Comparison of Two Approaches for the Metataxonomic Analysis of the Human Milk Microbiome.

Lorena Ruíz ,Lars Bode,Michelle K Mcguire,Courtney L Meehan,Rossina G Pareja,Debela Gindola K,Sophie E Moore,Andrew M Prentice,Mark A Mcguire,Cristina García-Carral,Claudio Alba,Egidioh W Kamundia,Juan M Rodríguez,Samwel Mbugua,Gloria E Otoo,Kimberly A Lackey,Elizabeth Kamau-Mbuthia ,James A Foster ,Daniel Sellen ,Esther Jiménez ,Janet E Williams

doi:10.3389/fcimb.2021.622550

Abstract

Recent work has demonstrated the existence of large inter-individual and inter-population variability in the microbiota of human milk from healthy women living across variable geographical and socio-cultural settings. However, no studies have evaluated the impact that variable sequencing approaches targeting different 16S rRNA variable regions may have on the human milk microbiota profiling results. This hampers our ability to make meaningful comparisons across studies. In this context, the main purpose of the present study was to re-process and re-sequence the microbiome in a large set of human milk samples (n = 412) collected from healthy women living at diverse international sites (Spain, Sweden, Peru, United States, Ethiopia, Gambia, Ghana and Kenya), by targeting a different 16S rRNA variable region and reaching a larger sequencing depth. Despite some differences between the results obtained from both sequencing approaches were notable (especially regarding alpha and beta diversities and Proteobacteria representation), results indicate that both sequencing approaches revealed a relatively consistent microbiota configurations in the studied cohorts. Our data expand upon the milk microbiota results we previously reported from the INSPIRE cohort and provide, for the first time across globally diverse populations, evidence of the impact that different DNA processing and sequencing approaches have on the microbiota profiles obtained for human milk samples. Overall, our results corroborate some similarities regarding the microbial communities previously reported for the INSPIRE cohort, but some differences were also detected. Understanding the impact of different sequencing approaches on human milk microbiota profiles is essential to enable meaningful comparisons across studies.Clinical Trial Registrationwww.clinicaltrials.gov, identifier NCT02670278.

Highlights

Despite the fact that human milk has long been considered sterile, research conducted over the last decade has provided convincing evidence that this biological fluid harbors a rich microbial community under all physiological circumstances (Martıń et al, 2006; Martıń et al, 2007; Solis et al, 2010; Fernández et al, 2013)
The overall analysis of the beta diversity, calculated according to the relative abundance of amplicon sequence variants (ASVs) (Bray-Curtis distance) and the presence/absence of ASVs sequences, indicated that the profiles of bacterial genera of the different cohorts apparently clustered into different groups (p < 0.001 and p < 0.001, respectively; PERMANOVA) and the ETR cohort was observed to be more clearly separated from the other locations with both distance metrics (Figures 2A, B)
Our findings provide, for the first time and across globally diverse populations, evidence of the impact of different DNA processing and sequencing approaches on the microbiota profiles obtained for human milk samples

Summary

Introduction

Despite the fact that human milk has long been considered sterile, research conducted over the last decade has provided convincing evidence that this biological fluid harbors a rich microbial community under all physiological circumstances (Martıń et al, 2006; Martıń et al, 2007; Solis et al, 2010; Fernández et al, 2013). Milk’s microbial community contains an important arsenal of bacterial and fungal species of substantial interest as they likely play crucial roles in the maintenance of maternal and infant health (as reviewed in Boix-Amorós et al, 2019; Moossavi et al, 2020; Stinson et al, 2020) They likely seed the breastfed infant’s gastrointestinal (GI) tract, initiating the assembly of a mature healthy human GI microbiota, and orchestrating the innate immunity maturation and programming that will condition infant health outcomes in the short and long term (as reviewed in Milani et al, 2017). A combination of HTS and culturomic approaches has further supported the existence of a diversity of viable bacterial cells in healthy human milk wider than previously anticipated, and has offered novel opportunities to conduct mechanistic studies on the metabolic potential of this microbial community (Schwab et al, 2019; Togo et al, 2019; Treven et al, 2019)

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