MICROBIOLOGICAL EVALUATION OF SELECTED CREAMS IN BENIN CITY

TABLE OF CONTENTS

Title …………………………………………………………………………..i

Certification ………………………………………………………………….ii

Dedication……………………………………………………………………..iii

Acknowledgement……………………………………………………………iv

Table of content…………………………………….........................................v

Abstract………………………………………………………………………viii

CHAPTER ONE

Introduction and literature review…………………………………….1

Contamination of creams………………………………………2

Microbial spoilage of topical preparations…………………….3

Manisfestation and mechanism of microbial spoilage…………4

Isolation of contaminants in pure culture…………………….15

Identification of contaminants…………………………17

Quality control of topical pharmaceutical products………….19

Justification of this study….………………………………….21

Objectives of the study…….………………………………….21

CHAPTER TWO

Experimental materials……………………………………………22

Bacteriological and fungal media……………………22

Equipment……………………………………………23

Methods ……………. ………………………..…………….24

Collection of samples .………………..…………….24

Preparation of sample…..……………………………24

2.2.3Sterilization of glasswares….……………………………24

Qualitative determination of micro-organism

in topical preparations…………...…………………………...25

Quantitative determination of micro-organism in 

topical preparation per given quantity………….…………….25

Identification of specific organism present……….………….26

CHAPTER THREE

Results………………………………………………………………..29

CHAPTER FOUR

Discussions…………………………………………………………...35

CHAPTER FIVE

Conclusion and Recommendations……………………………………..38

References……………………………………………………………….40

Appendix…………………………………………………………………42

LIST OF TABLES

Table I : characterization of the pharmaceutical products used for 

the experiment…………………………………………………………29

table II : density/mL of product of viable organisms………………………….…32

table III : identification of fungi on Saboraudagar………………………………33

table IV : identification of bacterial on nutrient agar and 

MacConkey agar plates…………………………………………………34

table V : characterization and identification of isolated bacterial

colonies on nutrient agar……………………………………………..…42

table VI : characterization and identification of isolated bacteria

colonies on MacConkey agar………………………………………….44

ABSTRACT

Creams need not be sterile,however they must not be unduly contaminated with micro-organisms and should remain in a stable state throughout the shelf life of the product (or when being used by the consumer).Samples of creams were randomly selected in Benin city and dilutions were made. The dilutions were cultured in agar medium plates for presence of contaminants

Bacterial and fungal growths were observed. The bacterial observed were mainly of Staphylococcus spp. and Enterobacteriaceae. Fungi isolated were mainly Mucor,Penicilliumand Tricophytonspecies. Total viable bacterial count was within the range of 12x104 to 37x104.Total yeast and mould counts were within the range of 4x104 to 14x104. 

Staphylococcus spp was the most predominant bacterial Contaminant while Penicillium was the most predominant fungal contaminant observed.

CHAPTER ONE

1.0INTRODUCTION AND LITERATURE REVIEW

Creamsare those preparations which are applied to skin for the purpose of maintaining the appearance of the skin. They are used in some other cases for treatment purposes. Their composition can range from simple ingredients to an array of complex substances.( Hugo et.al., 1998) 

Creams can be said to be topical preparations intended to be applied to/on the human body for cleaning or improving the appearance of the skin without negatively affecting the dermatological properties or structure of the skin. The warm and rather humid climatic conditions that prevail in most tropical countries including Nigeria would tend to support the survival and growth of many micro-organisms. In a situation whereby a nutritionally rich pharmaceutical/cosmetic product is severely contaminated, rapid growth and multiplication of micro-organisms would be expected. Contaminating micro-organisms in creams may cause spoilage of the produce and, when pathogenic, they represent a serious health risk for consumers worldwide (Becks and Lorenzoni, 1995;Behravanet al., 2005).

Due to their complex composition of substances, preservation of these preparations against microbial spoilage involves protection of the large number of different constituents with variable physical and chemicals properties.

Products contamination may arise from raw materials or water used in the formulation process or accidentally during use.

1.1 CONTAMINATION OF CREAMS AND LOTIONS

The hazards of inadequately preserved creams to human health have been demonstrated by reports of staphylococcal infections from use of contaminated hand creams (Campanaet al., 2006). Regardless of whether a cosmetic becomes contaminated during manufacture or during consumer use, the hazard is usually two fold, namely: 

(1) The Direct effect of micro-organism on human health e.g. allergic reactions, skin irritations and even neurotoxic manifestation;

(2)The Indirect effect also on human health due to production contamination and spoilage as well as formation of harmful/noxious microbial metabolic.

