BackgroundThe infant gut microbiota undergoes significant fluxes in microbial composition during the first few years of life. The intestine is colonized shortly after birth with facultative anaerobes, like as Enterococcus, Enterobacter, Streptococcus, and Staphylococcus, which reduce the intestinal oxygen content and create niches for strict anaerobes, like Bifidobacteria, Bacteroides, and Clostridia. The dominant factors which influence the infant gut composition are gestational age and diet. The gut microbiota of preterm infants is characterized by limited microbial diversity and increased levels of facultative anaerobes such as Enterococci, Staphylococci, and Enterbacteriacea (Enterobacter, Escherichia, and Klebsiellaspp.). The gut microbiota is also highly influenced by diet, specifically formula and human milk use. The intake of human milk is associated with increased Bifidobacteria, while the intake of formula correlates with increased Enterobacteriaceae. This study aimed to analyze next‐generation products including preterm formula, human milk‐oligosaccharide term formula, and preterm breastmilk using a culture‐based model.Methods & ResultsWe cultured 8 different Bifidobacteria, 8 Lactobacilli, 8 Streptococcus, 3 Enterococci and 4 Klebsiella strains in a fully defined bacteria media and monitored growth and pH after 20 hrs of anaerobic incubation. Significant differences were identified in strain growth and culture pH between human milk and formula. Human milk significantly elevated the growth of all Bifidobacteria and Lactobacilli. In contrast, human milk supported the growth of three Streptococci and only nominally supported pathobiont Enterococci and Klebsiella strains. Preterm formula elevated the majority of Bifidobacteria and Lactobacilli, as well as several Streptococci. Interestingly, preterm and full‐term formula significantly enhanced the growth of pathobiont strains. Full‐term formula only supported the growth certain commensal microbes, which was in marked contrast to human milk and preterm formula. Using computational modeling, a correlation was observed with human milk oligosaccharide (HMO) degrading glycosyl hydrolase profiles in the genomes of commensal microbes with human milk, but not with formula; confirming the HMO preference of our representative infant commensal microbes.ConclusionsThese findings indicate a unique profile of growth in response to human milk and formula. Since alterations in the infant gut microbiota have linked to childhood health and development, understanding how bacteria differentially utilize whole milk or formula may provide insights into which microbes drive diet‐related microbiota shifts.