How to Prove the Concept of Microbiotic Skin Care

When the Human Genome Project¹ was launched in autumn 1990 with the aim of identifying and mapping all of the genes of the human genome, no-one would have thought that we would discover a new microcosmos revolving around and mingling with our human cells.
Of course, already long before this project, it was well-known that our body is not sterile and there are many bacteria living within and on it. These bacteria were however mainly classified as being malicious, threatening our health and causing problems.² Until the 70s of the last century, a germ-free personal environment was considered as most desirable, and strong cleaning products became quite popular. Only starting in the early 1980s, these ideas and information were carefully reevaluated.
When the first results of the Human Microbiome Project³ that started in 2008 were published and we learnt that not only do we host an amazing amount of microbes in and on our body but that these tiny cohabitants at least equal or possibly even exceed the amount of our own human cells, intense research has been dedicated to understand their function for human health and disease.⁴
With the rapid development of novel metagenomic methods, microbiome analysis has become much easier, quicker, more comprehensive and reliable than with traditional techniques such as sampling and culturing that only allow the identification of certain isolated species that can be cultured.

Besides the composition of our gut microbiome, also the microbial colonization of our skin which serves as our outside border to the environment has become the subject of intense
research.^{5, 6}

While research of microorganisms on the skin in the past was mainly serving the purpose of identifying pathogens that cause disease and studying the means to fight them, only recently, the beneficial aspects of our skin commensals were discovered and further studied.

Many publications are describing their interaction to create a stable environment and protect their community and thereby also the skin from the invasion of foreign and potentially harmful microbes. Strong evidence of the interaction of the microbiome with the human immune system has been found.⁷ The microbiome seems to be involved even in protecting the body from UV radiation.⁸ On the other hand, many studies have proven that there is a close link between skin diseases such as acne⁹ and atopic dermatitis¹⁰ and the skin microbiome. Recent research indicates that “the microbiome is crucial for healthy skin, but so is healthy skin for the health of the microbiome” (Kristin Neumann¹¹).
From these insights, we can conclude that by strengthening and balancing the microbial symbiosis, skin condition and health can be improved. Contrary, it seems clear that the use of antibiotics, strong disinfectants and cleaners may lead to a disruption of the microbial balance and impair skin health.
When the first evidence of the beneficial effects of our microbiome was found, it did not take long for more and more publications coming up about the possible improvement of the skin and its microbiome by pre-, pro- and postbiotic additions to cosmetic products.

Classic claims, such as the microbe-eliminating effects of cleansers, antibiotics and disinfectants can easily be proven, often by traditional methods focusing on individual species. Also, products that claim to leave the microbiome uninfluenced can be tested by metagenomic analysis.
Claims of balancing or improving the microbiome with dedicated products cannot be verified that easily. It seems that the marketers of such claims are far ahead of the scientists. Even though the most common bacteria on the skin have been identified, the microbial profiles of different individuals are significantly different.¹² This is attributed to genetic and demographic properties, age, gender, ethnicity, skin type, lifestyle, hygiene, geographic differences, environmental stress by temperature, humidity, seasonal variation, radiation exposure and pollution, cohabitation with animals, profession and many more. Even applied products might cause shifts in their composition.
But not only between different people, even intra-individually, there is a huge variation of the skin conditions at different body sites.¹³ The face offers home to microbes that profit from or at least tolerate high skin surface moisture and increased sebum production. On the other hand, the extremities are rather dry and provide challenging conditions for most bacteria. The amount of microbes is much lower there than on the face, and a much higher variation between different persons has been found. Then, there are the arm pits and body folds with high moisture but moderate sebum content, again offering a completely different environment to microorganisms. And not only different moisture and sebum contents provide different microbial environments, there is also a huge impact of the skin pH. Bacteria regarded as beneficial usually prefer acidic conditions.
Because of all these influencing variables, until today there is no scientific consensus about the composition of the “ideal” microbiome. Microbiome analysis can show if the bacteria of probiotic products or the nutrition for special bacterial species in prebiotic formulations will lead to an undesired or a desired shift in the microbiome composition. Also, for some skin problems such as atopic dermatitis and acne, changes in the microbiome may be used to confirm the product efficacy. For cosmetic effects on healthy skin, however, it is almost impossible to interpret the result of such an examination.
If microbial analysis does not yield the desired information on the improvement of the skin condition, how else can we measure the efficacy of cosmetic claims for microbiome care products?
We have learnt that a healthy skin and a healthy microbiome are closely intertwined. Even though with our current knowledge, it is not possible to clearly quantify an improvement of the microbiome, skin health can be and has been measured for years. Different skin functions and conditions are ascribed to skin health.

