Microbiological Quality and Characterization of Potential Pathogens Associated with Selected Brands of Commercial Cosmetic Products in Nigeria

Aims: Seemingly innocuous commercial cosmetic products have been responsible for serious overt and covert skin infections, which were often ignored as the sources or vehicles of transmission of pathogens. Materials and Methods: Three (3) popular brands of commercial cosmetic products, consisting of fifteen (15) units each, of Hair straighteners, “Lip Gloss” and baby lotions, were randomly purchased from retail stores in Southwest Nigeria, and the microbiological qualities were evaluated, using the Aerobic plate count technique. Results: From the forty five (45) samples analyzed, thirty-eight (84%) were contaminated, from which a total number of seventy (70) microbial isolates belonging to eleven (11) bacteria genera (comprising 7 Gram positive rods, 27 Gram negative rods and 10 Gram positive cocci) and nine (9) fungal genera, were isolated. Of the bacteria isolates, 50% of the Gram negatives and 52.9% of the Gram positives were multidrug resistant. The bacteria and fungal diversity in the baby lotion exceeded the hair relaxer and the Lip gloss, respectively. Original Research Article Babalola and Eze; BMRJ, 9(5): 1-17, 2015; Article no.BMRJ.14083 2 Conclusion: The isolation of Buttiauxella agrestis, which had not been previously reported, in addition to Enterobacter gergoviae, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus flavus, Penicillium spp and Candida albicans (designated as objectionable microorganisms in cosmetics) indicated failures in the statutory microbiological standards. As such the products could serve as vehicles for transmission of these pathogens.


