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How microbiome science is leading to human nutritional innovations

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We all know food plays a critical role in health, but can anyone really answer why or how it is so important? Scientists have made numerous links between particular foods and health outcomes over the past few decades, enabling dietitians and other professionals to give guidance, with some level of certainty, toward dietary choices that will maximize their ability to live longer, fuller lives. But many of these links between diet and health remain associative, gleaned from epidemiological studies with little evidence of causation. One example is dietary fiber—while it is clear that a higher consumption of fiber is associated with a myriad of health benefits and even lower mortality, the underlying mechanisms have remained elusive.

Microbiome science is playing a role in changing this uncertainty. Population cohort studies show that, aside from medication, diet is the leading environmental factor that predicts the composition of the gut microbiome from person to person. Meanwhile, experimental studies are showing the gut microbiome influences various aspects of human immune and metabolic health. Thus, many see the potential for diet to become a powerful means of manipulating the human gut microbiome for better health.

Under this framework, a number of companies are already focusing on the development of foods that better support the microbiome and health at different points in the lifespan, from infancy to older adulthood. Microbiota-modulating ingredients may be deliberately included in foods or concentrated into supplements—and then, of course, tested for their measurable effects on the health of the host.

Thinkstock image, used with permission

Below are some examples of microbiome-enabled innovations that may be around the corner for nutrition, both for healthy populations and for those with disease:

Nutritional interventions that support brain health

Alzheimer’s disease places a significant burden on both health services and patients’ family members, and prevalence is estimated to increase dramatically in the coming decades. Could nutritional interventions play a part in preventing this condition? Jared D. Hoffman of the University of Kentucky (USA) is studying a microbiota-modulating prebiotic intervention as a possible way to prevent Alzheimer’s disease in those who carry the APOE4 gene; in a mouse model, he found that increased intake of inulin led to gut microbiota alterations (e.g. more Bacillus subtilis), increased scyllo-inositol (which moves through the blood-brain barrier into the brain), and decreased amyloid beta in the brain.

Supplements to change the microbiome for healthy aging

While extremely healthy older individuals show gut microbiota compositions that resemble much younger individuals, it’s not clear whether the gut microbiota itself confers youthfulness or good health. However, interventions targeting the gut microbiota may show promise. One company recently carried out a placebo-controlled clinical trial of its resistant starch product in older adults, testing both gut microbiome changes and the resultant health effect; they found increases in bifidobacteria, which accompanied significant differences in blood glucose, insulin levels, and insulin resistance.

Dietary therapeutic interventions for IBD

Anecdotally, many people with inflammatory bowel disease (IBD) report an influence of diet on their symptoms; yet dietary research in IBD has been inconclusive, and diet is currently not a part of the standard therapeutic regimen for IBD. The exception is with exclusive enteral nutrition (EEN) for certain cases of Crohn’s disease—this diet appears effective at inducing remission. Gut microbiota is under investigation as the mechanism behind this effect, with recent research suggesting the gut microbiota of the patient at baseline may predict the success of EEN. In future, the idea of using microbiome composition to predict responders may be extended to other food items or dietary patterns in IBD, expanding the therapeutic toolbox for both Crohn’s disease and ulcerative colitis.

Nutritional interventions that boost bacteria with therapeutic potential in obesity and metabolic disease

Akkermansia muciniphila is a member of the human gut microbiota that may have particular importance in metabolic health; it stimulates butyrate production and prevents the development of obesity in animal models, and is currently being tested in a human clinical trial. Many regulatory hurdles, however, will need to be cleared before bringing these “bugs as drugs” to market. In the meantime, research may help uncover specific nutritional interventions that can increase Akkermansia in the gut environment for potential effects on metabolic disease and obesity.

These are just some examples of experiments in food science that have shown manipulation of health by way of the gut microbiome. As such, leaders in the food industry are beginning to use microbiome science to develop products that better support health, but not all companies have the internal expertise to tackle the challenge. Microbiome Insights addresses this gap in expertise by working closely with clients to plan and execute all aspects of nutrition-related microbiome studies. Our team is collaborating with several industry clients to help advance new food products and supplements that enhance health through the microbiome—confident that microbiome science will help guide us toward knowledge of who should eat what, and when, for better health.

 

How to Design a Skin Microbiome Study, Part II: Amplicon Sequencing

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In this post, the second of a 2-part series on skin microbiome research, we will discuss technical issues surrounding sequencing of human skin microbes. Read the first blog post here.

