Abstract
Cosmetic products contain several components that are ideal for microbial growth, they exposed to contamination by pathogenic bacteria and fungi, and this may cause health risks such as skin and eye infections. In this investigation, 50 samples were obtained from various shops in Mecca region, Saudi Arabia. Collected samples include high-quality and low-quality brands of makeup. Results show that most cosmetics are contaminated with microorganisms. Bacterial and fungal isolates were identified by morphological and microscopic techniques, and confirmed by molecular methods: (16s rRNA) for bacterial isolates and (18s rRNA) for fungal isolates associated with cosmetics. In low-quality cosmetics, frequency of microbial growth is higher and more diverse than high-quality cosmetics. It has been observed the most contaminated product was in lip gloss and it follows by the lipstick. The most predominant species of bacteria are Staphylococcus aureus (27 %), E. coli (27 %), which follows by Streptococcus pneumonia (18 %), Staphylococcus epidermis (17 %), Bacilli subtilis (12 %), and Pseudomonas aeruginosa (5 %). Aspergillus sp is the most predominant fungi (57 %), which is followed by Penicillium sp. (29 %) and Rhizopus sp. (14 %). In high quality brands, the frequency of microbial growth was the highest in mascara, lip-gloss. The most predominant species of bacteria is Staphylococcus aureus (41 %), which follows by Bacilli subtilis and Pseudomonas aeruginosa and E. coli (17 %). Streptococcus pneumonia is the less dominant (5 %). There is no growth on media of fungi. Due to the large number of cosmetics brands in Mecca region and for consumer safety, this study is prepared.
Keywords: High-quality cosmetics, Low-quality cosmetics, Fungal and bacterial contamination
1. Introduction
Cosmetics are synthetic or natural substances for use on various regions of the human body skin including (lips – eyes) (Oliveira et al., 2020). Cosmetics are produced in large numbers of brands, with different prices and quality. They are used regardless of whether they are safe or unsafe. They may have been contaminated during manufacture or storage. The majority of cosmetics are not sterile and are derived from non-sterile basic materials (Choubey and Godbole, 2017). Most cosmetic products contain several components that are favorable for microbial growth since they are not often manufactured in a media that is sanitized against microbial growth. Items could be contaminated in the factory (Siegert et al., 2005). Bacteria, notably Enterobacteria and Staphylococci are the most studied microbes polluting composition (Bashir and Lambert, 2020, Dashen et al., 2011). Pathogenic bacteria found in beauty products include Pseudomonas aeruginosa and Staphylococcus aureus (Sreeparna et al., 2017, Detmer et al., 2010). The most common bacteria that cause human diseases, such as skin infections, were Staphylococcus epidermidis and Staphylococcus aureus. Pathogenic filamentous fungi are of particular interest since they are linked to opportunistic infections as well as mycotoxin toxicity (Peraica et al., 1999, Hayleeyesus and Manaye, 2014).
Raw materials for cosmetics may be polluted in the first place. Additionally, water used in non-sanitized production may be contaminated. Such circumstances provide an ideal setting for microbial growth. Makeup can have negative effects on women's health if not handled properly. Cosmetic contamination can cause some kinds of infections ranging from mild to severe. There are two key reasons for needing conservatives: to prevent goods from being spoiled by microbes and to prevent disease caused by microbes (Nasir and Qasim, 2020). Cosmetics also contain a wide range of chemicals such as fragrances, preservatives, pigments and other substances known as skin sensitizers which are potentially harmful on general health (Panico et al., 2019). Microbial contamination of cosmetic products is a serious concern for the business, and it has the potential to be a major cause of both economic and product losses. Cosmetics are prone to microbial growth due to the presence of nutrients and water. Microorganisms are frequently the cause of organoleptic changes such as changes in color, viscosity, and disagreeable odors (Becks and Lorenzoni, 1995, Behravan et al., 2000).
Furthermore, in rare situations, contaminated bacteria or their activities may cause human health concerns such as allergic contact dermatitis, skin irritation, and infection, particularly in the mouth, eyes, or wounds (Orús et al., 2015). Cosmetics are exposed to contamination by microorganisms and may cause health risks such as skin and eye infections. In fact, a lot of microbes can enter the product via hand and mouth (Siegert, 2013, Dadashi and Dehghanzadeh, 2016). Occurrence of microbes is caused by two major factors: The first is a result of improper application of cosmetics or a failure to follow adequate hygiene procedures. Even though the products contain preservatives, this enables the deposit of skin-derived detritus, which favors the development and spread of microorganisms. The dispersed particles in indoor air, which include dust, bacteria, and spores, are the second and most significant cause of microbial proliferation in cosmetics (Oliveira et al., 2020).