INTRODUCTION
The US Food, Drug and Cosmetic Act defines cosmetics as articles intended to be rubbed, sprinkled or sprayed on, introduced into, or otherwise applied to the human body or any part thereof for cleansing, beautifying, promoting attractiveness, or altering the appearance, while maintaining the structure and functions [1]. Included in this definition are products such as skin creams, lotions, perfumes, lipsticks, eye and facial make-up preparations, hair straighteners, conditioners, shampoos, permanent waves, hair colors, and deodorants. The US FDA and the EU Cosmetics Directive requires that the microbial population be low, stable and devoid of harmful organisms, particularly in products designed to be applied around the eye and other sensitive areas, or for use by babies, young children, the elderly, and the immunocompromised [2]. While no mandatory limits are given for microorganisms, industry guidance recommends a Total Viable Counts (TVC) of <100 c.f.u/g for higher risk products (eye, baby products, and others) and <1000 c.f.u /g for all other products. However, all cosmetic products must be devoid of pathogenic microorganisms, such as, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and E. coli [3].
Evidence of microbial contamination and spoilage. Microbial contamination of cosmetics renders them unfit for use as the products may develop various degree of aesthetic changes. The growth of bacteria that produces alcohols or degrades emulsifiers may lead to instability, splitting of the emulsion and eventual spoilage. Microbial growth can produce enzymes that causes degradation of active ingredients and changes in the pH. Cellulose polymers in cosmetics can act as targets for microbial attack, and support extensive growth under suitable conditions when converted from a stiff gel into a running liquid, thereby rendering the cosmetic unfit for use. Finally, changes in the products may become evident as unpleasant aromas and tastes, color changes, production of irritants, loss of activity, alterations in viscosity, bulging/leakage of products, and visible surface growth [4].
Impact of Microbial contamination may include the cost of disposal and replacement of contaminated products, if a recall is required, loss of reputation to the brand or the manufacturer, serious microbial skin infections and even blindness, if the product is applied around the eyes [5]. A link between outbreak of Pseudomonas aeruginosa in a neonatal intensive care unit was reported [6] in the USA with contaminated hand washing lotion, just as Kallings et al. [7] reported that hydrocortisone ointment contaminated by Pseudomonas spp resulted in severe eye infections when used in the treatment of an ophthalmic condition. An outbreak of invasive mycoses caused by Paecilomyces lilacinus from contaminated skin lotion was reported by Orth et al. [8] in Switzerland, the cutaneous manifestation of which was confirmed [9]. Consequently, the health implications of microbially contaminated cosmetic products necessitated the US Department of Health and Human Services to issue a serious Import Alert (code named "Detention without Physical Examination of Cosmetics Due To Microbiological Contamination") on some products. The scope of the products included the followings among others: (1) Import alert on 15th June 2010 on Baby Moisturizing Lotions, Creams and Powders contaminated by Enterobacter gergoviae, and manufactured by Bio Botanical Labs, Quebec, Canada; (2) Import Alert on 23rd June 2011, on eye shadow and Lip gloss cosmetics contaminated by Staphylococcus intermedius and S. warneri and manufactured by beauty vision limited, Gong ming shenzen, China; (3) Import Alert of 7th Dec 2010 on Alexia Lip gloss cosmetic contaminated by Sphingomonas paucimobilis and Pseudomonas aeruginosa, manufactured by Maesa, Jinwan Zhuhai [10].
Previous reports of microbial quality assessment of cosmetics and toiletries were mainly from developed countries [11] usually in response to outbreaks of infectious diseases [6]. In the African continent, particularly Nigeria, few studies have been carried out to evaluate the microbiological quality of many brands of commercial cosmetics products that flood the Nigerian market, from the local and International cosmetic producers. Of these studies, Hugbo et al. [12] evaluated the microbial quality of ten brands of commercially available creams and lotions in Benin city Nigeria and discovered Staphylococci, Penicillium, Aspergillus fumigatus and Microsporium species as the major contaminants. Similarly, Okorie [13,14] in separate studies reported the presence of Klebsiella, Pseudomonas aeruginosa, Bacillus spp, Staphylococcus aureus and Penicillium spp from some brands of commercially hawked cosmetics products in Nigeria. However, these previous reports were primarily focused on products consumed by adults, and none of them provided the morphological characteristics of the detected fungi.
Hair straightener otherwise known as ''relaxer'' is a type of lotion or cream generally used by people with "afro textured hair", to make hair less curly, easier to straighten or to create perms by chemically "relaxing" the natural curls by breaking down the proteins bonds of hair, temporarily or permanently.
Lip gloss is a brand of cosmetic product invented by Max Factor in 1930, primarily used to give lips a glossy lustre and subtle color. It is distributed as a liquid or a soft solid, completely clear, translucent, or various shades of opacity, including frosted, glittered, glassy, and metallic finishes, packaged in squeezable tube and wand applicator formats such as "Lip Smackers" [15].
The harmful microbes in cosmetics constitute a much greater risk to new born babies, infants, and young children, than to adolescents and adults [16,17]. Considering the neglected research on the ecology and colonization of cosmetics by pathogens, this study investigated the microbiological quality of popular brands of Baby lotions, Hair relaxers, and Lip gloss cosmetics in Nigeria, and determined the characteristics of the detected fungal and bacteria contaminants.

Samples
A total number of forty-five (45) cosmetic products representing (15) units each of; ''Oz'' hair relaxer, "Lip gloss" and ''P'' baby lotion brands of cosmetics, were randomly purchased from different retail outlets in Southwest, Nigeria. Some of the products had comparable batch numbers, date of production, expiry dates and National Agency for Food, Drug Administration and Control (NAFDAC) numbers on the products labels. None of the products was expired prior to analysis at the Microbiology Laboratory of Adekunle Ajasin University, Akungba Akoko, Ondo State, Nigeria. The physical attributes of the products were studied and other information obtainable from the product labels were recorded.

Microbiological Analysis of Samples
The methods of Hitchings et al. [18] was adopted in evaluating the viable bacteria and fungal colonies, under a laminar air flow cabinet. After surface sterilization of the container with 70% ethanol, using a sterile spatula or pipette, 1 gram (or 1 ml) of each sample was aseptically withdrawn and dissolved in 1 ml sterile Tween-80 diluent, vortexed, made up to 10 -1 dilution in 8 mls of sterile Modified Letheen Broth (MLB), from which 5 mls was diluted in 45 mls of MLB to obtain 10 -2 dilution, and further serially diluted to 10 -4 dilution. Employing the pour plate method, Inoculum (1 ml) was taken from 10 -2 dilution to seed sterile plates of replicated Tryptic Soy Agar (TSA), Modified Letheen Agar (MLA) and Mannitol Salt Agar (MSA) and Chloramphenicol supplemented Sabouraud Dextrose Agar (SDA). The plates with uninoculated growth controls were then allowed to solidify, inverted and incubated at 37°C for 48hrs (TSA, MSA and MLA) and SDA at 27°C for between 3-5 days.

