Abstract
Objective:
We evaluated and compared the longitudinal patterns of gut microbiome colonization in twin and singleton neonates over the first six months postpartum in a tertiary care setting in Pakistan, aiming to delineate differences in microbial diversity, community composition, and temporal dynamics.
Methods:
A prospective longitudinal cohort of 120 neonates (60 twins — including 30 twin pairs — and 60 singletons) was enrolled at the neonatal unit of a tertiary hospital in Karachi, Pakistan, between January 2023 and December 2024. Fecal samples were collected at birth (meconium), 1 week, 1 month, 3 months, and 6 months. Microbial profiling was conducted via 16S rRNA gene sequencing, and taxa were annotated against SILVA and Greengenes databases. Alpha and beta diversity metrics were computed, and differential abundance analyses were performed. Statistical significance was assessed using mixed-effects models controlling for delivery mode, feeding practices, antibiotic exposure, and gestational age.
Results:
Both twins and singletons exhibited increasing microbial diversity over time. Singleton neonates had significantly higher alpha diversity (Shannon index) at week 1 and month 1 compared to twin neonates (p < 0.05). Twin pairs demonstrated delayed colonization by Bifidobacterium and Bacteroides species, and higher relative abundance of Proteobacteria at early time points compared to singletons. Beta diversity analyses revealed distinct community trajectories between twin and singleton groups from birth to six months (PERMANOVA, p < 0.01).
Conclusion:
Gut microbiome colonization followed distinct trajectories in twin versus singleton neonates within the first six months postpartum. These differences were influenced by host factors such as delivery mode and early feeding practices. Understanding divergent colonization patterns may inform targeted microbiome-based interventions in neonatal care to mitigate later-life health disparities.