Cosmetics need to be clean and healthy by adding preservative substances both to prevent contamination and financial loss. The additives used in the products should not be harmful to customers because the majority of the beautifying agents were used in delicate areas (Siya et al., 2019). Additionally, particularly in humid conditions, poor air quality has a significant impact on the growth of microbes in cosmetics (Mohammed et al., 2021). Lack of attention to proper manufacturing practices, insufficient preservation systems, insufficient microbiological test procedures, and/or insufficient microbial limits for finished products are thought to be the causes of contamination (El-Bazza et al., 2009).
Therefore, a good cosmetic product will contain a good preservative system that will last for a given period of time indicated by expiry dates. Expiry date on cosmetics indicates that after the given date the product may be altered and is risky to use (Nuzhath, 2014). These preservatives are different ingredients added to cosmetics, some of them are synthetic organic compounds like petrolatum chemicals such as Parabens, Formaldehyde, Paraformaldehyde, Mercury, Butylated Hydroxy anisole (BHA), Butylated Hydroxytoluene (BHT), PEGs, cocamide DEA and toluene (EWG, 2020). Other ingredients are natural like water and oils (Halla et al., 2018).
Under the proper physicochemical circumstances all products including cosmetics containing water and inorganic/organic compounds are exposed to microbial contamination in order to ensure microbiological safety of cosmetics products. This inhibition should have a broad range of activity effectiveness and should last for a longer period of time than the cosmetic product itself, which would be similar to the projected shelf-life plus the consumption time. As a result, all possible sources of contamination must be identified and monitored (Choubey and Godbole, 2017).
Generally, microbial contamination of cosmetics can be one of these reasons (1) raw material evaluation and control, (2) manufacturing process, (3) final product delivery and finally, (4) consumer use to prevent microbial contamination without causing the product's properties (Halla et al., 2018). Due to the large number of cosmetics brands in Mecca region and for consumer safety, the study's primary goals are to: a) assess the level of microbial contamination in high-quality and low-quality cosmetics, b) isolation and identification of microorganisms from unused different brands of cosmetics.
2. Materials and methods
2.1. Samples collection
A total of 50 unused cosmetic samples were collected from different shops in Mecca region, it is situated in the western part of KSA, at a longitude of 39.826168 East and a latitude of 21.422510 North. Collected samples were divided into two groups regarding to their quality; Low- quality brands (Table 1) and High -quality brands (Table 2), the products were separated by number, from 1 to 28 for low quality products: (4) lipsticks, (5) lip-glosses, (5) powders, (3) mascara, (3) liquid foundation, (3) concealers, (2) eye- pencils, (1) eye-liner, (3) eye shadows. Samples from 29 to 50 are high quality products as a follow: (3) lipsticks, (3) lip-glosses, (3) powders, (3) mascara, (3) liquid foundation, (1) concealers, (2) eye- pencils, and (3) eye shadows. Samples were put in sterile plastic bags and transferred to the lab. Each category of cosmetics was numbered and labeled with information like brand, expiration date and country of origin.
Table 1.
Group (1): Cosmetics products - low quality brands.
| Sample number | Cosmetic category | Number of samples collected | Expiry date | Country of region |
|---|---|---|---|---|
| 1 | Lipsticks | 4 | 9/2024 | China |
| 2 | 9/2024 | China | ||
| 3 | – | China | ||
| 4 | – | China (P.R.C) | ||
| 5 | Lip-gloss | 5 | 7/2026 | China |
| 6 | 5/2024 | China | ||
| 7 | 4/2025 | China | ||
| 8 | 4/2026 | China | ||
| 9 | 4/2025 | China | ||
| 10 | Powders | 3 | 9/2024 | China |
| 11 | 8/2022 | China | ||
| 12 | After used (30 month) |
England | ||
| 13 | Plusher | 2 | 12/2023 | China |
| 14 | After used (24 months) |
Italy | ||
| 15 | Mascara | 3 | After used (6 months) |
China |
| 16 | 5/2026 | China | ||
| 17 | 9/2026 | China | ||
| 18 | Foundation | 3 | After used (12 months) |
France |
| 19 | After used (12 months) |
USA | ||
| 20 | After used (12 months) |
France | ||
| 21 | Concealer | 3 | 12/2023 | China |
| 22 | – | – | ||
| 23 | 7/2024 | China | ||
| 24 | Eye pencil | 2 | 4/2025 | China |
| 25 | 5/2025 | China | ||
| 26 | Eye liner | 2 | 6/2026 | China |
| 27 | Eye Shadow | 3 | 10/2026 | China |
| 28 | 3/2024 | China |
Table 2.
