Early life events significantly influence the developing gut microbiome, yet the response time and duration of microbiome changes to specific factors, such as vaccination or solid food introduction, remain unclear. Consequently, determining the optimal sampling frequency to monitor gut microbiome development is challenging. This study monitored gut microbiome plasticity using 16S rRNA gene sequencing almost daily in one infant (A) and weekly in 12 others (B-M) during their first year. Changes were linked to external factors and their duration analyzed. Three bacterial colonization groups emerged: "Early-life colonizers," "Re-appearing colonizers," and "Later-colonizers." Weekly sampling underestimated microbiome variability, as individual samples within the same week differed by over 1 Shannon index, and most of the weekly coefficients of variation of different alpha diversity indices in the first 23 weeks were higher than 10 %. Alpha diversity variability decreased with age, but beta diversity variability remained high. Key events like solid food introduction and probiotics caused gradual but significant bacterial composition changes, with effects varying among infants. Sparse weekly sampling hindered a detailed understanding of the impact of maternal microbiome, diet, probiotics, vaccinations, and unforeseen variables. Analysis of weekly variability in alpha and beta diversity suggests that such rare sampling may not be sufficient in terms of the outcomes of interest.

Department of Experimental Biology Faculty of Science Masaryk University Kotlářská 2 611 37 Brno Czech Republic

RECETOX Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

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