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We hypothesized that integrating laboratory surveillance for neonatal colonization and infection, real-time epidemiologic investigations, and whole-genome sequencing (WGS) could identify transmission pathways to guide targeted infection prevention and control (IPC) strategies. We conducted\n                    <jats:italic>Kpn<\/jats:italic>\n                    surveillance in a 36-bed neonatal unit in Botswana over 12 months (2022\u20132023). WGS was performed on\n                    <jats:italic>Kpn<\/jats:italic>\n                    isolates from bloodstream infections (BSIs), and MDR-\n                    <jats:italic>Kpn<\/jats:italic>\n                    isolates collected from environmental sampling during outbreaks and twice-monthly colonization screenings (skin and perirectal swabs) using culture media selective for MDR-\n                    <jats:italic>Kpn<\/jats:italic>\n                    (CHROMagar Extended-spectrum beta-lactamase [ESBL]\/SuperCarba). WGS data were analyzed using multilocus sequence typing (MLST), pangenome and reference-based single-nucleotide polymorphism (SNP) analyses, and Bayesian phylogenetics. We identified 55\n                    <jats:italic>Kpn<\/jats:italic>\n                    BSIs during the 12-month surveillance period and the median prevalence of MDR-\n                    <jats:italic>Kpn<\/jats:italic>\n                    colonization was 28%.\n                    <jats:italic>Kpn<\/jats:italic>\n                    was recovered from multi-use intravenous (IV) fluid bags during a\n                    <jats:italic>Kpn<\/jats:italic>\n                    outbreak (41 BSIs, 10 deaths), which was controlled by implementing a 24-hour discard policy for IV medications\n                    <jats:italic>.<\/jats:italic>\n                    Among 270\n                    <jats:italic>Kpn<\/jats:italic>\n                    isolates available (28 BSI, 232 colonizing, 10 environmental [six IV fluid, four sink drain]), WGS confirmed over half of BSI genomes (n\u2009=\u200917) and all six IV fluid isolates belonged to ST1414 and were closely related (&lt;25 SNPs). The ST1414 clone was susceptible to third-generation cephalosporins and was therefore not detected during MDR-\n                    <jats:italic>Kpn<\/jats:italic>\n                    colonization screening. This study reinforces the value of integrating WGS with real-time epidemiologic investigations to understand transmission dynamics and guide IPC. Colonization surveillance focused solely on MDR-\n                    <jats:italic>Kpn<\/jats:italic>\n                    may overlook drug-susceptible but outbreak-prone strains.\n                  <\/jats:p>","DOI":"10.1371\/journal.pgph.0006468","type":"journal-article","created":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T17:23:27Z","timestamp":1781544207000},"page":"e0006468","update-policy":"https:\/\/doi.org\/10.1371\/journal.pgph.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Integrated epidemiologic investigation and genomic confirmation of a Klebsiella pneumoniae neonatal sepsis outbreak in 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