Group (2): Cosmetics products high-quality brands.
| Sample number | Cosmetic category |
Number of Collected samples |
Expiry date | Country of region |
|---|---|---|---|---|
| 29 | Lipsticks | 3 | 10/2023 | France |
| 30 | 09/2023 | Italy | ||
| 31 | 11/2023 | USA | ||
| 32 | Lip-gloss | 3 | 2/2023 | UK |
| 33 | 11/2024 | France | ||
| 34 | 10/2023 | USA | ||
| 35 | Powders | 3 | 08/2024 | Italy |
| 36 | 09/2024 | USA | ||
| 37 | 11/2023 | UK | ||
| 38 | Plusher | 2 | 08/2024 | France |
| 39 | 09/2024 | USA | ||
| 40 | Mascara | 3 | 12/2023 | France |
| 41 | 11/2024 | USA | ||
| 42 | 01/2024 | Italy | ||
| 43 | Foundation | 3 | 09/2023 | France |
| 44 | 07/2024 | USA | ||
| 45 | 03/2024 | Hong Kong | ||
| 46 | Concealer | 1 | 09/2023 | Poland |
| 47 | Eye pencil | 2 | 08/2024 | Uk |
| 48 | 03/2024 | France | ||
| 49 | Eye Shadow | 2 | 08/2024 | |
| Germany | ||||
| 50 | 02/2024 |
2.2. Media for growing microorganisms associated with cosmetics
Three different media were used to isolate pathogenic bacteria. These media are: (1) Nutrient agar, NA is medium used for growing bacteria. The chemical composition of this medium is (g/L): NaCl (5.0), yeast extract (2.0), beef extract (1.0), peptone (5.0), and agar (15.0), pH 7.4; (2) MacConkey agar is a differentiated and selective medium used for growing gram-negative bacteria. It containing of (g/l): peptone (17), agar (15), toluene (0.03), protease peptone (3), NaCl (5), methyl violet (0.001), pH 7.1; (3) Blood agar is also a differentiated and selective medium used for growing bacteria. The chemical composition (g/l): blood agar base (9 5 0) that contains NaCl (5.0 g), pantone (10.0), agar (15.0 g), beef heart (3.0), cornstarch (1.0 g), and sheep blood (50.0), pH 7.4.
For fungi, Potato Dextrose agar is used for growing fungi and yeast. The chemical composition is (g/ liter): Potato infusion (2 0 0), Dextrose (20), and agar (20).
2.3. Isolation of microorganisms
To isolate microorganisms we follow procedures specified in Chapter 23 of the FDA's Bacteriological Analytical Manual (BAM) for isolating bacteria and fungi procedure associate with cosmetics (https://www.fda.gov/food/laboratory-methods-food/bam-chapter-23-methods-cosmetics) (FDA, 2022). In the beginning, to clean each cosmetic, a sterile cotton swab was utilized such as mascara, lip gloss, powders, and lipstick. Cotton swab was taken and added to 10 ml of sterile saline was used to dilute the solution, which was then mixed in the vortex for 10 min. To make 10-1 dilutions, 1 ml of sample solution and 9 ml of saline solution were added to a fresh test tube. Dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5 were created. For liquid product like foundation and concealer 1 ml transfer to10ml of sterile saline to make a dilute solution, and the same serial dilution procedure was followed until concentrtion10-5. The culture dishes were incubated at 37 °C for 48 h. Most culture media showed bacterial colony development after incubation, for fungi the culture dishes were incubated at 25 °C for one week after inoculation. The total number of colonies was determined as (CFU ml−1) after the incubation time. The colonies were then sub cultured on nutrient agar dishes. Each colony underwent this technique three times to confirm the purity of each isolated bacteria.
2.4. Identification of microorganisms associated with cosmetics
2.4.1. Bacteria
Bacteria were identified based on morphological traits as reported in the 8th edition of Bergey's Manual of Determinative Bacteriology (Bergey and Holt, 2000). Bacteria identified by describing colonies growing on different plates, Gram staining and shapes under the microscope (Noor et al.,2020). Oxidases, catalase, and the analytical profile index (API-20E kit) are further biochemical tests. The kits contain up to 20 biochemical tests, gelatinase, arginine Di hydrolase, â-galactosidase enzyme, Voges-Proskauer test, citrate test, lysine decarboxylase, urea hydrolysis, ornithine decarboxylase, H2S production, indole test, tryptophan deaminase, as well as sugars (amygdalin, mannose, rhamnose, sorbitol, inositol, sucrose, arabinose, glucose, and melibiose) fermentation.
