| Summary: | Dysbiosis is associated with acute and long-term consequences for neonates. Probiotics can be effective in limiting the growth of bacteria associated with dysbiosis and promoting the healthy development of the infant microbiome. Given its adaptation to the infant gut, and promising data from animal and <i>in vitro</i> models, <i>Bifidobacterium longum</i> subsp. <i>infantis</i> is an attractive candidate for use in infant probiotics. However, strain-level differences in the ability of commercialized strains to utilize human milk oligosaccharides (HMOs) may have implications in the performance of strains in the infant gut. In this study, we characterized twelve <i>B. infantis</i> probiotic strains and identified two main variants in one of the HMO utilization gene clusters. Some strains possessed the full repertoire of HMO utilization genes (H5-positive strains), while H5-negative strains lack an ABC-type transporter known to bind core HMO structures. H5-positive strains achieved significantly superior growth on lacto-<i>N</i>-tetraose and lacto-<i>N</i>-neotetraose. <i>In vitro</i>, H5-positive strains had a significant fitness advantage over H5-negative strains, which was also observed <i>in vivo</i> in breastfed infants. This work provides evidence of the functional implications of genetic differences among <i>B. infantis</i> strains and highlights that genotype and HMO utilization phenotype should be considered when selecting a strain for probiotic use in infants.
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