Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants

Abstract

BackgroundInfants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation.MethodsUsing ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a ‘gold-standard’.ResultsResults indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had altered diversity profiles. Both bioinformatics 16S rRNA pipelines used in this study (OTU and paired end) presented similar taxonomic profiles at genus level.ConclusionsWe determined that DNA extraction from ELBW faecal samples, particularly those infants receiving probiotic supplementation, should include a prolonged beat-beating step. Furthermore, use of the 16S rRNA (V1 + V2 + V3) and (V6 + V7 + V8) regions provides reliable representation of ELBW microbiota profiles, while inclusion of the (V4 + V5) region may not be appropriate for studies where Bifidobacterium constitutes a resident microbiota member.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4229-x) contains supplementary material, which is available to authorized users.

Microbiota supplementation withBifidobacteriumandLactobacillusmodifies the preterm infant gut microbiota and metabolome

“…Limitations of this study include that it is observational in nature. However, low bifidobacteria abundance has been consistently reported in preterm infants in NICUs without any supplementation use (Alcon-Giner et al, 2017;Gasparrini et al, 2019;Sim et al, 2015). This indicates that the primary finding of high proportions of bifidobacteria, and associated changes in gut metabolites, in supplemented infants in this study is unlikely to be due to chance.…”

Streaming histogram sketching for rapid microbiome analytics

“…In this case we have used this sketching approach to differentiate between those preterm infants that had received antibiotics, versus those that did not. This is important clinically as antibiotic treatment in preterm infants is associated with significant alterations in the gut microbiota, which may link to increase risk of development serious conditions such as necrotising enterocolitis or sepsis (Sim et al , 2015;Shaw et al , 2015;Alcon-Giner et al , 2017) . Thus a rapid and discriminatory microbiome profiling method for this fragile and at-risk patient cohort, or indeed for other clinical microbiome samples, could prove useful for intervention or treatment options.…”

“…This is part of a wider clinical study, that is longitudinally profiling the gut microbiota of preterm infants that are residing in neonatal intensive care units (NICUs) and correlating this to health data, including impact of antibiotics. Faecal samples from preterm infants were collected and their bacterial DNA extracted following the protocols described in Alcon-Giner et al, 2017(Alcon-Giner et al , 2017 . Shotgun metagenomics libraries were prepared from 500 ng of genomic DNA which was sheared into fragments of~450 bp.…”

Rapid profiling of the preterm infant gut microbiota using nanopore sequencing aids pathogen diagnostics

“…Whilst MinION allows generation of long reads, DNA extraction to maximise this type of data is more time-consuming, thus in this study we utilised a rapid DNA extraction protocol (including a bead-beating step). Furthermore, previous studies, indicate that incomplete DNA extraction significantly biases metagenomic profiles obtained 34,47 , which may in turn limit pathogen detection and AMR analysis. Therefore, we acknowledge that it may be possible to improve read length in future (sample P8 had N50 of 3,479 bp) through improved DNA extraction methods.…”

“…We performed sequencing on seven infants (three healthy, and four diagnosed with suspected sepsis or NEC, Supplementary Fig. 1-2, Fig 3a), blindly selected from a larger ongoing clinical study (BAMBI-BMC genomics 34 ). Analysis of faecal samples using PCoA indicated three distinct clusters, which appear to be driven by the presence of either Bifidobacterium, Enterobacter or Klebsiella (Fig.…”