2.4.2. Fungi
Colonies of fungi were identified based on their microscopic and macroscopic appearance, reference to manuals of Barnett and Hunter (1972). Fungal filaments were stained by lactophenol.
2.5. Molecular Identification of bacteria 16S rRNA sequences
-
A)
DNA extraction: by using protocol of Gene Jet genomic DNA purification Kit (Thermo Fisher Scientific) after harvesting up of bacterial (2x109) cells by centrifugation for 10 min at 5000 × g.
-
B)
PCR amplification:
PCR amplification was done utilizing forward primer (5′AGA GTT TGA TCC TGG CTC AG’3), reverse primer (5′GGT TAC CTT GTT ACG ACT T 3) (Lane, 1991). PCR was performed using the recommended thermal cycling conditions as a follow: initial step for 10 min at 95 °C. Denaturation for 30 sec. at 95 °C. Annealing at 65 °C for 1 min. 35 rounds of 90-second extension at 72 °C. The extension time was 10 min. at 72 °C. Utilizing PCR products on agarose 1 %agarose gel against 1 Kb plus ladder (Thermo Fisher Scientific). BLAST analysis was then performed on the bacterial isolates' DNA sequences (Clarridge, 2004).
2.6. Molecular Identification of fungi by 18S rRNA
-
A)
DNA Extraction:
Using molecular ITS regions, fungi were detected. Each fungal isolate was grown in PDA for one week at 25 °C. Each sample's DNA was isolated using a Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research) according to the manufacturer's instructions.
-
B)
ITS-PCR:
ITS4 was amplified using forward primers (5′-TCCGTAGGTGAACCTGCGG-3′) and reverse primers (5′-TCCTCCGCTTATTGATATGC-3′) in PCR reactions using 20 ng genomic DNA in a total volume of 25 L. The temperature profile for PCR amplification was as follows: a 2 min warm-up at 94 °C, at 94 °C for 60 sec. 40 cycle were done, 90 sec. at 52 °C, and 2 min at 72 °C, and a final step of 7 minutes at 72 °C. The 1 % agarose gel was prepared and ran as indicated above. The fungus isolates' DNA sequences were then BLAST-ed.
3. Results
A Total of 50 samples of cosmetics were collected from different shops in Mecca region. Saudi Arabia. Samples were isolated on four different media (Potato dextrose agar, Blood agar, MacConkey agar, and Nutrient agar). Collected samples were divided into two groups regarding to their quality. Low - quality brands in Table 1 and high-quality brands in Table 2.
Generally, results of microbial growth are shown in Table 3 and Table 4 the bacterial out number fungi in terms of growth percentage. Most of the bacteria that isolated were Gram- positive (Cocci and bacilli), and Gram-negative bacteria such as short bacilli. While fungal isolates were related to genera Aspergillus, Penicillium and Rhizopus.
Table 3.
Cosmetics samples products with microbial contamination in low-quality makeup.
| Cosmetics Category | Number of samples |
Media type |
|||
|---|---|---|---|---|---|
| Nutrient Agar | Blood Agar | MacConkey Agar | Potato dextrose Agar | ||
| Lipsticks | 1 | S.aureus | S.aureus | Pseudomonas aeruginosa | – |
| 2 | – | – | Pseudomonas aeruginosa | – | |
| 3 | – | Streptococcus pneumonia | Pseudomonas aeruginosa | + Aspergillus | |
| 4 | – | – | – | – | |
| Lip-gloss | 5 | Staphylococcus epidermidis | – | – | – |
| 6 | E. coli | Staphylococcus epidermidis | – | – | |
| 7 | Staphylococcus aureus | Streptococcus pneumonia | – | – | |
| 8 | Staphylococcus aureus | Staphylococcus aureus | – | – | |
| 9 | – | Staphylococcus aureus | – | + Aspergillus | |
| Powders | 10 | Staphylococcus epidermidis | – | – | + Penicillium sp |
| 11 | Streptococcus pneumonia | Staphylococcus aureus | Escherichia coli | – | |
| 12 | – | – | – | – | |
| Plusher | 13 | Staphylococcus aureus | Staphylococcus aureus | Escherichia coli | + Aspergillus |
| 14 | – | Staphylococcus aureus | Escherichia coli | – | |
| Mascara | 15 | – | – | – | – |
| 16 | – | – | – | – | |
| 17 | – | – | – | – | |
| Foundation | 18 | – | Bacilli (G + ) | – | +Rhizopus |
| 19 | – | – | – | – | |
| 20 | Staphylococcus epidermidis | – | E. coli | + Aspergillus | |
| Concealer | 21 | E. coli | – | E.coli | – |
| 22 | – | – | – | – | |
| 23 | Streptococcus pneumonia | – | – | – | |
| Eye pencil | 24 | – | – | – | – |
| 25 | – | – | E. coli | + Penicillium sp | |
| Eye liner | 26 | Staphylococcus aureus | Staphylococcus aureus | – | – |
| Eye Shadow | 27 | Streptococcus pneumonia | – | – | – |
| 28 | Staphylococcus aureus | – | – | – | |
No growth (−).
