1. Introduction
The skin provides a diversity of habitats for bacteria, yeasts, and mold, with different microbiota associated with different regions of the skin. Based on traditional cultivating methods, the predominant bacteria are
Propionibacterium acnes and
Staphylococcus spp. in oily sites, and
Corynebacterium spp. and
Staphylococcus spp. in moist sites. Fungal diversity is dominated by the genus
Malassezia [
1]. Several of these skin microorganisms are involved in dermatological diseases, such as seborrheic dermatitis, atopic dermatitis, and acne vulgaris [
2,
3,
4].
Malassezia and bacteria such as
Staphylococcus spp.,
Propionibacterium spp., and
Corynebacteria spp. secrete multiple lipases with a broad spectrum of activity that hydrolyzes almost all triglycerides in the sebum into fatty acids [
5,
6,
7]. With the exception of
M. pachydermatis, all
Malassezia species known so far require an external lipid source for growth, i.e., they are inevitably lipid dependent [
6]. The
Malassezia genome lacks the genes that encode the cytosolic fatty acid synthase complex (FAS), which explains why these yeasts cannot synthesize de novo C14 or C16 free fatty acids (FFAs) [
8]. To overcome this,
Malassezia utilizes fatty acid sources directly from their host and uses the lipases to obtain FFA. In contrast, most bacteria lack the nutritional advantages offered by the degradation of sebum triglycerides to their constituent free fatty acids. However, the FFAs produced by bacterial lipases promote cell–cell adhesion and the aggregation of cells promotes cooperative cell nutrition due to effects on the local micro-environment [
9]. In vitro studies have shown that purified lipases influenced the functions of different human immune cells, such as the chemotaxis of neutrophils and granulocytes, decreasing the phagocytotic killing of bacteria [
10]. In this manner, bacterial colonization can be achieved. On the other hand, it has been shown that environmental conditions, including substrates, can influence the fatty acid composition of some bacterial strains [
11].
The application of cosmetics may adversely impact skin if microbial numbers and diversity are changed [
12]. However, the interactions of cosmetics with microbiota is not yet fully understood. In relation to fungi and bacterial lipases, cosmetic ingredients based on fatty acid esters are of paramount importance. Mayser et al. reported that some emulsifiers and oils promoted the growth of one or more
Malassezia species [
13]. The same behavior was found by Koyama at al. for other topical vehicles [
14]. Growth stimulation and the formation of high amounts of FFAs may be of pathophysiological importance. Additionally, some FFAs produced by the hydrolysis of esters have comedogenic properties and play an important role in the pathogenesis of acne [
15]. However, to date no systematic studies have been performed.
In this study, we tested the ability of Malassezia species and different bacteria to assimilate ingredients frequently used in dermal formulations. The growth of microorganisms was determined. Additionally, the metabolism of tested substances was analyzed by High Performance Thin Layer Chromatography (HPTLC).
4. Discussion
The human skin is a complex ecosystem with various microenvironmental conditions and hosts many co-existing microorganisms. When the bacterial ecosystem is balanced and varied, the skin remains healthy. However, several environmental factors inclusive of the use of cosmetics can change this balance. Therefore, the influence of cosmetics on microbial diversity and their connection to skin diseases has been explored more extensively in recent years.
Cosmetic products contain many components, which can influence skin microbiome. Some of these, including carbohydrates, proteins, and lipids, can promote microbial growth. On the other hand, they often contain ingredients, e.g., preservatives, which have an inhibitory effect and disturb the microbiota. It has been shown, that cosmetic ingredients influence the skin microbiome and that microbial diversity can significantly change with the use of cosmetics [
18,
19,
20,
21,
22]. However, to date no systematic studies have been performed.