At this point, the microbial ecologist conducting a skin microbiome study has now collected all the skin samples she needs, and the DNA has been extracted. We turn to the question of how to decide on the sequencing strategy. Metagenome shotgun sequencing, in which the entire community of microbes is sequenced in an untargeted manner, can provide invaluable information about the functional potential of the microbiome, but – despite continually dropping sequencing costs – it is still expensive. The researcher in this case settles for 16S marker gene sequencing, which targets a specific region of the gene. Now, which primer pair should she choose?

The current dogma in the field is that primers targeting regions V1-V3 are better at describing skin bacterial communities than the V4 region primer pair. (The V4 region is commonly used for studying gut communities and other environments.) This is because V1-V3-sequenced communities better recapitulate the taxonomic composition and relative abundance of “mock community” controls (Meisel et al., 2016). And V4 primers poorly amplify typical skin microbes, notably Propionibacterium and some Staphylococcus species (Meisel et al., 2016). But should V4 be discarded in favor of V1-V3?

The reason behind the V4 region’s underestimation of Propionibacterium is a single mismatch at the end of the primer that prevents efficient binding to a specific group of bacteria. To evaluate if V4 region may be a suitable target for characterizing skin bacteria, our team re-designed the V4 primer pair and tested in silico its ability to improve the coverage of underrepresented propionibacteria. With these new candidate primers, we are able (theoretically) to increase the coverage of Propionibacterium to over 67%–from less than 3%–without losing coverage of the other bacterial groups. Our next step is to evaluate the accuracy of this approach using a mock community as the standard.

There are advantages to using existing V4 primers. They can detect the genera Finegoldia and Peptoniphilus, which are increased in persons with primary immunodeficiencies (Oh et al ., 2013). Zeeuwen et al., citing previous work, also pointed out that the 27F primer used for the V1-V3 region inefficiently amplifies Gardnerella and Lactobacillus, which have been found to be associated with females (Zeeuwen et al., 2012). In general, V1-V3 classifies fewer populations down to the genus level (Meisel et al., 2016). Because the V1-V3 region is longer than the V4 region, paired-end reads generated with the Illumina MiSeq will not fully overlap. And without full overlap, denoising of reads is not as effective. Using the V3 chemistry (a 600-cycle kit, longer than the 500-cycle kit of the V2 version) will not solve the problem and may even make it worse, because the sequence quality drops after 500 cycles.

In this two-part blog series, we have discussed how to collect enough microbial biomass to run a skin microbiome study, and how to deal with environmental contamination. We have seen that even relatively minor changes in primer sequences may improve the detection of bacteria relevant to skin microbiomes. Feel free to reach out to our team for more information on designing your own skin microbiome study!

How to Design a Skin Microbiome Study, Part I: Sampling

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In this post, the first of a 2-part series on skin microbiome research, we will discuss technical issues surrounding sampling of human skin microbes.

Let’s say a researcher sets out to study bacteria on the human skin—the body’s largest organ, which is teeming with microbes from each domain of life, and viruses. The scientific question has been identified and the funding to conduct a pilot study has been secured. Perhaps her group has some experience studying the gut microbiome. For the most part, she has discovered, getting bacteria out of stool is not too difficult; a small amount of material contains enough microbial DNA to sequence the most prominent members of the microbiome. But unlike the intestine, the skin does not support a high-biomass microbiome. If microbes on the skin are present in low abundance, how does our researcher decide on reasonable sampling and sequencing strategies that together capture a representative picture of bacterial diversity?

Before collecting samples, the researcher must consider key advantages and limitations of available sampling protocols. Commonly used methods involve variations of swabbing – the repeated rubbing of a defined area of the skin with a sterile, pre-moistened swab. When the objective is to obtain sufficient microbial DNA from skin sites with variable and/or low microbial biomass, swabbing can be complemented with scalpel scrapping (Oh et al., 2014). If access to deeper layers, including the dermis, is required, punch biopsies are a viable alternative, but require specialized expertise and are more invasive, reducing the number of sites that can be sampled from the same subject.

Because each method samples a slightly different environment, we would expect different microbial profiles to arise from variations in sampling method. This prediction has not been thoroughly tested (but see Chng et al., 2016). As part of ongoing efforts to improve sampling methods, the Microbiome Insights team is currently evaluating whether D-Squame and Sebutape tape stripping, used for peeling off epidermal layers and sebum, respectively, provide a reliable means of sampling skin microbes. Compared with swabbing, tape stripping recovers ~2- to 3-fold less bacterial DNA. We have yet to evaluate, via amplicon sequencing, whether lower bacterial yield results in different microbial profiles. We are also exploring if coating pre-wetted swabs with aluminum oxide particles maximizes bacterial DNA recovery. The results of this experiment will be made available in a forthcoming technical note.