Table 4.
Microorganisms in the samples from cosmetics products in low-quality makeup.
| Cosmetics category | Bacterial isolates | Fungal isolates |
|---|---|---|
| Lipsticks | Staphylococcus aureus., Streptococcus pneumonia, Bacillus subtilis. Pseudomonas aeruginosa | Aspergillus sp |
| Lip-gloss | Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Streptococcus pneumonia | Aspergillus sp |
| Powders | Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli. | Penicillium sp |
| Plusher | Staphylococcus aureus, Escherichia coli. | Aspergillus sp. |
| Mascara | – | – |
| Foundation | Bacilli subtilis, Staphylococcus epidermidis, Escherichia coli. |
Aspergillus sp, Rhizopus sp . |
| Concealer | Streptococcus pneumonia, Escherichia coli | – |
| Eye pencil | Escherichia coli. | Penicillium sp |
| Eye liner | Staphylococcus aureus | – |
| Eye Shadow | Staphylococcus aureus, Streptococcus pneumonia | – |
Table 3 and Chart 1 show the frequency of microbial growth in low quality makeup. It has been observed the most contaminated product was in lip gloss and it follows by the lipstick. It found also, the less growth were in Eye pencil, Eye liner and Eye shadow, and there's no growth in mascara. The bacterial isolates from low- quality cosmetics were Staphylococcus aureus, S. epidermis, Streptococcus pneumonia, Bacillus subtilis, and E. coli. The fungal isolates were, Rhizopus sp., Penicillium sp., and Aspergillus Sp. (Table 4).
Chart 1.
Frequency of microbial growth in low-quality makeup.
The percentages and diversity of bacterial growth is shown in (Chart 2). The most predominant genus of bacteria is Staphylococcus aureus and E. coli (27 %), which follows by Streptococcus pneumonia (18 %), Staphylococcus epidermis (17 %), Bacilli subtilis (12 %), and Pseudomonas aeruginosa (5 %). In Chart 3 Aspergillus sp. is the most predominant fungi (57 %), which follows by Penicillium (29 %) and Rhizopus (14 %). The highest fungal growth rate was in lip-gloss that isolated from the low-quality cosmetics. In Table 4, high quality cosmetics show less growth of bacteria then low-quality, while there was no growth at PDA. Chart 4 shows the frequency of microbial growth was the highest in mascara, lip-gloss which follows in order by Eye Shadow, Lipsticks, Powders, Plusher, and Concealer. There is no growth of bacterial species in Foundation and Eye pencil.
Chart 2.
Percentage of bacterial growth in low-quality makeup.
Chart 3.
Percentage of fungal growth in low-quality makeup.
Chart 4.
Frequency of bacterial growth in high-quality makeup.
Table 5, Table 6 and Chart 5 show the most predominant genus of bacteria is Staphylococcus aureus with 41 %, which follows by Bacilli subtilis and Pseudomonas aeruginosa and E. coli with 17 %. Streptococcus pneumonia is the less dominant with 5 %.
Table 5.
Microbial contamination in the samples from unused cosmetics products in high-quality makeup.
| Cosmetics category | Number of samples |
Media type |
|||
|---|---|---|---|---|---|
| Nutrient Agar | Blood Agar | MacConkey | Potato dextrose Agar | ||
| Lipsticks | 29 | Staphylococcus aureus | – | – | – |
| 30 | – | – | – | – | |
| 31 | Staphylococcus aureus | – | – | – | |
| Lip-gloss | 32 | Staphylococcus aureus | + Bacillus sp (G + ) | – | – |
| 33 | – | – | – | – | |
| 34 | – | – | – | – | |
| Powders | 35 | – | Staphylococcus aureus | – | – |
| 36 | – | – | – | ||
| 37 | Staphylococcus aureus | – | – | – | |
| Plusher | 38 | – | Staphylococcus aureus | – | – |
| 39 | – | – | Escherichia coli | ||
| Mascara | 40 | Staphylococcus epidermidis | – | – | – |
| 41 | – | – | Pseudomonas aeruginosa | ||
| 42 | – | Streptococcus pneumonia | – | – | |
| Foundation | 43 | Staphylococcus aureus | – | – | – |
| 44 | – | – | – | – | |
| 45 | Staphylococcus aureus | – | – | – | |
| Concealer | 46 | – | + Bacillus sp (G + ) | – | – |
| Eye pencil | 47 | – | – | Pseudomonas aeruginosa | – |
| 48 | – | – | Pseudomonas aeruginosa | ||
| Eye Shadow | 49 | – | Staphylococcus aureus | – | – |
| 50 | Staphylococcus aureus | – | – | – | |
*No growth (−).