Characterization and Identification of the Bacterial Isolates
The bacteria isolates were characterized on the basis of their cellular morphology, cultural, biochemical, and sugar fermentation according to Olutiola et al. [19] and identified according to Holt et al. [20,21].

Antibiotic Susceptibility of the Bacterial Isolates
The antibiotic susceptibility of the bacteria was determined using the standard method of CLSI [22]. The following antibiotics and their disk concentrations were tested against the isolates using the Kirby-Bauer disk diffusion method on Mueller-Hinton Agar (Oxoid

Characterization and Identification of the Fungal Isolates
Typical discrete pure culture of each isolate was selected, characterised and identified by combinations of the cultural and microscopic observations/characteristics. The isolates were further identified based on the microscopic fungal features such as Phialides, septate Sporangiophores, Conidiophores, and budding, as previously described [23]. In addition, typical isolates were confirmed by culturing the pure isolates on 2% Malt Extract Agar to achieve enough conidiation, from which suspensions were prepared and inoculated into the BIOLOG FF Microplate (BIOLOG, CA, USA) and incubated at 26°C for 5days. Characteristic fingerprints of each isolate were then read using the BIOLOG Microstation and confirmed with the FF Database. Furthermore, the fungal wet mounts were prepared using Lactophenol-incotton blue, viewed under the microscope at X400 mag. and the photomicrographs were digitally recorded. The observed characteristics were then compared and confirmed with reference to Ellis [24].

Microbiological Quality of the Products
Of the total number of forty five (45) samples representing three popular brands of selected cosmetic products that were analyzed in this research, thirty-eight samples (84%) were positive for various levels of microbial contaminants (Tables 1 and 6). The qualitative microbiological analysis showed that 15 samples of the Lip Gloss, 12 samples of the Baby Lotion, and 11 samples of the Relaxer, were contaminated to varying degrees with respect to the diversity, the counts, and presence of potential bacterial and fungal pathogens. The bacteria counts in the Baby lotion ranged between 3.5x10 2 and 55x10 2 cfu/ml, in the Relaxer the counts were between 3.5x10 2 and 7.7x10 2 cfu/ml, while the counts in the Lip Gloss were 20x10 2 to 38x10 2 cfu/ml respectively. No contaminant was detected in samples R5, R8, R11, R13, P2, P9 and P15. While the product labels of the baby lotion and the Hair Straightener had authentic production dates, batch numbers, expiry dates, and NAFDAC numbers, all these information were lacking on the Lip Gloss samples. There were no visible defect(s) on the samples prior to the analysis. Regarding the shelf life of the products, a high fungal and bacteria counts were recorded as some of them were closer to the expiry dates ( Table 1). The shelf life of the Lip Gloss could not be determined as the necessary information were lacking on the product labels, thereby making vital assessment and batch recalls (of such products) impossible.

Table 1. Physical attributes and the total viable counts (TVC) of the bacteria isolates from commercial Hair straightener (Relaxer), Lip gloss and baby lotion cosmetics
LG4A LG5A LG6A LG7A LG8A Bacillus licheniformis P14 P14A Erwinia amylovora LG8B Bacillus cereus

