Study of the Microbial Contamination of Cosmetic Creams before and after Use

Transcription

1 Biohealth Science Bulletin 011, (), 7 EL-Bazza Z E et al. 011 EL-Bazza Z E 1, Toama M A, Taher H A 1. 1 Department of Drug Radiation Research, National Center for Radiation Research and Technology, Cairo, Egypt. Department of Microbiology, Faculty of Pharmacy, Cairo University, Egypt. (Received 19 th July 011. Revised th March 01. Accepted th April 01. Published Online 1 th July, 01.) Correspondence: EL-Bazza ZE. ahmandour_z@yahoo.com Study of the Microbial Contamination of Cosmetic Creams before and after Use Objective: The purpose of this study was to evaluate the microbial contamination of some types of cosmetic creams before and during handling by consumers. Materials and Methods: Fifty cosmetic cream samples of different brands including foundation cream samples, 10 foundation moisturizing cream samples and 1 moisturizing cream samples were purchased from the markets in Egypt. The total microbial counts of the cream samples were determined by the plate count technique. Results: The percentage of contamination with bacteria were found to be 0, 0,.7, with fungi they were %, 0%,.7 while with bacteria and fungi they were 8%, 0%,.7 for foundation cream, foundation moisturizing cream and moisturizing cream, respectively. The order of contamination of foundation cream samples is gram positive cocci > gram positive rods > gram negative cocci and gram negative rods, while on foundation moisturizing cream samples the order is gram positive rods > gram positive cocci > gram negative rods/ On moisturizing cream samples the order is gram positive cocci > gram negative rods > gram positive rods. Aspergillus species predominate over penecillium species as the only fungal contaminants on the tested cream samples. The level of microbial contamination was found to increase with time during consumer handling of the choosen cosmetic cream samples. Key words: Cosmetic creams and microbial contamination INTRODUCTION The warm and rather humid climatic conditions that prevail in most tropical countries would tend to support the survival and growth of many microorganisms. In a situation whereby a nutritionally rich pharmaceutical/ cosmetic product is severely contaminated, rapid growth and multiplicati-ons would be expected. This could lead to biodegradation of the product and hence the risk of infection to the consumers of the product. Cosmetic products, in addition to being free from gross microbial contamination and pathogens at manufacture, should be capable of maintaining low contamination levels during use. Product contamination may arise from row materials or water used in formulation or accidentally during use {1, }. However, aqueous cosmetic products in multiple use containers are subject to microbial contamination and spoilage unless they are satisfactory and packaged. The causes of contamination are believed to be lack of attention to good manufacturing practices resulting in the development of house organisms, inadequate preservative systems and / or inadequate microbiological test methods and microbial limits for finished products {} MATERIAL AND METHODS 1-Microorganisms: A total of bacterial contaminants and fungal contaminants were isolated from facial cream samples. Contaminants were isolated, purified and maintained on nutrient agar for bacteria and Sabouraud ' s agar for fungi. All cultures were stored at ºC and subcultured monthly on the same medium. - Gream samples: Fifty facial cream samples were purchased from the market: foundation cream samples representing different brands, 10 foundation moisturizing cream samples representing different brands and 1 moisturizing cream samples representing different brands (Table I). - Chemicals: Peptone, lablemco were the product of oxoid. Yeast extract was the product of BBL. Agar agar and peptone were the product of Difco. Other chemicals used in the present study were of the reagent grade. Microbial detection tests: For detection of the possible contamination of cream samples. One ml of each sample was suspended in 9 ml sterile tweenpeptone {}, 0.1 ml of the diluted sample was streaked on nutrient agar (oxoid) plates. The plates were incubated at ± ºC up to 7 hour for 7

