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Wang et al. Microbiome Res Rep. 2025;4:23 https://dx.doi.org/10.20517/mrr.2024.94 Page 3 of 19

In this study, we compared various approaches for absolute microbiome quantification, including a DNA
spike-in strategy in which exogenous bacterial DNA is added to sample DNA to enable accurate estimation
of absolute bacterial abundances. We selected Pseudoalteromonas sp. APC 3896 and Planococcus sp. APC
3900-marine strains isolated from deep-sea fish that represent phylogenetically distinct phyla rarely found
in mammalian fecal microbiomes. These genera are evolutionarily distant from gut-associated microbes and
are absent not only at the species level but also at the genus level in the human gut microbiome under
typical physiological conditions. Moreover, they are easily distinguishable from endogenous bacteria
through 16S rRNA gene sequencing. Their use is also technically convenient, as well-characterized isolates
were readily available in our laboratory.

The other absolute quantification approaches, including flow cytometry, total DNA quantification, qPCR,
and plate count, were compared with the DNA spike-in quantification. We then applied the method to
quantify absolute bacterial counts in the feces obtained from infants and their mothers, which highlighted
the difference in microbial analysis between relative abundance and absolute abundance.

METHODS
Participant recruitment
[21]
Stool samples were obtained from mothers (n = 6) and their infants (n = 6) enrolled in the MIMIC study .
All samples were collected at 4 weeks after birth. The protocol was approved by the Clinical Research Ethics
[21]
Committee of the Cork Teaching Hospitals and has been previously published . Inclusion criteria included
full-term infants (gestational age ≥ 37 weeks) and availability of paired maternal and infant fecal samples
within the first month postpartum. Exclusion criteria were antibiotic use by the infant within 2 weeks prior
to sample collection, neonatal complications requiring intensive care, or any known gastrointestinal
disorders.

Flow cytometry
For flow cytometry (BD FACSCelesta, BD Science, USA) analysis, 0.05 g aliquots of fecal samples were
diluted 10,000-fold in 0.85% NaCl. This dilution ensured that bacterial concentrations fell within the
optimal range for accurate detection (10 -10 cells/mL), minimized background noise, and maintained
5
7
compatibility with viability dyes and microsphere calibration. To remove debris from the fecal solutions,
TM
samples were filtered using sterile syringe filters with a pore size of 5 μm. The LIVE/DEAD BacLight
TM
Bacterial Viability and Counting Kit (Invitrogen, USA) was used to distinguish and quantify live and dead
TM
bacteria. This kit utilizes a mixture of two nucleic acid stains: green-fluorescent SYTO 9 dye and red-
fluorescent propidium iodide, for viability determinations. Additionally, a calibrated suspension of
microspheres was employed for accurate sample volume measurements.

Plate count
For the quantification of viable bacterial populations, fresh stool samples from mothers and infants were
subjected to plate count on YCFA (Yeast Extract, Casitone, Fatty Acids) medium . Stool samples (~ 1 g)
[22]
were homogenized in 9 mL of sterile phosphate-buffered saline (PBS) to prepare a 1:10 dilution, followed by
serial dilutions to ensure countable colony formation. Aliquots (100 µL) of each dilution were plated onto
YCFA agar plates and spread evenly using sterile spreaders. Plates were immediately transferred into a
Whitley A20 anaerobic workstation (Don Whitley Scientific Limited, UK) and incubated at 37 °C for 48 h.
After incubation, colonies on plates containing 30-300 colony-forming units (CFUs) were enumerated, and
bacterial counts were calculated as CFUs per gram of stool. Negative controls with sterile PBS and YCFA
plates were included to monitor contamination.