Table 6.
Bacterial contamination in the samples from unused cosmetics products in high-quality makeup.
| Cosmetics category | Bacterial isolate |
|---|---|
| Lipsticks | Staphylococcus aureus |
| Lip-gloss | Staphylococcus aureus, Bacillus subtills |
| Powders | Escherichia coli |
| Plusher | Staphylococcus aureus, Escherichia coli |
| Mascara | Staphylococcus aureus, Streptococcus pneumonia |
| Foundation | Staphylococcus aureus |
| Concealer | Bacillus subtills |
| Eye pencil | Pseudomonas aeruginosa |
| Eye Shadow | Pseudomonas aeruginosa |
Chart 5.
Percentage of bacterial growth in high-quality makeup.
Most of the bacteria that isolated were Gram-positive (cocci and bacilli), and in Gram-negative bacterial isolates (Bacilli, and short bacilli). Table 7 shows the average of the total counts of colony in low quality brands as (CFU/ ml). In low quality brands, the lip gloss and lipstick recorded high numbers than other products for bacterial isolates with (300X10-5 and 251 × 10-5) while fungal isolates recoded low percentage number less than 10 colonies in most low-quality brands except plusher. Table 8 shows the average of the total counts of colony in high quality brands which indicates the decrease of microbial population compared to low quality brands, mascara recoded the highest numbers with (300 × 10−5) while no fungal growth observed in all products.
Table 7.
Average of bacterial counts (CFU /10-5) in low-quality brands of collected cosmetics.
| Cosmetics category |
Bacterial isolates CFU (10-5/ml) |
Fungal isolates CFU (10-5/ml) |
|---|---|---|
| Lipsticks | 251 | <10 |
| Lip-gloss | 300 | <10 |
| Powders | 212 | <10 |
| Plusher | 198 | 33 |
| Mascara | ND | ND |
| Foundation | 119 | <10 |
| Concealer | 73 | ND |
| Eye pencil | 21 | <10 |
| Eye liner | 39 | ND |
| Eye Shadow | 65 | ND |
*ND (not detected).
Table 8.
Average of bacterial counts (CFU /10-5) in high-quality brands of collected cosmetics.
| Cosmetics category |
Bacterial isolates CFU 10-5/ml |
|---|---|
| Lipsticks | 89 |
| Lip-gloss | 110 |
| Powders | 77 |
| Plusher | 95 |
| Mascara | 300 |
| Foundation | 103 |
| Concealer | <10 |
| Eye pencil | 33 |
| Eye liner | 57 |
| Eye Shadow | 68 |
According to morphological and microscopic characters, bacteria were identified based on the reaction of gram stain and the arrangement of bacteria. Gram positive bacteria that isolated from low-quality makeup are shown in Fig. 1, Fig. 2, Fig. 3. While fungi isolates were identified by morphological cultures and microscopic detection by observing mycelium and spores' arrangement on pores or sporangium or vesicles. Fungal cultures are shown in Fig. 4, Fig. 5.
Fig. 1.
Different genera of bacteria isolated from low-quality cosmetics products; (A) Streptococci, (B) Staphylococci, and C) Bacillus sp.
Fig. 2.
Fungal cultures isolated from low-quality cosmetics products in culture medium; (A) Penicillium sp., (B) Aspergillus, and (C) Aspegillus fumigatus.
Fig. 3.
Fungi isolated from low-quality cosmetic products; (A) Aspergillus sp., (B)Penicillium sp., and (C) Rhizopus spp. Development of zygospore and its germination.
Fig. 4.
(A) Bacillus subtilis and Staphylococcus aureus.
Fig. 5.
Gram positive bacteria isolated from high-quality products (A) Bacillus sp. and (B) Staphylococcus sp.
3.1. Identification of bacteria by biochemical tests
Bacteria were identified using the biochemical assays described in Table 9. and the findings were Bergey's handbook compared, which showed the following microbes: Escherichia coli, Streptococcus pneumonia, Bacillus subtilis, Staphylococcus aureus, pseudomonas aeruginosa, and Staphylococcus epidermidis.
Table 9.
