Analysis of microbial composition and sharing in low-biomass human milk samples: a comparison of DNA isolation and sequencing techniques

Abstract

Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.

Post Natal Microbial and Metabolite Transmission: The Path from Mother to Infant

“…In the context of human milk and vertical transmission via breastfeeding, previous analyses of shared genera between HM and IS in the Dutch population [72] agree with the presence of Pauljensenia, but not with the presence of Streptococcus and Veillonella, which are shared in our work. Also, it has been shown that Bifidobacterium is bound to sIgA in HM [73], and the bacterium is transmitted by breastfeeding [74].…”

GROND: a quality-checked and publicly available database of full-length 16S-ITS-23S rRNA operon sequences

“…As the most comprehensive rrn database to date, and incorporating a GTDB-based version that includes numerous metagenome-assembled genomes (MAGs), this database should prove to be a valuable resource for the rrn sequencing community. Indeed, the preprint version of this database has already been used to study bacterial transmission in low-biomass human milk samples [ 50 ].…”

“…The choice of classifier for phylogenetic analysis is significantly influenced by the type of long-read platform employed. The high accuracy of PacBio sequencing data makes it compatible with denoising or clustering pipelines, such as DADA2 [ 50 51 ] and vsearch [ 17 28 ], respectively. Meanwhile, the lower read quality from ONT demands specialised pipelines to account for its higher error rates.…”

The assessment of bacterial diversity in CO2 treated and untreated raw milk during cold storage using high‐throughput sequencing technology

“…Previous studies have shown that there is no evidence of harmful bacteria being selected in carbonated raw milk during refrigeration. However, next‐generation amplicon sequencing offers advantages such as greater sampling depth, less manual processing and more representative and reproducible results (Catozzi et al ., 2020; Spreckels et al ., 2023). Therefore, the aim of this current study is to analyse the bacterial composition of carbonated raw milk during cold storage using Illumina sequencing of the 16S rRNA gene amplicon (García‐González et al ., 2022; Baptista & Domingues, 2023; Busetta et al ., 2023).…”