Genomic Epidemiology of Multi-Modal ESBL Gene Transmission among Enterobacterales in a Neonatal Unit

Antimicrobial resistance genes (ARGs) can spread via horizontal transfer or clonal expansion. We investigated the genomic epidemiology of extended-spectrum beta-lactamase (ESBL)–producing Klebsiella pneumoniae and Escherichia coli in a neonatal unit. Between January and November 2023, 53 ESBL isolates were obtained from 23 neonates via routine screening and clinical sampling. Long-read nanopore sequencing identified bla _CTX-M-15 as the dominant ESBL gene, alongside bla _CTX-M-65 , and bla _CTX-M-27 . Among 49 bla _CTX-M-15 isolates, 33 carried the gene on plasmids and in the remainder it was located on the chromosome. ESBL isolates belonged to one of seven MLST sequence types; E. coli isolates were dominated by ST131/ST131-like/ST5640 lineages, while K. pneumoniae were predominantly ST13. Plasmids (ESBL and non-ESBL-associated) clustered into 18 communities, five of which contained plasmid bearing bla _CTX-M genes. The largest cluster comprised IncFIB(K) plasmids from K. pneumoniae ST13, although, these were predicted as “non-mobilizable” by MOB-suite, and belonged to isolates from a clonally disseminated strain. No evidence of bla _CTX-M dissemination via shared plasmids was identified. Meanwhile, chromosomal phylogenetic analysis identified four distinct clonal clusters with ≤7 SNP differences (n=10, 5, 5, and 2 patients). In this setting, genomic analysis supported clonal dissemination of several bla _CTX-M -associated strains as the main outbreak mechanism, affecting 20/23 neonates, rather than plasmid-mediated transmission.