Cohort description and recruitment
A total of 134 volunteers comprising babies (4–15 months old at nursery start, median 10 months, 18 male, 25 female) about to attend the first year of nursery school, their parents (29–50 years old, median 36 years old, 30 male, 39 female), siblings (2–21 years old, median 2 years old, 3 male, 4 female) and house pets (n = 5, 2 cats and 3 dogs), and educators (34–56 years old, median 38.5 years old, 10 female) were recruited and enroled across 3 nursery schools (here identified as A, B and C), each with 2 distinct classes, in the municipality of Trento (Italy) in June 2022. The classes within the same nursery shared few activities (that is, baby drop-off and pick-up) and spaces throughout the day, and were followed by different educators. The protocol of this study was approved by the Ethics Committee of the University of Trento (protocol number 2022-040) and by the Ethics Panel of the European Research Council Executive Agency after evaluation of the project (microTOUCH Grant agreement ID 101045015). Upon enrolment, volunteers were asked to provide informed consent and complete metadata questionnaires. Consent for participation of babies was obtained directly from parents.
Metadata collection and organization
Date of birth, sex, anthropometric data (weight, height), and antibiotic treatment in the 3 months preceding the start of the study or supplemented during its course, in addition to information regarding putative contacts with other volunteers preceding the beginning of nursery, were collected for volunteers of all ages. Metadata specifically collected for babies included gestation length, mode of delivery and general diet at nursery admission (breast or formula milk feeding and weaning date of start). Adult participants were also required to provide information regarding past or ongoing chronic conditions and relative treatments, and putative maternal anti-Streptococcus B prophylaxis during birth. Diet metadata for babies and adults are detailed in the next section.
Dietary information collection and analysis
In brief, most babies had begun weaning at T01 (weaned n = 38, not weaned n = 2, not available = 3) and received identical solid meals while in the nursery. The majority followed a mixed feeding approach during weaning, combining solid foods with any type of milk (mixed diet n = 24, exclusively solid food n = 14, not available = 5). Among those babies receiving milk supplementation, feeding types were relatively balanced (breast-fed n = 9, formula-fed n = 10, receiving both n = 5). Finally, adults detailed their long-term dietary habits via the compilation of the EPIC Food Frequency Questionnaire (FFQ). FFQs were used to calculate the healthy Plant-based Diet Index65. Quality and quantity of plant-based foods were derived from FFQs for a total of 18 food groups, and divided into quintiles and assigned positive or negative scores. Participants whose intake exceeded the highest quintile received a score of 5, whereas those below the lowest quintile received a score of 1. Healthy plant-based foods received positive scores, whereas less healthy or unhealthy plant-based and animal-based foods received a negative score. A final score was derived by summarizing the scores of each participant. Metadata were collected and utilized after pseudonymization of volunteers IDs.
Sample collection
Sample collection began a week before the start of the first term of nursery (August 2022) and ended after the Christmas holidays (January 2023) for all volunteers. During the first 2 weeks the nursery organized a ‘settling-in phase’, in which babies were gradually introduced to the nursery and attended it for about 3 hours per weekday. In the following weeks, babies attended the nursery for about 8 hours per weekday. Throughout the term length (about 14 weeks), stool samples of infant participants were collected weekly (from before nursery admission T01 to at the end of Christmas holidays T15) by the nursery staff or the researcher in the nursery from nappies stored at room temperature on the same day of use, using collection tubes for specimen collection containing 9 ml of DNA/RNA Shield buffer (Zymo). Sample collection was extended until the end of the second term of the year (about 30 weeks, ending July 2023) for all donors in group 1 of nursery A, including babies, parents, educators and pets, maintaining sampling time-point frequencies and modalities. Two follow-up time points were collected for all participants enroled, at the end of the year of nursery (July 2023, ‘TA’) and at the end of the summer break (August/September 2023, ‘TB’). The samples collected were moved to the lab and DNA-extracted within 2 weeks of delivery. Samples collection of babies during summer or winter breaks time points together with those of siblings and pets were performed directly at home by the parents and stored at room temperature until the beginning of nursery (maximum 2 weeks later). All adult participants’ samples were self-collected following detailed instructions, delivered to the lab and processed as previously. Educators donated monthly, whereas parents collected one additional sample halfway the study period, in addition to initial and final sample time points.
DNA extraction and sequencing
After vortex homogenization, DNA was extracted using the DNeasy PowerSoil Pro Kit (Qiagen), following the directions of the Human Microbiome Project protocol66. Additional homogenized aliquots were stored at −20 °C. DNA was quantified using Qubit 2.0 fluorometer (Thermo Fisher Scientific). Sequencing libraries were prepared using the Nextera DNA Library Preparation Kit (Illumina), as described by the manufacturer’s guidelines. The sequencing was performed on the Illumina NovaSeq 6000 platform following manufacturer’s protocols. The sequencing depth was set at 15 Gbp.
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