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Efficacy of Antimicrobial Preservatives

Abstract.
The aim of the experiment is to test the efficacy of antimicrobial preservatives by means of investigating the survival of one selected species of micro-organism which is deliberately introduced into the tested products.
Introduction.
Cosmetic products are not expected to be sterile as the raw materials used in their production, especially those originating from plants, animals or soil are often contaminated with micro-organisms. The extent of microbial contamination depends largely on the handling of bulk ingredients during manufacturing as well as on maintaining the hygienic conditions during storage and distribution (Dashen et al 2011; Kallinngs et al 1996). The Good Manufacturing Practice (GMP) is essential to minimize the intrinsic bacterial contamination during the production stage but low level of microbial contamination in cosmetic products has to be guaranteed also during consumer use when, in addition to the bacteria which might have been already present in a cosmetic product, the bacteria introduced by the consumer from their hands, saliva or from tap water or air may flourish, especially in humid and warm conditions of hot bathroom or shower room, ideal for the bacteria to grow (Rodford 1997). Microbial quality of cosmetics and toiletries were examined in countries with moderate climate (Malcolm 1976; Brannan and Dille 1990) as well as in tropical countries (Okore 1992; Okeke and Lamikanra 2001), often as a result of an outbreak of infectious disease (Becks and Lorenzoni 1995; Itin et al 1998).
Isolation of various pathogenic micro-organisms from cosmetics has also been reported by Anelich and Korsten (1996) and Morse et al (1967).
Most micro-organisms of clinical significance belong to Klebsiella, Enterobacter, Serratia and Pseudomonas ssp. and are ‘free-living’ gram negative bacilli. They have simple nutritional requirements and can multiply in an ambient temperature and have great ability to survive even in the presence of commonly used disinfectant and bacteriostatic agents (Parker 1972). Microbes in cosmetic products may not only be infectious agents as such but also cause spoilage or breakdown of the product by producing a gas, odour or discoloration, making the product unacceptable to consumer (Lalitha and Prasadarao 2014). Therefore, the presence of preservatives is usually necessary and often also required by regulators ( )
Preservatives are added to cosmetic products to extend their shelf-life and protect the health of the consumer but preservatives are biologically active agents which may pose a risk to consumer themselves (Rodford 1997). Therefore, it is essential to use the most appropriate preservative or their blend for the specific application when broader spectrum of antimicrobial activity is required and also maintain the lowest possible Minimal Inhibitory Concentratiom (MIC) that will inhibit the growth of the micro-organisms.
The micro-organisms present in cosmetic and pharmaceutical products
The aim of the experiment is to test the efficacy of antimicrobial preservatives by means of investigating the survival of one selected species of micro-organism which is deliberately introduced into the tested products.
This method is known as ‘challenge testing’ and consist of series of repeated inoculations of appropriate dilutions of tested sample of cosmetic or pharmaceutical product and performing the viable counts of micro-organisms which survived the application of the preservatives, efficaccy of which is the subject of the laboratory procedure.
Some experiments performed to evaluate the correlation of in vitro ‘challenge testing’ with consumer use testing established that it is a good predictor of the risk of consumer contamination of cosmetic product, therefore the in vitro methods may be regarded as reliable means to provide the accurate data, consistent with ‘real-life’ scenarios (Smart and Spooner 1972; Ludlov et al 2011; Baird 1977).
Maillard et al (2013) investigated the problem of increasing antimicrobial resistance due to use of microbicides and warned against indiscriminate use and overuse of preservatives in commercial domestic products. Periame et al (2014) studied Enterobacter gergoviae as an example of bacterial adaptation to preservatives commonly used in cosmetic industry. Their research suggests that the maximum allowed concentrations of preservatives permitted by regulators are often insufficient to control proliferation of bacterial pathogens in cosmetic products.
Indicator organism is an organism, the presence of which is an evidence of contamination originating from particular source. It is not practical to test for every possible pathogen which may be present , therefore some typical organisms were chosen as the indicators and their presence suggests the most likely contaminant of particular material or product. Escherichia coli inhabits the gastrointestinal tract of humans and animals and is, therefore, routinely used as an indicator organism tested for contamination of water with human or animal faeces (Madigan et al 2014). Salmonella spp., Staphylococcus aureus and Candida albicans are also used as bacterial indicator of water polluted with sewage (Efstratiou et al 1997).
Various special media are used in order to culture and isolate particular group or species of bacteria. Selective media either favour the growth of particular type of bacteria or repress the growth of undesirable species of micro-organisms. Differential media are the media facilitating and speeding up the visual identification of bacterial species often indicated by a colour change or characteristic appearance of colonies or surrounding media. TSA (Tripticase Soy Agar) is
Cosmetic products should be safe to use during its shelf-life and when used by the consumer i.e. when the original package is repeatedly opened allowing the possibility of microbial contamination of previously factory-sealed container. It is therefore, usually mandatory and required by regulations to add a preservative to a cosmetic product to eliminate the health risk the end user could be exposed to and guarantee the maximum efficacy of the product throught its shelf-life. The effective preservative added to a cosmetic product has to play a dual role: protect the factory product and also eliminate the contamination introduced by an actual consumer after the purchase.
Methods.
Students were working in a group of two following the procedures as detailed in PHCO 2304 Pharmaceutical Microbiology Practical Manual 2014/2015 [4] and British Pharmacopoeia 2015 [5] (Appendix XVI B. Microbiological Examination of Non-sterile Products 1) with some modifications necessary to adjust to group’s timetable.
In order to obtain a concentration of approximately 1×106 cfu/g of cream for the tests the suspension of Staphylococcus aureus of about 108 was diluted and each of the two tested creams as well as control buffer were inoculated with appropriate volume of microbial suspension. A series of 1 in 100 and/or 1 in 10 dilutions was prepared to assess the exact starting concentration of viable cells. The most suitable dilutions were subsequently chosen and the dilutions were adjusted when necessary to provide the most convenient range of likely numbers of cfu on the spread plates used in further proceedings.
To carry out the viable count the appropriately diluted bacterial suspensios were inoculated in duplicate each week and incubated at room tamperature till the next week.
To calculate the number of viable count in controlled suspension, the number of colonies on the spread plates was multiplied by the dilution factor.
In weeks 2-5 the viable count was performed on creams A, B and C using the same method as in previous week.
Results.
Table1. Viable counts of the microorganisms in tested creams

10-2 10-3 10-5 Original concentration
cream cfu cfu cfu
Week 3 A1 9 0 6
A2 TNTC 2 1
B1 6 4 0
B2 2 1 5
C1 TNTC TNTC 20
C2 TNTC 40 10
10-2 10-4 10-6
cream cfu cfu cfu
Week 4 A1 4 0 6
A2 11 6 1
B1 8 4 0
B2 1 2 2
C1 TNTC 36 0
C2 TNTC 30 1
10-2 10-4 10-6
cream cfu cfu cfu
Week 5 A1 3 1 1
A2 2 0 0
B1 3 2 2
B2 1 2 12
C1 TNTC TNTC 92
C2 TNTC TNTC 90
10-2 10-3 10-5
cfu cfu cfu
Week 6 A1 36 5 10
A2 35 8 4
B1 2 1 1
B2 11 1 1
C1 TNTC TNTC TNTC
C2 TNTC TNTC TNTC

Key: A1,A2 – tested cream A, B1,B2 – tested cream B, C1,C2 control
Discussion
References:
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