2 Biohealth Science Bulletin 011, (), 7 Study of the Microbial Contamination bacterial detection while, for fungal detection, 1 ml of the diluted sample was mixed with Sabouraud ' s agar (oxoid) in plates. The plates were incubated at 8± C for -7 days. Microbial evaluation of the tested cream samples: Samples were analysed after purchase, surfaces of sample containers were disinfected with 70% ethanol before opening of the package. One ml of each facial cream sample was mixed with 9 ml sterile tween peptone and ten fold serial dilutions were made in the same diluent. Total aerobic bacterial count: The spread plate technique was used, 0.1 ml was taken from each appropriate dilution and spread in duplicate sterile plates containing solidified nutrient agar using a sterile glass spreader for each dilution. Plates were incubated at ±ºC and examined daily up to 7 hours. Suitable dilutions were counted as CFU. Total fungal count: One ml was taken from each appropriate dilution and mixed with Sabouraud, s agar is sterile duplicate plates. Plates were incubated at 8±ºC and examined daily up to 7 days. Suitable dilutions were then counted as CFU. Microbial evaluations of the tested cream samples during in-use: Facial cream samples that showed no detected contamination with microorganisms were chosen and used in the present investigations. Samples were subjected to share use daily by different consumers and the level of contamination through days was determined. According to the morphological characters of the microbial isolates, different colonies were separated on nutrient agar for bacteria and on Sabouraud ' s agar for fungi, purified and kept on the same medium at C for further studies. Identification of the bacterial isolates: Identification of the isolated gram positive rods was done by the method of Cowan, S.T. and Steel, K. J. 198 {}. Gram positive cocci and gram negative rods were identified using automated "microscan" (Liederbach, Germany, Dade Behring), which contain a variety of biochemical tests. Identification of the fungal isolates: The fungal isolates grown on Sabouraud ' s agar medium were identified according to Moubasher (199) and Manual of clinical microbiology (001) {,7}. RESULTS AND DISCUSSION The warm humid conditions that are characteristic of a topical environment are conductive to the growth of microorganisms which are responsible for a number of infectious diseases and the spoilage of cosmetic and pharmaceutical products {8, 9}. Developing countries also tend to have lower levels of hygiene and sanitation than industrialized countries, factors that make it possible for such organisms to thrive. In the present study detection of the bacterial and fungal contaminations were performed on 0 cosmetic cream samples ( foundation cream samples, 10 foundation moisturizing cream samples and 1 moisturizing cream samples. The microbial contaminations differ between the different cream types and brands. Out of foundation cream samples representing brands, 1 samples (8%) were found to be contaminated. Our of 10 foundation moisturizing cream samples representing brands, samples (0%) were contaminated, while, out of 1 moisturizing cream samples representing brands, samples (.7%) were contaminated (Table II). The level of microbial contamination of the tested cosmetic cream samples differ between the different types and between samples of the same brands. Out of foundation cream samples, 10 samples were found to be contaminated with bacteria in the range of 1.0x10 to. x10 7 and 9 samples were contaminated with fungi in the range of 1. x 10 to 1. x 10 CFU/ml. Out of 10 foundation moisturizing cream samples, samples were found to be contaminated with bacteria in the range of.0x10 to.x10 7, while, samples contaminated with fungi in the range of 1.x10 to 7.0x10 CFU/ml. On the other hand, out of 