Effect of leave‐on cosmetic antimicrobial preservatives on healthy skin resident Staphylococcus epidermidis

The effect of high doses preservatives in the leave‐on cosmetic products to the skin microbiota is not clear. Studies have shown that the preservatives might alter the balance of the skin microbiota.


| INTRODUC TI ON
The global cosmetics market size was valued at $380.2 billion in 2019 and is projected to reach $463.5 billion by 2027 at an estimated annual growth rate of 5.3%. 1 Microorganism contamination presents a major challenge in the safety and quality of cosmetic products, thus preservatives are added to inhibit or kill the microbial infestation and extend their shelf-life. 2,3 To ensure cosmetic safety and limit overuse of preservatives, agencies have used toxicological data to enlist certain preservatives and their maximum doses that could be added in cosmetics. 2,4 Human skin is the largest epithelial surface (approximately 30 m 2 ) that interacts with microbes. 5 There is a wide spectrum of microorganisms on healthy human skin, which include bacteria, fungi, yeasts, viruses, archaea, and mites. 6 Data have shown that the skin microbiome play important roles in human health, such as host defense against pathogens, inflammation control, and modulation of adaptive immune pathways. 7 Among the skin microbes, Staphylococcus epidermidis has been shown to protect against inflammation, infections, and cancer through interactions with keratinocytes, T cells, and other constituents of the skin microbiota. 8 The effect of high doses preservatives in the leave-on cosmetic products to the skin microbiota is not clear.
Studies have shown that the preservatives might alter the balance of the skin microbiota, 9,10 but a recent short-term in vivo study has shown the skin microbiome is not impacted by preservative containing product. 11 Consumers may use cosmetic products on the skin every day, the effect of these cosmetic preservatives on the skin microbiota should be fully addressed. Especially the establishment of the maximum permitted doses of preservatives in cosmetics should consider the inhibitory effect on microorganisms and enlist the friendly preservatives and avoid overuse of preservatives.
Minimum inhibitory concentration (MIC), mutant prevention concentration (MPC), and killing curves are three commonly used parameters to study the potency of antimicrobials. Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that completely prevents visible growth of the test strain under strictly controlled in vitro conditions. 12 Clinical and Laboratory Standards Institute (CLSI) has developed standard broth microdilution and agar dilution method for S. epidermidis MIC measurement. 13 MPC is the lowest antimicrobial concentration required to inhibit the growth of the least susceptible cells that may present in high density bacterial populations. 14 Time-kill curve can monitor bacterial growth and death when antimicrobials are present and has been frequently used to evaluate the potency of antimicrobials over time. 15 To date, there is limited data on the common cosmetic preservative effect to S. epidermidis from healthy skin. The maximum permitted doses of preservatives in cosmetics were based on the toxicological data and the effect on skin microbiota of healthy skin is not considered. The evaluation of the antimicrobial effect of preservatives on S. epidermidis may lead to an improvement of current criteria for the use of preservatives in cosmetics.
Here, we evaluated the antimicrobial effect of nine cosmetic chemical preservatives that are used in the leave-on cosmetics by analyzing multiple S. epidermidis isolates. Besides, their antimicrobial effect was also examined at the maximum permitted doses.

| Bacterial strains
A total of 77 S. epidermidis isolates recovered from 46 healthy zygomatic skin samples in 2021 were included in this study. No more than two isolates were included from each person. The age of the sampled persons ranged from 12 to 65 years old, and the 50% percentile of age was 35 years old. The samples were streaked on TSA plates and identified by the matrix assisted laser desorption/ ionization time of flight mass spectrometry (Tianrui, China) and confirmed through the API STAPH test (BioMérieux, Beijing, China), following the manufacturer's instructions. The isolates were kept in brainheart infusion broth (BD, China) with 50% glycerol at −80°C freezer for further analysis.

| Multilocus sequence typing (MLST) of S. epidermidis isolates
MLST analysis was conducted by sequencing fragments of seven housekeeping genes (arcC, aroE, gtr, mutS, pyr, tpi, and yqiL) and STs were assigned by comparison with the S. epidermidis MLST database. 16

| Minimal inhibitory concentrations (MICs) analysis of the cosmetic chemical preservatives
The minimal inhibitory concentrations (MICs) of nine leave-on cosmetic chemical preservatives were assessed using agar dilution method following the CLSI standard protocol. 13 Briefly, the fresh S. epidermidis culture was taken from TSA agar using cotton swab and diluted in saline to 10 6 CFU/mL. The diluted culture was inoculated onto MH agar plates supplemented with twofold serial dilution of preservatives using a multi-point inoculation instrument (OXOID).
The plates were incubated at 35 ± 2°C for 22 ± 2 h. The lowest concentration of a preservative that completely prevented visible growth of the test strain was recorded as its MIC. The tested preservatives included benzyl alcohol, 2-bromo-2-nitro-1,3-propanedio l, ethyl 4-hydroxybenzoate, hexadecyltrimethylammonium bromide (CTAB), imidazolidinyl urea, 2-phenoxyethanol, sodium benzoate, sodium salicylate, and trans, trans-2,4-hexadienoic acid potassium salt ( Table 1). All preservatives were ordered from Sigma-Aldrich and dissolved in either ethanol or water. S. aureus ATCC 29213 was used as a quality control organism in the susceptibility assays.