In the present study, we tested the influence of many different cosmetic ingredients on microorganism growth. We decided to test the substances at concentrations that are used in cosmetic formulations to better simulate their real influence on skin microbiota. The selection of ingredients was based on the most frequently used cosmetic substances according to the Food and Drug Administration (FDA), Deutscher Arzneimittel Codex (DAC) [
23], and other previously published studies [
24]. A strong influence of certain groups of ingredients on
Malassezia spp. growth was found. Natural oils, which mostly consist of fatty acid triglycerides and fatty acid esters with fatty chain lengths above 12C-atoms, presented good growth mediums. It is known that
Malassezia cannot synthesize fatty acids, and therefore uses skin sebum as a FFA source. Human sebum is a complex mixture of triglycerides, FFAs, wax esters, sterol esters, cholesterol, cholesterol esters, and squalene [
25]. These sources are exploited by
Malassezia by secreting lipases and phospholipases to release FFAs from lipids with fatty acid ester bonds [
26]. However, there is no strict requirement for them to be glycerol esters [
17]. It was shown that besides natural skin components, various other esters can be used by
Malassezia as a lipid source. Thus, bacterial growth on natural vegetable oils, emulsifiers, and synthetic oils can also be observed [
8,
13,
14,
17]. Our investigations confirm this hypothesis. However, it was apparent that
Malassezia metabolism was more complex, and that the chemical structure of an ester can strongly influence its nutritional potential. Mayser at al. showed that the alcohol moiety of the ester strongly influences
Malassezia metabolism [
17]. The relationship between growth promotion and hydrolysis rate can be arranged in the following order: ethyl ester > isopropyl ester > decyl ester. Additionally, unsaturated fatty acids were more capable of stimulating growth than saturated fatty acids [
17]. By testing various esters, we found additional factors that can play a role in growth promotion. One of these factors is the presence of a hydroxyl group on fatty acid backbones. Thus, growth in the presence of PEG-40 hydrogenated castor oil and ricinus oil was observed only for the
M. furfur species. In all species, no growth was observed in the presence of macrogol 40 glycerolhydroxystearat. These substances belong to the group of hydroxy fatty acids esters. We tested an additional natural product, lanoline, which is a mixture of hydroxy fatty acid esters (about 30%) with other fatty acid esters (up to 60%) and alcohols. Little or no growth of
Malassezia spp. on lanoline was observed. Similar observations were made by Mayser at al. for hydroxy fatty acids, such as PEG glycol-35 castor oil and ricinoleic acid [
13]. Growth on this medium was only observed for
M. furfur. Therefore, it was assumed that
M. furfur is distinct from other
Malassezia species with regard to the metabolism of ricinoleic acid. We also demonstrated that these results cannot be extrapolated to other hydroxy fatty acid esters. It is likely that other substances belonging to this chemical group cannot promote the growth of
Malassezia. The slow growth of some
Malassezia strains on lanoline is probably related to the presence of fatty acid esters other than hydroxy fatty acid esters, which can be metabolized by this species.
Interesting results were obtained using oleic acid as well as esters containing oleic acid. Growth was observed mostly for
M. furfur and
M. sympodialis species. No growth was observed for
M. globosa. However, HPTLC analysis showed an increase in free fatty acids, which could be correlated with the lipase activity of all tested strains. It can be also assumed, that
M. globosa cannot used oleic acid as a nutrient. Furthermore, the fungistatic properties of oleic acid against
M. globosa and
M. restricta strains were reported in a previous study [
27]. It is known that
M. globosa cannot degrade unsaturated species such as oleic acid due to an absence of the enzyme 2,3-enoyl-CoA isomerase. In the same study, it was shown that
M. furfur, in contrast to
M. globose, can use oleic acid as a carbon source [
27]. However, in another study no growth of
M. furfur on oleic acid was observed [
28]. The different results may be related to the concentration of the fatty acid used. Mayser used a pure substance, whereas Gordon used concentrations of up to 0.2%. Oleates play an essential role in a number of cellular processes, but at higher concentrations they can have a negative influence on cell survival and cause a lipotoxic effect [
29]. In our experiments, apart from hydrolyzed products of fatty acid esters, an additional HPTLC band at an R
f of 0.56 was detected. This band was identified as an ethyl ester [
28]. The formation of fatty acid ethyl esters may be an escape mechanism from excess FFAs and was also observed in other strains of yeast [
29]. Interestingly, no detectable formation of fatty acid ethyl esters was found during incubation with isopropyl palmitate, indicating a lower toxic potential of palmitic acid compared to oleic acid.
No growth of
Malassezia spp. was found on fatty acid esters with fatty chain lengths shorter than 12C-atoms. Medium-chain fatty acids like capric acid (C10:0), caprylic acid (C8:0), or caproic acid (C6:0) are known to have broad antimicrobial activity [
30]. The effects of free medium-chain fatty acids on different
Malassezia spp. were analyzed by Mayser et al. and all strains tested showed growth reduction after exposure to the free fatty acids [
31]. In another study, a similar effect was observed for medium-chain triglycerides [
32]. Our experiments confirm these data. No growth was found with capric acid, caprylic/capric triglyceride, cetearyl isononanoate, and polyglyceryl-3 caprate. Using decyl oleate and coco-caprylate/caprate, variable growth was observed. Thus,
M. furfur and
M. sympodialis showed growth on these media, whereas no growth was observed for
M. globosa and
M. restricta. These observations are in accordance with the results obtained for oleic acid esters. We can assume that the hydrolysis of esters with a decyl alcohol moiety is faster than the hydrolysis of esters with a coconut alcohol moiety, because of a shorter chain length. Therefore, a higher concentration of oleic acid exists in the medium compared to caprylic/capric acid.
