Set up for life: How breastfeeding benefits preterm infants

Breastfeeding has long been understood to be good for infants. What’s new is evidence of how breast milk shapes the gut microbiome of newborns and how it might tackle the most common deadly disease in early preterm babies: necrotizing enterocolitis (NEC).

February 22, 2022

More than one in ten babies are born preterm and thanks to modern healthcare, most survive. Unfortunately, early preterm babies (22 to 32 weeks gestation) are at a disadvantage as they have an immature gut and gut microbiome, and an undeveloped immune system. This means they’re vulnerable to diseases such as NEC and late onset sepsis (LOS). However, new research shows that acquiring potentially beneficial gut bacteria quickly may protect neonates and accelerate their development. So by the age of three, they will have established a normal diet, healthy gut bacteria and a stable gut microbiome – like full-term babies – that changes very little for years to come.

These findings come from clinical studies published in several papers by Dr. Christopher Stewart, from Newcastle University’s Translational and Clinical Research Institute. He’s been researching this field for more than a decade.

Using a recently developed model that combines human preterm intestinal cells alongside viable microbes, his team studies host–microbial interactions to better understand how the microbiome contributes to health and disease in preterm infants. And one key discovery is that components of breast milk can quickly modify the infant microbiome, helping to tackle NEC and safeguard long-term health.

Early life microbiome research

Twelve years ago, the chance to research bacteria that colonize the intestines of preterm babies was a turning point for Christopher Stewart. This novel and crucial area of microbiome research started a lifelong fascination to discover the impact of microbiota on the health of such young infants. As new technologies developed, Dr. Stewart found he could combine his dual interests in benchwork (on patient samples) with IT (bioinformatics, NGS analysis, mass spectrometry) to great effect, revealing much about microbiota and early-life disease.

“Setting up a sequencing facility in Newcastle and using mass spectrometry was a great way to learn how profiling of small molecules and NGS could be combined,” says Dr. Stewart. “We found out far more about the complexity of clinical samples than by using one technique alone.”

A meeting with Prof. Joseph Petrosino, from the human genome and human microbiome projects, prompted Dr. Stewart to move to Baylor College of Medicine in Texas. “This gave me the chance to work in a major sequencing facility with experts in many different fields. Sequencing power and nearby patient access (from the largest medical center in the world) provided data on a huge scale, making this one of the best places for infant microbiome research,” he says.

“These days, in my lab at Newcastle University, which is joined to the local hospital, we are working closely with clinicians and have access to The Great North Neonatal Biobank, the largest biobank of clinical samples from preterm infants.” His team’s goal is to understand the underlying mechanisms involving disease and breast milk, and develop diagnostic and therapeutic interventions. To investigate the microbiome and host responses, they use:

  • Computational multi-omic investigations of the clinical samples
  • Novel intestinal organoid models to explain host–microbial crosstalk and the mechanisms of gastrointestinal disease

“We culture ‘organoids’ – also called enteroids – in our lab. This is an incredibly exciting technology we’ve been working on for six years, where you take tissue from a person’s gut and isolate stem cells from the epithelial crypts. These same cells used to regenerate your gut, self-organize into ‘3D mini guts’ in the lab, which are called organoids. In essence, this allows us to grow intestine from a person in a dish for experiments.”

Patient-derived organoids retain the genetic background of that person and contain most cell types of the epithelium. Most importantly, co-culturing them with human endogenous microbes allows for exciting new studies on microbial-host interactions, such as the team’s research on breast milk.

The co-culture system is a unique device that allows us to study the human organoid and bacteria at the same time. The gut is anerobic – most bacteria would die in the presence of oxygen. But the cells that line the gut need oxygen from our bloodstream. So the whole system is housed in an anaerobic chamber to be able to replicate this oxygen gradient, to see what’s going on in real time.
NEC and the preterm gut

For significantly premature babies, especially those born around 24 weeks gestation, there’s a high risk of developing NEC disease. In infants surviving only a few days, NEC is common and dangerous. It is, in fact, the leading cause of death in this age group.

“NEC is a really difficult disease to understand,” says Dr. Stewart. “In fact, it’s probably more than just one disease, and the underlying pathophysiology may well vary in different cases. We are trying to subtype NEC better, collecting really extensive phenotypic data on patients to see if there are different modalities to it, and which ones the microbiome plays into.”

Immature bowels are prone to “leaky gut,” poor blood circulation, digestion and infection, all of which increase a preterm baby’s chances of developing NEC. NEC can progress very quickly when the tissue in the inner lining of the small and/or large intestine becomes inflamed. This can damage parts of the bowel so much that tissue within it dies, causing a perforation and a medical emergency. “The most common site for NEC in premmies – or preemies – is at the end of ileum, although it can affect any part of the ileum and even the whole colon,” says Dr. Stewart.