API-20E kit was used for biochemical testing of bacterial isolates.
|
Bacterial Isolate |
Gram stain | Catalase test | Oxidase test | Citrate test | Indole test |
H2S production |
Urea test | Sugar Fermentation test |
Gelatin hydrolysis test |
|---|---|---|---|---|---|---|---|---|---|
| Bacillus subtilis | Gram positive | +ve | +ve | -ve | -ve | -ve | +ve | -ve | +ve |
| E. coli | Gram Negative |
+ve | -ve | -ve | +ve | -ve | -ve | +ve | -ve |
| Staphylococcus aureus | Gram positive | +ve | - ve | +ve | -ve | -ve | +ve | +ve | +ve |
| Staphylococcus epidermidis | Gram positive | +ve | -ve | +ve | -ve | +ve | +ve | +ve | -ve |
| Streptococcus pneumonia | Gram positive | -ve | -ve | +ve | -ve | +ve | -ve | +ve | +ve |
| pseudomonas aeruginosa | Gram Negative |
+ve | +ve | +ve | -ve | -ve | -ve | -ve | +ve |
3.2. Bacterial and fungi molecular identification
The resultant sequences of bacteria's 16 srRNA and fungi's ITS were aligned in the NCBI database, and matched sequences were supported and identified, by earlier biochemical studies. Based on the matching ratio of sequencing of bacterial and fungal isolates, as shown in Table 10 below, Phylogenetic tress for each isolate is shown in Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Fig. 15.
Table 10.
The matching ratio of bacterial and fungal isolates in the NCBI database.
| Match ratio | Organism |
|---|---|
| 99 % | Bacillus subtilis |
| 99 % | Escherichia coli |
| 99 % | Staphylococcus aureus |
| 99 % | Staphylococcus epidermidis |
| 99 % | Streptococcus pneumonia |
| 99 % | pseudomonas aeruginosa |
| 99 % | Aspergillus fumigatus |
| 99 % | Aspergillus niger |
| 99 % | Penicillium citrinum |
| 99 % | Rhizopus arrhizus |
Fig. 6.
Phylogenetic tree of bacterial strain B. subtills isolated from cosmetic product based on 16s rDNA sequence.
Fig. 7.
Phylogenetic tree of bacterial strain E. coli isolated from cosmetic product based on 16s rDNA sequence.
Fig. 8.
Phylogenetic tree of bacterial strain S. aureus isolated from cosmetic product based on 16s rDNA sequence.
Fig. 9.
Phylogenetic tree of bacterial strain S. epidermis isolated from cosmetic product based on 16s rDNA sequence.
Fig. 10.
Phylogenetic tree of bacterial strain S. pnenmoniae isolated from cosmetic product based on 16s rDNA sequence.
Fig. 11.
Phylogenetic tree of bacterial strain P. aeruginosa isolated from cosmetic product based on 16s rDNA sequence.
Fig. 12.
Phylogenetic tree of fungal strain A. fumigatus isolated from cosmetic product based on 18s rRNA sequence.
Fig. 13.
Phylogenetic tree of fungal strain A.niger isolated from cosmetic product based on 18s rRNA sequence.
Fig. 14.
Phylogenetic tree of fungal strain R. arrhizus isolated from cosmetic product based on 18s rRNA sequence.
Fig. 15.
Phylogenetic tree of fungal strain Penicillium citrinum isolated from cosmetic product based on 18s rRNA sequence.
4. Discussion
The majority of the isolated microorganisms in this investigation were pathogens found in unused cosmetics. Even though these products are new brands and have never been used, the risk of microbiological infection was considerable. The results showed that both high-quality and low-quality cosmetic samples were contaminated with fungus and germs. Gram positive cocci such Staphylococci, Streptococci, and Bacilli were among the isolated microorganisms. Moreover, short bacilli are preferred by gramme negative bacteria. While isolates of fungi from the Rhizopus, Penicillium, and Aspergillus genera.
In general, high-quality brands exhibit less contamination than low-quality ones; this may be related to the products' ingredients, the way preservatives are applied, the circumstances of transit and storage. The most prevalent genus was Staphylococci, which was discovered in both high- and low-quality samples with a frequency of (41 %) both. Numerous research has established that Staphylococcus sp. is present in various cosmetic items. Some of these strains are dangerous and bring on skin conditions like desquamate and acne (Dadashi & Dehghanzadeh, 2016) and (El-Tablawy, 2009). Additionally, a study by Noor et al., 2020 discovered that staphylococci were the most typical species isolated from a variety of cosmetic tests.
