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Linehan et al. Microbiome Res Rep. 2025;4:24 https://dx.doi.org/10.20517/mrr.2024.92 Page 3 of 20

METHODS
Study design, ethics and recruitment
This study was conducted at Cork University Maternity Hospital (CUMH), recruiting 63 healthy mother-
infant dyads born full term either naturally or via CS between November 2015 and February 2019 as part of
the Oral Placenta Infant Microbiota Study (OPIuM Study). Ethical approval was obtained from the Cork
Teaching Hospitals Clinical Research Ethics Committee [ECM 4 (v) 12/08/14]. Infants born < 35 weeks
gestation, admitted to the neonatal intensive care unit (NICU), or born to ill mothers were excluded. A
subset of 18 dyads with available samples from all sites (vaginal, maternal saliva, placenta, infant saliva, and
meconium) was selected for this analysis. Table 1 summarizes the characteristics of the 18 mother-infant
dyads. The infants were 56% male and 44% female, with 44% born via CS and 56% vaginally. The mean birth
weight was 3,667.2 g (SD ± 386.6), gestational age 39 weeks (SD ± 0.97), and Apgar score 9 (SD ± 1.15).
Premature rupture of membranes (PROM) occurred in 39% of births. Feeding included 56% breastfed, 33%
formula-fed, and 11.1% unspecified. None of the infants received antibiotics. Among mothers, 56% received

antibiotics (two specified as Amoxicillin and Flucloxacillin), and 44% did not. The mean gravida was 3 (SD
± 1.5), and parity was 2 (SD ± 1.3).

Sample collection
TM
[18]
Vaginal and saliva samples were collected using CatchAll swabs (Cambio, UK) . Mid-vaginal samples
were collected by midwives or gynecologists within 1 h before delivery and immediately placed on dry ice,
then stored at -80 °C. Placental samples were collected by obstetricians within 1 h of delivery; four random
cross-sectional pieces were excised to encompass both maternal and fetal sides, and then stored similarly.
Maternal saliva was pooled from the floor of the mouth within 24 h postpartum, and infant saliva was
collected within four days of delivery. Meconium samples were collected within four days and confirmed to
have typical characteristics (dark, sticky, tar-like). Hospital-collected meconium and saliva samples were
immediately stored on dry ice at -80 °C. Home-collected samples were stored at 4 °C until retrieved by the
research nurse and transported in a temperature-controlled case to the laboratory for long-term storage at
-80 °C.

DNA extractions
DNA extraction for swab samples (vagina and saliva) was performed using the MO BIO PowerSoil DNA
Isolation kit (Qiagen, Hilden, Germany), adapting the protocol to process swabs as previously
described [18,19] . Thawed swabs were cut above the swab head, which was inserted into PowerBead Pro tubes
containing 600 μL of solution C1. Samples were homogenized for 3 min at maximum speed using a Mini
Beadbeater (BioSpec), followed by incubation at 65 °C for 10 min. DNA was eluted in 50 μL solution C6 as
per the manufacturer’s instructions. Meconium samples underwent DNA extraction using a bead-beating
and column-based protocol , combined with the QIAamp Fast DNA Stool Mini kit (Qiagen, UK) . A
[20]
[21]
lysis buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.0, 50 mM EDTA, 4% w/v SDS) was added to bead-
beating tubes containing 0.25 g of meconium, followed by homogenization (3 min, maximum speed),
incubation at 70 °C for 15 min, and centrifugation (4 °C, 16,000 × g, 5 min). Supernatant pooling,
ammonium acetate treatment, ethanol washing, and RNAse/proteinase K treatments were performed before
DNA elution in 50 μL buffer AE. Placental DNA was extracted using the DNeasy Blood and Tissue Kit
[22]
(Qiagen) . Approximately 0.25 g of frozen tissue was diced with sterile scalpels and suspended in
microcentrifuge tubes with tissue lysis buffer (180 μL ALT) and a stainless steel bead. Tissue
homogenization was conducted using a TissueLyser II (Qiagen) at 30 Hz for 20 s. After adding proteinase K
(20 μL, 20 mg/mL), samples were incubated at 56 °C with intermittent vortexing. DNA was eluted in 50 μL
buffer AE. Extraction controls containing 250 μL sterile water were processed alongside samples. DNA
concentrations were measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific) with a detection
TM
limit of 10 pg/μL. All extracted DNA was stored at -30 °C.