Methods for exploring the faecal microbiome of premature infants: a review

Abstract

The premature infant gut microbiome plays an important part in infant health and development, and recognition of the implications of microbial dysbiosis in premature infants has prompted significant research into these issues. The approaches to designing investigations into microbial populations are many and varied, each with its own benefits and limitations. The technique used can influence results, contributing to heterogeneity across studies. This review aimed to describe the most common techniques used in researching the preterm infant microbiome, detailing their various limitations. The objective was to provide those entering the field with a broad understanding of available methodologies, so that the likely effects of their use can be factored into literature interpretation and future study design. We found that although many techniques are used for characterising the premature infant microbiome, 16S rRNA short amplicon sequencing is the most common. 16S rRNA short amplicon sequencing has several benefits, including high accuracy, discoverability and high throughput capacity. However, this technique has limitations. Each stage of the protocol offers opportunities for the injection of bias. Bias can contribute to variability between studies using 16S rRNA high throughout sequencing. Thus, we recommend that the interpretation of previous results and future study design be given careful consideration.

To Probiotic or Not to Probiotic: A Metagenomic Comparison of the Discharge Gut Microbiome of Infants Supplemented With Probiotics in NICU and Those Who Are Not

“…As for the limited taxonomic depth and limited sample size of the sub-cohort that underwent shotgun metagenomics, this could be overcome using shotgun metagenomics across the entire cohort. However, this technique, and others of similar resolution, are cost prohibitive at present ( 32 ).…”

“…Despite some heterogeneity between studies reported in the literature (31,86), probiotics have demonstrated efficacy in reducing the incidence of diseases, such as NEC (7)(8)(9), as well as positively modulating the infant gut microbiome (13,14), in the most premature of infants. The heterogeneity observed could result from the use of different probiotic species (8), variability in the microbiome detection methods used (32) or the many confounding variables that influence the developing gut microbiome. Nonetheless, several countries, such as Japan and Australia, use probiotic-treatment as part of standard care for the most premature of infants, and those at high risk of NEC.…”

Methodological challenges in neonatal microbiome research

“… 22 , 26–30 A recent systematic review of 92 studies found that freezing at −80°C is the most commonly used method, employed in 71.4% of studies. 23 Storage of neonatal stool samples at this temperature has been confirmed to largely preserve the microbiota for at least 2 years. 31 In addition, as rapid freezing of samples and transport via a cold chain is not always possible, various storage buffers have also been tested.…”

“…But it has been shown that experimental design and choice of methods can affect stool microbiota composition and bias downstream analysis. 22 , 23 This is the case for all stages of sample preparation, incorporating sample collection, sample storage, DNA extraction and sequencing method. This section will initially focus on aspects that bias molecular based approaches, namely 16S rRNA gene sequencing and metagenomics, before discussing impacts on culture-based approaches.…”

“…Timing of sample collections is therefore a key factor in any microbiome study. 23 This will impact comparisons that can be made both between samples within the study and already published datasets. Studying neonates creates challenges to this process as there is no ability to control precisely when a neonate has a bowel movement, they cannot communicate when they are anticipating one, and they are unable to collect their own samples.…”