635 THE ROLE OF THE SCALP MICROBIOME IN HEALTH AND DISEASE SKIN BIOLOGY Skin is a multilayered covering consisting of a “permanent” nonrenewing dermis covered by a continually renewing epidermis. The dermal layer is 2–3 mm thick, the epidermal layer 0.1–0.2 mm thick, and the overlying stratum corneum 10–20 μm. As the dermis is a nonrenewable layer, it ages along with our body and is primarily responsible for aging issues such as wrinkles and sagging. At the dermal or epidermal junction are stem cells that divide and differentiate into the tough outer stratum corneum, the sort of plastic wrap that covers our body. The entire epidermis will renew itself about every two months, but the stratum corneum should renew itself every 14 days. In “hyper proliferative” skin disorders such as dandruff, instead of renewing every 14 days, the stratum corneum will renew itself in only six or seven days (1). It is generally accepted that the microbiology on skin resides in the stratum corneum (2,3). However, skin consists of many different environmental niches. Forearm skin is dry, it does not have sebum, and it can be considered desert wasteland and challenging environment for microorganisms. But when we consider the scalp, we have heavy sebaceous secretions and hair occluding and holding in water. Thus, on the scalp, there is an optimal environment for microbiology (4,5). Another important consideration is we think of skin as a singular organ. If one were to remove the skin and spread it out, it would be about 2 m2, and, for many years, that is how it was considered. But if one considers the topography, follicular openings, invaginations, and ridges, the surface area is closer to 25 m2, on par with the gut and lung as a surface area for host–microbe communication (6). SKIN MICROBIOLOGY First, a few terms should be defined. A “microbiome” refers to the collection of microbial genomes present in a system. Hence, the “microbiome” does not do anything it is a means of identifying what organisms are present. The “microbial community” is the collection of organisms, living and dead, that functionally interact with the host environment, and each other. What is the microbiology living on or skin? There are on the order of 1–10 million microorganisms per cm2. One reason skin microbiology is considered an easier research is because it is easier to sample. You can use a swab, tape strip, cup scrub, or even a q-tip and sample right off the skin’s microbiome. It is also complicated by the specific environment, immune system, and sex, and many sites are completely structurally different. As one reads and learns about the skin microbiome, it is imperative to understand where people are sampling, what they are looking at, and how they are looking at it (3,7). After sampling, the primary method for microbiome analysis is nonculture-based next- generation DNA sequencing, usually by one of two methods: amplicon or metagenomic. In amplicon sequencing, DNA is isolated from the sample and amplified via PCR to isolate unique microbial genomic regions. The resulting sequences are compared to existing databases to identify and count the different genomes. The primary insufficiencies with amplicon sequencing are that there may be microbes that are not amplified, as they are too different, fall out of consideration because of amplification bias, or do not exist in the

636 JOURNAL OF COSMETIC SCIENCE database. In shotgun metagenomics, the entire DNA sample is putatively sequenced without amplification. The main issues with shotgun metagenomics are the lack of organisms in the current databases and the lack of any quantitative nature. The amplicon databases, referred to as 16S for bacteria and ITS (intergenic transcribed spacer region) for fungi, are much more comprehensive than full metagenomic databases, particularly for eukaryotic organisms. Keep in mind that microorganisms are being discovered at a phenomenal rate, with many still unknown. When sequences are not found in the databases, they are clustered into “dark matter” and disregarded. Hence, there is often more uncharacterized dark matter in metagenomics than in amplicon sequencing. Finally, in shotgun metagenomics, the output is diversity, the relative abundance of population members. This does not provide any information on quantity of microbes, which remains an important part of microbiology. Using both techniques, one finds that the human skin microbiome generally consists of 93–97% bacterial genomes and 3–7% fungal genomes, and this very much depends on the body site (with fungi populating the oily sebaceous areas, including scalp, face, and back). The vast majority of the fungal community are Malassezia, with a more diverse bacterial community dominated by Cutibacterium acnes various Staphylococci (4,8). FUNGI AND MALASSEZIA Fungi is a vast kingdom. There are many different types, including mushrooms, mold, smut, and rusts. A famous toxic mold is Stachybotrys chartarum, a very dangerous mold to have in one’s basement. Smuts and rusts are major agricultural pests, causing billions of dollars per year in damage to crops such as rice, corn, and citrus. Malassezia are yeasts, and, importantly, closely related to many plant pathogens. There are 18 known Malassezia species and dozens of functionally diverse strains (9). This article now returns to the topic of nonculture-based microbiome detection methods: amplicon and metagenomics sequencing. With either method, the percent of the microbiome defined above is based as the number of genomes, implying there are 10 to 100 times more bacterial cells per skin unit area than fungal cells (8,10). An important difference is that fungi are gigantic compared to bacteria. The volume of a sphere varies with the cube of the radius and the volume of the cylinder with the square of the radius. So, when something is a few microns larger in diameter, it is tremendously larger in volume or biomass. Malassezia globosa or Malassezia furfur is conservatively a thousand times larger than bacteria (11). If there are 100 times more bacterial cells, then there are almost certainly at least as much interactive biomass from fungi. The biomass interacts with us, not the genome. Another thing about mycology is that few people study it. Less than 10% of the microbiome publications consider fungi, and when one adds in skin, you get less than 1% of microbiome publications relating to skin fungi. In fact, fungi are so poorly investigated that in 2017, some students in California went to the grocery store and bought a packet of porcini mushrooms and sequenced their genomes. They identified seven previously undescribed species in a porcini packet on a grocery store shelf. Finally, mycology has been catching up in importance in large part because of public awareness and interaction, including since it became apparent that the Earth is a lot more like the Middle Earth in J.R.R. Tolkien’s world because plants actually talk to each other through an underground fungal mycelium.