10 moisturizing cream samples, samples were found to be contaminated with bacteria in the range of 7.x10 to 1.x10 8 and with fungi in the range of 9.x10 to.1x10 CFU/ml (Tables III and IV). The percentage of contamination with bacteria were found to be 0%, 0%,.7%, with fungi they were %, 0%,.7, while contamination with bacteria and fungi were 8%, 0%,.7 for foundation cream, foundation moisturizing cream and moisturizing cream, respectively (Table IV). In the present investigation, bacterial contaminants and fungal contaminant were isolated from the tested cream samples. A total of, 8 and 10 bacterial contaminants and 19, 10 and fungal contaminants were isolated from the different brands of foundation cream, foundation moisturizing cream and moisturizing cream, respectively (Tables IV and V). The presented data are consistent with those objected by Hugbo et al. (00) {} in Nigeria who stated that the tested cosmetic cream products are contaminated to varying digress. In the present study the results of identification of bacterial contaminants on the cosmetic cream samples revealed that the order of contamination on foundation cream samples is gram positive cocci > gram positive rods > gram negative cocci and gram negative rods. On foundation moisturizing cream the order is gram positive rods > gram positive cocci and gram negative rods > gram negative cocci. On the other hand, the order of contamination on moisturizing cream samples is gram positive cocci > gram negative rods > gram positive rods (Table V). 8

3 Biohealth Science Bulletin 011, (), 7 EL-Bazza Z E et al. 011 In other study, Compana et al. (00) {10} in Italy evaluated 7 tensiolytes, 1 aqueous pastes, in three different states of intact, in use and ending product. Total bacterial count, isolation and identification of pathogenic isolates were performed on the collected cosmetics. About 10.% of tensiolytes were contaminated with Staphylococcus warneri, Staphylococcus epidermidis and Pseudomonas putida. The results of evaluation of the fungal contamination show that Aspergillus species predominate over Penicillium species as the only fungal contaminants (Table VI). These results consisted with Becks and Lorenzoni (199) and Behravan et al. (00) {11, 1}. The level of microbiological contamination in a non sterile product, such as cosmetic formulations is made clear in the microbial limit standards. FDA (001) {1} stated that cosmetics of non eye areacounts should not be more than 1000 CFU/g or ml. These value should be maintained in the products during in use, in spite of the inevitable contamination by the users through the addition of suitable preservative in the product which guarantees the control of microbial growth even before they are marketed and during their use by consumers {1, 1, 1} Since microorganisms are ever in the home especially in woman and moist areas, cosmetics and toiletries are exposed to contamination with both spoilage and potentially hazardous microorganisms during in use. From the moment the product is opened until the consumer discards it, it is subjected to constant and variable microbial contamination from the domestic environment and the consumer's hands and body fluids {1}. In the present study the effect of consumer handling and time on the microbial levels of the tested cream samples (showing no detected contamination) is illustrated in (Table VII). The level of contamination through days, due to share use, was found to increase with time and during use. After days of use the bacterial contamination of foundation cream, foundation moisturizing cream and moisturizing cream reach to 1.x10-8 CFU/mL with gram positive rods;.0x10 7 CFU/mL with gram positive rods and gram positive cocci; and.0x10 7 with gram positive rods and gram positive cocci, respectively. On the other hand the level of contamination with fungi reaches to 7.x10 with Penicillium species and Aspergillus species;.x10 with Aspergillus species; and.x10 7 with Aspergillus species, respectively. The presented data are consistent with those obtained by other authors Dawson et al. (1981) {17} indicated that the microorganisms recovered from the cosmetic eye make up were mainly representative of the normal skin flora and early born contaminants. They stated that most contamination was probably introduced into the cosmetics by the frequent and common use of fingers and multiple use applicators. Misilivec et al. (199) {18} studied the potential health risk from shared use cosmetic caused by microorganisms. They found that fungi were present in 10.