Microbial contamination of cosmetics during manufacture was a major problem in the 1960’s and 1970’s. Since then, microbial contamination is still one of the major causes of product recall on the world, particularly in developing tropical countries (Okeke and Lamikanra 2001). However, significant progress has been made by cosmetic industry towards implementation of sanitary manufacturing practices, more rigorous microbiological control and the development of better preserved cosmetic products. Therefore, it is important to improve the preservation system. (Farrington et al., 1994; inter and Genet 1998) in order to inhibit the growth of contaminating micro-organisms during manufacturing, storage and use by consumers, also by using non-invasive package (Brannan and Dille 1990).

1.2 Microbial Spoilage of Topical Preparations

The formulation of an efficacious, elegant and safe product which will be both stable and acceptable to the consumer may require the use of a variety of ingredients in a complex physical state. This complexity in the composition or constitution of the product could create conditions conducive to the survival of micro-organisms that contaminate the product either during manufacture or consumer use. A topical preparation may be considered microbiologically spoilt if depending on it intended use it possess:

i.low levels of acutely pathogenic micro-organism or higher level of opportunistic pathogen are present

ii.Toxic microbial metabolic persist even after dealth of the original/primary contaminants or 

iii.Microbiological growth has initiated significant physical or chemical deterioration of the product. Such spoilage might result in financial loses for the manufacturer, either in the immediate loss of products or in the increasingly expensive cost of litigation should spoilage cause harm to the end user. 

1.3 Manifestation and Mechanisms of Microbial Spoilage

Before spoilage can occur, organisms which are capable of altering the components of a product in situ must first be introduced via raw materials, the processing plant, packaging materials, operatives or elsewhere in the environment. Although spoilage does not necessarily depend upon the growth of these contaminants it is generally facilitated if the formulation and the ambient conditions of temperature and humidity encourage their multiplication. When these criteria are satisfied changes in the product will occur and may ultimately manifest themselves to the user in one or more of the following ways:

Microbial toxins: 

Several species of micro-organisms produce molecules and may render a product dangerous if they grow in it under conditions supporting toxin production. Endotoxins, produced by Gram-negative bacteria such as Escherichia coli, are intimately bound to the cell, lipopolysaccharide in nature and are not necessarily inactivated by sterilization as they are heat stable. Toxins of this type are poorly absorbed by the oral route but are very important in connection with injectable products, particularly perfusion fluids. Exototins are much more highly lethal and are bound less rigidly to the cell so that they are readily liberated into the growth medium. The outstanding example, of course, is that produced by Clostridium botulinumwhich is lethal to mice in doses of the order of 0.1ng. Fortunately, conditions for growth and toxin production are quite strict; anaerobiosis, the presence of suitable pH and nutrients and of few competing bacteria is required. Such conditions are not often attained in pharmaceuticals and cosmetics and we know of no case of botulism arising from their use. Certain strains of staphylococcusaureus produce a toxin, characterized as a specific polysaccharide, but the organism must grow to a density of several million cells per gram before its toxin becomes a problem. The evidence in connection with other bacterial, e.gClostridium perfringens, Bacillus cereus, streptococeusfaecalis, proteus and pseudomonas species is less clear, but poisonous metabolites are certainly produced by a variety of fungi. Over the last decade there has been much interest shown in the afflatoxins produced by Aspergillusflavus(Barnes,1970). These heat-stable compounds exhibit potent toxic and carcinogenic properties in animals. A. flavuscommonly infects peanuts, cotton seed and grain which are all components of animal foods. Under poor storage conditions mould growth occurs and toxic doses of aflatoxin accumulate in the food stuff.While it is difficult to visualize this occurring with cosmetics or pharmaceuticals, it is wise to ensure that ingredients such as talc, kaolin or starch are not stored for long periods under conditions supporting mould growth.

Metabolic Products:

In addition to microbial toxins, which are complex molecules and may be looked upon as biosynthetic products, simpler catabolic products such as organic acids and amines, which can be toxic to man, may be produced. Indeed, many microbial metabolites exhibit pharmacological activity (Perlman and Peruzzotti, 1970). As these compounds are considerably less toxic than are the classic bacterial toxins, relatively high concentrations have to be attained before a spoiled product causes illness and the sense often detect that something is wrong before food spoiled to this extent is swallowed. This may not apply to medicines, as they are expected to be unpleasant and, indeed, frequently contain a flavouring agent in order to mask an unpleasant taste. However, well-documented examples incriminating specific metabolic products in pharmaceuticals are not easy to find.

Irritancy:

Incidents of irritation following the application of cosmetics occasionally occur, and the offending preparation may subsequently be shown to contain a high level of microbial contamination. Direct evidence that irritation is caused by the presence of the micro-organisms is lacking but it is reasonable to suppose that, on some occasions, the contaminants provide a source of foreign protein evoking an allergic contact dermatitis reaction or that high levels of a microbial metabolite will cause a primary irritant reaction. The eye, of course, is particularly susceptible to infection from contaminated cosmetics and it is also at risk from the direct effect of irritant metabolites left in a product even after the organisms producing them have been eradicated.

