Preterm infants harbour diverse populations, including atypical species that encode and produce an array of antimicrobial resistance- and virulence-associated factors.

spp. are frequently enriched in the gut microbiota of preterm neonates, and overgrowth is associated with necrotizing enterocolitis (NEC), nosocomial infections and late-onset sepsis. Little is known about the genomic and phenotypic characteristics of preterm-associated , as previous studies have focused on the recovery of antimicrobial-resistant isolates or culture-independent molecular analyses. The aim of this study was to better characterize preterm-associated populations using phenotypic and genotypic approaches. Faecal samples from a UK cohort of healthy and sick preterm neonates (=109) were screened on MacConkey agar to isolate lactose-positive . Whole-genome sequences were generated for spp., and virulence and antimicrobial resistance genes identified. Antibiotic susceptibility profiling and macrophage and iron assays were undertaken for the strains. Metapangenome analyses with a manually curated genome dataset were undertaken to examine the diversity of and related bacteria in a publicly available shotgun metagenome dataset. Approximately one-tenth of faecal samples harboured spp. (, 7.3 %; , 0.9 %; , 2.8 %; , 1.8 %). Isolates recovered from NEC- and sepsis-affected infants and those showing no signs of clinical infection (i.e. 'healthy') encoded multiple β-lactamases. No difference was observed between isolates recovered from healthy and sick infants with respect to siderophore production (all encoded enterobactin in their genomes). All , , and faecal isolates tested were able to reside and persist in macrophages, indicating their immune evasion abilities. Metapangenome analyses of published metagenomic data confirmed our findings regarding the presence of in the preterm gut. There is little difference in the phenotypic and genomic characteristics of isolates recovered from healthy and sick infants. Identification of β-lactamases in all isolates may prove problematic when defining treatment regimens for NEC or sepsis, and suggests that healthy preterm infants contribute to the resistome. Refined analyses with curated sequence databases are required when studying closely related species present in metagenomic data.