cosmetics Article Salicylate UV-Filters in Sunscreen Formulations Compromise the Preservative System Efficacy against Pseudomonas aeruginosa and Burkholderia cepacia Noa Ziklo, Inbal Tzafrir, Regina Shulkin and Paul Salama * Innovation department, Sharon Laboratories Ltd., Odem St. Industrial zone Ad-Halom, Ashdod 7898800, Israel; [email protected] (N.Z.); [email protected] (I.T.); [email protected] (R.S.) * Correspondence: [email protected]; Tel.: +972-54-2166476 Received: 15 July 2020; Accepted: 1 August 2020; Published: 3 August 2020 Abstract: Contamination of personal-care products are a serious health concern and therefore, preservative solutions are necessary for the costumers’ safety. High sun protection factor (SPF) sunscreen formulations are known to be difficult to preserve, due to their high ratio of organic phase containing the UV-filters. Salicylate esters such as octyl salicylate (OS) and homosalate (HS) are among the most common UV-filters currently used in the market, and can undergo hydrolysis by esterase molecules produced by contaminant microorganisms. The hydrolysis product, salicylic acid (SA) can be assimilated by certain bacteria that contain the chorismate pathway, in which its final product is pyochelin, an iron-chelating siderophore. Here, we show that OS and HS can compromise the preservative efficacy against two pathogenic important bacteria, Pseudomonas aeruginosa and Burkholderia cepacia. Challenge tests of formulations containing the UV-filters demonstrated that only bacteria with the chorismate pathway failed to be eradicated by the preservation system. mRNA expression levels of the bacterial pchD gene, which metabolizes SA to produce pyochelin, indicate a significant increase that was in correlation with increasing concentrations of both OS and HS. These data suggest that certain UV-filters can provide a source for bacterial resistance against common preservatives in sunscreen formulations. Keywords: sunscreen; UV-filters; preservation; salicylate; Pseudomonas aeruginosa; Burkholderia cepacia 1. Introduction Preservation of non-sterile personal-care products containing water and organic/inorganic compounds is necessary in order to prevent spoilage by contaminants microorganisms, which subsequently can cause severe health risk for the consumers [1]. High sun protection factor (SPF) sunscreen formulations in particular are known to be difficult to preserve, due to the presence of hydrophobic UV-filters in high concentration, thus enhancing the size of their oil phases. As most commercially available preservatives display poor water solubility themselves, they will tend to migrate towards the oily phase, rendering the water phase where the bacteria are present, unprotected. Salicylate esters were the first UV-filters used in sunscreen formulations and yet remain highly popular in the market [2]. Such esters as octyl salicylate (OS) and homosalate (HS) can be hydrolyzed to salicylic acid (SA) by esterases molecules, possibly produced by the contaminant microorganisms [3,4]. Some bacterial species such as Pseudomonas aeruginosa and Burkholderia cepacia contains genes for the chorismate biochemical pathway, in which SA is a precursor to produce pyochelin (Figure1). As part of the pathway, SA can be adenylated at the carboxyl group by PchD (2,3-dihydroxybenzoyl adenylate synthase), which is encoded by the pchD gene, found in both above mentioned bacterial species [5–7]. Cosmetics 2020, 7, 63; doi:10.3390/cosmetics7030063 www.mdpi.com/journal/cosmetics Cosmetics 2020, 7, x FOR PEER REVIEW 2 of 13 adenylate synthase), which is encoded by the pchD gene, found in both above mentioned bacterial species [5–7]. Growing evidences for microbial tolerance and resistance towards common preservatives and disinfectants are accumulating in the past two decades, specifically in regard to Pseudomonas and Burkholderia spp., and represent a major health concern [8–12]. Nosocomial outbreaks of contaminated personal-care products have been repeatedly reported in hospitals [13–16]. A recent report exposed that 11.76% of the products were recalled because of a contamination with a potentially pathogenic microorganism, while P. aeruginosa was the most frequently found microorganism (35%) [17]. In addition to the classical scheme representing the inherent physico-chemical reasons for which high SPF sunscreens are difficult to preserve, we now would like to report an additional difficulty of finding its source in a specific microbiological phenomenon and shedding a new light on sunscreen Cosmetics 2020, 7, 63 2 of 13 preservation. FigureFigure 1.1. ChorismateChorismate cyclecycle inin PseudomonasPseudomonas aeruginosaaeruginosa andand BurkholderiaBurkholderia cepaciacepacia incorporatedincorporated withwith thethe hypothesizedhypothesized hydrolysis of octyloctyl salicylatesalicylate ultravioletultraviolet (UV)-filter(UV)-filter fromfrom sunscreensunscreen formulationsformulations toto salicylicsalicylic acid,acid, aa precursorprecursor forfor pyochelinpyochelin biosynthesis.biosynthesis. 