DISCUSSION
Statutory Cosmetic Industry standards stipulated a total viable count of aerobic bacteria, yeast and moulds of less than 100 c.f.u/g (or ml) for eye and baby products and 1000 c.f.u/g (or ml) for other products, at completion of manufacture, while potentially pathogenic microorganisms such as Pseudomonas, Candida and Staphylococcus should not be present in the product [3]. The results of this study clearly indicated that the microbial diversity and population of the detected organisms exceeded the stipulated permissible levels, particularly in the Baby lotion and the "Lip gloss". These findings therefore disqualify these products as meeting the stipulated qualitative microbial limits in Official monographs [3]. The manufacturers needed to evolve adequate measures towards the equipment cleaning and sanitation, as the observed contamination could be due to periodic dislodgements from equipment surfaces, crevices, elbows and joints. Although, the bacteria diversity was highest in the lip gloss, it however contained the least fungal diversity.
Four (4) fungal genera were detected from the Hair Relaxers, as opposed to the eight (8) and two (2) genera that were detected in the Baby lotions and the Lip gloss respectively. This may be attributed to the alkalinic nature of the products. While fungi grows best in acidic milieu as obtained in the Baby lotion, the bacteria prefers alkaline and neutral medium as obtainable in the Hair relaxers and the Lip gloss. Hence, the bacteria diversity exceeded the fungal diversity in all the product categories as presented in Tables 1, 2 and 5.
Fungal colonization and multiplication in cosmetic products are more rapid and evident than bacteria because of fungi nutritional and enzymatic versatilities, proliferation and eventual spoilage of the products. Three fungal enzymes (exo-β-1, 4-glucanase, endo-β-1,4-glucanase and β-glucosidase) usually synthesized by three fungal species, belonging to the genera Aspergillus, Fusarium and Penicillium, are involved in the degradation of crystalline cellulose in cosmetics and toiletries [25]. These groups of fungi were typically isolated in this study (Table 5). Cladosporium species may present opportunistic infection in the user as it was reported to cause infections of the skin and toenails, and may effect spoilage of the hair relaxer product as it was incriminated in the transformation of some steroidal drug to inactive forms and offensive metabolites [26].  Conidiophores, slender and simple bearing phialides, with macroconidia held in small mont head that are several celled and slightly curved at the pointed end. Septate, conidiophores absent, conidia hyaline. One-celled, short, cylindrical with truncate ends, formed by segmentation of hyphae.
Geotrichum spp   The detection of these fungal contaminants (Figs. 1a -1u), particularly the pathogenic species comprising Candida, Fusarium, Aspergillus and Trichoderma cannot be condoned in the "P" Baby lotion as this brand is a popular product applied in the personal care of the newborn (> 28 days old), the infants (1year old) and the children (1-4 year old), whose immune developments were still rudimentary. The recent increase in numbers of people with immunodeficiencies has caused an equally dramatic surge in the incidences of fungal infections particularly Candida, Aspergillus and Penicillium species. Aspergillosis is recognized as the most common fungal infection of the immunocompromised patients, while Candida sp. is one of the notorious pathogens responsible for septicemia and superficial mycotic infections of the skin, the nail, the hair, and mucous membranes. Physiologically, the acidic pH of the human skin ranges between 4-5, while the acidic growth condition of fungi ranges between 2.2 -4.5. These pH ranges potentially provide favorable conditions for superficial infection and invasive establishment of fungal diseases in the tender skin of the susceptible user. Furthermore, majority of the detected fungi have the capacity to elicit mycotic keratitis, an opportunistic fungal infection of the eye that causes ulceration and inflammation, usually following a trauma to the cornea by vegetative matter, contaminated cosmetics, soil, surgery, or prolonged treatment with corticosteroids by some users who also apply toning or bleaching creams [27]. The etiological agents include various saprophytic fungi especially Aspergillus fumigatus, A. flavus, A. niger, Fusarium solani and Candida albicans, that were typically isolated in this present study. It is also possible that epithelial damage caused by Aspergillus could release nutrients for fungal growth in infected tissues.
Aside the direct pathogenic infection of these fungal contaminants, the isolates synthesizes secondary metabolites directly into their growth milieu. Of these metabolites is gliotoxin of Aspergillus fumigatus, with potent cilioinhibitory, genotoxic and cytotoxic properties, produced by various Penicillium, Aspergillus, Gliocladium, Thermoascus, and Candida species [28]. Intrinsic contamination and in-use growth of these contaminants overtime, will certainly elicit the metabolites. Although it is not yet known how common the production of gliotoxin exist in the actual cosmetics from which A. fumigatus, Penicillium and Candida were isolated, or to what extent and by what kind of mechanism the gliotoxin could be mobilized from the products. However, the fact remains that the contaminated cosmetics present potential risk to the health of the consumer.
