Data_Sheet_2_Evaluation of Human Milk Microbiota by 16S rRNA Gene Next-Generation Sequencing (NGS) and Cultivation/MALDI-TOF Mass Spectrometry Identification.docx

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.

本研究旨在通过16S rRNA基因扩增子下一代测序和培养/基质辅助激光解吸/电离-飞行时间质谱（MALDI-TOF MS）鉴定方法对人类乳汁微生物群（HMM）进行特征描述。我们分析了31份来自健康斯洛文尼亚母亲的乳汁样本。为了验证MALDI-TOF MS鉴定的准确性，我们将代表最多产属和那些无法通过MALDI-TOF可靠识别的菌株的16S rRNA基因进行了Sanger测序。我们证明了培养/MALDI-TOF MS是文化依赖性确定HMM的适宜工具。在这两种方法中，我们发现葡萄球菌和链球菌是HMM中的优势属，其中葡萄球菌的丰度与微生物多样性降低相关。此外，我们还描述了可能影响HMM的因素。使用吸奶器与HMM组成、较高的微生物负荷和可培养葡萄球菌丰度降低显著相关。此外，我们的研究还表明，给哺乳婴儿补充益生菌可能通过增加乳酸杆菌的丰度和在人类乳汁中存在可存活的益生菌细菌来影响HMM。然而，鉴于我们的研究为观察性研究且样本量相对较小，需要更多的针对性研究来研究通过外部途径将益生菌转移到乳腺的可能性和这些事件的生理相关性。