Comparison of gut microbiome in the first 6 weeks of postnatal age in NICU-admitted preterm neonates versus term neonates: A hospital-based one-year prospective cohort study.

Preterm birth is a significant contributor to neonatal illness and mortality worldwide. Premature infants have an immature gastrointestinal tract, with a gut epithelium that has diminished barrier function and increased permeability. This allows for translocation of bacteria from the gut to the bloodstream, leading to systemic inflammation or sepsis. Beyond the immediate neonatal period, being born prematurely is known to have lasting health effects that can persist into adulthood. Studies have shown that individuals born preterm face an increased risk of developing chronic conditions such as diabetes, hypertension, and obesity when compared to those born at full term.

 In premature infants, the combination of frequent antibiotic exposure and immature immune and gastrointestinal systems is believed to foster the growth of gut microbiota that are less diverse and more prone to harbor potentially harmful pathogens. This altered microbial environment, alongside prematurity, has been linked to heightened systemic inflammation, increasing the likelihood of serious complications such as sepsis and necrotizing enterocolitis (NEC).

 Notably, the microbial species absent in the intestines of preterm infants often include those typically found in healthy, full-term infants—species known to support immune development, metabolic regulation, and protection against chronic conditions like obesity and allergies. The underlying causes of these differences in microbial colonization remain unclear, but structural and functional immaturity of the gut in preterm infants may play a key role in promoting abnormal microbial patterns from birth.

Objective of the study

Primary objective :

Comparison of gut microbiome in first 6 weeks of postnatal age in preterm neonates admitted in NICU vs Term infants admitted in hospital.

Secondary objective :

1.     To study factors affecting the gut microbiome in preterm infants and term infants in first 6 weeks of life.

2.     To analyze the progression and alterations in gut dysbiosis among neonates from the time of birth through the first six weeks of life, in order to better understand early microbial colonization patterns and their potential implications for infant health

Data collection procedure:

The participants antenatal and perinatal history will be recorded in a structured proforma including gestational age, mode of delivery, antenatal risk factors, PROM, TORCH profile. Samples will be collected only from neonates,whose parents consent for this study.

Stool samples (S-1, S-2, S-3) at admission (S1) ,, at discharge (S2), will be collected from the neonates from nappies and stored in sterile containers at room temperature. The samples will be sent to the microbiology laboratory at KLE’s Dr. Prabhakar Kore Hospital within 4 h of collection.

At the time of the patient’s discharge from the hospital, we will provide a stool collection container along with a plastic zip-lock bag. The caregivers will be given detailed instructions on how to collect the stool sample at home during the 6th week (S3)following discharge. They will be taught proper hygiene practices and the correct method of collecting and handling the sample to avoid contamination.

Caregivers will also be advised to store the collected sample in a refrigerator immediately after collection, in order to preserve its integrity for analysis.

We will then arrange for the stool sample to be collected from their home within 48 hours of collection. The sample will be transported under appropriate conditions to the microbiology laboratory, where it will be stored for further molecular analysis.

            During the baby’s scheduled visit to the Outpatient Department (OPD) for vaccination and routine clinical review, we will collect a stool sample if the baby passes stool while at the hospital. The caregivers will be instructed to inform the clinical staff immediately if the baby has a bowel movement during the visit. A sterile stool collection container will be provided on-site, and the sample will be collected promptly and securely. This approach ensures the sample is fresh and reduces the need for home collection and storage, while also making the process convenient for the family.

  All collected stool samples from preterm infants will be subjected to comprehensive microbiological evaluation. The first aliquot of each sample will be utilized for phenotypic identification of key indicator bacteria, focusing specifically on aerobic Escherichia coli and anaerobic Bacteroides species. These organisms are selected due to their relevance in early-life gut colonization and their known associations with health outcomes such as dysbiosis, inflammation, and susceptibility to infections including sepsis and necrotizing enterocolitis (NEC) in premature infants.

 The second aliquot of each stool sample will be preserved in RNAlater, a stabilization reagent that allows for the long-term preservation of nucleic acids at room or cold storage temperatures without degradation. This aliquot will be reserved for molecular analysis, including nucleic acid extraction and sequencing-bas ed studies.

 To assess the gut microbial diversity and composition across various degrees of prematurity, 10% of the samples from each gestational age category (e.g., extremely preterm, very preterm, and moderate to late preterm) will be randomly selected and processed for next-generation sequencing (NGS). These samples will undergo 16S rRNA gene amplicon sequencing, as well as shotgun metagenomic sequencing where appropriate