Change of activity:

An interesting aspect, but perhaps not one of great significance, is the inactivation of biologically active molecules by organisms contaminating a formulation. Several examples have now been demonstrated in the laboratory and in some cases have been observed to occur in practice. A classic example is the destruction of penicillins by penicillinases, enzymes produced by a broad range of micro-organisms. Microbial enzymes which inactive chloramphenicol are also known (Smith and Worrel, 1949) and the destruction of preservatives and disinfectants is established (Hugo, 1965). Pharmacologically active substances can also be degraded. For instanceKedzia, Lewon and Wisniewski (Kedzia, et.al.,1961) found that a loss of atropine of up to 20% in eye drop could be caused by Corynebacterium and Pseudomonas spp. isolated from the eye drops and atropine itself. Recently, Grante, de Szors and Wilson (Grant, et.al., 1970) have shown that in the laboratory, a strain of AcinetobacterIwoffi, obtained from distilled water, utilized aspirin as a sole carbon source in a mineral salt solution. The same organism metabolized other active esters; for instance it could degrade heroin to morphine. Another organism, Corynebacteriumhoffnaii, which was isolated from laboratory dust, metabolized analgesics, aspirin, phenacetin and paracetamol.

Loss of useful activity is not restricted to pharmaceutical products. For instance emphasis on the need for detergents which are biodegradable has had some repercussion and shampoos have been known to lose their surface-active properties due to degradation of the surfactants by contaminating bacterial.

Visible Growth: 

When micro-organisms can actually be observed in or on a product then there is obviously no doubt that microbial spoilage has occurred. In fact, this is probably the most common way in which it is manifest. In liquid formulations contaminants may be seen as a sediment, turbidity or a pellicle while on more solid preparations colonies, often coloured, of bacteria, yeasts or mould may form.

Colour changes

Sometimes visible spoilage is more striking, particularly if a colour change is involved. Colour changes due to alternations in the components of a product may result from pH.Redox or other changes caused by the metabolic activities of an organism, or to pigment production by the contaminants themselves. 

Members of the Pseudomonas genus are often implicated in spoilage of this type. These organisms metabolize a very broad range of compounds, and can also produce soluble pigments ranging in colours from blue-green to brown. In addition, they can render conditions suitable for less adaptable spoilage organisms; for example they can create conditions favouring the growth of anaerobes. Similarly, in an acidic product, oxidative yeasts can cause a rise in pH by utilizing organic acids and this will encourage bacterial growth.

Gas production

If microbial metabolism produces gas in a sufficient amount to exceed its solubility in a product, visible bubbles, frothing and other manifestations of an increase in pressure occur. Products containing carbohydrates or other fermentable substrates are particularly susceptible to this type of spoilage. Of the latter, glycerol, an essential ingredient in many cosmetic preparations, is fermented particularly readily by some common waterborne organisms.

Other changes

Microbial metabolism can result in the composition of a homogeneous product becoming visibly heterogeneous. Emulsions, for instance, are notoriously susceptible to changes in physicochemical conditions; hydrolysis of the oil phase or changes in the pH of the aqueous phase will upset the equilibrium and thus cause visible changes. In liquid product changes of viscosity can be seen to occur when contaminants have broken down large molecules, utilized sugars or caused the aggregation of particles in suspension.

Olfactory effects

It has long been known that many micro-organisms produce characteristicodours and as early as 1923 a variety of aroma producing bacteria had been listed (Omelianski,1923). These aromas include the highly characteristic ones of sulphur-containing metabolites such as hydrogen sulphide, the sickly smells of the fatty acids, the ‘fishy’ odours of the amines and the astringency of ammonia. Often these are combined to produce the ‘off’ odours of a spoiled product. Changes in the aroma of a product due to contaminants vary from the production of a nauseating smell to a slight change in the bouquet but all can be disastrous, particularly to cosmetic and toiletry preparations which depend so much upon their specific perfumes. One of the most common olfactory warnings of spoilage is the typical smell of mould. The responsible aromatic elements have not been clearly identified but some actinomycetes which taint water with undesirable earthy odours have recently been shown to produce goesmin, a strongly earth-smelling, neutral oil (Gerber, and Lechevalier,.1965). An alcoholic odour, produced from fermentable substrates, is typical of spoilage by yeast.

Taste

Reports that products taste ‘peculiar’ are often the first indications that they may be spoiled. The sense of taste varies widely between individuals and these reports do not invariably indicate microbial contamination. For this reason, and because of the hazards involved, taste is not a practicable control procedure with which to detect spoilage at an early stage. Nevertheless, the combined sense of smell and taste are highly perceptive to changes in flavor, particularly in bland, unflavoured, preparations where the presence of microbial metabolites is not masked. Margalith and Schwartz (Margalith, and Schwartz, 1970) have listed over 100 organic compounds involved in the production of flavor by micro-organisms. These consist mainly of alcohols, aldehydes, ketones, acetals, acids, amines, esters and phenols.