2. MaterialsGrowing and evidences Methods for microbial tolerance and resistance towards common preservatives and disinfectants are accumulating in the past two decades, specifically in regard to Pseudomonas and Burkholderia2.1. Microorganismsspp., and Strains represent and Growth a major Conditions health concern [8–12]. Nosocomial outbreaks of contaminated personal-care products have been repeatedly reported in hospitals [13–16]. A recent report exposed Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), P. aeruginosa (ATCC 9027), B. that 11.76% of the products were recalled because of a contamination with a potentially pathogenic cepacia (ATCC 25416), Candida albicans (ATCC 10231), and Aspergillus brasiliensis (ATCC 16404) were microorganism, while P. aeruginosa was the most frequently found microorganism (35%) [17]. obtained from ATCC and cultured according to the manufacturer's instructions. These In addition to the classical scheme representing the inherent physico-chemical reasons for microorganisms are routinely used for minimum inhibitory concentration (MIC) and challenge tests which high SPF sunscreens are difficult to preserve, we now would like to report an additional according to the US and EU recommendations for the efficacy of preservatives in the cosmetic difficulty of finding its source in a specific microbiological phenomenon and shedding a new light on industry. Bacterial strains were maintained in tryptic soy agar (TSA) (Hy-labs, Rehovot, Israel), while sunscreen preservation. yeast and mold strains were maintained in sabouraud dextrose agar (SDA) (Hy-labs, Israel), 2.supplemented Materials and with Methods oxytetracycline at a concentration of 1% (Hy-labs, Israel). Preservative efficacy assays (challenge tests) were performed in order to enumerate the number of microorganisms present 2.1.in a Microorganismsformulation containing Strains and the Growth preservative Conditions system, while the diluent solution was supplemented withEscherichia neutralizers coli containing(ATCC 8739), 4% StaphylococcusTween 20 and aureus 0.5% lecithin(ATCC 6538),(Hy-labs,P.aeruginosa Israel). (ATCC 9027), B. cepacia (ATCC 25416), Candida albicans (ATCC 10231), and Aspergillus brasiliensis (ATCC 16404) were obtained from ATCC and cultured according to the manufacturer’s instructions. These microorganisms are routinely used for minimum inhibitory concentration (MIC) and challenge tests according to the US and EU recommendations for the efficacy of preservatives in the cosmetic industry. Bacterial strains were maintained in tryptic soy agar (TSA) (Hy-labs, Rehovot, Israel), while yeast and mold strains were maintained in sabouraud dextrose agar (SDA) (Hy-labs, Israel), supplemented with oxytetracycline at a concentration of 1% (Hy-labs, Israel). Preservative efficacy assays (challenge tests) were performed in order to enumerate the number of microorganisms present in a formulation containing the preservative system, while the diluent solution was supplemented with neutralizers containing 4% Tween 20 and 0.5% lecithin (Hy-labs, Israel). Cosmetics 2020, 7, 63 3 of 13 2.2. Preservative System and Formulations For the challenge tests, creams having SPF values of 30 and 45 and basic cream with or without OS were used and the constitution of compounds are listed in Table1. A typical representative preservative system, Sharomix™ Amplify AM-25 was used in this study at a final concentration of 0.5%. The preservative blend contained 80% Phenoxyethanol, 5% Didecyldimethylammonium chloride, 10% Chlorophenesin, and 5% Caprylyl glycol. Table 1. Ingredients within each of the formulations used in this study, SPF 30 with octyl salicylate (OS) (+OS), SPF 45 with OS (+OS), and basic cream with or without OS or homosalate (HS) (+/ OS/HS). − SPF 30 (+OS) with Preservative AM-25 Phase A Trade Name INCI Name %w/w Water Aqua Up to 100% Xanthan gum Xanthan gum 0.5 Phase B Montanov 68 Cetearyl Glucoside (and) Cetearyl Alcohol 3 Emulgade® 1000 NI Cetearyl Alcohol (and) Ceteareth-20 3 IPM Isopropyl myristate 5 Octyl Salicylate Octyl Salicylate 5 Octyl Methoxycinnamate Ethylhexyl Methoxycinnamate 7.5 Benzophenone-3 Benzophenone-3 4 Avobenzone Butyl Methoxydibenzoylmethane 1.6 Antaron™ V-220F VP/Eicosene Copolymer 3 Phase C Vitamin E Tocopheryl acetate 0.2 Sharomix™ Amplify AM25 Phenoxyethanol, Chlorphenesin, Caprylyl 0.5 (Preservative) glycol, Didecyldimethylammonium chloride Final pH 5.0–5.5 SPF 45 (+OS) with Preservative AM-25 Trade Name INCI Name