Microbiome Movement Skin Health & Dermatology Conference Summary: Days 1 & 2

Microbiome Insights was a proud founding partner of the Microbiome Skin Health and Dermatology conference, a part of the Hanson Wade Microbiome Movement Series—held September 10th to 12th, 2018. The aim of this event was to explore ways of understanding the mechanisms underlying microbial interactions with skin health, and to discuss standardization of metagenomics research and how to develop effective products. Over 100 leaders in the skin microbiome field were on site at this exciting event.

We’re pleased to present a summary of the highlights from both the pre-conference workshop and the two-day main event.

Pre-conference workshop

The September 10th workshop, called “Harnessing the gut-skin-brain axis in health and disease”, was led by Lionel Breton, L’Oreal Advanced Research, Scientific Director. Breton spoke about what we know today regarding the connections between the skin microbiota, gut microbiota, and neurological disorders. Scientists are puzzling through these links, on the path to developing commercially viable therapeutics for skin health and brain health.

Day 1 summary

The first day of the conference began with opening remarks by Larry Weiss of Persona Biome: he acknowledged that the skin microbiome represents an exciting therapeutic frontier in dermatology, but encouraged the audience to be cautious and exercise humility—because after all, “we still don’t know what we don’t know”.

Richard Gallo of UCSD then spoke about “Targeted Design of Microbial Biotherapy for Skin Disease”. Gallo began with an overview of how microbes relate to human health in general: he argued that researchers need to look at the ‘hologenome’, as it takes into account that human functions are based on genes that are expressed via microbes and the environment.

Gallo turned specifically to the microbial life surrounding hair follicles on the skin. He noted that human follicles greatly increase the surface area of the skin; when the microbes in follicles were analyzed using 16S DNA sequencing, it appeared that most of the microbial DNA did not exist on the surface, but rather, it existed deep down in the follicle. A very small percentage of the microbes were further down, penetrating the fat and tissues under the follicle. The composition of the microbes in the fat was similar to that found on the surface.

Gallo emphasized the capacity for systemic interactions linked to the skin microbiome, as the blood vessels on the skin can have extensive communication with, and impact on, the rest of the body. He gave the example of atopic dermatitis: not only do people with this condition have a higher abundance of Gram-positive bacteria (S. aureus) than other people, but Gallo’s work showed that they also have an immune abnormality that inhibited the development of antimicrobial peptides to fight the disease. The presence of S. aureus drives a Th2 immune response, which further decreases the antimicrobial peptides and amplifies the disease. So what appears to be a skin disease is actually a complex condition with systemic influences.

Next up was a talk by Curtis Huttenhower of the Harvard TH Chan School of Public Health, a member of the Microbiome Insights Scientific Advisory Board. He presented on “Structure, Function and Diversity of the Healthy Human Microbiome”. Huttenhower began by explaining the phases of the Human Microbiome Project (HMP): while HMP 1 had the goal of identifying the baseline microbiota of a healthy cohort of individuals, HMP 2 included more multi-omics analyses, and had a longitudinal focus, aiming to uncover interactions in disease. HMP1-II was a recent follow-up on the HMP 2 data; it involved 300 people (half men, half women). Huttenhower noted that the field has seen great advancements since the kick-off of HMP 1, and argued that we are getting to the point where scientists can do meta-analyses to see which methods are more reproducible and reliable.

Specifically related to the skin microbiome, Huttenhower said we have a long way to go—not only in characterizing the ‘healthy’ skin microbiome, but also in defining its biochemistry. Novel functionality is associated with certain strains. Ongoing work is looking at how the skin microbiome may be acquired in the first place, by examining mother-infant pairs. Initial analyses indicate the strains of most bugs on the infant do not originate from the mother.

Julia Oh of The Jackson Laboratory spoke next, on “The Human Skin Microbiome: From Metagenomes to Therapeutics”. Oh emphasized the differences in composition of the skin microbiome, depending on body site: oily sites, for example, are very different from dry sites.

She advised that when looking at therapeutics, skin microbiome researchers should consider factors like site specificity, diversity of the microbiome, and stability of the community. For instance, in those with atopic dermatitis, the skin microbiome looks different depending on whether you measure it at baseline, during a disease flare (when relative abundance of S. aureus increases), and post-flare.

Oh offered some general criteria for what organisms to focus on in the development of new skin therapeutics:

  1. An organism that grows well on the skin—that is, on a graph of time versus growth, an organism that has a U-shaped trajectory (rather than flat).
  2. An organism that is beneficial to the immune system. Their group looked at T cells and measured the rate of MAIT activation with 4000 different species.
  3. An organism that is innate. Most skin sites are stable over time, so the challenge is integrating a foreign species into a community. Previous experiments show it’s difficult to get something to colonize on the skin.