M. furfur and
M. sympodialis use oleic acid as nutrition, whereas
M. globosa cannot use this acid, as explained above. The amount of caprylic/capric acid medium may be too low to have a toxic effect on the species.
Unexpectedly, the growth of some
Malassezia spp., especially
M. furfur, was observed in the presence of a primary fatty alcohol, cetylstearyl alcohol. No growth was observed with a secondary fatty alcohol, octyldodecanol. Until now, the results of the metabolism of fatty alcohols by
Malassezia were not known. Mayser et al. observed no growth of
Malassezia spp. on Lanette N and postulated that fatty alcohols cannot be used by this species as nutrition [
13]. Lanette N is a mix of ionic surfactant sodium stearyl sulfate and cetylstearyl alcohol. It is possible that the ionic surfactant negatively influences the growth of this species, and therefore no growth was observed. The toxicity of ionic surfactants has been previously reported [
33]. Our data suggests that in some cases the metabolism of fatty alcohols by
Malassezia may be possible.
No growth of most
Malassezia species was observed in the presence of fatty alcohol ether, paraffin- and silicon-based substances, polymers, polyethylene glycols, and quaternary ammonium salts. Thus, the substances based on these chemical groups cannot be assimilated by
Malassezia spp. because they either lack fatty acids or the bound fatty acids cannot be cleaved by the
Malassezia lipases. However, some exceptions are found for
M. furfur. This species is able to grow in the presence of paraffin liquidum and squalene, but its growth is significantly slower than on other growth media. The mechanism of assimilation is not known, but has been previously studied by other research groups [
14].
The results obtained with pure substances were evaluated using complex formulations containing several tested ingredients. The results confirm previous observations. Thus, no growth was observed in the presence of formulations containing inert ingredients. In contrast, strong growth was observed for formulations containing natural oils. We assumed that the results obtained for pure substances can be extrapolated to complex formulations.
The utilization rate of cosmetic ingredients on skin is relevant to their potentially harmful effects. Therefore, the hydrolysis rate, its dependence on cell number, and incubation time were estimated for isopropyl palmitate incubated with M. sympodialis. The hydrolysis rate is proportional to cell number, indicating that lipase concentration is cell number dependent. The loss of palmitic acid after a longer incubation time pointed to the assimilation of this substrate by Malassezia. This explains the strong growth of Malassezia on isopropyl palmitate, as well as on other fatty acid esters.
Most of skin’s bacteria are lipid-independent and do not use primarily skin lipids as nutrition. However, some produce lipases, which can hydrolyze skin lipids as well as other fatty acid esters. The hydrolysis of synthetic emulsifiers by
staphylococci and
corynebacteria has been reported [
34]. The lipolytic activity of bacteria is strongly dependent on the strain and on the fatty acid nutrition source [
35,
36,
37,
38]. The fatty acids produced by lipases can give some strains of bacteria benefits by occupying skin and by changing the environment pH. Skin bacteria that produce lipases are
Staphylococcus spp.,
Corynebacterium spp., and
Propionibacterium spp., among others. We tested the influence of cosmetic ingredients on the growth of resident skin bacteria. In most cases no influence on growth was found; however, some interactions were observed. Capric acid was found to inhibit the growth of all tested strains and oleic acid inhibited the growth of
C. minutissimum. The toxic effect of some fatty acids on bacteria is well-documented. Capric acid and lauric acid display a higher bactericidal activity than other saturated fatty acids [
39,
40]. Fatty acids demonstrate selective bactericidal activity and their influence on bacterial strains is complex [
41,
42,
43]. Nakatsuji et al. found that lauric acid shows a stronger bactericidal activity against
S. aureus than that against
S. epidermidis [
44]. Moreover, Hsuan et al. reported that oleic acid preferentially kills
S. aureus and suggested that gram-positive bacteria are more susceptible to fatty acids than are gram-negative bacteria [
45]. Furthermore, it was shown that the antimicrobial activity of fatty acids is strongly dependent on pH and concentration. Thus, less concentrated fatty acids, such as oleic acid, can promote growth, whereas high concentrations have antimicrobial effects [
43]. We assumed that the concentrations used in our experiments were above the MIC values (minimum inhibitory concentration) for
C. minutissimum, causing growth inhibition of this species. In contrast, growth was observed for oleic acid esters: ethyl oleate, oleyl oleate, decyl oleate, glycerol oleate, and polyglyceryl-3 oleate. In this case, oleic acid was bound as an ester and the ester bond must first be cleaved by a lipase to release it. Therefore, the concentration of free oleic acid is considerably lower and can be used by bacteria for growth promotion.
The growth of all bacterial strains observed in the presence of sucrose stearate is attributed to the sucrose moiety, which can be used as an additional carbon source.
In accordance with the results obtained with pure substances, no growth of tested bacterial strains was observed on complex W/O and O/W formulations.