The viewpoints expressed in this article are those of the authors and do not necessarily reflect those of any Competent Authority or their respective companies. The purpose of this article is to guide and inform the reader. The reader is encouraged to verify any opinions and facts the authors present. All inquiries about the methodology should by directed to Craig Weiss, CRWeiss@cptclabs.com.


The term “microbiome” refers to the microflora that shares our body.¹ An association of “skin” with “microbiome/microbiota,” as can be best estimated from PubMed keyword searching, first appeared in the scientific literature in 1969. Since then, thousands of references to this association have appeared in numerous publications. Awareness of human “microbiome/microbiota” and its benefits to human health has grown during the past 10 years. Last year, the term became a major point of discussion within the beauty industry and even among savvy consumers. This explains why more beauty brands are seeking to capitalize on this trend by making “microbiome-friendly” claims for their products.

The skin microbiome is composed of diverse communities of viruses, archaea, bacteria, protozoa, fungi and mites, forming interconnected, yet distinct niches, on the skin. Bacteria being the most abundant that can be found from the top external layers of the stratum corneum down to the dermal layers.^2,3,4,5 This microflora interacts with the skin to form stable communities that are in harmony with each other and the skin. This harmony maintains a functional skin barrier and skin.^5,6 However, this harmony may be disrupted by using topical cosmetics, soaps and toiletries that may alter the skin microflora communities and adversely affect the skin. Such products may have a crushing impact on the microbiome. An impact that has yet to be fully elucidated.⁷

For anyone wishing to evaluate a new or existing product to assess microbiome impact, we suggest the following three sequential steps: 

• Assess the formula’s potential to support microbial growth;
• Conduct an in vitro microbiological assessment of the formula; and
• Conduct an in vivo one-day microbiome assessment study.


Each of these suggested steps is discussed below.

Supporting Microbial Growth

Start by reviewing the product formula to determine if it is composed mostly of organic or inorganic ingredients. Organic (carbon-based) ingredients are potentially biodegradable which can serve as a source of nutrients for microorganisms, thereby supporting their growth. Inorganic (non-carbon based) ingredients on the other hand (e.g., silicone-based) will not effectively support microbial growth. Any data that you already have on the ingredients in your formulation, such as biodegradability for example, will assist in this evaluation. If most of the ingredients in your formula are carbon-based, you should be encouraged that it may potentially be microbiome-friendly.


If your formula contains amino acids, fatty acids and electrolytes, the potential is high that they will support and replenish a healthy skin microbiome. Amino acids are essential as a protein-building nutrient for microorganisms. Botanical extracts also contain nutrients or may degrade into nutrients. A formula containing a “cocktail” of fermented ingredients, extracts, ceramides, vitamins and ingredients identical to the form naturally present in skin, plays a vital role in helping maintain skin hydration levels and lipid content. These are also good indicators of a microbiome-friendly product. Ceramides occur naturally in skin, protecting against water loss. Fatty acids further maintain skin hydration. Polysaccharides and hyaluronic acid may also be used for moisturizing the skin and they could be a readily available nutritional source for the skin microbiome.⁸ Hydration is essential in the maintenance of the skin microbiome since microorganisms require water to survive and reproduce. Even if preservatives are present in the formulation, they may not negatively affect the skin microbiome since preservatives are organic compounds. They are typically present at low levels in the formula which can be metabolized by the microorganisms in the microbiome. As the product is applied, the relatively small number of preservative molecules are dispersed over the applied relatively large area of the skin and are overwhelmed by the vast number of organisms on the skin surface.⁹

The objective of evaluating the formula is to determine if it will positively affect, or at the very minimum, not disturb the skin microbiome. To explore this, one may choose to conduct exploratory in vitro testing to characterize the influence of the formula on the microbiome.

Following is a description of testing that was conducted using a preserved, currently marketed face product having the above-described characteristics. This product shall be referred to as the “Sample.”