Skin barrier assessment

The skin works as a barrier between our inner body and the environment. The strength of the skin barrier is closely linked to skin health: healthy skin will provide a strong barrier. Assessment of the barrier quality is usually performed by measuring the so-called TransEpidermal Water Loss (TEWL).¹⁴ This is the amount of water that evaporates through the skin into the environment. Healthy skin at most skin sites, measured at ideal ambient conditions of 20–22°C and 40–60% of relative humidity, will show a TEWL between 5–15 g/h/m2. If the barrier function is not optimal, a higher loss of water can be measured. Different probes for this application are available.

Skin surface hydration measurement

The water holding capacity of the stratum corneum is depending on the presence of natural moisturizing factors and an intact skin barrier function. It is easily affected by many external influences such as dry air, absence of sebum, frequent washing etc. as this skin layer interacts strongly with the environment.
Dry skin will not only look scaly, but can also lead to small fissures in the skin surface where pathogens may settle. On the other hand, in a continually wet environment, the corneocytes that are forming the bricks in our skin wall will macerate which can also lead to a disruption of the skin integrity. The stratum corneum hydration therefore should be balanced, not too high but neither too low.
The water content will not only vary between persons with different skin types, but also between different skin sites on the single individual. At dryer skin sites like the arms less bacterial species can be found than in areas with higher moisture such as the face.

The Corneometer® is recognized throughout the industry as the gold standard to determine the stratum corneum hydration in the most important upper layers by capacitance. The measurement takes less than one second, thus not influencing the skin at all. Also, the L’Oréal Skinchip® or the MoistureMap® devices are based on capacitance measurement. They display the distribution of the water on the skin by capacitance imaging. Other equipment available on the market uses impedance or conductance methods.

Skin pH-value

Skin pH is essential for the well-being of specialized bacterial communities. Many bacteria associated with a healthy microbiome prefer acidic conditions and cooperate with the skin and amongst each other to maintain this microenvironment. Skin pH measurement can show if an applied product supports this aspect or if it leads to a shift of the pH value.

From neutral pH values in the lower epidermis, there is a steep decrease of pH values towards the skin surface. Depending on the body site, age, gender and many other intrinsic and external factors, values at the skin surface mainly range between pH 4–6.¹⁵ Higher pH values at certain body sites like the axilla or intertriginous areas promote the colonization of certain odour-producing or harmful bacteria and fungi.^{16, 17}
Cleaning the skin with soaps will lead to a short-term shift of the pH value. Even pure water with its neutral pH of 7.0 will influence the surface pH for a short period. Healthy skin will be able to balance this disruption within a certain time span. Frequent washing can however lead to cumulative effects and can seriously harm the skin barrier.¹⁸
Especially atopic dermatitis is often linked to increased pH-values and at the same time a different composition of the bacterial associations with an increased presence of staphylococcus aureus.¹⁹ Skin pH measurement in that case can show whether a shift of the pH value could be obtained by the product application.

Skin sebum

The Cambridge Dictionary describes the lipids excreted by the sebaceous glands as “an oil-like substance produced by the sebaceous glands in the skin that makes hair shiny and prevents skin from becoming dry”.²⁰ The role of sebum in atopic dermatitis has been dismissed by different investigations in the past. More recent studies however, have shown that in diseased skin conditions such as atopic dermatitis, sebum levels are considerably lower than in healthy individuals.¹⁹ On the other hand, increased sebum production is correlated to the occurrence of acne.²¹

Sebum is very different at different skin areas. In the face and on the scalp sebum levels are highest.²² Other skin sites show much lower values. The sebum content has a high impact on microbial life on the skin. Especially the facultative anaerobe cutibacterium acnes prefers sebum rich environments.
The Sebumeter® is the standard device to measure the sebum secretion on the skin surface. The special matte tape will become transparent by absorbing sebum present on the skin surface. The transparency will be measured – the higher, the higher the sebum level.
Other methods such as Sebufix® or Sebutape® use special microporous tapes that show the sebum pits on the skin surface as dark blotches of different sizes.
You can also study the presence of acne bacteria by using specialized camera equipment. The Cutibacterium acnes produces porphyrins that will show a fluorescence when exposed to UV light of special wavelengths.²³ Even though these bacteria can be found on most people at oily skin sites, their activity poses a problem for acne-prone skin. Assessment of the porphyrin fluorescence is useful to test the efficacy of special anti-acne products. This can be done by using cameras providing the needed wavelengths. Special software will evaluate the number, area and intensity of fluorescence.

