Chlorinated Drinking Water Exposure Enriches Antimicrobial Resistance Pathways in the Infant Gut Microbiome: A Randomized Trial

ABSTRACT Background Water chlorination is essential for controlling harmful microbes in drinking water; however, the antimicrobial effects of chlorine-based disinfectants present in tap water may influence early life gut microbial ecology. Objective To investigate the functional and compositional impact of chlorinated drinking water on the gut microbiome of infants. Design The waTer qUality and Microbiome Study (TUMS) was an Australian-based double-blinded, randomised controlled trial. Six-month old infants (n=197) received either de-chlorinated drinking water via benchtop filtration (treatment, n=99), or regular chlorinated water (control, n=98) for twelve months. Tap water and stool samples were collected at baseline and at end of intervention. Metagenomic sequencing was used for faecal microbiome analysis. Primary outcomes were differences in gut microbiota alpha and beta diversity. Secondary outcomes included changes in the differential abundance of species-level genome bins (SGBs) and functional profiles. Results 170 baseline (83 control, 87 intervention), and 130 end of intervention (65 control, 65 intervention) stool samples were collected. Overall community structure was similar between groups after the intervention, including beta diversity (95% CI; 0.4, 0.13, p=0.35), richness (-4.25, 95% CI; -14.85, 6.35, p=0.43) or Shannon Index (0.06, 95% CI; -0.14, -0.32, 0.04, p=0.45). The chlorinated water group showed enrichment of antibiotic resistance MetaCyc groups and pathways (adjusted p < 0.05). Conclusion Chlorinated drinking water may enhance resistance functions in the infant gut microbiome. While remaining vital for public health, future studies should explore whether adjusting the timing or method of drinking water disinfectants into the infant diet can reduce selective pressures. KEY MESSAGES What is already known on this topic Chlorination of drinking water is a cornerstone public health intervention to prevent waterborne disease. Chlorine-based disinfectants possess antimicrobial properties that may influence gut microbial ecology, particularly in early life when microbial communities are still developing. Recent studies in low- and middle-income settings have demonstrated that exposure to chlorinated water can increase the abundance of antimicrobial resistance genes (ARGs) in children’s gut microbiota. However, data from high-income settings with advanced water treatment practises, and from randomized controlled trials remain scarce. What this study adds This randomized controlled trial provides the first evidence from a high-income setting that chlorinated drinking water, while not substantially altering gut microbial diversity in infants, is associated with enrichment of ARG-related functional pathways. The findings indicate that routine water disinfection may exert selective pressures that enhance resistance functions in the early-life gut microbiome, even in the absence of overt taxonomic changes. How this study might affect research, practice or policy These findings underscore the need to consider AMR implications when evaluating infant feeding guidelines and drinking water disinfection strategies. Further research should investigate whether timing or mode of drinking water introduction can minimize selective pressures and consider alternative disinfection strategies.