Maturation of the gut microbiota (GM) in infants is critically affected by environmental factors, with potential long-lasting clinical consequences. Continuous low-dose antibiotic prophylaxis (CAP) is the standard of care for children with vesico-ureteral reflux (VUR), in order to prevent recurrent urinary tract infections. We aimed to assess short-term GM modifications induced by CAP in infants. We analyzed the GM structure in 87 infants (aged 1-5 months) with high-grade VUR, previously exposed or naive to CAP.</p><p>Microbial DNA was extracted from stool samples. GM profiling was achieved by 16S rRNA gene-based next-generation sequencing. Fecal levels of short- and branched-chain fatty acids were also assessed. 36 out of 87 patients had been taking daily CAP for a median time of 47 days, while 51 out of 87 had not. In all patients, the GM was predominantly composed by Bifidobacteriaceae and Enterobacteriaceae. Subgroup comparative analysis revealed alterations in the GM composition of CAP-exposed infants at phylum, family and genus level. CAP- exposed GM was enriched in members of Bacteroidetes and Enterobacteriaceae, especially in the genera Bacteroides, Parabacteroides and Klebsiella, the latter often associated with antibiotic resistance. In contrast, the GM of CAP-naive children was mostly enriched in Bifidobacterium. No differences were found in fatty acid levels. In infants with VUR, even a short exposure to CAP definitely alters the GM composition, with increased relative abundance of opportunistic pathogens and decreased proportions of health-promoting taxa. Early low-dose antibiotic exposure</p><p>might bear potential long-term clinical risks.