Evaluation of skin blemishes

Blemishes are usually an indication that the skin condition is not ideal. They especially occur in acne and are often correlated to an increased sebum production.
The occurrence of pimples and blackheads can be visually assessed and graded. This can be done based on direct inspection or for better comparison by the evaluation of high-resolution photographs.²¹ The comparability of photos is strongly influenced by ambient light not only in reference to the light colour and intensity but also to the angle of light. Additionally, the position of the face and the angle of photographing as well as the distance between camera and face will have a huge impact on the outcome.
Therefore, simple mobile phone cameras or even high-end consumer cameras are not the best-suited tools for taking such pictures.

There is dedicated equipment available on the market that provides a homogeneous illumination, sometimes even the use of different light sources and a fixed positioning of face and other body sites for taking standardized photos. With such equipment, very reproducible and comparable images can be taken that are evaluated either by subjective scoring or by automatic calculation of the size of the pores and the amount of blemishes.

Skin healing properties

Skin healing can be assessed by creating small wounds by punch biopsy, excision or damaging the skin in a different way. The extent of the wound and wound closure time can be assessed using visual methods.

An elegant solution not involving an open wound is the measurement of the TransEpidermal Water Loss (TEWL), already described above, after standardized repetitive skin stripping with special tapes such as Corneofix® or D-Squame®. The damage will show in an increased TEWL and can be evaluated together with the time the skin takes to recover to healthy TEWL values.

Desquamation

The skin continuously sheds dead skin cells from the outmost layer of the stratum corneum to make room for new cells growing from the base of the epidermis. This desquamation process is triggered by enzymatic activities that are induced in different skin layers, often depending on special pH values.²⁵ Desquamation has been related to skin health and barrier function. In healthy skin, the shedding of corneocytes should be more of less imperceptible and very regular. When the desquamation process is disrupted, the shedding tends to become inhomogeneous, leaving islands of rather thick layers of dry corneocytes that can sometimes even be perceived by the eye as scaliness.
Desquamation is easily assessed by using the same type of tapes as for standardized tape stripping, e.g. the Corneofix® or the D-Squame®. These sticky tapes remove lose corneocytes. Combined with special cameras and evaluation software, you can quantify the degree of desquamation. In the resulting picture, accretions of corneocytes appear in whitish colour, whereas the regular desquamation will show in grey colours. Homogeneity or irregularities in desquamation can be easily displayed by using false colours.

Measurement of skin irritation

Skin irritation will show in increased microcirculation visible as an increased redness.²⁴ Its intensity is often assessed by subjective scoring performed by experienced scientists. The human eye is, however, not able to evaluate very small differences and compare them to colours that have been previously seen at a different examination day. Photographs, taken at standardized conditions, will assist the researchers in the grading of redness.
Instead of subjective grading, image colour analysis can also be automatically performed by special software.
Besides comparing pictures, colour measurement to assess redness is often done by probes that are quicker and easier to use.
Also, other parameters such as skin surface topography or elasticity may offer information on skin health.

Conclusions

Research on the skin microbiome is an interesting and trendy topic that will probably evolve further. Despite the intense research of the last years, there is until now no scientific consensus on the perfect composition since the microbiome shows huge both inter- and intra-individual variation. Microbiotic analysis will therefore in most cases not yield satisfying prove of the efficacy of pre- and pro-biotic formulations for improved skin health.
You can easily prove skin health claims that are attributed to an improved microbiome by using traditional biophysical testing methods and imaging.
Some aspects that still need more research effort are the influence of cosmetic products on the resident microbiome. There are some concerns that microbiome influencing products may shift the scope of the microbiome by favouring only special microorganisms, thereby disturbing the balance of a perfectly healthy microbiome.
The role of environmental influences on the skin microbiome also offers a wide field of research.
These doubts need to be addressed in future tests and might even lead to the definition of new skin types, taking into account their main microorganisms.

References

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