His extensive research on the ileum and other samples has shown “strong associations” of harmful and beneficial microbes with disease. For the latter, Bifidobacterium microbes and Human Milk Oligosaccharides (HMO) are associated with protection from both NEC and LOS. Now, new tools such as lab-grown organoids are helping to move his research on from associations to discovering specific mechanisms of disease and prevention.

Diet and the benefits of breast milk

In a sterile incubator, preterm infants have very limited environmental exposure. So the gut microbiome is colonized only with a few bacteria from the environment (mother and hospital-acquired) and from diet (formula or breast milk). Often the undeveloped gut’s mucus layer is depleted and thin, so the epithelial cells are vulnerable.

If the immune system recognizes bacteria at the cell surface, it can mount a huge inflammatory response. Although designed to clear infection, this response has damaging side effects like reducing the oxygen flow to the gut, causing necrosis. So how can this be prevented?

“We first looked at breast milk in 2013,” says Dr. Stewart. “With its beneficial effects in mind, and following up on seminal work by Prof Lars Bode at UCSD, we looked at it again more recently – this time studying the many bioactive components in it that can directly improve health. And most importantly, how breast milk components such as HMOs and IgA (secretory immunoglobulin A) modulate the microbiome to improve health.

At Newcastle, we have over 20,000 breast milk samples to work with, and what we learned was that certain sugars, or HMOs, were incredibly important to the microbiome. Our research moved from 90% infant focus and 10% mother focus to 50/50. We validated that DSLNT (disialyllacto-N-tetraose) is one of the sugars that seems to be particularly beneficial, and expanded previous work to show it can modulate infant microbiome and potentially promote Bifidobacterium. We’re looking for ways to increase the quantity of this component.

Unfortunately, DSLNT is in low abundance in breast milk and it’s expensive to synthesize with current technology, so we can’t produce it at scale yet. But we’re researching how to boost it by mother’s diet, for example, and by screening donor breast milk.”

“In the infant microbiome, we’ve found Bifidobacterium microbes resulting from breast milk that might protect preterm infants from disease. But we’ve also identified a community of microbes we think could be harmful to preterm infants. The organoids allow us to do a high volume of experiments, testing how microbes respond to each other while tracking gut inflammation – and thankfully all without the use of animals. Being patient-specific, our experiments also take into account the genetic and epigenetic backgrounds of individuals,” Dr. Stewart says.
Current research

“At the moment, we’re starting to see supplements being used in neonatal units as therapies. About 30% of units in the UK administer probiotics, including ours, which contain Bifidobacterium. So the importance of the microbiome is being recognized and probiotics are readily available, relatively cheap and overall considered safe. But we’ve still a lot to learn about probiotics and prebiotics – like HMOs. The bacteria need to feed on these to fully colonize the gut,” says Dr. Stewart.

In terms of biomarkers, DSLNT holds great promise. By screening mother’s milk it’s possible to identify babies most at risk of developing NEC based on this one sugar. In a recent paper, Dr. Stewart’s team reported they were able to predict this with a specificity and sensitivity of 90%.

In Newcastle University, the team has access to the Great North Neonatal Biobank containing 12 years’ worth of samples from local hospitals. Aside from breast milk, it includes over 30,000 stool samples from babies, as well as urine, blood and baby tissue samples collected during surgery for NEC, milk-curd obstructions and stoma reversals. Working closely with clinicians, the team can get daily samples from early preterm babies, who can be in incubators beyond the first 100 days of life.

“Historically, we processed samples manually using QIAGEN’s DNeasy PowerLyzer PowerSoil Kit. We tested several kits but found this to be optimal in terms of yield, robust microbiome profiles, plus the spin columns never clogged. These days in our high-volume microbiome work, we rely on automation using the DNeasy 96 PowerSoil Pro QIAcube HT Kit with the QIAcube HT for nucleic acid extraction, and the QIAgility for PCR setup. The PowerSoil Kits give very good yields and consistent results, and we have comparability with everything we’ve done in the past. We also use RNeasy Mini Kits to extract RNA from our organoid monolayers for RNA sequencing,” says Dr. Stewart.

To find out more about NEC, breast milk and the preterm infant gut, you can view Dr. Stewart’s presentation given at QIAGEN’s Human Microbiome Days, 2021.

About Dr. Christopher Stewart

Dr. Christopher Stewart has researched the early life microbiome in health and disease for more than a decade, specializing in infants born prematurely (<32 weeks gestation). During that time, he’s published over 70 peer-reviewed manuscripts and has regularly presented his work at international conferences.

Dr. Stewart is a team leader at Newcastle University (UK) and his lab is in the Translational and Clinical Research Institute (Faculty of Medical Sciences). Applying state-of-the-art sequencing to clinical samples from a large biobank, collected at the Royal Victoria Infirmary (Newcastle upon Tyne), his research has shown that specific microbes are associated with protection from both NEC and LOS.