471 - Co-twin Comparison of Gut Microbiota Using Nanopore 16S rRNA Sequencing: Association Between Early Postnatal Environment and Microbial Composition

Publication Number: 2459.471

Teahyen Cha, Gangnam Severance Hospital, Seongdong-gu, Seoul-t'ukpyolsi, Republic of Korea; Jihyun Keum, Hanyang university, Seoul, Seoul-t'ukpyolsi, Republic of Korea; Min-Jin Kwak, Kookmin University, Seoul, Seoul-t'ukpyolsi, Republic of Korea; Gulam Mustafa, Hanyang University, Seoul, Seoul-t'ukpyolsi, Republic of Korea; Jeong Kyu Hoh, Hanyang University Medical School, Seoul, Seoul-t'ukpyolsi, Republic of Korea; Byong-Hun Jeon, Hanyang University, Gwangjin-gu District, Seoul-t'ukpyolsi, Republic of Korea; Hyun-Kyung Park, Hanyang University College of Medicine, Seoul, Korea, Seoul, Seoul-t'ukpyolsi, Republic of Korea

Background: The gut microbiota is influenced by both genetic and environmental factors. In neonates, particularly those in the neonatal intensive care unit (NICU), microbial assembly is highly dynamic and vulnerable to clinical interventions such as antibiotics, parenteral nutrition, and feeding delay. Using a co-twin control design allows direct assessment of how subtle, short-term clinical exposures shape early gut colonization under shared genetic and environmental conditions.

Objective: To determine the within-pair impact of short-term clinical exposures on early gut microbiota composition in cesarean-delivered dizygotic twins using a co-twin control design.

Design/Methods: This pilot study included dizygotic twin pairs delivered by cesarean section and admitted to the NICU at Hanyang University Medical Center (2022-2024). Only stool samples collected within the first month of life were analyzed. Each twin pair provided same-day samples to minimize temporal variability. A total of 76 samples underwent 16S rRNA gene sequencing using the Oxford Nanopore platform. Clinical variables during the week prior to sampling included NPO duration, antibiotic exposure (Day of Therapy; DOT), feeding advancement rate (mL/kg/day), and time to full enteral feeding. Microbial diversity and relative abundances were compared within twin pairs using paired statistical tests to minimize genetic and environmental confounding.

Results: A total of 23 dizygotic twin pairs (46 infants) were enrolled in the study. Clinical exposures were largely similar between twins, except for higher birth weight in the first baby (p = 0.014). Proteobacteria and Firmicutes dominated the gut microbiota, showing borderline trends toward higher Proteobacteria in the 2nd baby (p = 0.061) and higher Firmicutes in the 1st baby (p = 0.065). At the genus level, Raoultella and Enterococcus were enriched in the 1st baby, whereas Klebsiella and Staphylococcus were more abundant in the 2nd baby. Alpha diversity did not differ significantly within pairs, whereas beta diversity showed a trend toward compositional divergence without reaching statistical significance.

Conclusion(s): Despite minimal clinical differences, the 2nd baby consistently exhibited higher Proteobacteria abundance. This suggests that early gut colonization can diverge even under shared genetic and environmental conditions, reflecting the ecological sensitivity of the neonatal microbiome. Further co-twin studies integrating metabolomic analyses are warranted to clarify the functional impact of these compositional shifts.

Clinical characteristics and short-term exposures of dizygotic twin pairs
Except for birth weight, which was significantly higher in the first baby (p = 0.014), no significant differences were observed in gestational age, sex, delivery type, or early clinical exposures including antibiotic use (Day of Therapy; DOT), fasting duration (NPO), and feeding parameters.
Data are presented as mean ± SD or median (IQR) for continuous variables, and as n (%) for categorical variables. p-values were calculated using the paired t-test or Wilcoxon signed-rank test, as appropriate.

Comparison of gut microbiota composition between twin pairs
(A) Phylum-level relative abundance of gut microbiota in the 1st and 2nd babies.
(B) Genus-level relative abundance (Top 10 genera) in the 1st and 2nd babies.
Proteobacteria and Firmicutes were dominant across both groups, with a borderline trend toward higher Proteobacteria in the 2nd baby (p = 0.061) and higher Firmicutes in the 1st baby (p = 0.065). At the genus level, Raoultella and Enterococcus were enriched in the 1st baby, while Klebsiella and Staphylococcus were more abundant in the 2nd baby.
Bars represent mean relative abundance (%); comparisons were performed within pairs using paired statistical tests.