, cots, and the swaddling cloths). Utilising epidemiological and SNP data, Kpn appeared to be transmitted to neonates primarily from cots, ward surfaces (sinks and oxygen delivery equipment) and from other neonates. Network analysis implicated cots, antibiotics and oxygen delivery in ESBL-Kpn transmission. Interpretation An unsafe hospital environment is strongly implicated in neonatal invasive infection and stool colonisation with ESBL Kpn. IPC interventions should focus on containing neonatal stool, hand hygiene, cot decontamination, single use oxygen delivery and surface cleaning, particularly sinks.

The authors have declared no competing interest.

This work was funded by the Antimicrobial Resistance Cross-Council Initiative through grants from the Medical Research Council, a Council of UK Research and Innovation and the National Institute for Health Research (MR/R015074/1 & MR/S004793/1); and the Bill and Melinda Gates Foundation (INV-005692). Malawi-Liverpool-Wellcome Research Programme (MLW) is core-funded by Wellcome (206545/Z/17/Z).

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College of Medicine Research Ethics Committee of Kamuzu University of Health Sciences gave ethical approval for this work (COMREC P.10/18/2499) Liverpool School of Tropical Medicine Research Ethics Committee of Liverpool School of Tropical Medicine gave ethical approval for this work (19-018)

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