Another potentially hazardous organism was identified during this examination, called Bacillus. Bacillus causes localized necrotizing cellulitis in the skin, making it risky to use eye cosmetics that have Bacillus contamination (Dadashi & Dehghanzadeh, 2016). According to a 2019 study by Bashir and Lambert, cosmetic regulations expressly state that products shouldn't contain pathogenicity's organisms. Between 70 and 90 percent of the products were contaminated with germs. Fungi also are pathogenic organisms, in low quality cosmetics items Aspergillus, Rhizopus and Penicillium were recovered. Penicillium sp. was the most abundant organism in other research, appearing in 75 % of samples in the study of (De oliveira et al., 2020). Due to their involvement in the synthesis of mycotoxins, particularly ochratoxin A and citrinin, Penicillium represent a risk to human health. Additionally, both mycotoxins are extremely nephrotoxic at micromolar doses (Geisen et al., 2018). Filamentous fungus can utilize the humidity of the environment to assure growth on the surfaces of cosmetics with low quantities of water, such foundation, qualifying these goods as semi-aqueous preparations. This implies that moisture promotes the growth of filamentous fungus. Despite the fact that there was a lot of water in those preparations (Oliveira et al., 2020).
In Saudi Arabia, Nasser (2008) investigated the microbial contamination in 75 cosmetic samples. She discovered that lip cosmetic products had the highest fungal counts, and the samples were infected with 13 and 24 mesophilic and thermophilic fungus species from 2 and 6 genera, respectively. The most common fungal genus was Aspergillus, and 36.7 % of the analyzed samples were contaminated with E. coli, while Bacillus and Pseudomonas were commonly observed (Okeke and Lamikanra, 2001). From a cosmetic manufacturing facility, Pseudomonas aeruginosa and Enterobacter gergovia were identified, and these microorganisms demonstrated increased resistance to formaldehyde-releasing preservatives and parabens (Ferrarese et al., 2003). The risk of infection from these germs, which can infect the lips or eyes, is very high (Bashir and Lambert, 2020, Burleson and Martinez-Vaz, 2011).
On the other hand, because nature can encourage microbial growth in cosmetics, which can lead to infection, these products are more likely to be contaminated by microorganisms than those that don't contain water. (Halla et al., 2018 & Skowron et al., 2017). Water that hasn't been sterilized or treated can encourage bacterial growth, which can contaminate cosmetics. Additionally, cosmetics can serve as a nutritious environment for the growth of microorganisms. (Alwan, 2018). Microbial contamination, according to (Choubey & Godbole, 2017) can occur during the manufacturing process, in raw materials, during consumer usage of the product, or as a result of dust in retail marketplaces. After a product has been opened, the consumer's hands or the surroundings may contaminate it further.
Due to the creation of hazardous bacteria metabolites and product degradation, cosmetic product contamination can directly affect human health. Therefore, maintaining the sanitary standards of the product and ensuring consumer safety depend on the microbiological preservation of cosmetics (Choubey & Godbole, 2017). It is generally known that microorganisms have the capacity to multiply and proliferate in cosmetic products. Cosmetics include a variety of components, including sugar, vitamins, proteins, oils, and water, all of which are necessary for the growth of microbes (Ahmida et al., 2018). According to (Wilson et al., 1975), climatic variables like temperature variation and moisture from humidity promote microbial growth in cosmetic goods.
(Ahmida et al., 2018) recommended Keep cosmetics in a dry, cool environment to prevent spoiling, shield consumers from infections, and lengthen the shelf life of your items. A good preservative system, as described by (Nuzhath, 2014 will prevent quick postproduction contamination, resulting in acceptable low levels of microorganisms in the preparation and so making it safe for consumers, which in this case is ineffective. Cosmetics that have been contaminated by microbes run the risk of breaking down and costing consumers money. As a result, it's crucial to guarantee that cosmetic goods are bacteria free, and the preservatives used in them should be able to kill any potential microbes (Siya et al., 2019).
5. Conclusion
Microbial contamination of cosmetic products is a matter of great importance to the industry, and it is potentially a major cause of economic losses. Water and some ingredients support microbial growth. The results of this study indicated that all samples in high-quality and low-quality brands show bacterial and fungal growth more than the acceptable number prescribed by FDA and other legislations. The high-quality of cosmetics doesn’t mean the high price only, it means selection a good raw material, offering a good storage and transport, and have preservation system which may reduce microbial growth. For consumers, we recommend sterilizing these items with a good sanitizer. Our vision is to keep searching for more studies in cosmetics microbiology discuss the microbial contamination and solutions to reduce it.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
The author would like to thank Umm AL Qura university for giving opportunity for scientific sabbatical leave (decision number 4401092524).
Footnotes
Peer review under responsibility of King Saud University.
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