In Vitro Microbiological Assessment

To build on the formula’s potential to support or not harm the skin microbiome we suggest conducting in vitro experiments with Staphylococcus epidermidis, a microorganism representative of skin microflora. Other skin microflora candidates will also suffice. One way is to explore the potential of your formula to serve as nutrient for growth of a low-level inoculum. To demonstrate this, Figure 1 shows the results of a face product formula determined to be “microbiome-friendly.”



When a new nutrient source is introduced, microorganisms typically take some time to establish new mechanisms to utilize that new energy source. During this time, they do not proliferate. This is known as the lag phase of a growth curve. S. epidermidis may have remained in a lag phase longer than that of the control, before moving into the log phase (growth/proliferation) and may explain the lower enumeration. More noteworthy was that the Sample, after a 24-hour incubation period, showed significant growth; i.e., 101 → 108, indicating it may have moved into the log phase. This indicates that the Sample did support growth. This data alone indicates that the Sample formula is not deleterious to growth and may have supported growth by acting as nutrient.

To build on this, one might repeat this experiment with recovered log phase S. epidermidis and/or a less nutritive option to potentially allow S. epidermidis to use the test sample as a nutrient source and perhaps show comparable growth to SDW.

Finally, one may want to conduct a more direct experiment to gauge the potential of the formula to serve as nutrient for growth by directly inoculating it with S. epidermidis at a much higher concentration of S. epidermidis (>106), that would be more indicative of populations on the skin or what would be recovered from a typical skin swab. A square centimeter of skin may contain up to a billion microorganism.¹⁰

Under non-experimental, i.e., “real life” conditions, skin S. epidermidis or skin microflora constantly exposed to a mainly carbon-based formula for an extensive period would eventually adjust to use it to its advantage.

In Vivo One-Day Assessment

Assessing the potential of a formula to support microbial life by conducting an in vitro evaluation may provide support for a very general claim. However, it will not sufficiently support a claim for “microbiome-friendly.” For such a claim, we suggest conducting in vivo testing. Unlike others who have reported lengthy, time consuming, complicated, and uncontrolled indirect methods,^11,12,13,14 we describe a simple direct-control method which may be conducted in one day and that can provide support for such a claim. The objective of this test is to directly test on one’s skin the impact the formula has on the individual microbiome. To best demonstrate impact, is to first compromise the microbiome.

We used the same face product Sample as above, to apply directly on the skin during a 24-hour interval by repeated applications on a single subject. If you suspect, but are not certain, your formula is microbiome-friendly, we recommend conducting an exploratory test on 1-3 subjects. This could potentially give you an indication of impact on the microbiome at minimal cost.

In this exploratory study, the subject washed his face with soap to decrease the number of microbes on the skin. Starting the test with a lower number of microorganisms gives them impetus to grow. This established time zero (T0) baseline. A swab was taken from each side of the face to establish the T0 count and plated to determine the baseline microbial count. Half the face received a predetermined amount of product based on use instructions. Application on the same side of the face was repeated at regular intervals throughout the day (6-8 hours). A total of seven applications were performed between 9 am and 3pm. Application intervals were, at a minimum, approximately 40 minutes apart. Three and six hours after the T0 swab, swabs were taken to establish the T3 and T6 time points and enumerated by plate count. It is important to note that the subject was not isolated but was allowed to go about his daily activities. The subject was not prevented from exposing his face to the sun or other environmental elements and may or may not have touched his face. For perspective, a pre-wash count was also determined. Data for each time point are shown in Figure 2. 



To see if there is a pattern, the highlighted data (T0, T3, and T6) were plotted as in Figure 3.

As can be seen from Figure 3, the treated side had a steeper recovery and reached a CFU count above the starting point meaning it reached the normal CFU count region before the wash. The untreated side did not even surpass its starting point CFU count.


Another way to look at this data is by considering the percent CFU change from time zero (T0) as shown in the table below (Figure 4). It details the Colony Forming Unit (CFU) Count Percent Difference from Baseline (T0).

