The Gut Bacterial Resistome in the First Two Years of Life: Protocol for a Longitudinal Observational Birth Cohort Study

Background: Antimicrobial resistance (AMR) is a global health threat that increases the burden of infectious diseases and disproportionately affects communities of low socioeconomic status. Despite the call for community-level AMR data, prospective studies from rural sub-Saharan African communities to inform appropriate targeted interventions remain scarce. Given the role of enteric bacteria in AMR transmission dynamics, there is a need to understand the timing, risk factors, and ecological drivers of gut resistome acquisition and development during infancy. Objective: This study aimed to characterize the temporal dynamics of enteric bacterial resistomes during the first 2 years of life and to identify drivers of AMR acquisition and development in a community-based, prospective, observational birth cohort study in a rural South African community. Methods: The study aims to enroll 200 newborns and their mothers within 17 days post partum. Data on key exposures and variables include sociodemographics; perinatal and anthropometrics; feeding practices and dietary exposures; illness, medication, and vaccination history; breast milk metabolomic profiles; household socioeconomic status; maternal psychosocial and behavioral factors; hygiene and sanitation practices; and environmental exposures including hydro-meteorological variables, in-house livestock and pets, and drinking water quality. Biological samples include stools from monthly collections and diarrhea episodes for metagenomic analysis and breast milk for metabolomics. Planned analyses include assessing the infant microbiome and resistome structure (diversity, abundance, and composition) across time points and modeling associations between risk factors and AMR outcomes. Additionally, a cross-sectional community survey on knowledge, attitudes, and practices regarding antimicrobial use is conducted to inform knowledge translation through responsive dialogues, thereby developing ethnographically relevant packages for community-level AMR stewardship. Results: Participant identification and enrollment began in August 2023. By October 2025, 167 newborns had been enrolled, with 20 having completed the 24-month follow-up. The characteristics of the enrolled participants are presented in this protocol. Conclusions: This study will offer a unique opportunity to generate longitudinal resistome data from a rural sub-Saharan African setting. The study is expected to contribute knowledge on the microbiome and resistome structure dynamics and trajectories associated with key risk factors of acquisition and development. In addition, co-produced ethnographically tailored educational packages, informed by knowledge, attitudes, and practices and bacterial resistome data, will drive sustainable community-centered AMR awareness interventions.