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The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples ( n  = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM ( n  = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by  Enterobacteriaceae ,  Enterococcaceae , and  Staphylococcaceae . The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance ofEscherichia    sp.  preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducingEscherichia    sp.  and increasing other  Enterobacteriaceae .  This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics.

Development of the Preterm Gut Microbiome in Twins at Risk of Necrotising Enterocolitis and Sepsis