Texture 

The feel of topical preparations, particularly cosmetic and toiletry ones, is vital to their acceptability but texture may be marred by contaminants. For instance, creams can become lumpy or ‘gritty’ and changes in viscosity of liquid preparations, which can be detected when applied to the skin, may occur.

Audible effects

Apart from immediate manifestations of toxicity, which happily appear to be rare, audible manifestations of spoilage are the most dramatic. If visible effects of spoilage are obscured by the pack, an explosion can be the first indication that a gas-producing micro-organism has successfully adapted itself to what may have been considered inimical conditions. 

Types of Susceptible Product

The range and composition of pharmaceutical and cosmetic products is so varied and the species, and even strains, of micro-organism capable of causing spoilage are so multifarious that, as we have already emphasized, each spoilage incident tends to be unique. Generalizations about susceptible products are therefore likely to be inaccurate and are made more difficult today because of the inclusion, particularly in cosmetics, of increasingly sophisticated and often highly biodegradable ingredients. Nevertheless, some types of product are more susceptible than others to spoilage by specific organisms and those of which we have experience are described below.

Liquids 

Water:

Water is a major constituent of living material and participates in many metabolic reactions. Bacteria, in particular, require high concentrations of water in their immediate environment and may be regarded as aquatic organisms. Hence, all products containing large amounts of free water can be particularly susceptible to spoilage by bacteria.

Water supplied by water undertakings in this country is of high micro-biological quality and is generally suitable for the manufacture of pharmaceuticals and cosmetics. Low-conductivity water, whether prepared by distillation or deionization, may be chemically purer but can constitute a greater microbiological hazard. Distilled water leaving the still can readily pick up organisms from pipes and tubing and ion-exchange columns may actually serve as a reservoir of organisms because nutrient organic residues are not removed by the process. Without effective treatment to minimize contamination, water can, within a few days, contain large numbers of initially Gram-negative and Gram-positive bacteria, and subsequently a wide variety of bacterial, moulds and yeasts. At this stage, visible and olfactory spoilage occurs and a foul taste may develop. Indeed, contaminated deionized water has often been incriminated as the original source of spoilage in a formulation. Often the responsible organism arepesudomonas which are not only highly resistant to preservatives but are also able to use the widest range of organic compounds as substrates.

1.4 Isolation of Contaminant in Pure Culture

In order to study the properties of a given organism, it is necessary to handle it in pure culture free of all other types of organism by isolating a single cell colony from all others and cultivated in such a manner that is collectively progeny also remain isolated.

Since in the majority of cases, the organism to be isolated is part of a mixed population, the isolation of a single species from the environment is crucial. The isolation of single species from a mixture of micro-organisms is called the pure culture technique. Two methods are routinely used in the laboratory to obtain pure cultures, each can be enhanced by isolation of selective media if appropriate. They include:

(a)Pour Plate Method

In this method, the mixed population of micro-organism is first diluted and a small quantity is transferred to the bottom of a sterile petri-dish. Melted agar is then poured into the petri-dish to cover the micro-organisms and gently tilted to ensure uniform distribution of organism. Colonies will develop on the surface as well as within the Agar.

(b)Streak Plate Method

In the streak method, a small quantity of the mixed bacteria population in a specimen is streaked directly on the surface of an already set agar medium. The streaking is done with a wire loop. The procedure thins out the microbial population on the surface of the Agar, and where an organism has been deposited, a colony will develop after a suitable incubation. From one of these colonies is streaked onto the surface of a second plate and the colonies that develop there are also examined. This subculture procedure ensures that only one kind of micro-organism was present in the initially isolated colony

1.4.1Identification of Contaminants (Micro-organism)

The various metabolic activities of bacteria and their responses to immediate environmental changes have been explored in the design of special diagnostic and selective media.

Such media are used in public health laboratories and hospitals for identifying organisms found in samples believed to be contaminated by them and as an aid to their diagnosis and treatment. 

Micro-organisms have specific shapes and structures that can be identified and characterized. The following are some of the techniques used in the identification of micro-organism in the laboratory.

(a)Examination of stained preparations: for example Gram-staining procedure: This is one of the first important identification procedures after isolation of colonies and enhances the divison of the bacteria world to at least four (4) categories, namely:

i.Gram Positive cocci

ii.Gram Positive bacilli

iii.Gram Negative cocci

iv.Gram Negative bacilli

(b)Examination of biochemical characteristics, for example: 

Catalase Test

In some micro-organism, the transfer of hydrogen to oxygen in respiration results