The treated side showed a decreased CFU count of 53% at 3 hours followed by a 22% increase at 6 hours. The untreated side only showed a 40% or greater decrease at both 3 hours and 6 hours. This exploratory test using one subject showed that the formula had a dramatic influence on the growth of face microflora after insult with soap. Based on this, we were encouraged to conduct a larger, more controlled study as discussed here. Eleven men and women, aged 31-59 years were recruited and not allowed to leave the facility. The overall protocol was a single center, monadic, baseline controlled clinical study designed for approximately 7-hour duration to look at the effects the product will have over a 6-hour period. A swabbing technique was used to collect surface skin microflora. Swabbing was performed after washing to establish baseline (T0) and at 3 and 6 hours after initial application of the Sample. The side of face receiving product was randomized so that approximately half of the subjects received it on the right side and half on the left.



The overall procedure was as follows: Subjects reported to the facility without washing or using any facial product. In the facility they washed with soap for approximately 10 seconds, thoroughly rinsed with cold water for approximately 30 seconds, patted dry using a paper towel, and let dry for at least 15 minutes before collection of the first swab (T0). Product application was repeated 1 and 2 hours after the first application which was after washing and swabbing to establish T0. Product application was again repeated 3, 4 and 5 hours post-initial application. Approximately 3 hours (T3) and again 6 hours (T6) after the first application, subjects had microbial samples collected.

The CFU data for all 11 subjects is shown below. Note the average count and the CFU increase or decrease from baseline. Baseline is T0. The data show a similar pattern as that of the exploratory test previously conducted.



The table data show that the average CFU count for the untreated side showed a steep decrease by the 3-hour time point (T3) followed by a gradual recovery but not reaching the baseline count (T0) by the 6-hour time point (T6). The Sample treated side, on the other hand, had almost no decline from baseline (T0) at the 3-hour time point (T3), followed by a gradual recovery that greatly surpassed the baseline by the 6-hour time point (T6). To better visually demonstrate the data, we determined the CFU magnitude change from baseline (T0). This is shown in the last row of the table here (Figure 5) and plotted for visualization as shown in Figure 6.



As in the exploratory test detailed earlier, the data showed that the Sample had a dramatic influence on the growth of face microflora after insult with soap. In this case, the T0 to T3 data also strongly suggest that the product had a saving effect on the skin microflora from insult. Soap (surfactants in general) are known to disrupt cellular membranes leading to cell kill. It may be that this Sample product rescued some microbes from this effect.

A Word About Statistical Significance

When conducting microbiome studies like these it is very hard to achieve statistical significance with a relatively small sample size (i.e., number of subjects) and large variance. That is due to several variables, including personal habits, hygiene, exposure to sun and diet. Most important, there is a great human variability in skin microbial numbers—even at different sites on the same subject. As such, microbial enumeration may be vastly different on individual split face analysis. Also, the swabbing is not a precise technique and very much depended upon the experimenter. Additionally, the fact that swabbing is repeated in the same area may affect subsequent microbial populations.

For figures 5 and 6 the raw counts and percent difference from baseline were analyzed separately. Raw counts, when compared to baseline values, did not show a statistical significance. Percent difference did not show a statistical significance at T3. However, it did show statistical significance at at T6.

The Final Word

In conclusion, based on all the available data detailed here, the weight of evidence may allow one to substantiate a microbiome related claims from any of the below themes:

• Microbiome friendly/kind
• Supports and replenishes a healthy microbiome
• Creates a supporting microbiome environment for growth
• Microbiome rescue
• Protects the microbiome from surfactants or soap use
• Microbiome recovery after soap wash.

The beauty of the described method is that it is quick and yet flexible for add-on tests or modifications such as enumerating the undisturbed microorganisms before and after product application; identifying the microorganisms in a macro-scale (e.g., Gram positive or negative, yeasts; molds; etc.) or micro-scale (genus and species) that were most impacted. 

References:

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3. Y.E. Chen., M.A..Fischbach, and Y. Belkaid. 2018. Skin microbiota-host interactions. Nature, 553(7689): 427-436. 
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