The set of talks was rounded out by Microbiome Insights CEO Malcolm Kendall, on the topic: “From Swab to Data: Considerations for Designing Skin Microbiome Studies.” He emphasized looking at skin microbiome studies from many angles: study design (including the power), collection, stabilization, transport, sequencing, and analysis.

He noted the in-house work of Microbiome Insights that investigated the total DNA as well as the microbial content while using swabs versus tape for a skin microbiome sample. Results showed little difference in total DNA content across systems, but higher microbial content (less host content) on premoistened swabs. Of course, this approach is useful for skin surface microbiome but may not be the best method to look at the follicle microbiome.

Kendall says he has noticed a lot of debate surrounding the variable region that will provide the most fruitful information for skin microbiome analysis using 16S. Microbiome Insights has done some internal R&D work to address this issue: the outcome of which is a new ‘V4_skin’ primer that provides better species-level identification of skin taxa while maintaining low error.

An intriguing panel discussion was held later that morning: “Where will the Skin Microbiome Need Standardization to Advance Science & Future Products?” It was led by Huttenhower, joined by Amanda Nelson of PennState.

Participants emphasized the problem of contamination, since skin microbiome samples have a low microbial biomass; researchers need to include positive and negative controls for ‘human’ and body site. The panelists covered the relative advantages of different collection techniques, sequencing techniques, and culture libraries. For bacteria and skin fungi, libraries are improving, but viruses change so quickly that they won’t be covered in reference libraries. Integrating different data (using multi-omics analyses) will lead to better insights.

In the afternoon of day one, participants heard from Huiying Li of UCLA, on “The Human Skin Microbiome in Health and Disease”. Li spoke about acne in particular: they found individuals with acne and those with healthy skin had significant differences in the facial microbiome at the strain level; there are genes that are differentially expressed.

Later, Pieter Dorrestein of UCSD moved the topic to metabolomics—he spoke about “Microbial Metabolites of the Skin Microbiome – Identifying Skin Chemistry to Search for Function.” He described how his group is taking several hundred swabs from the bodies of two individuals and translating the mass spectrometry data onto a 3D model—with the ultimate aim being to find the origin of the molecules. A combined metabolomics and 16S analysis showed no changes when the face and arms were treated with certain personal care products; however, there appeared to be a deodorant-dependent change in the armpit skin. In general, it appears that the profile is resilient, but that certain lifestyle factors can indeed impact the skin.

Day 2 summary

The second day of the Skin Health & Dermatology meeting featured another great lineup of speakers addressing different aspects of the skin microbiome. Amitabha Majumdar, Senior Research Scientist at Unilever, presented on “Commercializing Microbiome-based Beauty & Personal Care Products”.

Majumdar spoke about some of the major challenges in microbiome-focused product development:

  1. Finding the right target: In looking for the cause of malodour, the company found certain microbes, molecules, and pathways that were responsible; when they had the right target, they developed products (like Dove deodorant) to address the problem.
  2. Hitting a target better: In terms of acne, Majumdar says they found that lesions were reduced after a particular 3-week treatment that included natural oils. The company then took two natural oils that were part of this treatment and added them to two facial products already on the market.
  3. ‘Rebalancing’ the microbiota: One of the company’s toothpaste products were studied clinically—and they found that after 14 weeks, some of the bacteria associated with oral health were altered.

Majumdar’s talk was followed by one from Alex Goddard, VP, Research & Development at AOBiome: “Using Ammonia Oxidizing Bacteria to Restore Healthy Skin”. Goddard started with the premise that we are being deprived of certain metabolites because of the nature of our hygiene practices, and this may be altering our overall immune system and general health. He reported that David Whitlock, one of the company founders, noticed animals like horses roll in the soil to relieve an itch on the skin. This led to an investigation of whether the soil somehow had a therapeutic effect—and to the potential of ammonia oxidizing bacteria. These bacteria produce nitric oxide (which is anti-microbial and anti-inflammatory).

The company’s general approach is to discover new environmental bacteria that can be used on the skin. Goddard cited some of the challenges in developing new therapeutics: determining mechanism of action (whether direct or indirect); dosing; and variability in patients (from a physiological and microbial standpoint).

Then, Livia Zaramela, postdoc at UCSD, spoke on “The Role of the active Microbiome in Skin: Emphasis on Atopic Dermatitis”. Zaramela addressed the connection between food allergy and atopic dermatitis, as about one third of children with atopic dermatitis have food allergy. Their group uses a multi-omics approach to identify whether food allergies are intrinsically linked to atopic dermatitis or not.

Zaramela discussed how to overcome the challenges of low biomass samples for metatrascriptomics analysis: they maximize collection, extraction, and mRNA enrichment, and they work to minimize contamination. In terms of contamination, it’s necessary to reduce human content in the samples, but also reagent contamination.

Another speaker in the morning session was Magali Moreau, Associate Principal Scientist, Open Research at L’Oreal. She spoke on “Human Skin Microbiome: Opportunities for Healthy Skin with Aging.”

