2.1. Study Subjects and Sample Collection

From April to October 2015, at the Unit of Gynecology and Obstetrics of Fatebenefratelli Hospital of Rome in Italy, 116 healthy newborns were consecutively enrolled in this observational cross-sectional study.

The inclusion criteria regarded healthy newborns that were vaginally-delivered at term (37–42 gestational weeks) with a normal birth weight (2.5–4.5 kg), breastfed, and with an age range of 0–180 days. The exclusion criteria included caesarean section delivery, antibiotic intake, and acute or chronic gastrointestinal diseases that were registered in the 30 days before the starting point of both the stool and saliva collection and also during the entire time course.

The Hospital Ethics Committees approved the study (“Protocol 784_OPBG_2014”), and parents signed the informed consent for this study. The saliva samples were collected by gently swabbing the infants’ cheeks with a sterile cotton swab at 7, 15, 30, 90, and 180 days of time life. The saliva samples at birth were not collected to avoid the disturbance of mother and neonate privacy during the baby’s first day of life, causing a potential stressful situation that might compromise the breastfeeding. The stool samples were collected at birth (meconium) and 7, 15, 30, 90, and 180 days from infant diapers (Table 1). To increase the study feasibility and reduce the risk of dropout, we enrolled different babies at each time point.

The general characteristics of the neonate cohort.

^a SEM, standard error of the mean; ^b at 180 days 10/15 infants started weaning mostly with fruit (mainly apple and pear).

All the samples were collected during routine visits at Bambino Gesù Children’s Hospital and Research Institute of Rome, except for meconium samples that were collected at birth at the Unit of Gynecology and Obstetrics of Fatebenefratelli Hospital, Rome, Italy.

The samples were daily stored at 4 °C at the clinical unit and then, as soon as possible, they were transported at a controlled temperature and were stored long-term at −80 °C, at the BBMRI Biobank Microbiome node of the Human Microbiome Unit of Bambino Gesù Children Hospital, until laboratory processing.