Evaluation of Human Milk Microbiota by 16S rRNA Gene Next-Generation Sequencing (NGS) and Cultivation/MALDI-TOF Mass Spectrometry Identification.

Primož Treven,Majda Golob,Tina Pirš,Petra Mohar Lorbeg,Aleksander Mahnič,Bojana Bogovič Matijašić,Maja Rupnik

doi:10.3389/fmicb.2019.02612

Abstract

The aim of the present study was to characterize human milk microbiota (HMM) with 16S rRNA gene amplicon next-generation sequencing and cultivation/matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) identification approaches. We analyzed 31 human milk samples from healthy Slovenian mothers. To check the accuracy of MALDI-TOF MS identification, several colonies representing most abundant genera and those, which could not be reliably identified by MALDI-TOF, were subjected to Sanger sequencing of their 16S rRNA gene. We showed that cultivation/MALDI-TOF MS was a suitable tool for culture-dependent determination of HMM. With both approaches, Staphylococcus and Streptococcus were found as predominant genera in HMM and the abundance of Staphylococcus was associated with decreased microbial diversity. In addition, we characterized factors that might influence HMM. The use of a breast pump was significantly associated with composition of HMM, higher microbial load, and lower abundance of cultivable staphylococci. Moreover, our study suggests that administration of probiotics to the suckling infant might influence HMM by increased abundance of lactobacilli and the presence of viable probiotic bacteria in human milk. However, since our study was observational with relatively small sample size, more targeted studies are needed to study possible transfer of probiotics to the mammary gland via an external route and the physiological relevance of these events.

Highlights

Majority of bacteria found in breastfed infants’ gut, including gut-associated obligate anaerobes, originate from human milk (Jost et al, 2013b; Pannaraj et al, 2017)
Our systemic approach and usage of MALDITOF mass spectrometry (MS) for identification enabled us to cover the major representatives of cultivable human milk microbiota (HMM)
Since accuracy of MALDITOF MS determination is limited to the quality of the spectral database (Sauget et al, 2017), we tested its accuracy with Sanger sequencing of 16S rRNA isolated from selected colonies

Summary

Introduction

Majority of bacteria found in breastfed infants’ gut, including gut-associated obligate anaerobes, originate from human milk (Jost et al, 2013b; Pannaraj et al, 2017). Individual factors that shape the human milk microbiota (HMM) are still unclear since reports seem to be contradictory. Some authors reported no differences in HMM regarding mode of delivery, geographic location (Pannaraj et al, 2017), maternal BMI (Li et al, 2017), and stage of lactation (Sakwinska et al, 2016; Li et al, 2017). Beside different study protocols in these reports, the human milk collection protocol seems to be an important factor that could contribute to inconsistencies (Sakwinska et al, 2016)

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Conclusion