Handling samples with low microbial biomass is challenging. Even if the sampling method affects how much microbial biomass is collected, the amount of DNA recovered from skin is always low. (Of course, the DNA extraction method affects DNA yield and microbial composition from study to study. But because most studies are comparative in nature, methodological consistency is vitally important.) As most of the DNA is human, obtaining enough genetic material for microbial profiling can be difficult. Microbial load can be increased by instructing participants not to wash with soap or bathe at least 24 hrs prior to sampling, although the effect of cleansing is probably minor compared with the combined influence of sampling and extraction.

Another challenge of low microbial biomass samples is dealing with environmental contamination. Contamination can be introduced during sample collection, DNA extraction, and sequencing library preparation. For instance, bacterial DNA is often found in DNA extraction kits and in other reagents used for preparing samples. And while it is tempting to create lists of “usual suspect” contaminants, this may be futile when studying skin microbes or other human-associated bacteria because, for example, Staphylococcus, a common skin inhabitant, and Escherichia have been identified as potential kit reagent contaminants (Salter et al., 2014).

Processing negative controls alongside low-biomass specimens is critical, because the proportion of microbial DNA attributable to contamination is higher in low-biomass samples compared with high-biomass samples. Usually, we include at least four replicates for each of two types of negative controls on each 16S sequencing run: (1) DNA extraction controls, to assess if kit reagents carry a detectable signal, and (2) template-free PCR blanks, to pinpoint contamination that may arise during downstream processing. For skin microbiome analysis, sterile swabs opened at the site of sample collection are co-processed with the swabbed samples. In general, the number of sequencing reads in our negative controls is about 3- to 4-fold lower than the average in samples derived from skin sites. This is what we would expect for samples containing little to no DNA.

Stay tuned for the second post in this series: amplicon sequencing in skin microbiome studies.

Microbiome Insights’ Year in Review: 2017 by the numbers

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This year, 2017, will go down in Microbiome Insights history as the year the company hit its stride and established itself as a leader in the microbiome testing field. The challenging work our team has been doing since we were founded in 2015 clearly paid off—as evidenced by our expanding client list, our strong financial position, and our team’s awards and recognitions.

In the past twelve months we have had to more than double the size of our Vancouver-based team, hiring some of the best technicians, scientists, and bioinformaticians to meet the needs of our clients.

 

Here are some highlights of our company’s 2017, by the numbers:

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Place out of 150+ companies in the 17th annual BCIC-New Ventures Competition, the largest and longest-running competition for tech companies in BC. The judges, who award $300,000 in cash and prizes to early-stage start-up companies, honoured Microbiome Insights with second place overall and with the Center for Drug Research and Development (CDRD) Life Sciences prize.

 

8

Number of conferences Microbiome Insights attended as exhibitors or sponsors. Our team enjoyed meeting new and existing clients at important academic and industry conferences in all corners of North America—from ASM Microbe 2017 in New Orleans to the Microbiome R&D and Business Collaboration Forum in San Diego. We also trailblazed as one of the first microbiome-centered companies to attend two genomics events: ASHG 2017 in Orlando and the 3rd annual Understand your Genome conference in Boston.

9

British Columbia (BC) life sciences companies (including Microbiome Insights) on the ‘Ready to Rocket’ 2017 Life Science Emerging Rocket List—a business recognition program that profiles technology companies in BC with the greatest potential for revenue growth. These “Emerging Rocket” companies are recognized as having a clear business model and go-to-market strategy that will prove attractive to investors.

 

125

Number of microbiome studies our team supported this year—across a broad range of human, animal, agricultural, and environmental applications. We are very proud that many of these were repeat clients.

 

750,000
Number of dollars invested in our company by Genome BC, following successful equity financing. In combination with funds from the company’s recent round of equity funding, this will help build out our new CLIA-certified lab facility, grow our team and capabilities, and develop and launch new services and tests in 2018.

 

Here are some of the technical insights from our team this year:

  • The staying power of amplicon-based analyses: For studies whose primary objective is to get a comprehensive picture of the bacteria in an environment, or to build models of disease classification (for many diseases) data derived from 16S genes is as good as it gets
  • The power of multi-omics integration: Although it still poses technical challenges, integration of multiomics datasets can reveal things far beyond what each technology can show separately

And finally, here are our team’s picks for the hot areas to watch in microbiome science in the coming year:

  • The contribution of microbes to “inflammaging”—the progressive increase in pro-inflammatory status that occurs with age
  • Progress toward microbiome therapies that modulate the brain through the gut-brain axis
  • Larger and longer studies on the skin microbiome, looking at lifestyle factors and making more definitive associations between skin microbes (composition and function) and healthy skin
  • Tracking the presence of viruses in the gut microbiome and gaining insights about health implications

A very happy 2018, from our team to you!