% of products. Orth and Kobara (1998) {19} reported that adequately preserved cosmetic and drug products may become contaminated if they are diluted or repeatedly exposed to microorganisms during use. On the other hand, EL-Bazza et al. (009) {} reported that the contamination of cosmetic eye make up-samples increase during use. After 8 days of consumption the bacterial contamination level reach to 7x10 to 8x10 CFU/mL. CONCLUSION The real problem was not only the heavy contamination on the cosmetic cream samples but also, the contamination with pathogens. The consumers may play and important role in contaminating their cosmetic cream samples during in-use. Cosmetic cream samples were generally found to be contaminated (intact and in-use) with (and, or) gram positive cocci, gram positive rods, gram negative cocci, gram negative rods, Aspergillus species and Penicillium species. REFERENCES 1- Okeke, I. N. and La mikanara, A. Bacteriological of skin moisturizing creams and lotions distributed in a tropical developing country. J. Appl Microbiol., 001; 91:pp Hugbo, Peter G.; Onyekwelli, O. Anthony; and Igwe, Ijoma. "Microbial contamination and preservative capacity of some brands of cosmetic creams." Tropical journal of Pharmaceutical research., 00; : pp Orth, D. S.; Dumatol C. and House organisnlszia, S. "Dealing with the "bug in the plant", Cosm & Toil, 199; 111: pp. 9- and pp El-Bazza, Z. E.; EL-Tablawy, S. Y.; Hashem, A. E. and Nasser, H. H. Evaluation of the microbial contamination of some Eye-make up products before and after use. Biohealth Science Bulletin Malysia., 009; 1: pp Cowan, S. T. and Steel, K.J. Manual for the identification of medical bacteria., 198; Cambridge University press, London, U.K. - Moubasher, A. H. Soil fungi on Qatar and other arab countries. Published by the center for scientific and Applied Research University of Qatar. 199; The Daha modern printed press. 7- Manual of clinical microbiology Eighth Edition by American Society for Microbiology., 001; (Corporate Author), Patrick R.; Ph.D. Murray; Baron Ellen Jo ; James H. Jorgensen; Pfaller Michael A. and Yolken Robert, H. 8- Ballereau, F.; Prazack, T.; Schrive, I.; Lafleuriel, M.; Rozec, D.; Fisch, A. and Lafaix, C. Stability of 9

4 Biohealth Science Bulletin 011, (), 7 Study of the Microbial Contamination essential drugs in the field results of a study conducted over a two-year period in Burkina Faso. Am. J. Trop. Med. Hyg., 1997; 7: pp Rosas, I.; Salinas, E.; Yela, A.; Glava, E.; Elsava, C. and Cravioto, A. Escherichia coli in settled-dust and air samples collected in residential environments in Mexico City. Appl. Environ. Microbiol., 1997; : pp Campana R., Scesa C.; Patrone V.; Vittoria E. and Baffone, W. "Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems". Lett. Appl. Microbiol., 00; : pp Becks, V. and Lorenzoni, N. Pseudomonas aeruginosa outbreak in a neontal intensive care unit: a possible link to contaminated hand lotion.am T Infect control, 199; : pp Beheravan, J.; Bazzaz, F and Malaekeh, P. "Survey of bacteriological contamination of cosmetic creams in Iran (000)." Int. J. Dermatol., 00; : pp FDA. Food and drug Administration, Bacteriological analytical manual 8 th edition Revision A, 1998" "Chapter " By: Tran, T. T.; Hitichins, A. D. and McCarron, E. J., Food and Drug Administration, AOAC International, Arlington, VA. (001). 