15 ml centrifuge tube

The NSS and SS from the 10 volunteers (five women and five men) between 25 and 67 years of age were collected in the morning between 9 and 11 a.m. and for 5 min (Figure 1). The NSS was naturally collected in 15 ml centrifuge tubes (VWR, PA, USA), while for the SS, the volunteers chewed a piece of Parafilm™ during the collection (30 (link)). After this, the saliva samples were centrifuged at 15,000 g and 4°C during 15 min (31 (link)). The supernatants from the NSS and SS were pooled into two different tubes. The pH was determined in both the saliva mixtures SS (pH = 7.7) and NSS (pH = 7.6). Then, aliquots of 1.5 ml of saliva were prepared in Eppendorf tubes (VWR, PA, USA) and were frozen (−80°C) until the experiments. This storage technique has been previously proven not to affect the saliva EA (32 (link)). Figure 1 shows a schematic summary of the experimental procedure.
The volunteers did not report oral diseases. The donors were not allowed to eat or drink anything 1 h before the saliva collection. The participants were informed about the purpose of the study, and they gave their written consent to participate. These experiments were authorized by the Bioethical Committee of the Spanish National Council of Research (CSIC), Spain.

In total, five mother-infant pairs were enrolled. Fecal samples and breast milk were collected for all pairs at 3 months (time point 1); additional samples were collected for pair 4 and pair 5 at 10 months (time point 2) and for pair 5 only at 16 months (time point 3) (see Table S1 and Fig. S1 in the supplemental material). All aspects of recruitment and sample and data processing were approved by the local ethics committee. Fecal samples were collected from mothers and infants in sterile feces tubes (Sarstedt, Nümbrecht, Germany) and immediately stored at −20°C. In those cases where metatranscriptomics was applied, a fecal aliquot was removed prior to freezing the remaining feces. This aliquot was stored at 4°C, and the RNA was extracted within 2 h of sampling to preserve RNA integrity. Milk was expressed and collected midflow by mothers into 15-ml centrifuge tubes (VWR, Milan, Italy) and immediately stored at −20°C. Within 48 h of collection, all milk samples and feces samples were moved to storage at −80°C until processed.

For the biofilm formation, conventional 96-well plates (Corning®, Amsterdam, The Netherlands) were used. Three colonies of each strain were suspended in 2 mL tryptic soy broth enriched with 1% glucose (TSB-G) in a 15 mL centrifuge tube (VWR International, Radnor, PA, USA) and incubated at 37 °C in a moist chamber under constant circular shaking (Edmund Bühler GmbH, Hechingen, Germany) at 200 rpm for 24 h. After incubation, the bacterial solutions were diluted in fresh TSB-G to a 10⁵ bacteria/mL concentration. Afterwards, 100 µL of the diluted bacterial solutions were added in the wells, each containing a sterile stainless-steel disc (DIN9021, stainless steel A2, size M2, diameter 5.9 mm) as the substrate for biofilm growth. The 96-well plates were incubated in a moist chamber under constant circular shaking at 37 °C for 48 h for the obtainment of biofilms. These experiments were carried out in triplicate.