Recently the Humbugs crew sat down to watch a webinar entitled: “Characterising the microbiome of neonates and infants to explore associations with health and disease” by Dr Christopher Stewart. Not only is the topic ideally aligned to our research group, but the presenter was instrumental in setting up the network and agreements that allows us to do our research. Chris is currently a Post Doctoral Associate at the Baylor College of Medicine (Petrosino Lab). He gained 2nd prize in the 2015 Microbiome Awards (for more information on the 2017 Microbiome Awards please see www.mobio.com/microbiome ) for his work relating to the microbial impact on infant gut function and health in preterm infants. There are so many different areas of research in this wide-ranging topic, www.neonatalresearch.net provides more information about the programmes in place and the progress made to date. There are so many amazing parents that have allowed samples to be collected from their infants, some of which are healthy and others that are very ill – we are so grateful, and their generosity spurs us on!
But back to the webinar… Chris gave a clear overview of the techniques and methods used to analyse preterm infant stool samples:
• DNA extraction – always remembering that each kit has its’ own microbiome and therefore a kit negative is imperative to ensure viable downstream analysis
• PCR – usually targeting the V4 region 16S rRNA
• MSeq 2 x 250bp paired end sequencing – after which the poor-quality reads are removed and aligned to a database such as SILVA. But this statistical analysis can be far reaching and touch on new approaches, such as: FishTaco and Tax4Fun – which not only prove that coders have a mean sense of humour as well as a slightly cruel streak… more on this area in a later blog I think 😊
• Dielectrophoresis – where a force is exerted on a dielectric particle, which can be uncharged, when subjected to a non-uniform electric field – used for separation.
• Antibiotic capture to find distinguishing features which are then modelled.
• Models: Cell culture, Enteroids – model cell types normally found in the intestinal epithelium, Mice / Germ free mice, Bioreactors, Caenorhabditis elegans animal model
The relevance of using stool samples to interrogate Necrotising Enterocolitis (NEC) which occurs in the terminal ileum was also discussed – investigating infant stool microbiome has relevance so long as Alpha diversity, Beta diversity and Taxonomic abundance can be related to bacterial functionality in order to draw causative conclusions.
There are patterns in the preterm microbiome which have already been drawn and give useful insight into prospective treatments:
• Reduced: Diversity, Stability, Bifidobacterium sp. (higher abundance in breast milk fed infants), Lactobacillus sp., Bacteroides sp.
• Increased: Klebsiella sp., Staphyloccocus sp., Escherichia sp., Enterococcus sp.
These patterns infer the existence of a ‘healthy’ microbiome as Cassy is investigating; also, that there is a role for Bifidobacteria especially in relation to how oligosaccharides in breast milk influence a ‘healthier’ microbiome as I am researching; and the maternal role of the placenta as the starting point for the infants’ microbiome as Rochelle is elucidating.
So, all in all a very useful webinar!