1- Farrington, J. K.; Martz, E. L.; Wells, S. J.; Ennis, C. C.; Holder, J.; Levchuk, J.W.; Avis, K. E. and Hoffman, P. S. "Ability of laboratory methods to predict in-use efficacy of antimicrobial preservatives in an experimental cosmetic". Appl. Environ. Microbiol., 199; 0: pp Linter, K. and Genet, V. "A physical method for the preservation of cosmetic products". Int. J. Cosmet. Sci., 1998; 0: pp Perry, Brian. Microbiology Today" Cosmetics microbiology" 001; 8: pp Dawson N. L. and Rheinhartdt D. J. "Microbial flora of in use, display eye shadow testers and bacterial challenges or unused eye shadows" Appl. Environ. Microbiol.,1981; : pp Misilivec, P. B.; Bandler, R. and Allen, G. Incidence of fungi in shared used cosmetics available to the public., J. AOAC Fnt., 199; 7: pp Orth, D. S. and Kobara, J. J. Preservative-free and self-preserving cosmetics and drugs. Cosm & Tiol., 1998; 11: pp Table I: Cosmetic cream samples used in the present investigations. Cosmetic cream Brand code Number of samples Foundation cream Total F.C. 1 Foundation moisturizing cream Total F.M.C moisturizing cream Total M.C. 1 1 Total Types 10 0 Samples F.C.: foundation cream F. M. C.: foundation moisturizing cream M.C.: moisturizing cream A B C D E F G H I J 0

5 Biohealth Science Bulletin 011, (), 7 EL-Bazza Z E et al. 011 Table II: Percent of the microbial contamination in the tested cosmetic creams of different brands. Cream Type brand No. of *S. No. of **C.S. % of C.S Foundation cream A B C D E Total 1 8 Foundation moisturizing cream F G Total 10 0 moisturizing cream H I J Total 1.7 *S: samples. **C.S.: contaminated samples Table III: Total microbial counts and number of microbial isolates of the tested cream samples of different brands. Type of cream Foundation cream Foundation moisturizing cream Moisturizing cream Brand A B C D F G H I No. of C.S Bacteria Fungi L.C. (CFU/ml. No. is L.C. (CFU/ml) No. is.7x10-1.9x x10,.x10.0x10,.x x10 1.0x10-8.x10.x10.x x10 7,.0x x10, 1.x x10-1.x10.x10, 8.x x10,.0x x10 7.0x10 1.x10.0x10 9.x10,.1x10 1.x10, 1.8x10 Total C.S.: contaminated samples L.C.: level of contamination CFU: colony forming unit No. is: number of isolates 1

6 Biohealth Science Bulletin 011, (), 7 Study of the Microbial Contamination Table IV: Survey on the microbial contamination of the tested cosmetic cream samples of different brands. Type F. cream F. M cream M. cream Brands Samples 10 1 C.S.B (%) L.C.B. (cfu/ml) 1.0 x x x x x x 10 8 C.S. F (%) L.C.F (cfu/ml) 1. x 10-1.x 10 1.x10 7x10 9.x10 -.1x10 S with B & F (%) NCS (%) CSB: contaminated samples with bacteria LCB: level of contamination with bacteria CSF: contaminated samples with fungi LCF: level of contamination with fungi NCS: non contaminated samples CFU: colony forming units Table V: Evaluation of the bacteria contaminating each type of the tested cosmetic creams. Cream type No. of C.S. Total No. of bacteria Foundation cream 10 Foundation moisturizing cream 8 moisturizing cream 10 Identification No. of B.C. Percent (%) + ve cocci ve rods ve cocci 1 - ve rods 1 + ve cocci + ve rods. - ve rods ve cocci ve rods ve rods 0 C.S: contaminated samples B.C.: bacterial contaminants

7 Biohealth Science Bulletin 011, (), 7 EL-Bazza Z E et al. 011 Table VI: Evaluation of the fungi contaminating each type of the tested cosmetic creams. Cream Type No of C. S. Total No. of fungi Identification No. of F.C. Percent (%) Foundation cream 9 19 Foundation moisturizing cream 10 Aspergillus. sp Penecillium.sp Aspergillus. sp Penecillium.sp Moisturizing cream C.S.: contaminated samples F.C.: fungal contaminants Aspergillus. sp Penecillium.sp Table VII: Survey for the microbial isolates on the tested cosmetic cream samples during handling for days. Cosmetic cream Foundation cream Foundation moisturizing cream Moisturizing cream CFU: colony forming units. Brand Bacteria Fungi Sample Count range Count range No. Type Type CFU/ml CFU/ml B 9.x10-1.x10 G + ve rods 1x x10 Penecillum sp. D 18 x10 1.1x10 7 G + ve rods.0x x10 Aspergillus sp. E 1.0x10 1.x10 8 G + ve rods 1.x10 1 Aspergillus sp. -7.x10 Penecillum sp. G 1 x10 7 G + ve rods x10 G + ve cocci 0.x10 -.x10 Aspergillus sp. H 0.x10 1x10 7 G + ve rods 1.0x x10 7 Aspergillus sp. I 1.0x10.0x10 7 G + ve cocci 0.x x10 Aspergillus sp.