Isolates and sequence data included in the study.

Klebsiella pneumoniae is a leading cause of neonatal sepsis in low- and middle-income countries, with antimicrobial resistance (AMR) significantly contributing to mortality. We used whole genome sequencing to explore the impact of an infection prevention and control (IPC) intervention on K. pneumoniae strains and transmission dynamics responsible for sepsis in a Zambian neonatal unit. Blood culture isolates were collected during the Sepsis Prevention in Neonates in Zambia (SPINZ) study, including a 7-month baseline period and 12 months following implementation of a low-cost IPC bundle. K. pneumoniae genomes associated with 411 neonatal infections were characterised, comprising 24 unique sequence types (STs) and dominated by ST307 (69.3%, n = 285). Nearly all isolates (99.0%) carried extended spectrum beta-lactamases, but few carried carbapenemases (2.7%). Most infections (95.6%) were associated with probable transmission clusters, ranging in size from 2–202 patients and spanning durations of 2–232 days. Most K. pneumoniae (n = 228, 70%) were isolated during the 7-month baseline period and formed six clusters, including one cluster of >200 neonates infected with ST307. Transmission of all strains was periodically suppressed by an IPC bundle; however not all strains were eliminated, and some were able to re-emerge later to re-establish infection and transmission, alongside newly introduced strains that formed additional transmission clusters. Some clusters were associated with rapid onset of disease (within 2 days of admission) and others with delayed onset, suggesting different sources of contamination (e.g., reagent vs environmental). These findings reinforce the need for sustained IPC efforts, and better understanding of environmental reservoirs of opportunistic pathogens in neonatal units to inform such efforts.