Moreau described how the company is looking at the skin microbiomes of women of all ages. At four different skin sites across two age groups, the distribution of Staphylococcus appeared to change significantly, while the proportion of Cutibacterium at each site decreased with age. Diversity was higher in the older group, across all sites. Thus, the trend with aging seems to involve a decrease in sebum and an increase in skin microbiome diversity, with some oral bacteria increasing on the skin.

She then addressed how to translate this growing microbiome knowledge into products. Approaches include fostering the growth of beneficial bacteria, controlling the community metabolites to bring back ‘equilibrium’, or perhaps using the virome for precision modulation.

Stephen France, Business Development at SkinBioTherapeutics, was the next speaker, on “Harnessing the Power of the Microbiome for Skin Health.” He focused particularly on some interesting observations on lysates (i.e. fluids containing the contents of lysed cells). They found lysates could inhibit pathogens and change the skin barrier; pretreatment of the skin with lysates protected against S. aureus invasion.

A talk from Greg Hillebrand, Senior Principal Scientist at Amway, capped off the morning sessions: “Changes in the Facial Skin Microbiome: A One-Year Longitudinal Study in Normal Healthy Men and Women.”

Hillebrand began by describing the company’s investigations into the meaning of skin health, with input from 70 individuals. The resulting points were: “it has to perform, but needs to be resilient when stressed; it needs to look even color-toned, and fairly unremarkable (you don’t notice your skin)”.

Hillebrand described the company’s ‘Cinco de mayo’ study, done in collaboration with Microbiome Insights. Skin samples from the forehead and cheek were taken from 150 Amway employees (aged 20 to 60) in 2017, with a repeated measure on 137 of the individuals in 2018. They measured other parameters like elasticity. Microbiome analysis using 16S V4 and V1-V3 revealed overlap in the microbial communities of the forehead and cheek, with some differences. Individuals’ skin microbiomes were relatively stable, and the individuals who had low diversity of their skin microbiome in 2017 also had low diversity the following year.

Barrier function was stronger with a more diverse skin microbiome; as water loss went up, barrier function went down. The other striking finding was that bacteria from the genus Corynebacterium increased with age. Amway is looking to build on these findings, with Microbiome Insights acting as an external R&D arm, to rapidly develop microbiome-focused solutions for skin health.

Better skin microbiome analyses using new 16S V4 region primers developed by Microbiome Insights’ scientific team

Over the past several years the Microbiome Insights team has invested in the development of new tools and techniques for obtaining high-quality, actionable skin microbiome data for our partners and clients in the cosmetics and dermatology industry.

When designing a new skin microbiome study, we always have an important discussion: which variable region should be sequenced? Although many assume that, for characterizing skin bacteria, primers targeting regions V1-3 are superior to those targeting the V4 region, it’s not so straightforward.

All current primers have their limitations—namely, that they underestimate the abundance of some skin-dwelling bacteria, poorly capturing skin commensals.

Our team members Pedro Dimitriu and Hilary Leung redesigned the V4 primer pair under the direction of Microbiome Insights co-founder Dr. Bill Mohn, and found that the new primers resulted in the detection of more bacterial genera, while improving error rates. The new primer also addressed a main limitation of common primers used for the v4 region: it can detect Propionibacterium acnes—the most abundant human skin bacterium.

Thus, we are now pleased to offer our clients this exclusive V4_skin primer in order to help them make the most of their skin microbiome surveys.

Improved bacterial 16S rRNA gene (V4 region) primers for skin microbiome surveys

Download the PDF version of this v4_skin poster.

If you’re thinking about designing a skin microbiome study, be sure to read our blog posts on both sampling and amplicon sequencing.

Contact our scientific team to learn more, or catch us in person at the upcoming Hanson Wade Skin Health & Dermatology Conference, September 10th to 12th in San Diego!

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

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!

About the company

Microbiome Insights provides state-of-the art microbiome analysis and bioinformatics.

Our end-to-end service starts with experimental design and sample collection and extends to data analysis and bioinformatics interpretation.

Microbiome Insights is focused on providing our clients with a deeper understanding of functions and interactions of microbial communities across a range of human, animal, agricultural, and environmental research applications. Our team of experts and testing methods combine to provide fast, dependable, cost-effective results with highly comprehensive, publication quality bioinformatics. To learn more, see here.

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

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.

About the company

Microbiome Insights provides state-of-the art microbiome analysis and bioinformatics.

Our end-to-end service starts with experimental design and sample collection and extends to data analysis and bioinformatics interpretation.

Microbiome Insights is focused on providing our clients with a deeper understanding of functions and interactions of microbial communities across a range of human, animal, agricultural, and environmental research applications. Our team of experts and testing methods combine to provide fast, dependable, cost-effective results with highly comprehensive, publication quality bioinformatics. To learn more, see here.