A considerable growth area in marketing cosmetics is specialized ethnic populations, of products targeting the African-American, Hispanic, and Asian-American markets. In the ethnic market, products that target skin decoloration are experiencing the best consumer response [29,30]. However, it was a common phenomenon and gory sight to behold in some consumers, who in the name of civilization, apply bleaching and toning creams to achieve lighter complexion [16], but eventually acquire patchy, scaly, chameleonic complexion, attributable to the synergistic impact of the chemical constituents and microbial contaminants in the formulations. As creams consist of emulsions of water and oil, stabilized with emulsifiers, they are prone to contamination with Pseudomonas, Burkholderia cepacia, Micrococci, Staphylococcus aureus, and aerobic spore formers [4]. These conditions may have been responsible for the detection of Pseudomonas aeruginosa and high prevalence of Enterobacter gergoviae, Bacillus subtilis and Serratia marcescens in the Baby lotion. The mesophilic nature of Bacillus is enhanced by the storage temperature and retailed conditions of the products. Some microorganism survive by forming endospores, biofilms, capsules, extracellular enzymes and by exhibiting acid tolerance [31]. All these characteristics are exceptionally exhibited by the isolated bacteria species in this study and could have been responsible for their survival in the cosmetic products, particularly Buttiauxella agrestis which is hereby reported for the first time in the Hair straightener (Relaxer) brand.
Generally, these products from where Pseudomonas, Bacillus, Klebsiella, Staphylococcus, Escherichia, Enterobacter and Candida were isolated should not have been released for consumers' use, in view of the pathogenic potentials. Similar discovery of these potentially pathogenic species prompted the US Department of Health and Human Services to issue a serious ''Import Alert'' on some cosmetic products [10]. The detection of Escherichia hermannii, Staphylococcus aureus, Bacillus cereus, and Enterobacter spp in the ''Lip Gloss'' portends a critical risk to health as the cosmetics are usually applied on the lips and may eventually be ingested and elicit gastroenteric infections [32] in the susceptible user. Typical microbial contamination by objectionable microorganisms that necessitated product recalls of some baby lotions, creams, shampoos  [33], were similarly identified in this study. In addition, Klebsiella pneumoniae, Penicillium and Aspergillus were detected in the tested products thereby lending credence to the earlier reports of Osungunna et al. [14] and Okeke and Lamikanra [34]. However, contrary to the report of Razooki et al. [35], Staphylococcus and fungal contaminants were readily isolated from this present study, and this report provide a comprehensive knowledge on the diversity and characteristics of the fungal and bacterial contaminants, and potential pathogens associated with commercial cosmetic products. Considering the antibiogram of the detected bacteria, this present work is in tandem with the previous report of Gopalkrishna et al. [36]. Serratia rubidaea was resistant to Nitrofurantoin, Nalidixic acid, Colistin, Ampicillin and Streptomycin ( Table 3).
The resistance pattern observed in the S. rubidaea is attributable to inducible chromosomal beta lactamase of the cephalosporinase type, thereby engendering resistance to Ampicillin and Cephalotin. The observed resistance by the bacteria isolates could be plasmid mediated or induced by the biocides that were employed in the sanitation of the factory equipment as well as the preservatives in the products. In many cosmetics, preservatives may loose activity [37], and such product becomes a potential risk for microbial contamination [38]. In this study, the observed high level of contaminants may signify the failure of the incorporated preservatives to inhibit the contaminants, as earlier indicated [39]. These findings imply that the cosmetics do not meet the stipulated microbiological quality standards in official monographs and can therefore serve as vehicles for the transmission of the detected pathogenic organisms. The products constitute potential health risks to unsuspecting consumers, and the manifestation of the risks are highly probable, considering the immune fragility of babies, inadvertent ingestion of microbially tainted ''Lip gloss'', and the potential inoculation of pathogens, subsequent upon possible skin breaches and the in-use chemical tenderization or "burns" of the adult' scalp.

CONCLUSION
The isolation of Buttiauxella agrestis, which had not been previously reported, in addition to Enterobacter gergoviae, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus flavus, Penicillium spp, and Candida albicans (designated as objectionable microorganisms in cosmetics) indicated that the cosmetics do not meet the stipulated microbiological quality standards in official monographs and can therefore serve as vehicles for the transmission of the detected pathogenic organisms. Therefore, Good Manufacturing Practice (GMP) should be strengthened, the efficacy, and continued use of the adopted preservatives should be reviewed, to ensure wholesomeness of the products through their shelf life.