| Mutant prevention concentration (MPC) analysis of the cosmetic chemical preservatives
Since each preservative showed the same MIC to all the 77 S. epidermidis isolates except for CTAB, two S. epidermidis isolates (FC6609 and FC6636) of two sequence types (ST59 and ST73) were randomly selected to conduct MPC analysis with all the leave-on preservatives, and another five isolates with different CTAB MIC were tested only for the MPC of CTAB ( Table 2). The MPC of each leave-on cosmetic chemical preservative was defined as the lowest concentration of a preservative that could inhibit the emergence of mutants from ≥10 10 viable cells on Mueller-Hinton (MH) agar. 17

| DISCUSS ION
Our data demonstrated that the maximum permitted doses of certain preservatives were significantly higher than the MICs and MPCs of S. epidermidis isolates. At the maximum permitted doses, two preservatives could completely kill 10 7 CFU/mL S. epidermidis in less than 1 h in MH broth. Because S. epidermidis has been shown to be beneficial to the skin health, 19,20 the inhibitory effect of preservatives in the cosmetic products to skin S. epidermidis should be extensively re-evaluated to maintain the balance of the skin microbiota.
To increase the robustness of our data, 77 S. epidermidis isolates of more than 17 sequence types from 46 healthy zygomatic skin samples were analyzed to examine the antimicrobial effect of nine cosmetic preservatives. 21,22 Inclusion of more isolates might tolerate intraspecific genetic variation and could strengthen the outcome of the antimicrobial effects of the preservatives. Out data showed each tested preservative had a specific MIC against all the tested S. epidermidis isolates (n = 77) except CTAB which indicated the tolerance of these preservatives might be hard to be developed.
The inhibitory mechanisms of these preservatives to bacteria were diverse. Organic acid salts could inhibit bacterial growth through acidification, changing the plasma membrane fluidity, chelation key nutrients, or inhibition of cellular metabolism enzymes. 2,23,24 Benzyl alcohol and phenoxyethanol could denature the structure of the proteins and induce membrane lysis. 25 Parabens may perturb membrane transport processes and inhibit DNA and RNA synthesis. 2,26,27 On the other hand, formaldehyde releasing agents, imidazolidinyl urea, and 2-bromo-2-nitro-1,3-propanediol can inhibit bacterial growth through oxidation of enzyme thiol. 28,29 CTAB ruptures the bacterial membrane integrity and induce leakage of cellular contents. 30 These mechanisms differ from common antibiotics which have welldefined action sites, such as cell wall or topoisomerases. 31,32 The main purposes for the use of preservatives in cosmetics were to inhibit or kill contaminating microorganisms and prolong the shelf-life of the cosmetics. The nine cosmetic preservatives analyzed in this study had similar maximum permitted doses as in the criteria from different countries ( Table 1). The establishment of the maximum permitted doses of the preservatives were mainly based on the toxicology data, 33 and the susceptibility data of skin microorganisms were not considered. Our data showed that the maximum permitted doses of three preservatives were 4-64 times higher than their MICs, and a significantly higher difference was observed for CTAB.
A recent study showed adverse effects of personal care products which could change the bacterial and archaeal ecosystem on the skin. 9 Since S. epidermidis has been demonstrated to be a major skin defense mechanism, there is need to reevaluate the maximum permitted doses of preservatives in the leave-on cosmetics to ensure a balanced skin microbiota.
MPC indicates the emergence of spontaneous resistant mutants within a population of 10 10 cells. 34  Besides, establishment of the preservative maximum permitted doses should not only be based on the toxicological data, but also on the antimicrobial susceptibility of the skin microorganisms. Cosmetic companies should consider S. epidermidis friendly preservatives and avoid their overuse to keep a healthy skin with balanced microbiota.

AUTH O R CO NTR I B UTI O N S
All authors have read and approved the final manuscript. W.Z., X.W., and L.Z. performed the research. Y.G. and Y.C. designed the research study. N.L. and L.A. analyzed the data. Y.L. and S.C. wrote the paper.

ACK N OWLED G M ENTS
This study was supported by the Young and Middle-aged Fund (2022C2) from the National Institutes for Food and Drug Control, China.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request

I N FO R M E D CO N S E NT A N D E TH I C A L S TATE M E NT
Not applicable.