639 THE ROLE OF THE SCALP MICROBIOME IN HEALTH AND DISEASE century knew there were asymptomatic carriers of tuberculosis, and now, we know that likely more than 90% of microorganisms are unculturable. Today, it is not easy to put disease causing microorganisms back on healthy people, making it more complicated. We need to understand that in all cases Koch’s postulates do not always have to be fulfilled (16). Let us return to this concept of pathogenesis and Malassezia. How can Malassezia cause dandruff if they are on everyone? If we consider susceptible individuals, people who have the possibility of getting dandruff for whatever reason: • If you remove the fungi, the dandruff goes away. • If you remove the bacteria, it does not. • If you remove both the bacteria and the fungi, the dandruff gets better but at the same magnitude as if you removed only the fungi. • If you create a resistant Malassezia and put it back on the same subjects that are under antifungal treatment, that dandruff returns. These were experiments conducted and published in the 1960s and 70s and are high- quality studies (20–22). Additionally, in the experiment in which an isolated toxic metabolite is added back, even in the absence of Malassezia, dandruff returns (15). This makes a pretty strong case that Malassezia can be a pathogen, and we can prove that in susceptible individuals. If one looks exclusively at subjects who are susceptible to dandruff, the pathogenic mechanism can be described: Malassezia is on skin. They eat break down sebaceous lipids by secreting a lipase that breaks down the triglycerides. They consume a subset of saturated fatty acids, actually eating what is good for our skin. They then can proliferate this is a commensal cycle. But that is not how it works. Unfortunately, Malassezia are unable to consume unsaturated fatty acids, and they are left behind on the scalp. They penetrate and break down the scalp’s barrier, inducing a repair response, proliferation, and flaking (13). MUTUALISM (PROTECTION) Mutualism is a relationship in which both parties benefit. A recent example in the human–Malassezia relationship is in atopic dermatitis, a hyperproliferative skin condition characterized by redness, scales, and itching, usually on the appendages or trunk. In a longitudinal study comparing unaffected control individuals, affected AD lesions, and nonlesional sites in affected individuals, Malassezia were compared with shotgun metagenomics. The Malassezia as a group showed little difference. Even with Malassezia restricta, the species most commonly identified on skin, there is no real difference. However, upon examination of Malassezia globosa, the species that is the primary player in dandruff, it was found that there is not that much M. globosa in the lesions, and M. dermatis and M. sympodialis are measurably (but not significantly) higher. In this instance, it was apparent that different Malassezia species have very different activities (23). The hypothesized M. globosa protective mechanism AD involves secretion of an aspartyl protease, MGSAP1. Proteases are designed to break down proteins, and fungi usually employ them to prepare proteins for digestion. So, being a carnivore, Malassezia secretes a battery of proteases to eat our skin. However, it has now been demonstrated in vitro that secreted MFSAP1 is able to break down Staphylococcus aureus biofilms. S. aureus biofilm creation is closely linked to atopic dermatitis. While it is still open as to whether S. aureus biofilms cause atopic dermatitis, they are likely involved, and people with AD lesions have a significantly increased S. aureus

640 JOURNAL OF COSMETIC SCIENCE numbers and S. aureus biofilms, which are very hard to break down with treatment. So, Malassezia may be secreting an enzyme making life more difficult for S aureus (24). This sounds like a mutualist benefit, with Malassezia, in some cases, being beneficial for skin. MOVING FORWARD Now what? Malassezia represent many different species, some awaiting cultivation and others even awaiting identification. They do different things: they can do nothing, they can be good, or they can be bad. How do we move forward to understand and to treat dandruff and other common skin diseases impacted by Malassezia? How will we replace the broad- spectrum antifungal materials in shampoos? How are we going to design treatments that work? Heinrich Koch’s vision of microbiology remains the dogma, starting with parallel group studies comparing affected and unaffected populations, identifying microorganisms on affected subjects, culturing microbes from the infected individuals and putting them back into unaffected individuals to induce lesions, and then reisolating those microorganisms to confirm the correct affliction. Then, the investigator would design molecular entities to interrupt this cycle. I remain skeptical that this paradigm will be effective in most cases, and it has previously and repetitively been shown so. I think we need newer, more creative ways to be able to understand how the microbiota works. The dogma is that there are fungi and bacteria on the skin. They secrete stuff and that affects the host. If one removes that microorganism, the impact on the host is also removed. Unfortunately, it is more complicated than that. Microbes talk to us, and they talk to each other. “Health” is when there is a healthy homeostasis in which the bacteria, fungi, and even viruses are in balance with the host (3). It is also become increasingly clear in that in fungal-mediated disease, the damage may not come from the fungi but from the host— where we have made an aberrant reaction to an otherwise benign organism and damaged ourselves (25). A clear example is COVID-19. The people who are sickest with COVID are not necessarily sick because of the virus. They are sick because their immune system has overreacted and attacked their lungs. Instead of simplistic models in which we identify a fungus or bacteria and remove it from the system and everything will get better, we need to figure out what these communication molecules, “soluble modulatory factors,” are. We know the host secretes antimicrobial peptides that affect bacterial and fungal populations, fungi reduce the skin pH, which is difficult for some bacteria, and fungi secrete antibacterial compounds and short chain fatty acids. Now, a number of labs are working on these soluble factors, in which microbes secrete materials that cause inflammation, block immune responses, and may even be anti- inflammatory. Only by tackling microbe–host interactions as a larger homeostasis are we going to get away from broad-spectrum antimicrobial materials. In summary, we see the tip of an iceberg. We understand that skin is more complicated than we thought. We understand skin microbiology is complicated, and it is not just direct 1:1 pathogenesis or secretion. The microbes and host communicate with each other. To understand microbiology, one has to pay attention to the details and think about what the sample is, how to analyze that sample, and, very importantly, the clinical design. The human skin microbiome is important and involved in human health. Ignoring the microbiome in skin treatment design will not get the right answer. Malassezia are the